WO2019149115A1 - 核酸适配体在识别并结合碱性磷酸酶异源二聚体或肿瘤检测中的应用 - Google Patents

核酸适配体在识别并结合碱性磷酸酶异源二聚体或肿瘤检测中的应用 Download PDF

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WO2019149115A1
WO2019149115A1 PCT/CN2019/072749 CN2019072749W WO2019149115A1 WO 2019149115 A1 WO2019149115 A1 WO 2019149115A1 CN 2019072749 W CN2019072749 W CN 2019072749W WO 2019149115 A1 WO2019149115 A1 WO 2019149115A1
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alkaline phosphatase
nucleic acid
acid aptamer
sample
tested
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PCT/CN2019/072749
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French (fr)
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上官棣华
邴涛
沈璐瑶
刘祥军
张楠
汪俊彦
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中国科学院化学研究所
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Priority claimed from CN201810105373.3A external-priority patent/CN109554369B/zh
Priority claimed from CN201910001280.0A external-priority patent/CN109682973B/zh
Application filed by 中国科学院化学研究所 filed Critical 中国科学院化学研究所
Priority to US16/967,102 priority Critical patent/US20200362349A1/en
Publication of WO2019149115A1 publication Critical patent/WO2019149115A1/zh

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    • C12N2310/00—Structure or type of the nucleic acid
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    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/914—Hydrolases (3)
    • G01N2333/916—Hydrolases (3) acting on ester bonds (3.1), e.g. phosphatases (3.1.3), phospholipases C or phospholipases D (3.1.4)

Definitions

  • the invention belongs to the technical field of biotechnology and clinical medicine, and particularly relates to the application of a nucleic acid aptamer in recognizing and binding an alkaline phosphatase heterodimer or tumor detection.
  • a aptamers are a class of single-stranded DNA, RNA, peptide nucleic acids or chemically modified nucleic acid sequences that specifically interact with a target substance, usually consisting of 15-80 nucleotides.
  • the nucleic acid aptamer can form a specific three-dimensional structure with high affinity binding to the target molecule, such as hairpin, pseudoknot, G-quadruplex, etc.
  • the high specific binding is through van der Waals force, hydrogen bonding, electrostatic interaction and hydrophobic interaction. And other intermolecular interactions are realized.
  • Nucleic acid aptamers are called "chemical antibodies” because of their high affinity, good specificity, non-immunogenicity, easy synthesis, modification and modification, good biochemical stability, reversibility and renaturation.
  • Nucleic acid aptamers can be used in the diagnosis and detection of some diseases, drug target localization, new drug development and transportation-related drug molecules.
  • nucleic acid aptamers for the treatment of diseases such as cancer and AIDS are also emerging.
  • a VEGF-targeting nucleic acid aptamer (trade name Macugen) developed by Eyetch/Pfizer has been approved by the FDA in 2004 for successful treatment of age-related macular degeneration.
  • the method for screening specific nucleic acid aptamers by using cell-SELEX technology, and then discovering tumor markers has a good application prospect.
  • the bottleneck problem lies in the purification/identification of nucleic acid aptamer target molecules located on the cell membrane.
  • Alkaline phosphatase is an enzyme widely distributed in the liver, bones, intestines, kidneys and placenta of the human body through the liver to the outside of the gallbladder. It can directly participate in phosphorus metabolism, in the digestion and absorption of calcium and phosphorus. The process of secretion and ossification plays an important role. This enzyme catalyzes the removal of the 5' phosphate group by the nucleic acid molecule, thereby converting the 5'-P terminus of the DNA or RNA fragment to the 5'-OH terminus. But it is not a single enzyme, but a group of isozymes.
  • Human isozymes are currently known to include: tissue non-specific alkaline phosphatase (TNAP), intestinal alkaline phosphatase (IAP), placental alkaline phosphatase (PALP) and placental alkaline phosphatase (GCAP). .
  • Serum alkaline phosphatase The alkaline phosphatase in normal human serum is mainly from the liver and bone.
  • the alkaline phosphatase assay is mainly used to diagnose hepatobiliary and skeletal diseases. It reflects extrahepatic biliary obstruction, intrahepatic space-occupying lesions and rickets. Important indicator.
  • Alkaline phosphatase heterodimer is highly expressed in tumor tissues such as colorectal cancer, breast cancer, hepatocellular carcinoma, and cervical cancer. Free alkaline phosphatase heterodimers, exosomes containing alkaline phosphatase heterodimers or circulating tumor cells can be released from the primary tumor or metastatic lesion into the bloodstream. Therefore, detection of free alkaline phosphatase heterodimers, exosomes or circulating tumor cells will help early diagnosis and screening of tumors, monitoring tumor recurrence and metastasis in postoperative patients, and assessing the sensitivity of antitumor drugs. Sex and patient prognosis and strategies for selecting individualized treatments.
  • Circulating tumor cells refer to tumor cells that spread during the development of malignant tumors and survive in peripheral blood, which are closely related to tumor metastasis and prognosis.
  • Circulating tumor cell detection refers to the method of analyzing circulating tumor cells in peripheral blood of tumor patients. Detection of circulating tumor cells in peripheral blood is the most direct and important method for predicting tumor metastasis. Clinical diagnosis and prognosis of early metastasis of tumors It is of great significance to judge and monitor the efficacy. The discovery of circulating tumor cells is expected to change the current state of clinical dependence on imaging and traditional tumor markers. Since the number of circulating tumor cells in the peripheral blood is extremely rare, extremely high requirements are placed on the sensitivity and selectivity of the detection technique.
  • the circulating tumor cells are separated and enriched by density gradient centrifugation, cell filtration or adhesion technology, immunomagnetic bead separation technology and microfluidic chip technology, and then subjected to immunocytochemistry. Circulating tumor cells were detected by techniques, reverse transcriptase polymerase chain reaction, and flow cytometry.
  • FDA US Food and Drug Administration
  • the exosomes are a nano-scale lipid inclusion structure with a diameter of 30-100 nm, which is surrounded by proteins, mRNA and microRNA. Almost all types of cells, including tumor cells, can produce and release exosomes. Exosomes are secreted by cells, spread in body fluids such as blood, and finally phagocytized by other cells, and are important mediators of cell-to-cell communication. More and more studies have found that exosomes secreted by host cells or tumor cells are involved in tumorigenesis, growth, invasion and metastasis, so the detection and research of exosomes have received more and more attention.
  • the nucleic acid aptamer or derivative thereof provided by the present invention is any one of the following 1) to 7):
  • nucleic acid aptamer shown in 1) 2) deleting or adding one or several nucleotides to the nucleic acid aptamer shown in 1) to obtain a derivative of a nucleic acid aptamer having the same function as the nucleic acid aptamer;
  • nucleic acid aptamer shown in 1) subjecting the nucleic acid aptamer shown in 1) to nucleotide substitution or modification to obtain a derivative of a nucleic acid aptamer having the same function as the nucleic acid aptamer;
  • the skeleton of the nucleic acid aptamer shown in 1) (refers to the stem formed by the 1st-7 and 37-44 nucleic acids of SEQ ID NO: 1) is transformed into a phosphorothioate skeleton to obtain an aptamer with the nucleic acid a derivative of a nucleic acid aptamer having the same function;
  • RNA molecule encoded by the nucleic acid aptamer shown in 1) to obtain a nucleic acid aptamer derivative having the same function as the nucleic acid aptamer;
  • a peptide nucleic acid encoded by the nucleic acid aptamer shown in 1) which gives a derivative of a nucleic acid aptamer having the same function as the nucleic acid aptamer;
  • nucleic acid aptamers shown in 1) to 6) indirectly or indirectly with a signal molecule and/or an active molecule and/or a functional group and/or a radionuclide to obtain an aptamer with the nucleic acid A derivative of a nucleic acid aptamer having the same function.
  • the derivative of the nucleic acid aptamer is for removing or changing the nucleotide sequence of the nucleic acid aptamer shown in SEQ ID NO: 1 from the first nucleotide of the 5' end, including the first nucleotide residue at the 5' end.
  • the nucleotide sequence of the nucleic acid aptamer shown in the removal sequence 1 includes the first nucleotide residue at the 3' end from the 1st nucleotide of the 3' end 1-7 nucleotides; or the derivative of the nucleic acid aptamer is a nucleotide or modification added to the 5' or 3' end of the nucleotide sequence of the nucleic acid aptamer shown in SEQ ID NO: 1.
  • the group does not affect the structure formed by the 10th to 36th positions of the sequence 1 (G-quadruplex structure), and the nucleic acid aptamer composed of the remaining nucleotide residues.
  • the derivative of the nucleic acid aptamer is any one of the following 1) to 6):
  • the nucleic acid aptamer derivative is a fluorescent group, a biotin group or a radionuclide labeled at the 5' end or the 3' end of the nucleic acid aptamer shown in any of 1) to 6).
  • nucleic acid aptamer or derivative thereof in at least one of, for example, 1) to 4) is also within the scope of protection of the present invention:
  • the target to be detected or diagnosed or extracted or captured is alkaline phosphatase itself, alkaline phosphatase heterodimer, cells containing alkaline phosphatase or a heterodimer thereof, or alkaline phosphatase or An exosome of a heterodimer, a tissue section containing an alkaline phosphatase or a heterodimer thereof, and an animal living body containing an alkaline phosphatase or a heterodimer.
  • the sample to be detected or diagnosed is whole blood, serum, culture solution, saliva, urine, tissue section or living body;
  • the detection or diagnosis method is fluorescence imaging, such as fluorescence imaging of cells, fluorescence imaging of tissue sections, fluorescence imaging of living bodies, and then microscopic examination.
  • nucleic acid aptamer or derivative thereof in at least one of a1 to a26 is also within the scope of protection of the present invention:
  • A12 preparing a substance product that detects binding to an antibody against alkaline phosphatase
  • A14 preparing a product for diagnosing and/or treating an alkaline phosphatase-related disease
  • A15 capturing and/or detecting expression or high expression of alkaline phosphatase-expressing cells or exosomes
  • A16 preparing a product for capturing and/or detecting expression or high expression of alkaline phosphatase cells or exosomes
  • A20 detecting or capturing circulating tumor cells expressing or highly expressing alkaline phosphatase in the sample to be tested;
  • A23 preparing a tumor or tumor cell product for detecting or capturing an alkaline phosphatase expressed or highly expressed in the sample to be tested;
  • A24 preparing a circulating tumor cell product for detecting or capturing an alkaline phosphatase expressed or highly expressed in the sample to be tested;
  • Another object of the invention is to provide a kit.
  • the kit provided by the present invention comprises the above nucleic acid aptamer or a derivative thereof, and a vector for immobilizing or coupling the nucleic acid aptamer or a derivative thereof.
  • the kit has at least one of the following functions b1-b11:
  • the carrier for immobilizing or coupling the nucleic acid aptamer or derivative thereof is a nanoparticle or a microparticle or a chip.
  • the carrier in this embodiment is a magnetic nanoparticle, and is a superparamagnetic magnetic nanoparticle (200 nm) modified with streptavidin on the surface, which not only functions for size amplification, but also can be used for magnetic separation operation to achieve high Efficiency capture. It is not limited to a magnetic ball, and may be a substrate such as a chip.
  • the immobilized or coupled nucleic acid aptamer or derivative thereof is carried out by coupling, and the coupling means that they are linked together by covalent coupling, hydrophobic interaction or intermolecular force, and in this embodiment, the streptavidin is involved.
  • the interaction between the element and the biotin is carried out by coupling, and the coupling means that they are linked together by covalent coupling, hydrophobic interaction or intermolecular force, and in this embodiment, the streptavidin is involved.
  • the carrier for immobilizing or coupling the nucleic acid aptamer or a derivative thereof is a nanoparticle or a microparticle or a chip.
  • the nanoparticles are modified nano/micro particles modified
  • nanoparticles are magnetic nanoparticles
  • the modification is streptavidin, biotin, carboxyl, amino or sulfhydryl.
  • the kit further includes a chromogenic substrate that reacts with alkaline phosphatase;
  • the chromogenic substrate is a fluorescent substrate molecule, a chemiluminescent substrate molecule or a visible light substrate molecule.
  • the chromogenic substrate is a fluorescent substrate molecule, a chemiluminescent substrate molecule or a visible light substrate molecule or other alkaline phosphatase substrate.
  • Embodiments of the present invention relate to p-nitrophenylphosphoric acid disodium (pNPP) which reacts with alkaline phosphatase to produce p-nitrophenol which is yellow under alkaline conditions and which can be tested for absorbance at 405 nm. It also involves the reaction of BCIP/NBT with alkaline phosphatase to produce a blue-violet precipitate. It also involves the reaction of fluorescein diphosphate with alkaline phosphatase to produce green fluorescence.
  • pNPP p-nitrophenylphosphoric acid disodium
  • the above kit further includes a red blood cell lysate and a magnetic separation rack; if the sample to be tested is whole blood, the red blood cell lysate is included, and if the sample to be tested is serum or plasma or saliva, the lysate is not required.
  • the expression or high expression alkaline phosphatase-expressing cells are tumor cells expressing or highly expressing alkaline phosphatase;
  • the tumor cells expressing or highly expressing alkaline phosphatase are human cervical cancer cells, human breast cancer cells, human colon cancer cells or human hepatocyte cancer cells;
  • the circulating tumor cells expressing or highly expressing alkaline phosphatase are human cervical cancer circulating tumor cells, human breast cancer circulating tumor cells, human colon cancer circulating tumor cells or human hepatocellular carcinoma circulating tumor cells.
  • nucleic acid aptamer or a derivative thereof and a vector which immobilizes or couples the nucleic acid aptamer or a derivative thereof, in the preparation of a product having at least one of the following functions b1 to b11 is also a scope of protection of the present invention :
  • the sample to be tested is peripheral blood whole blood, serum, plasma, cell culture fluid or saliva.
  • the sample to be tested corresponding to circulating tumor cells expressing or highly expressing alkaline phosphatase is peripheral blood whole blood;
  • the sample to be tested corresponding to the exosome expressing or highly expressing alkaline phosphatase is peripheral blood serum or plasma;
  • test sample corresponding to the free protein expressing or highly expressing alkaline phosphatase is peripheral blood serum or plasma, or saliva.
  • a third object of the present invention is to provide a method for capturing and/or detecting whether a sample to be tested contains tumor cells expressing or highly expressing alkaline phosphatase, comprising the steps of:
  • the sample to be tested is peripheral blood whole blood
  • the preparation of the nucleic acid aptamer magnetic nanoparticle is to connect the magnetic nanoparticle and the alkaline phosphatase nucleic acid aptamer to obtain the nucleic acid aptamer magnetic nanoparticle;
  • the preparation of the nucleic acid aptamer magnetic nanoparticle is performed by coupling the above streptavidin-modified magnetic nanoparticle with the biotin-labeled alkaline phosphatase nucleic acid aptamer to obtain a nucleic acid aptamer magnetic nanoparticle;
  • the product containing circulating tumor cells is stained with a substrate of alkaline phosphatase (such as pNPP), and then the absorbance is detected; the control nucleic acid aptamer sequence is used as a control, and the absorbance after capture with the control nucleic acid aptamer sequence If the values are significantly different, the sample to be tested contains or can be selected to contain tumor cells expressing or highly expressing alkaline phosphatase; if there is no significant difference in absorbance values after capture from the control sequence, the sample to be tested does not contain or the candidate does not contain expression or high. Tumor cells expressing alkaline phosphatase;
  • the product containing circulating tumor cells is stained with a substrate of alkaline phosphatase (such as BCIP/NBT) and observed under a microscope. If a cell with a blue-violet precipitate is observed, the sample to be tested contains or can be expressed or expressed. A tumor cell with high expression of alkaline phosphatase; if there is no surface blue-violet cell, the sample to be tested does not contain or the candidate does not contain tumor cells expressing or highly expressing alkaline phosphatase.
  • alkaline phosphatase such as BCIP/NBT
  • a fourth object of the present invention is to provide a method for capturing and/or detecting whether a sample to be tested contains exosomes expressing or highly expressing alkaline phosphatase, comprising the steps of:
  • the preparation of the nucleic acid aptamer magnetic nanoparticle is to connect the magnetic nanoparticle and the alkaline phosphatase nucleic acid aptamer to obtain the nucleic acid aptamer magnetic nanoparticle;
  • the preparation of the nucleic acid aptamer magnetic nanoparticle is performed by coupling the above streptavidin-modified magnetic nanoparticle with the biotin-labeled alkaline phosphatase nucleic acid aptamer to obtain a nucleic acid aptamer magnetic nanoparticle;
  • Collecting the tumor cell exosomes of the sample to be tested is collecting tumor cell exosomes from serum or plasma of the sample to be tested;
  • the method for detecting the absorbance containing the exosome product to determine whether the sample to be tested contains tumor cells expressing or highly expressing alkaline phosphatase is as follows:
  • the exosome product is stained with pNPP for color development, and then the absorbance is detected; the control nucleic acid aptamer sequence is used as a control, and if the absorbance value is significantly different after the capture sequence of the control nucleic acid aptamer is captured, the sample to be tested contains or Candidates contain exosomes that express or highly express alkaline phosphatase; if there is no significant difference in absorbance values after capture from the control sequence, the sample to be tested does not contain or candidates do not contain exosomes that express or highly express alkaline phosphatase;
  • the method for detecting whether a sample containing a surface of an exosome product forms a precipitate to determine whether a sample to be tested contains or expresses alkaline phosphatase is as follows:
  • the sample to be tested contains or can be selected to contain exosomes expressing or highly expressed alkaline phosphatase; , the sample to be tested does not contain or the candidate does not contain exosomes that express or highly express alkaline phosphatase.
  • a fifth object of the present invention is to provide a method for capturing and/or detecting the presence of alkaline phosphatase in free protein of a sample to be tested, comprising the steps of:
  • the preparation of the nucleic acid aptamer magnetic nanoparticle is to connect the magnetic particle and the alkaline phosphatase nucleic acid aptamer to obtain a nucleic acid aptamer magnetic particle;
  • the preparation of the nucleic acid aptamer magnetic nanoparticle is performed by coupling the above streptavidin-modified magnetic nanoparticle with the biotin-labeled alkaline phosphatase nucleic acid aptamer to obtain a nucleic acid aptamer magnetic nanoparticle;
  • Collecting free protein of the sample to be tested is collecting free protein from serum or plasma of the sample to be tested;
  • the detection of the absorbance containing the free protein product determines whether the free protein in the sample to be tested contains alkaline phosphatase as follows:
  • the free protein product is stained with pNPP for color development, and then the absorbance is detected; the control nucleic acid aptamer sequence is used as a control, and if the absorbance value of the control nucleic acid aptamer sequence is significantly different, the free protein of the sample to be tested is Containing or candidate containing alkaline phosphatase; if there is no significant difference in absorbance value after capture with the control sequence, the free protein of the sample to be tested does not contain or the candidate does not contain alkaline phosphatase;
  • the detection whether the free protein product is capable of fluorescent staining determines whether the free protein in the sample to be tested contains alkaline phosphatase as follows:
  • the product is developed with fluorescein diphosphate, and then the fluorescence is measured. If fluorescence is observed, the free protein of the sample to be tested contains or may contain alkaline phosphatase; if there is no fluorescence or no obvious fluorescence, Then, the free protein of the sample to be tested does not contain or the candidate does not contain alkaline phosphatase.
  • the alkaline phosphatase is a heterodimer alkaline phosphatase
  • the heterodimeric alkaline phosphatase is PALP (placental alkaline phosphatase), IAP (intestinal alkaline phosphatase), GCAP (germ cell alkaline phosphatase) or any heterogeneous Dimer.
  • PALP placental alkaline phosphatase
  • IAP intestinal alkaline phosphatase
  • GCAP germ cell alkaline phosphatase
  • Figure 1 is a graph showing the secondary structure of the nucleic acid aptamer BG2, the apparent binding constant of the nucleic acid aptamer BG2 and its derivatives, and competition experiments.
  • Figure 2 is a graph showing the expression of a cell surface alkaline phosphatase heterodimer detected by a nucleic acid aptamer.
  • Figure 3 shows the binding of the nucleic acid aptamer BG2 after knockdown of placental alkaline phosphatase (PALP) and intestinal alkaline phosphatase (IAP), respectively.
  • POP placental alkaline phosphatase
  • IAP intestinal alkaline phosphatase
  • Figure 4 is a nucleic acid aptamer BG2 capture alkaline phosphatase heterodimer
  • POP placental alkaline phosphatase
  • IAP intestinal alkaline phosphatase
  • GCAP germ cell alkaline phosphatase
  • Figure 5 is a measurement of alkaline phosphatase activity extracted using the nucleic acid aptamer BG2.
  • Figure 6 is a cell imaging and tissue section immunostaining for detecting phosphatase heterodimer expression by nucleic acid aptamer BG2;
  • Figure 7 shows BG2 aptamers imaged with live animals (left: live animals; right: isolated tumors).
  • Figure 8 is a diagram showing the capture of target cells in a sample to be tested by a nucleic acid aptamer micromagnetic ball
  • Figure 9 shows the results of nucleic acid aptamer magnetic nanoparticles capturing and microscopically detecting target cells in exosomes.
  • Figure 10 is a nucleic acid aptamer magnetic nanoparticle detection of soluble alkaline phosphatase in a sample to be tested;
  • results of detection of soluble alkaline phosphatase for nucleic acid aptamer magnetic nanoparticles are results of detection of soluble alkaline phosphatase in cell culture medium by nucleic acid aptamer sugar spheres.
  • Binding buffer solution 1 containing 137 mM NaCl, 5 mM MgCl 2 , 2.7 mM KCl, 2 mM KH 2 PO 4 , 10 mM Na 2 HPO 4 , 25 mM glucose, 1 ⁇ g/ml BSA, 0.1 ⁇ g/ml salmon sperm DNA and 0.01% (v/ v) Tween-80, the rest is water.
  • streptavidin-modified magnetic nanoparticles in the following examples were manufactured by Xiamen Puruimag Biotech Co., Ltd. at a concentration of 10 mg/mL.
  • Human cervical cancer cells (Hela), human hepatocyte cancer cells (SMMC-7721), human breast cancer cells (MCF-7), human embryonic kidney cells (HEK-293), and human neuroblasts in the following examples
  • Tumor cells (SH-SY5Y) and human leukemia cells (Jurkat E6-1) were purchased from Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences; human colon cancer cells (LoVo), human hepatocellular carcinoma cells (HepG2), human colon cancer cells (HCT116).
  • Human prostate cancer cells (PC3) were purchased from the Cell Bank of the Committee of Culture Collection of the Chinese Academy of Sciences.
  • the PBS buffer in the following examples: containing 12 mM NaH 2 PO 4 , 8 mM Na 2 HPO 4 , 122 mM NaCl, and 5 mM KCl, the balance being water.
  • PBST buffer in the following examples: a solution containing 12 mM NaH 2 PO 4 , 8 mM Na 2 HPO 4, 122 mM NaCl, 5 mM KCl, and 0.01% (v/v, volume percent) Tween-80, the rest For water.
  • Binding buffer 2 in the following examples: containing 12 mM NaH 2 PO 4 , 8 mM Na 2 HPO 4 , 122 mM NaCl, 5 mM KCl, 1 ⁇ g/ml bovine serum albumin, 0.1 ⁇ g/ml salmon sperm DNA and 0.01% (v /v, volume percent) Tween-80 solution, the rest is water.
  • red blood cell lysate in the following examples is a product of Roche Corporation, catalog number is 11814389001.
  • pNPP p-nitrophenyl phosphate disodium salt hexahydrate
  • BCIP 5-bromo-4-chloro-3-indolyl phosphate p-toluidine salt
  • NBT nitrotetrazolium chloride Lan
  • FDP Fluorescein Diphosphate
  • the red blood cell lysate in the following examples is a product of Sigma Aldrich (Shanghai) Trading Co., Ltd., catalog number is 11814389001.
  • Human colon cancer cells LoVo, human breast cancer cells MCF-7, and human cervical cancer Hela were cultured with RPMI 1640 (containing 10% fetal bovine serum, 1% cyan/streptomycin). All cells were routinely cultured in an incubator (37 ° C, 5% CO 2 ) and passaged every two to three days.
  • a random library consisting of 20 fixed nucleotides and 45 nucleotides in between is designed as follows: 5'-ACGCTCGGATGCCACTACAG TYRRRRRRNN GGGNNNGGNNNGGNNGGNNNNNNNNNNNN GGNYYYYYYRT CTCATGGACGTGCTGGTGAC (sequence 8)-3'; N stands for A, T, C or G, Y Represents T or C, and R stands for G or A.
  • a 10 nmol random nucleic acid library (synthesized in step 2) was dissolved in binding buffer, denatured at 95 ° C for 5 min, cooled on ice for 10 min, and reconstituted at room temperature for 30 min.
  • Each 1 ⁇ 10 6 human colon cancer cells LoVo, human breast cancer cells MCF-7 and human cervical cancer Hela were digested with PBS containing 5 mM EDTA for 10 min, mixed, washed once with washing buffer solution, and then added with the above DNA.
  • the library was incubated. After incubation for 30 min, the supernatant was removed by centrifugation and washed twice with a washing buffer solution, and the DNA molecules bound to the cells were directly used for PCR.
  • the forward primers for PCR amplification are:
  • PCR amplification procedure 94 ° C for 3 min; 94 ° C for 30 s, 60 ° C for 30 s, 72 ° C for 30 s, 10 cycles; 72 ° C, 5 min.
  • ssDNA FAM-labeled single-stranded DNA sequence was isolated from the PCR product using streptavidin agarose beads. The resulting ssDNA was desalted using a NAP-5 column (General Electric Medical Group, Sweden) and vacuum dried for the next round of screening.
  • the number of washings is gradually increased and the number of positive sieve cells is gradually decreased during the screening process to increase the pressure of the screening. After 5 rounds of screening, high throughput sequencing was performed.
  • the aptamer BG2 obtained after removing the primer is as follows:
  • the apparent dissociation constant of the BG2 nucleic acid aptamer was determined to be 2.5 ⁇ 0.3 nM (Fig. 1B).
  • the affinity of the primer sequence was further determined and the sequence was:
  • the aptamer was truncated and its affinity was reduced to some extent (9.3 ⁇ 1.6 nM).
  • GGGGTCGGTGTGGGTGGTTATGATTGG is the core region of the aptamer and the target.
  • sequence 4 The loop region sequence is retained and the stem of sequence 1 is randomly replaced, for example, sequence 4:
  • nucleic acid aptamer BG2 was thio-modified, and the sequence was as follows:
  • the thio-modified nucleic acid aptamer was determined to still retain good affinity with an apparent dissociation constant of 3.5 ⁇ 0.6 nM (Fig. 1G).
  • BG2 aptamer represented by SEQ ID NO: SEQ ID NO: 6 or a derivative thereof can bind to colon cancer cell LoVo.
  • the fluorescein-labeled nucleic acid aptamer BG2 (BG2-FAM, 100 nM) was separately mixed with the BG2 nucleic acid aptamer and its derivative (4 ⁇ M) of the unlabeled fluorescent molecule, and about 5 ⁇ 10 4 LoVo cells were added, respectively.
  • the mixture was separately obtained, and the mixture was incubated on ice for 30 min, washed twice with washing buffer, passed through a 400 mesh screen, and tested by an upflow cytometer.
  • the BG2 loop region sequence is also included:
  • Sequence 7 5 '-TAGGGGTCGGTGTGGGTGGTTATGATTGGC-3' (SEQ ID NO: 7);
  • the nucleotide sequence of the control nucleic acid sequence L45 NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN
  • Example 2 Study on the binding of nucleic acid aptamer BG2 to different types of cells
  • the nucleic acid aptamer BG2 is synthesized by a DNA synthesizer.
  • the nucleotide sequence of the nucleic acid aptamer BG2 is as follows: 5'-CAAGGAATAGGGGTCGGTGTGGGTGGTTATGATTGGCTTCCTTG-3' (SEQ ID NO: 1), and different molecules can be labeled on the nucleic acid aptamer BG2 according to the test requirements. .
  • DNA deprotection after deprotection with cold ammonia, and then dissolve the DNA in the TEAA solution;
  • DNA purification purification by PAGE or high performance liquid chromatography
  • the fluorescein-labeled nucleic acid aptamer BG2 was obtained by coupling the fluorescein group FAM at the 5' end of the nucleic acid aptamer BG2, and the BG2-FAM was dissolved in the binding buffer according to the ultraviolet absorption calibration concentration (200 nM). Heat at °C for 5 min, place on ice for 5 min, and let stand at room temperature for 15 min to obtain BG2-FAM solution.
  • the fluorescein-labeled control nucleic acid sequence L45 was obtained by coupling the fluorescein group FAM at the 5' end of the control nucleic acid sequence L45, and the L45-FAM was dissolved in the binding buffer according to the UV absorption calibration concentration (100 nM). After that, it was heated at 95 ° C for 5 min, placed on ice for 5 min, and left at room temperature for 15 min to obtain a L45-FAM solution.
  • the nucleotide sequence of the control nucleic acid sequence L45 TTTNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN.
  • human cervical cancer cells Hela
  • human hepatocyte cancer cells SMMC7721
  • human breast cancer cells MCF-7
  • human embryonic kidney cells HEK- 293
  • human neuroblastoma cells SH-SY5Y
  • human colon cancer cells LiVo
  • human hepatocyte cancer cells HepG2
  • human colon cancer cells HCT116
  • PC3 human prostate cancer cells
  • the cells were digested with 5 mM EDTA in PBS to form a monodisperse cell suspension, and washed twice with washing buffer. Then, the above cells were separately added with 10 ⁇ g/mL of anti-IAP antibody (Cat. No. GTX60746, GeneTex) or 10 ⁇ g/mL of anti-PALP antibody (Cat. No.: MA1-20245 for 30 min, after washing once, 4 ⁇ g/mL was added for anti-small).
  • the murine m-IgG ⁇ BP-PE antibody (sc-516141) was incubated for 30 min, and once washed, the cells were resuspended and examined by flow cytometry.
  • the BG2-FAM solution and the L45-FAM solution prepared in the first step of Example 2 were mixed with 10 cell lines of different origins (5 ⁇ 10 4 cells each), respectively, to obtain a mixed solution, and the mixed solution was Incubate on ice for 30 min, wash twice with wash buffer, pass through a 400 mesh screen, and test with an upflow cytometer.
  • the fluorescence intensity data of the first channel was collected using a FACSCalibur flow cytometer from BD Corporation as the fluorescence intensity on the cell surface.
  • the fluorescence intensity of each sample was measured by deducting the autofluorescence of the cells to obtain the fluorescence intensity of the aptamer bound to the cell surface of each sample.
  • Human colon cancer cells (LoVo), human breast cancer cells (MCF-7), and human cervical cancer cells were confirmed with anti-IAP antibody (Cat. No. GTX60746, GeneTex) or anti-PALP antibody (Cat. No.: MA1-20245).
  • Hela human hepatocellular carcinoma cells (SMMC7721) and human hepatocellular carcinoma cells (HepG2) express alkaline phosphatase heterodimer, HCT116 cells underexpress alkaline phosphatase heterodimer, and PC-3 cells Jurkat cells, SH-SY5Y cells and HEK293 cells did not express alkaline phosphatase heterodimers.
  • the fluorescein-labeled nucleic acid aptamer BG2 is associated with human colon cancer cells (LoVo), human breast cancer cells (MCF-7), human cervical cancer cells (Hela), and human hepatocytes.
  • the cancer cells (SMMC-7721) bind to human hepatocyte cancer cells (HepG2) and weakly bind to HCT116 cells, and do not bind to PC-3 cells, Jurkat cells, SH-SY5Y cells, and HEK293 cells.
  • LoVo cells were seeded in 6-well plates containing 2 mL of FBS and double-antibody 1640 medium.
  • RNAiMAX reagent Cat. No. 13778-075
  • add 40 pmol of siRNA to 125 ⁇ L of serum-free Opti-MEM (where ALPI siRNA is: ALPI-homo-1288, sense sequence (5'-3') :GCAAAGCCUACACGUCCAUTT (SEQ ID NO: 12), antisense sequence (5'-3'): AUGGAGUGUAGGCUUUGCTT (SEQ ID NO: 13);
  • siRNA for PALP is: PALP-homo-947, sense sequence (5'-3'): GAGACAUGAAAUACGAGAUTT (sequence 14) , antisense sequence (5'-3'): AUCUCGUAUUUCAUGUCUCTT (sequence 15)) mix.
  • RNAiMAX reagent was diluted with 125 ⁇ L of serum-free Opti-MEM medium and mixed.
  • RNAiMAX reagent was mixed and mixed 1:1 and allowed to stand at room temperature for 5 minutes.
  • RNAiMAX RNAiMAX mixture
  • the RNAiMAX mixture is added to the wells of the culture plate containing the cells and the medium, and the cell culture plate is shaken back and forth.
  • the cells were cultured in a CO2 incubator at 37 ° C for 72 hours.
  • the cells were digested with 5 mM EDTA in PBS to form a monodisperse cell suspension, and washed twice with washing buffer. Then, the above cells were separately added with 200 nM BG2-FAM solution, and the mixture was incubated on ice for 30 min, washed twice with washing buffer, passed through a 400-mesh sieve, and tested by an upflow cytometer; another cell was added with 10 ⁇ g/ mL anti-IAP antibody (Cat. No. GTX60746, GeneTex) or 10 ⁇ g/mL anti-PALP antibody (Cat. No.: MA1-20245 for 30 min, after washing once, add 4 ⁇ g/mL anti-mouse m-IgG ⁇ BP-PE antibody ( Inc. was incubated for 30 min. After one wash, the cells were resuspended and examined by flow cytometry.
  • nucleic acid aptamer BG2 binds to the IAP protein or PALP protein of the target cell LoVo.
  • the 3000 reagent was diluted with 125 ⁇ L of serum-free Opti-MEM medium and mixed.
  • the cells were cultured in a CO 2 incubator at 37 ° C for 48 hours.
  • the cells were digested with 5 mM EDTA in PBS to form a monodisperse cell suspension, and washed twice with washing buffer. Then, the above cells were separately added with 200 nM BG2-FMA solution, and the mixture was incubated on ice for 30 min, washed twice with washing buffer, passed through a 400-mesh sieve, and tested by an upflow cytometer; another cell was added with 10 ⁇ g/ mL anti-IAP antibody (Cat. No. GTX60746, GeneTex) or 10 ⁇ g/mL anti-PALP antibody (Cat. No.: MA1-20245 for 30 min, after washing once, add 4 ⁇ g/mL anti-mouse m-IgG ⁇ BP-PE antibody ( Inc. was incubated for 30 min. After one wash, the cells were resuspended and examined by flow cytometry.
  • a nucleic acid aptamer is capable of binding to BG2 only when its IAP protein and the PALP protein or the IAP protein and the GCAP protein are simultaneously expressed.
  • nucleic acid aptamer BG2 can bind to an IAP/PALP or IAP/GCAP heterodimer.
  • the biotin-labeled nucleic acid aptamer BG2 is obtained by coupling biotin bio containing a disulfide bond at the 5' end of the nucleic acid aptamer BG2, and dissolving BG2-SS-bio in a binding buffer, and calibrating the concentration according to ultraviolet absorption ( After 200 nM), it was heated at 95 ° C for 5 min, placed on ice for 5 min, and allowed to stand at room temperature for 15 min to obtain a BG2-SS-bio solution.
  • the precipitate was removed by centrifugation at 2000 rpm, and the supernatant was collected, and streptavidin-modified agarose microspheres (GE, Cat. No. 17-5113-01) were added and incubated for 1 hour to extract the target protein.
  • the membrane was washed 5 times with PBST, HRP-labeled secondary antibody (1:5000 dilution, Santa) was added and incubated for 1 hour at room temperature.
  • the membrane was washed 5 times with PBST, and SuperSignal West Femto Maximum Sensitivity Substrate reagent (Thermo Fisher Scientific) was added and imaged using a fully automated chemiluminescence image analysis system (Tianneng).
  • the nucleic acid aptamer BG2 can capture an in situ cross-linked alkaline phosphatase heterodimer.
  • nucleic acid aptamer BG2 can bind to an IAP/PALP or IAP/GCAP heterodimer.
  • Nucleic acid aptamer BG2 specifically extracts alkaline phosphatase
  • Biotin-labeled nucleic acid aptamer BG2 (BG2-Bio) was obtained by coupling biotin Bio group at the 5' end of nucleic acid aptamer BG2, and BG2-Bio was dissolved in binding buffer, and the concentration was determined according to ultraviolet absorption ( After 200 nM), it was heated at 95 ° C for 5 min, placed on ice for 5 min, and allowed to stand at room temperature for 15 min to obtain a BG2-Bio solution.
  • the biotin-labeled control nucleic acid sequence L45 (L45-Bio) was obtained by coupling the biotin group Bio at the 5' end of the control nucleic acid sequence L45, and the L45-Bio was dissolved in the binding buffer, and the concentration was determined according to the ultraviolet absorption (200 nM). After that, it was heated at 95 ° C for 5 min, placed on ice for 5 min, and left at room temperature for 15 min to obtain a L45-Bio solution.
  • the nucleotide sequence of the control nucleic acid sequence L45 TTTNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN.
  • Nucleic acid aptamer BG2 extracts alkaline phosphatase
  • the results are shown in Fig. 5.
  • the left picture shows the phosphatase activity color of the extracted protein, and the right picture shows the absorption of the color solution at 405 nm. It can be seen that the protein extracted by the biotin-labeled nucleic acid aptamer BG2 can extract the cells.
  • the alkaline phosphatase in the protein that is, the binding of the nucleic acid aptamer BG2 to the alkaline phosphatase in the cell.
  • nucleic acid aptamer BG2 extracts alkaline phosphatase and interacts with it
  • Heavy-duty isotope-labeled LoVo cells Add heavy-duty isotopically labeled lysine ([ 13 C 6 , 15 N 2 ]-L-lysine to the medium containing RPMI 1640 without lysine and arginine, article number :211604102) and heavy isotopically labeled arginine ([ 13 C 6 ]-L-arginine, Cat. No.
  • Light isotopically labeled LoVo cells light isotopically labeled lysine ([ 12 C 6 , 14 N 2 ]-L-lysine, respectively, added to RPMI 1640-free medium without lysine and arginine, :L8662) and light isotope-labeled arginine ([ 12 C 6 ]-L-arginine, Cat. No. A8094) (sigma), allowing light isotopically labeled lysine and light isotopically labeled arginine The concentrations in the medium were 0.274 mM and 0.575 mM, respectively. LoVo cells were cultured in this medium for 6-7 passages and used to obtain light isotopically labeled LoVo cells.
  • Biotin-labeled nucleic acid aptamer BG2 (BG2-Bio) was obtained by coupling biotin group Bio at the 5' end of nucleic acid aptamer BG2, and BG2-Bio was dissolved in binding buffer, and the concentration was determined according to ultraviolet absorption ( After 200 nM), it was heated at 95 ° C for 5 min, placed on ice for 5 min, and allowed to stand at room temperature for 15 min to obtain a BG2-Bio solution.
  • the biotin-labeled control nucleic acid sequence L45 (L45-Bio) was obtained by coupling the biotin group Bio at the 5' end of the control nucleic acid sequence L45, and the L45-Bio was dissolved in the binding buffer, and the concentration was determined according to the ultraviolet absorption (200 nM). After that, it was heated at 95 ° C for 5 min, placed on ice for 5 min, and left at room temperature for 15 min to obtain a L45-Bio solution.
  • the nucleotide sequence of the control nucleic acid sequence L45 TTTNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN.
  • DTT reduction a light isotopically labeled mixed protein extracted from a biotin-labeled nucleic acid aptamer BG2 and a light isotope-labeled mixed system extracted from a control nucleic acid sequence L45, a light-weight extracted from a biotin-labeled nucleic acid aptamer BG2
  • the isotope-labeled protein and the heavy-duty isotopically labeled mixed system extracted from the control nucleic acid sequence L45 were added with 200 ⁇ L of 20 mM dithiothreitol (DTT), and reacted at 56 ° C for 45 min.
  • DTT dithiothreitol
  • IAA alkylation The product of the step (1) was centrifuged, the supernatant was discarded (DTT was removed), and 200 ⁇ L of 55 mM iodoacetamide (IAA) was added to the precipitate, and the reaction was carried out at 37 ° C for 30 min in the dark.
  • step (3) Centrifuge the product of step (2), discard the supernatant (removal of IAA), add 5 ⁇ g of mass spectrometry to trypsin (Promega, catalog number: V5111), and digest overnight at 37 °C to obtain enzyme digestion. Peptide.
  • step (4) The product of the step (4) was analyzed and identified using an LTQ-Orbitrap Velos mass spectrometer (Thermo Fisher Scientific, San Jose, CA) to obtain the original mass spectrometry data.
  • LTQ-Orbitrap Velos mass spectrometer Thermo Fisher Scientific, San Jose, CA
  • the raw mass spectral data obtained in step (5) was searched in the uniprot protein database using the MaxQuant search engine (version number: 1.5.5.1).
  • Some parameters of the database search are as follows: the immobilization modification is an alkylation modification on cysteine, the variable modification is an oxidative modification on methionine, and the N-terminal acetylation modification of the protein. Two missed cut sites were allowed, the parent ion tolerance was 20 ppm, and the MS/MS fragment ion mass error was 0.5 Da.
  • nucleic acid aptamer BG2 is capable of binding to the proteins shown in Table 1, including alkaline phosphatase ALPI, ALPP and ALPPL2, and proteins interacting with these alkaline phosphatases; Experiments have also demonstrated that the nucleic acid aptamer BG2 can be used to detect alkaline phosphatases such as ALPI, ALPP and ALPPL2.
  • the biotin-labeled nucleic acid aptamer BG2 was obtained by coupling a biotin bio group at the 5' end of the nucleic acid aptamer BG2, and BG2-bio was dissolved in a binding buffer, and the concentration was determined according to ultraviolet absorption (200 nM). Heat at °C for 5 min, place on ice for 5 min, and let stand at room temperature for 15 min to obtain BG2-bio solution.
  • the biotin-labeled control nucleic acid sequence L45 was obtained by coupling the biotin bio group at the 5' end of the control nucleic acid sequence L45, and the L45-bio was dissolved in the binding buffer, and the concentration was determined according to the ultraviolet absorption (200 nM). After that, it was heated at 95 ° C for 5 min, placed on ice for 5 min, and left at room temperature for 15 min to obtain a L45-bio solution.
  • the nucleotide sequence of the control nucleic acid sequence L45 TTTNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN
  • LoVo cells (alkaline phosphatase detected) were cultured on a 35 mm confocal culture dish for 1 day, washed once with a washing buffer solution, and then incubated with 400 ⁇ l of BG2-bio solution at 37 ° C for 30 min, L45-bio solution. The dyeing method is the same, and the blank is not stained;
  • BG2 can bind to LoVo cells.
  • the fluorescein-labeled nucleic acid aptamer BG2 was used to stain the colon cancer tissue (altered with alkaline phosphatase).
  • the section was first incubated with a binding buffer solution containing 20% FBS and 1 mg/ml salmon sperm DNA for 60 min at room temperature;
  • BG2 can bind to colon cancer tissue.
  • Example 6 the application of BG2 in the imaging of live animals
  • Alexa Fluor 647-labeled nucleic acid aptamer BG2 is a nucleotide thio modification of the Alexa Fluor 647 group, 5' end and 3' end 1-7 coupled to the 5' end of the nucleic acid aptamer BG2 (sequence 6)
  • BG2-AF647 is a nucleotide thio modification of the Alexa Fluor 647 group, 5' end and 3' end 1-7 coupled to the 5' end of the nucleic acid aptamer BG2 (sequence 6)
  • Alexa Fluor 647-labeled control nucleic acid sequence L45 is a nucleotide thio modification of the Alexa Fluor 647 group, 5' end and 3' end 1-7 coupled to the 5' end of the nucleic acid aptamer BG2, using binding buffer Dissolve L45-AF647, according to the UV absorption calibration concentration (3 ⁇ M), heat at 95 ° C for 5 min, place on ice for 5 min, and let stand at room temperature for 15 min to obtain L45-AF647 solution.
  • the nucleotide sequence of the control nucleic acid sequence L45 sNsNsNsNsNsNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNsNsNsNsNsNsNsN. (sN is thio-modified A, T, G or C, and N is random A, T, G or C)
  • mice are housed for 3-4 weeks, and the diameter of the tumor is as long as 0.8–1.2 cm.
  • BG2-AF647 can achieve in vivo imaging of LoVo tumor-bearing mice, and has good specificity in vivo, and BG2-AF647 cannot bind to PC-3 tumor-bearing mice.
  • Biotin-labeled nucleic acid aptamer BG2 (BG2-Bio) was obtained by coupling biotin group Bio at the 5' end of nucleic acid aptamer BG2, and BG2-Bio was dissolved in binding buffer, and the concentration was determined according to ultraviolet absorption ( After 200 nM), it was heated at 95 ° C for 5 min, placed on ice for 5 min, and allowed to stand at room temperature for 15 min to obtain a BG2-Bio solution.
  • BG2-Bio solution and magnetic microspheres were then incubated at room temperature for 30 min and then washed twice with PBS to obtain BG2-modified magnetic microspheres.
  • the biotin-labeled control nucleic acid sequence L45 (L45-Bio) was obtained by coupling the biotin group Bio at the 5' end of the control nucleic acid sequence L45, and the L45-Bio was dissolved in the binding buffer, and the concentration was determined according to the ultraviolet absorption (200 nM). After that, it was heated at 95 ° C for 5 min, placed on ice for 5 min, and left at room temperature for 15 min to obtain a L45-Bio solution.
  • the nucleotide sequence of the control nucleic acid sequence L45 TTTNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN.
  • nucleic acid aptamer functionalized magnetic microspheres capture cells
  • Nucleic acid aptamer magnetic microspheres are incubated with cells:
  • the magnetic rack was then washed 3 times for 3 minutes, the cells not bound to the magnetic spheres were removed, and the magnetic sphere-cell complex was resuspended in PBS, repeatedly dispersed, observed under a microscope and photographed.
  • BG2-modified magnetic microspheres can capture LoVo cells expressing alkaline phosphatase heterodimer, but cannot capture PC-3 cells negatively expressed by alkaline phosphatase heterodimer.
  • the magnetic ball modified by the control sequence cannot capture any kind of cells. This indicates that the BG2 nucleic acid aptamer can be used for the capture or enrichment of alkaline phosphatase heterodimer positive expression cells.
  • Nucleic acid aptamer magnetic nanoparticles are incubated with cells:
  • the cultured LoVo cells were digested with EDTA and washed twice with PBST buffer. Different numbers of cells (25, 50, 100, 200, 400, 600) were added, 1 mL of binding buffer was added, and 10 ⁇ L of the above (1) was added. Nucleic acid aptamer magnetic nanoparticles were incubated for 30 min at 4 °C with shaking.
  • the above-mentioned incubated products were placed on a magnetic separation rack for magnetic separation to further remove non-specific cells, washed twice with PBST buffer, and magnetically separated to obtain desired tumor cells.
  • Fig. 8B(a) A positive correlation was established between the absorbance value and the number of cells, and the results are shown in Fig. 8B(a). It can be seen from Fig. 8B(a) that as the number of cells increases, the absorbance value increases with a positive correlation, which proves the feasibility of the method.
  • the detection limit is 5 cells.
  • the tumor cells obtained in the above (1) were directly observed under a microscope, and the results are shown in Fig. 8B(b). It can be seen from Fig. 8B(b) that the surface of the target cell LoVo cells is surrounded by nucleic acid aptamer magnetic nanoparticles, demonstrating the effectiveness of the capture method.
  • Nucleic acid aptamer magnetic nanoparticles capture and detect target cells in mixed cells
  • Nucleic acid aptamer magnetic nanoparticles are incubated with cells:
  • the above-mentioned incubated products were placed on a magnetic separation rack for magnetic separation to further remove non-specific cells, washed twice with PBST buffer, and magnetically separated to obtain desired tumor cells.
  • the absorbance value of the positive cell sample (LoVo cell) captured by the BG2 nucleic acid aptamer was significantly different from that of the positive cell sample captured by the control nucleic acid aptamer sequence (P value ⁇ 0.01), and the BG2 nucleic acid.
  • Negative samples (PC3 cells) captured by the aptamer and control nucleic acid aptamer sequences showed no significant change in absorbance. This method can be used to detect whether a sample to be tested contains tumor cells expressing or highly expressing alkaline phosphatase.
  • Fig. 8D(b) the tumor cells obtained in the above (1) were directly observed under a microscope, and as a result, as shown in Fig. 8D(b), it can be seen that LoVo cells (by dye 4', 6-diamidino-2-phenyl) After sputum staining, it is blue. After mixing with PC3 cells (green stained with dye fluorescein diacetic acid), LoVo cells can be seen to encapsulate nucleic acid aptamer magnetic nanoparticles, but non-specific cells PC3 surface non-magnetic nanoparticles. . The specificity of the method was demonstrated.
  • Nucleic acid aptamer magnetic nanoparticles are incubated with cells:
  • LoVo cells were digested with EDTA and washed twice with PBST buffer. Different numbers of cells were taken. LoVo cells were added to 1 mL of whole blood, 2 mL of red blood cell lysate was added, and the mixture was gently pipetted and lysed at room temperature for 10 min, 500 g. Centrifuge for 5 min, discard the red supernatant. If the red blood cell lysis is incomplete, repeat 1-2 times, wash twice with PBST buffer, leave the pellet as a cell, add 1 mL of binding buffer to the pellet, and add 10 ⁇ L of the obtained nucleic acid. The aptamer magnetic nanoparticles were incubated for 30 min at 4 °C with shaking.
  • the above-mentioned incubated products were placed on a magnetic separation rack for magnetic separation to further remove non-specific cells, washed twice with PBST buffer, and magnetically separated to obtain desired circulating tumor cells.
  • Rate (%) absorbance value produced by reaction of the cells with the chromogenic substrate after capture / absorbance value produced by reaction of pure cells with the chromogenic substrate x 100).
  • Nucleic acid aptamer magnetic nanoparticles capture and microscopically target cells in human whole blood
  • BCIP/NBT working solution BCIP concentration of 50 mg/mL dissolved in 100% dimethylformamide, NBT concentration of 50 mg/mL dissolved in 70% dimethylformamide, buffer buffer per 1 mL 4 ⁇ L of NBT was added to the solution, and 4 ⁇ L of BCIP was added after mixing. The mixture was mixed again. The reagent was prepared and used within 1 h. When reacted with alkaline phosphatase, a blue-violet precipitate was produced.
  • Nucleic acid aptamer magnetic nanoparticles are incubated with cells:
  • the cultured LoVo cells were digested with EDTA and washed twice with PBST buffer. 50 LoVo cells were added to 1 mL of healthy individual whole blood, 2 mL of red blood cell lysate was added, gently mixed by pipetting, lysed at room temperature for 10 min, and centrifuged at 500 g for 5 min. The red supernatant was discarded. If red blood cell lysis was found to be incomplete, repeat 1-2 times, wash twice with PBST buffer, add 1 mL of binding buffer, add the above 10 ⁇ L of nucleic acid aptamer magnetic nanoparticles, and incubate for 30 min at 4 °C with shaking.
  • the above-mentioned incubated products were placed on a magnetic separation rack for magnetic separation to further remove non-specific cells, washed twice with PBST buffer, and magnetically separated to obtain desired circulating tumor cells.
  • Fig. 8F shows the circulating tumor cells captured in the whole blood of the tumor
  • Fig. 8F(b) shows the circulating tumor cells captured in the whole blood of the actual cancer patient. It can be seen that the alkaline phosphatase Highly expressed circulating tumor cells reacted with BCIP/NBT to produce a blue-violet precipitate on the cell surface, demonstrating that this method is expected to be a microscopic examination of clinical samples. Therefore, this method of high efficiency, high selectivity and rapid capture is expected to be used for the detection of circulating tumor cells in clinical samples, thus contributing to the early diagnosis and prognosis evaluation of cancer.
  • the nucleic acid aptamer and the magnetic nanoparticle can be used to detect whether the sample to be tested contains circulating tumor cells, and the kit used for the detection includes the following substances:
  • a streptavidin-modified magnetic nanoparticle a biotin-labeled BG2 nucleic acid aptamer, wherein the nucleotide of the BG2 nucleic acid aptamer is the sequence 1 in the sequence listing;
  • PBST buffer binding buffer
  • red blood cell lysate and magnetic separation rack
  • stains that bind to alkaline phosphatase such as chromogenic substrate pNPP or BCIP/NBT
  • fluorogenic substrates such as fluorogenic substrate pNPP or BCIP/NBT
  • fluorogenic substrates such as fluorogenic substrate pNPP or BCIP/NBT
  • spectrophotometers such as spectrophotometers, or microscopy.
  • a or B The above method for detecting a product containing circulating tumor cells is as follows: A or B:
  • the product containing circulating tumor cells is stained with pNPP for colorimetry, and then the absorbance is detected; the control nucleic acid aptamer sequence is used as a control, and if the absorbance value of the control nucleic acid aptamer sequence is significantly different, the sample to be tested contains Or the candidate contains circulating tumor cells; if there is no significant difference in absorbance values after capture with the control sequence, the sample to be tested does not contain or the candidate does not contain circulating tumor cells;
  • the product containing circulating tumor cells is stained with BCIP/NBT and observed under a microscope. If a cell with a blue-violet precipitate on the surface is observed, the sample to be tested contains or may contain circulating tumor cells; if there is no surface blue-violet cell, The sample to be tested does not contain or the candidate does not contain circulating tumor cells.
  • control nucleic acid aptamer L45 biotin was labeled at the 5' end of the control nucleic acid aptamer aptamer.
  • Example 8 nucleic acid aptamer magnetic nanoparticles capture and detection of exosomes
  • Streptavidin-modified magnetic nanoparticles are coupled to a recognition BG2 nucleic acid aptamer:
  • the exosomes are prepared by the commonly used ultra-high speed centrifugation method.
  • the LoVo cells are cultured in a medium containing normal serum. After the cell density reaches 70-80%, the original medium is removed, replaced with a serum-free medium, and cultured for 48 hours. After that, the cell supernatant was collected, 800 ⁇ g, centrifuged at 4° C. for 10 min, carefully aspirated, 2000 ⁇ g, centrifuged at 4° C. for 20 min, the supernatant was collected, 10000 ⁇ g, centrifuged at 4° C. for 30 min, and the supernatant was collected to ensure cells or The cell debris was removed.
  • the supernatant was centrifuged at 120,000 ⁇ g, centrifuged at 4 ° C for 120 min, the supernatant was discarded, the pellet was resuspended in PBS buffer, 120,000 ⁇ g, centrifuged at 4 ° C for 120 min, and the resulting exosomes were dispersed in 200 ⁇ L of PBS buffer.
  • nucleic acid aptamer magnetic nanoparticles were incubated with exosomes: different concentrations (0.25, 0.5, 1, 2, 4, 8 ⁇ g/mL) of the exosomes obtained in (2) above were added, and 1 mL of binding buffer was added. The above (1) nucleic acid aptamer magnetic nanoparticles were added and incubated at 4 ° C for 30 min with shaking.
  • Streptavidin-modified magnetic nanoparticles are coupled to a recognition BG2 nucleic acid aptamer:
  • 1 ⁇ L of 10 mg/mL streptavidin-modified 200 nm magnetic nanoparticles (binding biotin-labeled oligonucleotide 300 pmol/mg) and 10 ⁇ L of 1 ⁇ M biotin-labeled BG2 nucleic acid aptamer were buffered in 1 mL PBST. The solution was incubated for 30 min at room temperature, magnetically separated for 1 min, and washed twice with PBST buffer to obtain nucleic acid aptamer magnetic nanoparticles.
  • alkaline phosphatase 4, 8, 16, 30, 60, 120, 240 microvibrity units
  • nucleic acid aptamer magnetic nanoparticles were added and incubated at 4 °C with shaking. 30min.
  • binding buffer 1 mL was added to 10 ⁇ L of serum/plasma, and the above (1) nucleic acid aptamer magnetic nanoparticles were added and incubated at 4 ° C for 30 min with shaking.
  • the absorbance value measured by 10 ⁇ L of the serum sample was taken to the above standard curve to obtain a concentration of soluble alkaline phosphatase in the serum of 20 U/L.
  • Example 10 Detection of free alkaline phosphatase heterodimeric protein in cell culture medium
  • alkaline phosphatase can be captured in the alkaline phosphatase-expressing cell line (LoVo) culture medium, but not in the culture medium of the negative expression cells (PC3). This demonstrates that the method can be used for the capture and detection of free alkaline phosphatase heterodimers in actual samples.
  • the present invention finds for the first time that the nucleic acid aptamer BG2 can specifically recognize and bind to an alkaline phosphatase heterodimer.
  • the nucleic acid aptamer BG2 of the present invention has high affinity, specificity, no immunogenicity and no toxicity.
  • the method for detecting an alkaline phosphatase heterodimer based on the nucleic acid aptamer BG2 can be used for the detection of alkaline phosphatase heterodimer expression and the diagnosis of related diseases.
  • the nucleic acid aptamer-based magnetic particle technique of the present invention is applied to a method for capturing and detecting circulating tumor cells with high expression of alkaline phosphatase in peripheral blood, and can achieve high-selective capture and detection of target cells.
  • Magnetic particles can be used for size amplification, for enrichment of circulating tumor cells, and for magnetic separation for high efficiency capture.
  • the capture and detection method of the invention can be used for the detection of clinical samples without complicated modification and operation process, simple steps, high efficiency, rapidity and low cost.
  • the captured tumor cells can be used for further culture or genetic testing and the like.

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Abstract

提供核酸适配体在识别并结合碱性磷酸酶异源二聚体中的应用。提供了一种核酸适配体或其衍生物，其核苷酸序列为序列表中序列1。还提供了试剂盒，包括核酸适配体或其衍生物，以及，固定或偶联所述核酸适配体或其衍生物的载体，所述核酸适配体为序列1所示的单链DNA分子。基于核酸适配体磁性纳米颗粒技术用于外周血中高表达碱性磷酸酶的循环肿瘤细胞、外泌体和自由碱性磷酸酶捕获和检测的方法，可以实现对目标细胞、外泌体或自由蛋白高选择性的捕获及检测。

Description

核酸适配体在识别并结合碱性磷酸酶异源二聚体或肿瘤检测中的应用 技术领域
本发明属于生物技术与临床医学技术领域，具体涉及核酸适配体在识别并结合碱性磷酸酶异源二聚体或肿瘤检测中的应用。
背景技术
核酸适配体(aptamer)是一类能与靶标物质特异性相互作用的单链DNA、RNA、肽核酸或化学修饰的核酸序列，通常由15-80个核苷酸组成。核酸适配体可以形成特定的三维结构与靶标分子高亲和力结合，如发卡、假结、G-四链体等结构，高特异性地结合作用是通过范德华力、氢键、静电作用和疏水作用等分子间相互作用实现的。由于核酸适配体具有亲和力高、特异性好、无免疫原性、易合成、改造与修饰、生物化学稳定性好、能可逆变性与复性等特性，所以被称之为“化学抗体”。
核酸适配体可被应用在一些疾病的诊断和检测、药物靶点定位、新药研发和运输相关药物分子等领域，目前，用于治疗癌症、艾滋病等疾病的核酸适配体也不断涌现。例如，由Eyetch/Pfizer开发的靶向VEGF的核酸适配体(商品名Macugen)2004年已获得FDA的批准，成功地用于治疗年龄相关的黄斑变性。近年提出的利用细胞-SELEX技术筛选特异性核酸适配体，进而发现肿瘤标志物的方法具有好的应用前景。但目前只有极少数成功的例子，其中的瓶颈问题就在于位于细胞膜上的核酸适配体靶分子的纯化/鉴定。
碱性磷酸酶(ALP或AKP)是广泛分布于人体肝脏、骨骼、肠、肾和胎盘等组织经肝脏向胆外排出的一种酶，可直接参与磷代谢，在钙、磷的消化、吸收、分泌及骨化的过程发挥了重要作用。这种酶能催化核酸分子脱掉5’磷酸基团，从而使DNA或RNA片段的5’-P末端转换成5’-OH末端。但它不是单一的酶，而是一组同功酶。目前已知人类同工酶包括：组织非特异性碱性磷酸酶(TNAP)，肠型碱性磷酸酶(IAP)，胎盘型碱性磷酸酶(PALP)和类胎盘型碱性磷酸酶(GCAP)。血清碱性磷酸酶正常人血清中的碱性磷酸酶主要来自肝和骨骼，碱性磷酸酶测定主要用于诊断肝胆和骨骼系统疾病，是反映肝外胆道梗阻、肝内占位性病变和佝偻病的重要指标。
碱性磷酸酶异源二聚体在结直肠癌、乳腺癌、肝细胞癌、宫颈癌等肿瘤组织中高表达。游离的碱性磷酸酶异源二聚体、含有碱性磷酸酶异源二聚体的外泌体或循环肿瘤细胞可以从原发性肿瘤或转移病灶释放到血流中。因此，检测游离的碱性磷酸酶异源二聚体、外泌体或循环肿瘤细胞将有助于肿瘤的早期诊断和筛查、监测术后患者肿瘤的复发与转移、评估抗肿瘤药物的敏感性与患者预后以及选择个体化治疗的策略。
循环肿瘤细胞是指恶性肿瘤在发展过程中传播并存活于外周血中的肿瘤细胞，与肿瘤的转移和预后密切相关。循环肿瘤细胞检测是指对肿瘤患者外周血中的循环肿瘤细胞进行分析的方法，在外周血中检到循环肿瘤细胞是预示肿瘤转移最直接、重要的方法，在肿瘤早期转移的临床诊断、预后判断、监测疗效等方面具有重要意义。循环肿瘤细胞的发现有望改变临床上仍依赖于影像学检查及传统肿瘤标志物的现状。由于外周血中循环肿瘤细胞的数量极其稀少，故对检测技术的灵敏度和选择性提出了极高的要求。目前用于循环肿瘤细胞检测的方法很多，首先通过密度梯度离心法、细胞过滤或黏附技术、免疫磁珠分离技术和微流控芯片技术等方法分离富集得到循环肿瘤细胞，再通过免疫细胞化学技术、逆转录聚合酶链反应和流式细胞术等对循环肿瘤细胞进行检测。目前美国食品和药品管理局(FDA)唯一批准的检测循环肿瘤细胞的方法是CellSearch系统，虽然能够实现循环肿瘤细胞的捕获与检测，但仍然要进一步提高灵敏度和特异性，并且实现方法的快速简便高通量。
外泌体是一种直径为30-100nm的纳米级脂质包裹体结构，内部包裹了蛋白、mRNA和microRNA等物质。包括肿瘤细胞在内的几乎所有类型的细胞，都可以产生并释放外泌体。外泌体由细胞分泌释放出来，在血液等体液内传播，最后又可被其他细胞吞噬，是细胞间通讯的重要介质。越来越多的研究发现，宿主细胞或肿瘤细胞分泌的外泌体参与了肿瘤发生、生长、侵袭和转移，所以外泌体的检测和研究得到了越来越多的关注。
发明公开
本发明一个目的是提供一种核酸适配体或其衍生物。
本发明提供的核酸适配体或其衍生物，为如下1)-7)中任一种：
1)序列1所示的单链DNA分子；
2)将1)所示的核酸适配体删除或增加一个或几个核苷酸，得到与所述核酸适配体具有相同功能的核酸适配体的衍生物；
3)将1)所示的核酸适配体进行核苷酸取代或修饰，得到与所述核酸适配体具有相同功能的核酸适配体的衍生物；
4)将1)所示的核酸适配体的骨架(指序列1的第1-7和37-44个核酸所形成的茎)改造为硫代磷酸脂骨架，得到与所述核酸适配体具有相同功能的核酸适配体的衍生物；
5)由1)所示的核酸适配体编码的RNA分子，得到与所述核酸适配体具有相同功能的核酸适配体衍生物；
6)由1)所示的核酸适配体编码的肽核酸，得到与所述核酸适配体具有相同功能的核酸适配体的衍生物；
7)将1)-6)任一所示的核酸适配体的一端或中间接上信号分子和/或活性分子和/或功能基团和/放射性核素，得到与所述核酸适配体具有相同功能的核酸适配体的衍生物。
上述核酸适配体或其衍生物中，
所述核酸适配体的衍生物为去除或改变序列1所示的核酸适配体核苷酸序列自5’端第1个核苷酸起包括5’端第一个核苷酸残基的1-7个核苷酸；和/或，去除序列1所示的核酸适配体的核苷酸序列自3’端第1个核苷酸起包括3’端第一个核苷酸残基的1-7个核苷酸；或所述核酸适配体的衍生物为将序列1所示的核酸适配体核苷酸序列5’端或3’端增加若干个核苷酸或修饰基团且不影响序列1第10-36位形成的结构(G-四链体结构)，保留的核苷酸残基组成的核酸适配体。
上述核酸适配体或其衍生物中，
所述核酸适配体的衍生物为如下1)-6)中任一种：
1)序列2所示的单链DNA分子；
2)序列3所示的单链DNA分子；
3)序列4所示的单链DNA分子；
4)序列5所示的单链DNA分子；
5)序列6所示的单链DNA分子。
6)序列7所示的单链DNA分子。
上述核酸适配体或其衍生物中，
核酸适配体衍生物为在1)-6)任一所示的核酸适配体的5’端或3’端标记荧光基团、生物素基团或放射性核素。
上述的核酸适配体或其衍生物在如1)-4)中至少一种中的应用也是本发明保护的范围：
1)检测或诊断碱性磷酸酶；
2)制备检测或诊断碱性磷酸酶产品；
3)提取或捕获碱性磷酸酶；
4)制备提取或捕获碱性磷酸酶产品；
所述检测或诊断或提取或捕获的靶标为碱性磷酸酶本身、碱性磷酸酶异源二聚体、含有碱性磷酸酶或其异源二聚体的细胞、含有碱性磷酸酶或其异源二聚体的外泌体、含有碱性磷酸酶或其异源二聚体的组织切片、含有碱性磷酸酶或异源二聚体的动物活体。
上述应用中，所述检测或诊断的样本为全血、血清、培养液、唾液、尿液、组织切片或活体；
或，所述检测或诊断方式为荧光成像，比如细胞的荧光成像、组织切片的荧光成像、活体的荧光成像，进而镜检。
上述的核酸适配体或其衍生物在如下a1-a26至少一种中的应用也是本发明保护的范围：
a1)富集提取碱性磷酸酶；
a2)识别并结合或辅助识别并结合碱性磷酸酶；
a3)识别并结合或辅助识别并结合表达碱性磷酸酶的细胞；
a4)检测待测样品中碱性磷酸酶含量或活性；
a5)检测待测样品中是否含有碱性磷酸酶；
a6)检测与抗碱性磷酸酶的抗体结合的物质；
a7)检测与碱性磷酸酶相互作用的蛋白；
a8)制备富集提取碱性磷酸酶产品；
a9)制备识别并结合或辅助识别并结合碱性磷酸酶产品；
a10)制备检测待测样品中碱性磷酸酶含量或活性产品；
a11)制备检测待测样品中是否含有碱性磷酸酶产品；
a12)制备检测与抗碱性磷酸酶的抗体结合的物质产品；
a13)制备检测与碱性磷酸酶相互作用的蛋白产品；
a14)制备诊断和/或治疗与碱性磷酸酶相关疾病的产品；
a15)捕获和/或检测表达或高表达碱性磷酸酶表达细胞或外泌体；
a16)制备捕获和/或检测表达或高表达碱性磷酸酶细胞或外泌体的产品；
a17)制备用于靶向碱性磷酸酶的动物成像的探针；
a18)制备用于靶向碱性磷酸酶治疗的产品；
a19)检测或捕获待测样品中表达或高表达碱性磷酸酶的肿瘤或肿瘤细胞；
a20)检测或捕获待测样品中表达或高表达碱性磷酸酶的循环肿瘤细胞；
a21)检测或捕获待测样品本中表达或高表达碱性磷酸酶的外泌体；
a22)检测或捕获待测样品中的可溶性碱性磷酸酶；
a23)制备检测或捕获待测样品中表达或高表达碱性磷酸酶的肿瘤或肿瘤细胞产品；
a24)制备检测或捕获待测样品中表达或高表达碱性磷酸酶的循环肿瘤细胞产品；
a25)制备检测或捕获待测样品中表达或高表达碱性磷酸酶的外泌体产品；
a26)制备检测或捕获待测样品中的可溶性碱性磷酸酶产品。
本发明的另一个目的是提供一种试剂盒。
本发明提供的试剂盒，包括上述核酸适配体或其衍生物，以及，固定或偶联所述核酸适配体或其衍生物的载体。
上述试剂盒中，
所述试剂盒具有如下b1-b11中至少一种功能：
b1)富集提取碱性磷酸酶；
b2)识别并结合或辅助识别并结合碱性磷酸酶；
b3)识别并结合或辅助识别并结合表达碱性磷酸酶的细胞；
b4)检测待测样品中碱性磷酸酶含量或活性；
b5)检测待测样品中是否含有碱性磷酸酶；
b6)检测与抗碱性磷酸酶的抗体结合的物质；
b7)检测与碱性磷酸酶相互作用的蛋白；
b8)检测或捕获待测样品中表达或高表达碱性磷酸酶的肿瘤或肿瘤细胞；
b9)检测或捕获待测样品中表达或高表达碱性磷酸酶的循环肿瘤细胞；
b10)检测或捕获待测样品中表达或高表达碱性磷酸酶的外泌体；
b11)检测或捕获待测样品中的可溶性碱性磷酸酶.
上述试剂盒中，
所述固定或偶联核酸适配体或其衍生物的载体为纳米颗粒或微米颗粒或芯片。本实施例中的载体为磁性纳米颗粒，是表面带有链霉亲和素修饰的具有超顺磁性的磁性纳米颗粒(200nm)，既起到尺寸放大作用，还可用于磁分离操作，实现高效率捕获。不限于磁球，也可以是芯片等基质。
所述固定或偶联核酸适配体或其衍生物是通过偶联进行，偶联是指通过共价偶联、疏水作用或者分子间作用力连接在一起，本实施例中涉及到链霉亲和素和生物素相互作用连接。
上述试剂盒中，所述固定或偶联核酸适配体或其衍生物的载体为纳米颗粒或微米颗粒或芯片。
上述试剂盒中，
所述纳米颗粒为修饰物修饰的纳米/微米颗粒；
或，所述纳米颗粒为磁性纳米颗粒；
或，所述修饰物为链霉亲和素、生物素、羧基、氨基或巯基。
上述试剂盒还包括与碱性磷酸酶反应的显色底物；
所述显色底物为荧光底物分子、化学发光底物分子或发可见光底物分子。
所述显色底物为荧光底物分子、化学发光底物分子或发可见光底物分子或其它碱性磷酸酶底物。本发明实施例涉及到对硝基苯磷酸二钠(pNPP)，其与碱性磷酸酶反应产生对硝基苯酚，在碱性条件下呈黄色，可以在405nm处检测吸光度。还涉及BCIP/NBT与碱性磷酸酶反应后，产生蓝紫色沉淀。还涉及荧光素二磷酸与碱性磷酸酶反应后，产生绿色荧光。
上述试剂盒还包括红细胞裂解液和磁分离架；若待测样本为全血，则包括红细胞裂解液，若待测样本为血清或血浆或唾液，则不需要此裂解液。
上述中，所述表达或高表达碱性磷酸酶表达细胞为表达或高表达碱性磷酸酶的肿瘤细胞；
和/或，所述表达或高表达碱性磷酸酶的肿瘤细胞为人宫颈癌细胞、人乳腺癌细胞、人结肠癌细胞或人肝细胞癌细胞；
和/或，所述表达或高表达碱性磷酸酶的循环肿瘤细胞为人宫颈癌循环肿瘤细胞、人乳腺癌循环肿瘤细胞、人结肠癌循环肿瘤细胞或人肝细胞癌循环肿瘤细胞。
上述核酸适配体或其衍生物，以及，固定或偶联所述核酸适配体或其衍生物的载体在制备具有如下b1-b11至少一种功能的产品中的应用也是本发明保护的范围：
b1)富集提取碱性磷酸酶；
b2)识别并结合或辅助识别并结合碱性磷酸酶；
b3)识别并结合或辅助识别并结合表达碱性磷酸酶的细胞；
b4)检测待测样品中碱性磷酸酶含量或活性；
b5)检测待测样品中是否含有碱性磷酸酶；
b6)检测与抗碱性磷酸酶的抗体结合的物质；
b7)检测与碱性磷酸酶相互作用的蛋白；
b8)检测或捕获待测样品中表达或高表达碱性磷酸酶的肿瘤或肿瘤细胞；
b9)检测或捕获待测样品中表达或高表达碱性磷酸酶的循环肿瘤细胞；
b10)检测或捕获待测样品中表达或高表达碱性磷酸酶的外泌体；
b11)检测或捕获待测样品中的可溶性碱性磷酸酶。
上述中，所述待测样品为外周血全血、血清、血浆、细胞培养液或唾液。
在本发明的实施例中，
表达或高表达碱性磷酸酶的循环肿瘤细胞对应的待测样本为外周血全血；
表达或高表达碱性磷酸酶的外泌体对应的待测样本为外周血血清或血浆；
表达或高表达碱性磷酸酶的自由蛋白对应的待测样本为外周血血清或血浆，或唾液。
本发明第3个目的是提供了一种捕获和/或检测待测样本是否含有表达或高表达碱性磷酸酶的肿瘤细胞的方法，包括如下步骤：
1)制备核酸适配体磁性纳米颗粒和去除待测样本的红细胞；
所述待测样品为外周血全血；
所述制备核酸适配体磁性纳米颗粒为将磁性纳米颗粒和碱性磷酸酶核酸适配体连接，得到核酸适配体磁性纳米颗粒；
所述制备核酸适配体磁性纳米颗粒为将上述链霉亲和素修饰的磁性纳米颗粒和上述生物素标记的碱性磷酸酶核酸适配体偶联，得到核酸适配体磁性纳米颗粒；
2)将所述去除红细胞外周血全血样本与所述核酸适配体磁性纳米颗粒结合后再进行磁分离，去除非特异性细胞，得到含有循环肿瘤细胞的产物；
3)检测含有循环肿瘤细胞的产物，根据吸光度或细胞表面是否生成沉淀判断待测样本是否含有表达或高表达碱性磷酸酶的肿瘤细胞。
上述根据吸光度判断待测样本是否含有表达或高表达碱性磷酸酶的肿瘤细胞的方法为如下A：
A、将含有循环肿瘤细胞的产物用碱性磷酸酶的底物(如pNPP)染色显色，然后检测吸光度；以对照核酸适配体序列作为对照，若与对照核酸适配体序列捕获后吸光度值有显著差异，则待测样本含有或候选含有表达或高表达碱性磷酸酶的肿瘤细胞；若与对照序列捕获后吸光度值无显著差异，则待测样本不含有或候选不含有表达或高表达碱性磷酸酶的肿瘤细胞；
上述根据细胞表面是否生成沉淀判断待测样本是否含有表达或高表达碱性磷酸酶的肿瘤细胞的方法为如下B：
B、将含有循环肿瘤细胞的产物用碱性磷酸酶的底物(如BCIP/NBT)染色后显微镜观察，若观察到表面带有蓝紫色沉淀的细胞，则待测样本含 有或候选含有表达或高表达碱性磷酸酶的肿瘤细胞；若无表面蓝紫色细胞，则待测样本不含有或候选不含有表达或高表达碱性磷酸酶的肿瘤细胞。
本发明第4个目的是提供了一种捕获和/或检测待测样本是否含有表达或高表达碱性磷酸酶的外泌体的方法，包括如下步骤：
1)制备核酸适配体磁性纳米颗粒和收集待测样本的肿瘤细胞外泌体；
所述制备核酸适配体磁性纳米颗粒为将磁性纳米颗粒和碱性磷酸酶核酸适配体连接，得到核酸适配体磁性纳米颗粒；
所述制备核酸适配体磁性纳米颗粒为将上述链霉亲和素修饰的磁性纳米颗粒和上述生物素标记的碱性磷酸酶核酸适配体偶联，得到核酸适配体磁性纳米颗粒；
所述收集待测样本的肿瘤细胞外泌体为从待测样本的血清或血浆中收集肿瘤细胞外泌体；
2)将所述外泌体与所述核酸适配体磁性纳米颗粒结合后再进行磁分离，去除非特异性结合，得到含有外泌体产物；
3)检测含有外泌体产物，通过检测含有外泌体产物的吸光度或产物表面是否生成沉淀，判断待测样本是否含有表达或高表达碱性磷酸酶的肿瘤细胞。
所述检测含有外泌体产物的吸光度判断待测样本是否含有表达或高表达碱性磷酸酶的肿瘤细胞方法为如下A：
A、将含有外泌体产物用pNPP染色显色，然后检测吸光度；以对照核酸适配体序列作为对照，若与对照核酸适配体序列捕获后吸光度值有显著差异，则待测样本含有或候选含有表达或高表达碱性磷酸酶的外泌体；若与对照序列捕获后吸光度值无显著差异，则待测样本不含有或候选不含有表达或高表达碱性磷酸酶的外泌体；
所述检测含有外泌体产物表面是否生成沉淀判断待测样本是否含有表达或高表达碱性磷酸酶的肿瘤细胞方法为如下B：
B、将产物用BCIP/NBT染色后显微镜观察，若观察到表面带有蓝紫色沉淀，则待测样本含有或候选含有表达或高表达碱性磷酸酶的外泌体；若无表面蓝紫色沉淀，则待测样本不含有或候选不含有表达或高表达碱性磷 酸酶的外泌体。
本发明第5个目的是提供了一种捕获和/或检测待测样本的游离蛋白中是否含有碱性磷酸酶的方法，包括如下步骤：
1)制备核酸适配体磁性纳米颗粒和收集待测样本的游离蛋白；
所述制备核酸适配体磁性纳米颗粒为将磁性颗粒和碱性磷酸酶核酸适配体连接，得到核酸适配体磁性颗粒；
所述制备核酸适配体磁性纳米颗粒为将上述链霉亲和素修饰的磁性纳米颗粒和上述生物素标记的碱性磷酸酶核酸适配体偶联，得到核酸适配体磁性纳米颗粒；
所述收集待测样本的游离蛋白为从待测样本的血清或血浆中收集游离蛋白；
2)将所述游离蛋白与所述核酸适配体磁性纳米颗粒结合后再进行磁分离，去除非特异性蛋白，得到含有游离蛋白产物；
3)检测含有游离蛋白产物，通过检测含有游离蛋白产物的吸光度或游离蛋白是否能够荧光染色判断待测样本的游离蛋白中是否含有碱性磷酸酶。
所述检测含有游离蛋白产物的吸光度判断待测样本的游离蛋白中是否含有碱性磷酸酶为如下A：
A、将含有游离蛋白产物用pNPP染色显色，然后检测吸光度；以对照核酸适配体序列作为对照，若与对照核酸适配体序列捕获后吸光度值有显著差异，则待测样本的游离蛋白中含有或候选含有碱性磷酸酶；若与对照序列捕获后吸光度值无显著差异，则待测样本的游离蛋白中不含有或候选不含有碱性磷酸酶；
所述检测含有游离蛋白产物是否能够荧光染色判断待测样本的游离蛋白中是否含有碱性磷酸酶为如下B：
B、将产物用荧光素二磷酸显色，然后测定其荧光，若观察到有荧光产生，则待测样本的游离蛋白中含有或候选含有碱性磷酸酶；若无荧光或无明显荧光产生，则待测样本的游离蛋白中不含有或候选不含有碱性磷酸酶。
上述中，所述碱性磷酸酶为异源二聚体碱性磷酸酶；
和/或，所述异源二聚体碱性磷酸酶为PALP(胎盘型碱性磷酸酶)、IAP(肠型碱性磷酸酶)、GCAP(生殖细胞碱性磷酸酶)或任意的异源二聚体。
附图说明
图1为核酸适配体BG2的二级结构、核酸适配体BG2及其衍生物的表观结合常数、竞争实验。
图2为用核酸适配体检测细胞表面碱性磷酸酶异源二聚体的表达情况。
图3为胎盘型碱性磷酸酶(PALP)和肠型碱性磷酸酶(IAP)分别敲降后，核酸适配体BG2的结合情况。
图4为核酸适配体BG2捕获碱性磷酸酶异源二聚体；
A)为PC-3细胞表面分布表达胎盘型碱性磷酸酶(PALP)、肠型碱性磷酸酶(IAP)、生殖细胞碱性磷酸酶(GCAP)或其异源二聚体蛋白后，核酸适配体BG2的结合情况；B)为核酸适配体BG2对碱性磷酸酶异源二聚体阳性细胞的捕获情况。
图5为利用核酸适配体BG2提取的碱性磷酸酶活性测定。
图6为核酸适配体BG2检测磷酸酶异源二聚体表达的细胞成像和组织切片免疫染色；
A)为核酸适配体BG2实现LoVo细胞碱性磷酸酶异源二聚体表达的细胞成像；B)为BG2适配体用于组织切片免疫染色(上图：对照序列L45；下图：适配体BG2)。
图7为BG2适配体用活体动物成像(左侧：活动物；右侧：离体肿瘤)。
图8为核酸适配体微米磁球对待测样品中靶细胞的捕获情况；
A)为核酸适配体微米磁球对细胞的捕获情况；B)为核酸适配体磁性纳米颗粒捕获及检测靶细胞结果；C)为核酸适配体磁性颗粒捕获细胞的碱性磷酸酶的活性；D)为核酸适配体磁性纳米颗粒在混合细胞中捕获及检测靶细胞结果；E)为核酸适配体磁性纳米颗粒在人全血中捕获及检测靶细胞结果；F)为核酸适配体磁性纳米颗粒在人全血中捕获及镜检靶细胞结果。
图9为核酸适配体磁性纳米颗粒在外泌体中捕获及镜检靶细胞结果。
图10为核酸适配体磁性纳米颗粒检测待测样品中可溶性碱性磷酸酶；
A)为核酸适配体磁性纳米颗粒检测可溶性碱性磷酸酶的结果；B)和C)为核酸适配体糖球检测细胞培养基中可溶性碱性磷酸酶的结果。
实施发明的最佳方式
下述实施例中所使用的实验方法如无特殊说明，均为常规方法。
下述实施例中所用的材料、试剂等，如无特殊说明，均可从商业途径得到。
结合缓冲溶液1：含137mM NaCl、5mM MgCl 2、2.7mM KCl、2mM KH 2PO 4、10mM Na 2HPO 4，25mM葡萄糖，1μg/ml BSA，0.1μg/ml鲱鱼精DNA和0.01％(v/v)吐温-80，其余为水。
洗脱缓冲液(pH＝8.0)：含137mM NaCl、5mM MgCl 2、2.7mM KCl、2mM KH 2PO 4、10mM Na 2HPO 4和25mM葡萄糖，其余为水。
下述实施例中的链霉亲和素修饰的磁性纳米颗粒是厦门普睿迈格生物科技有限公司的产品，其浓度为10mg/mL。
下述实施例中的人宫颈癌细胞(Hela)、人肝细胞癌细胞(SMMC-7721)、人乳腺癌细胞(MCF-7)、人胚胎肾细胞(HEK-293)、和人神经母细胞瘤细胞(SH-SY5Y)和人白血病细胞(Jurkat E6-1)购自中国医学科学院基础医学研究所；人结肠癌细胞(LoVo)、人肝细胞癌细胞(HepG2)、人结肠癌细胞(HCT116)、人前列腺癌细胞(PC3)购自中国科学院典型培养物保藏委员会细胞库。
下述实施例中的PBS缓冲液：含12mM NaH 2PO 4、8mM Na 2HPO 4、122mM NaCl和5mM KCl，其余为水。
下述实施例中的PBST缓冲液：含12mM NaH 2PO 4、8mM Na 2HPO 4、122mM NaCl、5mM KCl、和0.01％(v/v，体积百分含量)吐温-80的溶液，其余为水。
下述实施例中的结合缓冲液2：含12mM NaH 2PO 4、8mM Na 2HPO 4、122mM NaCl、5mM KCl，1μg/ml牛血清白蛋白，0.1μg/ml鲱鱼精DNA和0.01％(v/v，体积百分含量)吐温-80的溶液，其余为水。
下述实施例中的工作缓冲液(pH＝9.5)：含100mM Tris-HCl，100mM NaCl和5mM MgCl 2。
下述实施例中的红细胞裂解液是Roche公司的产品，产品目录号是11814389001。
下述实施例中对硝基苯磷酸二钠盐六水合物(pNPP)、5-溴-4-氯-3-吲哚基磷酸酯对甲苯胺盐(BCIP)和氯化硝基四氮唑兰(NBT)，均为百灵威科技有限公司的产品，产品目录号分别是254303、338560和151804。荧光素二磷酸(Fluorescein Diphosphate，FDP)是Thermo Fisher Scientific公司的产品，产品货号是F2999。
下述实施例中的红细胞裂解液是西格玛奥德里奇(上海)贸易有限公司的产品，产品目录号是11814389001。
实施例1、核酸适体的筛选和制备
一、细胞的培养
人结肠癌细胞LoVo、人乳腺癌细胞MCF-7、人宫颈癌细胞Hela用RPMI1640(含10％胎牛血清、1％青/链霉素)培养。所有细胞均在培养箱中进行常规培养(37℃，5％CO 2)，每两-三天传代一次。
二、随机核酸文库的设计
设计两端包括20个固定核苷酸、中间包括45个核苷酸的随机文库如下：5’-ACGCTCGGATGCCACTACAG TYRRRRRRNN GGGNNNGGNNNGGNNGGNNNNNNNN GGNYYYYYYRT CTCATGGACGTGCTGGTGAC(序列8)-3’；N代表A、T、C或G，Y代表T或C，R代表G或A。
三、核酸适体的筛选和表征
1、文库预处理
将10nmol随机核酸文库(步骤二中合成)溶于结合缓冲液，95℃变性5min，冰上冷却10min，室温复性放置30min。
2、正筛
将各1×10 6人结肠癌细胞LoVo、人乳腺癌细胞MCF-7和人宫颈癌细胞Hela用含5mM EDTA的PBS消化10min后，混匀，用洗涤缓冲溶液洗涤1次后，加入上述DNA文库孵育。孵育30min后离心去掉上清，用洗涤缓冲溶液洗涤2次，结合在细胞上的DNA分子直接用于PCR。PCR扩增的正向引物为：
5'-FAM-ACGCTCGGATGCC ACTACAG-3'(序列9)；
反向引物：
5'-biotin-GTC ACC AGC ACG TCC ATG AG(序列10)
PCR扩增程序：94℃3min；94℃30s，60℃30s，72℃30s，10个循环；72℃，5min。
用链霉亲和素琼脂糖球从PCR产物中分离得到FAM标记的单链DNA(ssDNA)序列。将得到的ssDNA用NAP-5柱子(通用电气医疗集团，瑞典)脱盐，真空干燥，用于下一轮的筛选。
为了提高核酸适体的亲和力和特异性，在筛选过程中逐步增加洗涤次数、减少正筛细胞数量，以增加筛选的压力。5轮筛选后，进行高通量测序。
去掉引物后得到的核酸适体BG2如下：
5'-CAAGGAATAGGGGTCGGTGTGGGTGGTTATGATTGGCTTCCTTG-3'(序列1)。
3、核酸适配体亲和力表征
取一皿对数生长期的结肠癌细胞LoVo，用含0.2％EDTA的PBS消化成单分散细胞悬液后平均分成若干份，分别与标记荧光分子的核酸适配体探针溶液孵育30min后，用洗涤缓冲液洗涤两遍，用BD公司的FACSCalibur流式细胞仪测定细胞表面的荧光强度。以细胞表面平均荧光强度和核酸适体浓度作图，利用公式Y＝B maxX(K d+X)计算核酸适体的平衡解离常数K d。
4、BG2核酸适体及其衍生物结合情况
经测定BG2核酸适配体表观解离常数为2.5±0.3nM(图1B)。为了研究序列1延伸后对其结合的影响，进一步测定了加上引物序列的亲和力，其序列为：
5'-ACGCTCGGATGCCACTACAGt CAAGGAATAGGGGTCGGTGTGGGTGGTTATGATTGGCTTCCTTG tCTCATGGACGTGCTGGTGAC-3'(序列2,BG2核酸适体衍生物)
经测定其仍然保持很好的结合力，其表观解离常数为2.9±0.4nM(图1C)。
如图1A所示，序列1经结构分析后，发现可以形成环-茎结构，设计并合成一系列经截短的核酸序列，通过荧光染料修饰，然后考察它们与LoVo的结合能力，挑选结合能力最强的序列用于进一步的应用，最终得到的截短核酸适体序列如下：
核酸适体BG2c:
GGGGTCGGTGTGGGTGGTTATGATTGG(序列3,BG2核酸适体衍生物)
由图1D所示，适配体截短，其亲和力有一定程度的降低(9.3±1.6nM)。
说明GGGGTCGGTGTGGGTGGTTATGATTGG是该适配体与靶标作用的核心区域。
保留环区序列，对序列1的茎进行了随机的替换，例如序列4：
5‘-TAAGAAATAGGGGTCGGTGTGGGTGGTTATGATTGGC TTTCTTA-3’(序列4,BG2核酸适体衍生物)；
序列5：
5 ‘-GATAACATAGGGGTCGGTGTGGGTGGTTATGATTGGC TGTTATC-3’(序列5,BG2核酸适体衍生物)
经测定序列4和序列5的表观解离常数分别为：5.7±0.4nM(图1E)和 3.0±0.5nM(图1F)，说明仍然保持良好的亲和力。
为了提高核酸适配体的稳定性，对核酸适配体BG2进行了硫代修饰，序列如：
5'-sCsAsAsGsGsAsATAGGGGTCGGTGTGGGTGGTTATGATTGGCsTsTsCsCsTsTsG-3'(序列6,BG2核酸适体衍生物)，其中sA，sT,sG，sC分别为硫代修饰
经测定硫代修饰的核酸适配体仍然保持很好的亲和力，其表观解离常数为3.5±0.6nM(图1G)。
上述表明，序列1-序列6所示的BG2核酸适体或其衍生物均能结合结肠癌细胞LoVo。
5、BG2核酸适体衍生物与BG2核酸适配体的竞争
将荧光素标记的核酸适配体BG2(BG2-FAM，100nM)分别与未标记荧光分子的BG2核酸适配体及其衍生物(4μM)混合，分别加入约5×10 4个LoVo细胞数，分别得到混合液，将混合液在冰上孵育30min，用洗涤缓冲液洗涤两遍，过400目筛网后，上流式细胞仪进行检测。除上述BG2衍生物外，还包括BG2环区序列：
序列7：5‘-TAGGGGTCGGTGTGGGTGGTTATGATTGGC-3’(序列7)；
对照核酸序列L45的核苷酸序列： NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN(序列11)。
结果如图1H所示，未标记荧光BG2核酸适体(序列1)及其代表性衍生物(序列2-7)都能够与荧光素标记的BG2核酸适配体竞争性与目标细胞结合，说明BG2的衍生物具有与BG2核酸适配体相同的功能。
实施例2、核酸适配体BG2与不同类型细胞结合情况的研究
一、核酸适配体BG2及其衍生物的制备
1、核酸适配体BG2的合成
通过DNA合成仪合成核酸适配体BG2，核酸适配体BG2的核苷酸序列如下：5’-CAAGGAATAGGGGTCGGTGTGGGTGGTTATGATTGGCTTCCTTG-3’(序列1)，根据试验需要可以在核酸适配体BG2上标记不同的分子。
2、DNA脱保护：用冷氨水脱保护后，然后把DNA溶解在TEAA溶液当中；
3、DNA纯化：通过PAGE或高效液相色谱仪纯化；
4、DNA干燥：通过离心浓缩干燥；
5、溶解测定浓度备用。
二、荧光素标记的核酸适配体BG2溶液(BG2-FAM)的制备
1、荧光素标记的核酸适配体BG2-FAM溶液的制备
荧光素标记的核酸适配体BG2为在核酸适配体BG2的5’端偶联荧光素基团FAM得到的，用结合缓冲液溶解BG2-FAM，依据紫外吸收标定浓度(200nM)后，95℃加热5min，冰上放置5min，室温放置15min，得到BG2-FAM溶液。
2、荧光素标记的对照核酸序列溶液(L45-FAM)(200nM)的制备
荧光素标记的对照核酸序列L45(L45-FAM)为在对照核酸序列L45的5’端偶联荧光素基团FAM得到的，用结合缓冲液溶解L45-FAM，依据紫外吸收标定浓度(100nM)后，95℃加热5min，冰上放置5min，室温放置15min，得到L45-FAM溶液。
对照核酸序列L45的核苷酸序列：TTTNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN。
三、细胞株的预处理
分别取如下9种生长对数期的细胞株各一皿：人宫颈癌细胞(Hela)、 人肝细胞癌细胞(SMMC7721)、人乳腺癌细胞(MCF-7)、人胚胎肾细胞(HEK-293)、人神经母细胞瘤细胞(SH-SY5Y)、人结肠癌细胞(LoVo)、人肝细胞癌细胞(HepG2)、人结肠癌细胞(HCT116)、人前列腺癌细胞(PC3)，用5mM EDTA的PBS消化成单分散细胞悬液后，用洗涤缓冲液洗涤2遍，分为若干份，每份细胞数为5×10 4个；将悬浮生长的人白血病细胞(Jurkat E6-1)直接吹散后用洗涤缓冲液洗涤2遍，平均分为若干份，每份细胞数为5×10 4个。
四、用抗体表征细胞系异源二聚体表达情况
用5mM EDTA的PBS消化细胞成单分散细胞悬液后，用洗涤缓冲液洗涤2遍。然后上述细胞分分别加入10μg/mL的anti-IAP抗体(货号：GTX60746，GeneTex公司)或10μg/mL的anti-PALP抗体(货号：MA1-20245孵育30min，洗涤一次后，加入4μg/mL抗小鼠m-IgGκBP-PE抗体(sc-516141)孵育30min，洗涤一次后重悬细胞，上流式细胞仪检测。
五、核酸适配体BG2检测细胞株
将实施例2的步骤一制备的BG2-FAM溶液、L45-FAM溶液分别与10种不同来源的细胞系混合(每份细胞数为5×10 4个)，分别得到混合液，将混合液在冰上孵育30min，用洗涤缓冲液洗涤两遍，过400目筛网后，上流式细胞仪进行检测。
用BD公司的FACSCalibur流式细胞仪收集第一通道的荧光强度数据，作为细胞表面的荧光强度。每个样品的仪器测得荧光强度扣除细胞自发荧光，得到每个样品结合在细胞表面的核酸适体的荧光强度。
用anti-IAP抗体(货号：GTX60746，GeneTex公司)或anti-PALP抗体(货号：MA1-20245)分别证实人结肠癌细胞(LoVo)、人乳腺癌细胞(MCF-7)、人宫颈癌细胞(Hela)、人肝细胞癌细胞(SMMC7721)和人肝细胞癌细胞(HepG2)表达碱性磷酸酶异源二聚体，HCT116细胞低表达碱性磷酸酶异源二聚体，而PC-3细胞、Jurkat细胞、SH-SY5Y细胞和HEK293细胞则不表达碱性磷酸酶异源二聚体。
结果如图2所示，可以看出，荧光素标记的核酸适配体BG2与人结肠癌细胞(LoVo)、人乳腺癌细胞(MCF-7)、人宫颈癌细胞(Hela)、人肝细胞癌细胞(SMMC-7721)和人肝细胞癌细胞(HepG2)结合，而与HCT116 细胞弱结合，与PC-3细胞、Jurkat细胞、SH-SY5Y细胞和HEK293细胞等不结合。
实施例3、核酸适配体BG2与碱性磷酸酶异源二聚体结合的研究
一、核酸适配体BG2及其衍生物的合成
与实施例2的一相同；
二、荧光素标记的核酸适配体BG2溶液(BG2-FAM)的制备
与实施例2的二相同。
三、碱性磷酸酶的敲降实验
1、转染前一天，约3×10 5个LoVo细胞接种在6孔板中，含有2mL的FBS和双抗的1640培养基。
2、待细胞生长至70-90％时，更换为2mL无双抗的1640完全培养基。
3、按照
Figure PCTCN2019072749-appb-000001
RNAiMAX试剂(货号：13778-075)的相关说明，在125μL的无血清Opti-MEM培养基中加入40pmol的siRNA(其中ALPI的siRNA为：ALPI-homo-1288，正义序列(5‘-3’)：GCAAAGCCUACACGUCCAUTT(序列12)，反义序列(5’-3’):AUGGACGUGUAGGCUUUGCTT(序列13)；PALP的siRNA为：PALP-homo-947，正义序列(5‘-3’)：GAGACAUGAAAUACGAGAUTT(序列14)，反义序列(5’-3’):AUCUCGUAUUUCAUGUCUCTT(序列15))混匀。
4、8μL 
Figure PCTCN2019072749-appb-000002
RNAiMAX试剂中加入125μL无血清Opti-MEM培养基稀释，混匀。
5、将上述稀释好的2种碱性磷酸酶siRNA分别与
Figure PCTCN2019072749-appb-000003
RNAiMAX试剂1：1混合并混匀，室温放置5分钟。
6、将250μL的siRNA和
Figure PCTCN2019072749-appb-000004
RNAiMAX混合物加到含有细胞和培养基的培养板的孔中，来回摇晃细胞培养板。
7、细胞在CO2培养箱中37℃中培养72小时。
8、用5mM EDTA的PBS消化细胞成单分散细胞悬液后，用洗涤缓冲液洗涤2遍。然后上述细胞分别加入200nM BG2-FAM溶液，将混合液在冰上孵育30min，用洗涤缓冲液洗涤两遍，过400目筛网后，上流式细胞仪进行检测；取另一部分细胞分别加入10μg/mL的anti-IAP抗体(货号：GTX60746， GeneTex公司)或10μg/mL的anti-PALP抗体(货号：MA1-20245孵育30min，洗涤一次后，加入4μg/mL抗小鼠m-IgGκBP-PE抗体(sc-516141)孵育30min，洗涤一次后重悬细胞，上流式细胞仪检测。
结果如图3所示，分别用IAP蛋白的siRNA(siIAP)或PALP蛋白的siRNA(siPALP)蛋白敲降后，核酸适配体BG2与靶细胞LoVo的结合都降低。
推测，核酸适配体BG2结合靶细胞LoVo的IAP蛋白或PALP蛋白。
四、碱性磷酸酶过表达实验
1、转染前一天，约4×10 5个PC-3细胞接种在6孔板中，含有2mL的FBS和双抗的1640培养基。
2、待细胞生长至80-90％时，更换为2mL无双抗的1640完全培养基。
3、按照
Figure PCTCN2019072749-appb-000005
3000试剂(货号：L3000008)的相关说明，在125μL的无血清Opti-MEM培养基中分别加入3μg的IAP、PALP或GCAP质粒(将IAP(P09923，uniprot数据库基因的ID)或PALP(P05187，uniprot数据库基因的ID)序列插入pCMV-myc载体(优宝生物公司)的Xho1 and EcoR1酶切位点间；将GCAP(P10696，uniprot数据库基因的ID)序列插入pcDNA3.1(-)载体(优宝生物)的Xho1和BamH1酶切位点之间)，5μL P3000 TM试剂混匀。
4、5μL 
Figure PCTCN2019072749-appb-000006
3000试剂中加入125μL无血清Opti-MEM培养基稀释，混匀。
5、将上述稀释好的质粒和
Figure PCTCN2019072749-appb-000007
3000试剂 1：1混合并混匀，室温放置5分钟。
6、将250μL的质粒和
Figure PCTCN2019072749-appb-000008
3000试剂混合物加到含有细胞和培养基的培养板的孔中，来回摇晃细胞培养板。
7、细胞在CO 2培养箱中37℃中培养48小时。
8、用5mM EDTA的PBS消化细胞成单分散细胞悬液后，用洗涤缓冲液洗涤2遍。然后上述细胞分别加入200nM BG2-FMA溶液，将混合液在冰上孵育30min，用洗涤缓冲液洗涤两遍，过400目筛网后，上流式细胞仪进行检测；取另一部分细胞分别加入10μg/mL的anti-IAP抗体(货号：GTX60746，GeneTex公司)或10μg/mL的anti-PALP抗体(货号：MA1-20245孵育30min，洗涤一次后，加入4μg/mL抗小鼠m-IgGκBP-PE抗体(sc-516141)孵育 30min，洗涤一次后重悬细胞，上流式细胞仪检测。
结果如图4A所示，将阴性细胞分别转染PALP质粒、IAP质粒或GCAP质粒后，用其抗体测得它们的蛋白在细胞膜表面都有所表达，但是适配体BG2仍不与细胞结合，只有其IAP蛋白和PALP蛋白或IAP蛋白和GCAP蛋白同时表达时，核酸适配体才能够与BG2结合。
上述结果说明该核酸适配体BG2可以与IAP/PALP或IAP/GCAP异源二聚体结合。
五、碱性磷酸酶原位交联-捕获实验
生物素标记的核酸适配体BG2为在核酸适配体BG2的5’端偶联含有双硫键的生物素bio得到的，用结合缓冲液溶解BG2-SS-bio，依据紫外吸收标定浓度(200nM)后，95℃加热5min，冰上放置5min，室温放置15min，得到BG2-SS-bio溶液。
1、取5×10 6个指数生长期的LOVO细胞，用含5mM EDTA的PBS消化，PBS溶液洗涤2次.
2、加入200nM BG2-SS-bio溶液在冰上孵育30分钟。
3、然后加入25μL of 100mM的双交联试剂disuccinimidyl suberate(DSS,Thermo Fisher Scientific公司)，冰上孵育。
4、孵育2h后，加入25μL的1M Tris-HCl缓冲溶液(pH 7.0)终止交联反应。
5、PBS洗涤2遍，加入0.3mL的细胞裂解液(sigma公司)，裂解细胞。
6、2000rpm离心去除沉淀，收集上清，加入链霉亲和素修饰的琼脂糖微球(GE公司，货号：17-5113-01)，孵育1小时，提取靶标蛋白。
7、上述提取物加入4×SDS上样缓冲液(Bio-Rad公司)加热60℃变性 10min。
8、然后用6％SDS-PAGE电泳分离。
9、转膜至PVDF膜((Millipore,公司)，然后用含5％的脱脂牛奶(上海生工)和0.1％Tween-20的PBS室温封闭1h。
10、分别加入1:5000的anti-IAP抗体(ab186422，Abcam公司)或anti-PALP抗体(ab133602，Abcam公司)在4℃孵育过夜。
11、用PBST洗涤膜5次，加入HRP标记的二抗(1:5000稀释,Santa公司)，室温孵育1小时。
12、用PBST洗涤膜5次，加入SuperSignal West Femto Maximum Sensitivity Substrate试剂(Thermo Fisher Scientific公司)，用全自动化学发光图像分析系统(天能公司)成像。
如图4B所示，核酸适配体BG2可以捕获原位交联的碱性磷酸酶异源二聚体。
上述实验说明该核酸适配体BG2可以与IAP/PALP或IAP/GCAP异源二聚体结合。
实施例4、核酸适配体BG2结合细胞的碱性磷酸酶的研究
一、核酸适配体BG2特异性提取碱性磷酸酶
1、生物素标记的核酸适配体BG2和生物素标记的对照核酸序列L45的制备
(1)生物素标记的BG2(BG2-Bio)
生物素标记的核酸适配体BG2(BG2-Bio)为在核酸适配体BG2的5’端偶联生物素Bio基团得到的，用结合缓冲液溶解BG2-Bio，依据紫外吸收标定浓度(200nM)后，95℃加热5min，冰上放置5min，室温放置15min，得到BG2-Bio溶液。
(2)生物素标记的对照核酸序列L45(L45-Bio)
生物素标记的对照核酸序列L45(L45-Bio)为在对照核酸序列L45的5’端偶联生物素基团Bio得到的，用结合缓冲液溶解L45-Bio，依据紫外吸收标定浓度(200nM)后，95℃加热5min，冰上放置5min，室温放置15min，得到L45-Bio溶液。
对照核酸序列L45的核苷酸序列：TTTNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN。
2、核酸适配体BG2提取碱性磷酸酶
(1)取2×10 8个指数生长期的LOVO细胞，PBS洗涤后，分别与200nM BG2-Bio溶液和200nM L45-Bio溶液(4℃)孵育30分钟，然后加入甲醛固定10分钟。
(2)PBS洗涤2遍，加入1mL的细胞裂解液(R0278-50ML，Sigma)，孵育1小时。
(3)2000rpm离心去除沉淀，收集上清，加入链霉亲和素修饰的琼脂糖微球(GE公司，货号：17-5113-01)，孵育1小时，提取靶标蛋白，得到孵育后的链霉亲和素修饰的琼脂糖微球。
(4)用PBS洗涤上述步骤(3)孵育后的链霉亲和素修饰的琼脂糖微球，洗涤5次，分别得到生物素标记的核酸适配体BG2提取的蛋白、对照核酸序列L45提取的蛋白。
(5)用碧云天生产的碱性磷酸酶检测试剂盒(P0321)测定生物素标记的核酸适配体BG2提取的蛋白、对照核酸序列L45提取的蛋白中碱性磷酸酶的活性。
结果如图5所示，左图为提取蛋白的磷酸酶活性显色照片，右图为显色液在405nm的吸收，可以看出，生物素标记的核酸适配体BG2提取的蛋白可以提取细胞蛋白中碱性磷酸酶，也就是证明核酸适配体BG2与细胞中的碱性磷酸酶结合。
二、核酸适配体BG2提取碱性磷酸酶以及与其相互作用蛋白
1、LoVo细胞的同位素标记
重型同位素标记的LoVo细胞：向不含赖氨酸和精氨酸的RPMI 1640的培养基中加入重型同位素标记的赖氨酸([ 13C 6, 15N 2]-L-赖氨酸，货号：211604102)和重型同位素标记的精氨酸([ 13C 6]-L-精氨酸，货号：201204102)(Silantes GmbH公司，德国)，使重型同位素标记的赖氨酸和重型同位素标记的精氨酸在培养基中的浓度分别为0.274mM和0.575mM；用该培养基培养LoVo细胞6-7代后备用，得到重型同位素标记的LoVo细胞。
轻型同位素标记的LoVo细胞：向不含赖氨酸和精氨酸的RPMI 1640的培养基分别加入轻型同位素标记的赖氨酸([ 12C 6, 14N 2]-L-赖氨酸，货号：L8662)和轻型同位素标记的精氨酸([ 12C 6]-L-精氨酸，货号：A8094)(sigma公司)，使轻型同位素标记的赖氨酸和轻型同位素标记的精氨酸在培养基中的浓度分别为0.274mM和0.575mM。用该培养基培养LoVo细胞6-7代后备用，得到轻型同位素标记的LoVo细胞。
2、生物素标记的核酸适配体BG2和生物素标记的对照核酸序列L45的制备
(1)生物素标记的BG2-Bio溶液
生物素标记的核酸适配体BG2(BG2-Bio)为在核酸适配体BG2的5’端偶联生物素基团Bio得到的，用结合缓冲液溶解BG2-Bio，依据紫外吸收标定浓度(200nM)后，95℃加热5min，冰上放置5min，室温放置15min，得到BG2-Bio溶液。
(2)生物素标记的对照核酸序列L45(L45-Bio)
生物素标记的对照核酸序列L45(L45-Bio)为在对照核酸序列L45的5’端偶联生物素基团Bio得到的，用结合缓冲液溶解L45-Bio，依据紫外吸收标定浓度(200nM)后，95℃加热5min，冰上放置5min，室温放置15min，得到L45-Bio溶液。
对照核酸序列L45的核苷酸序列：TTTNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN。
3、用核酸适配体BG2碱性磷酸酶相互作用蛋白
(1)分别取2×10 8个指数生长期的重型同位素标记的LoVo细胞和轻型同位素标记的LoVo细胞，PBS洗涤后，分别与200nM BG2-Bio溶液和L45-Bio溶液孵育30分钟，然后加入甲醛固定10分钟。
(2)PBS洗涤2遍，加入1mL的细胞裂解液，孵育1小时。
(3)2000rpm离心去除沉淀，收集上清，加入链霉亲和素修饰的琼脂糖微球(GE公司，货号：17-5113-01)，孵育1小时，提取靶标蛋白。
(4)用PBS洗涤上述步骤(3)孵育后的链霉亲和素修饰的琼脂糖微球，洗涤5次，分别得到生物素标记的核酸适配体BG2提取的重型同位素标记的蛋白、对照核酸序列L45提取的轻型同位素标记的蛋白、生物素标记的核酸适配体BG2提取的轻型同位素标记的蛋白和对照核酸序列L45提取的重型同位素标记的蛋白。
4、正向和反向实验
(1)正向实验：将生物素标记的核酸适配体BG2提取的重型同位素标记的蛋白与对照核酸序列L45提取的轻型同位素标记的蛋白混合，得到生物素标记的核酸适配体BG2提取的重型同位素标记的蛋白和对照核酸序 列L45提取的轻型同位素标记的混合体系。
(2)反向实验：将生物素标记的核酸适配体BG2提取的轻型同位素标记的蛋白与对照核酸序列L45提取的重型同位素标记的蛋白混合，得到生物素标记的核酸适配体BG2提取的轻型同位素标记的蛋白和对照核酸序列L45提取的重型同位素标记的混合体系。
5、蛋白的酶解和LC-MS鉴定
(1)DTT还原：分别向生物素标记的核酸适配体BG2提取的重型同位素标记的蛋白和对照核酸序列L45提取的轻型同位素标记的混合体系、生物素标记的核酸适配体BG2提取的轻型同位素标记的蛋白和对照核酸序列L45提取的重型同位素标记的混合体系中加入200μL 20mM二硫苏糖醇(DTT)，56℃反应45min。
(2)IAA烷基化：将步骤(1)的产物离心，弃上清(去除DTT)，向沉淀中分别加入200μL 55mM碘乙酰胺(IAA)，在37℃避光反应30min。
(3)将步骤(2)的产物离心，弃上清(去除IAA)，向沉淀中加入5μg质谱用胰蛋白酶(Promega公司，产品目录号：V5111)，37℃酶切过夜，得到酶切后的多肽。
(4)酶切后的多肽经过真空浓缩后，加入100μL水，利用Ziptip C 18微柱脱盐。质谱分析前，放置-20℃冰箱。
(5)利用LTQ-OrbitrapVelos质谱仪(Thermo Fisher Scientific,San Jose,CA)对步骤(4)的产物进行分析鉴定，得到原始的质谱数据。
(6)数据搜索分析
利用MaxQuant搜索引擎(版本号：1.5.5.1)将步骤(5)获得的原始的质谱数据在uniprot蛋白数据库中进行检索。数据库搜索的一些参数如下：固定化修饰为半胱氨酸上的烷基化修饰，可变修饰为甲硫氨酸上的氧化修饰和蛋白质N端的乙酰化修饰。允许2个漏切位点，母离子容错量为20ppm，MS/MS碎片离子质量误差为0.5Da。
结果如表1所示，可以看出，核酸适配体BG2能够结合表1所示的蛋白，其中包括碱性磷酸酶ALPI、ALPP和ALPPL2，以及与这些碱性磷酸酶相互作用的蛋白；这个实验也证明了，核酸适配体BG2能够用于检测碱性磷酸酶如ALPI、ALPP和ALPPL2。
表1、利用SILAC鉴定出的核酸适配体BG2靶蛋白与其相互作用蛋白
Figure PCTCN2019072749-appb-000009
实施例5、BG2在细胞荧光成像和组织切片免疫荧光中的应用
一、BG2在细胞荧光成像中的应用
1、生物素标记的核酸适配体BG2-bio溶液(200nM)的制备
生物素标记的核酸适配体BG2为在核酸适配体BG2的5’端偶联生物素bio基团得到的，用结合缓冲液溶解BG2-bio，依据紫外吸收标定浓度(200nM)后，95℃加热5min，冰上放置5min，室温放置15min，得到BG2-bio溶液。
2、生物素标记的对照核酸序列溶液(L45-bio)(200nM)的制备
生物素标记的对照核酸序列L45(L45-bio)为在对照核酸序列L45的5’端偶联生物素bio基团得到的，用结合缓冲液溶解L45-bio，依据紫外吸收标定浓度(200nM)后，95℃加热5min，冰上放置5min，室温放置15min，得到L45-bio溶液。对照核酸序列L45的核苷酸序列：TTTNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN。
3、细胞染色
1)将LoVo细胞(已检测表达碱性磷酸酶)在35mm的共聚焦培养皿上培养1天后，用洗涤缓冲溶液洗涤1次后，然后与400μlBG2-bio溶液 37℃孵育30min，L45-bio溶液染色方法相同，空白不染色；
2)用洗涤缓冲液洗涤2次；
3)加入200μL 10nmol/L的链霉亲和素修饰的量子点(Q1104/Q1104a，北京纳晶生物科技有限公司)，孵育20min；
4)用洗涤缓冲液洗涤2次；
3)利用激光共聚焦扫描显微镜或荧光纤维镜观察。
结果如图6A所示，可以看出，BG2可以与LoVo细胞结合。
二、BG2在组织切片染色荧光的应用
应用荧光素标记核酸适体BG2对结肠癌组织(已确诊含有碱性磷酸酶)切片染色。
1、组织切片脱蜡水化
1)烤片：切片于60℃烤箱中烘烤60min；
2)立刻置于第一缸二甲苯中15min，而后置入第二缸二甲苯中15分钟；
3)依次放置入无水乙醇中10min，95％乙醇5min，70％乙醇5min；
4)自来水冲洗5min(缓慢流动的水盆中)，蒸馏水润洗一遍。
2、切片染色抗原修复
利用微波热修复法修复抗原：取适量TE buffer(EDTA 0.292g,Tris-base 6.05g,溶于1000mL蒸馏水，pH＝8.0)，将切片放入盛有修复液的容器中，置微波炉内加热至沸腾，停止加热，使容器内液体温度下降，保持在 95℃～98℃之间并持续15分钟。取出容器，自然冷却至室温，取出切片，蒸馏水冲洗后，再用洗涤缓冲液浸泡，5分钟×3次(保证第一次浸泡的是新配的洗涤缓冲液)。
3、适配体孵育染色步骤
1)切片先与含20％FBS和1mg/ml鲱鱼精DNA的结合缓冲溶液室温孵育60min；
2)然后与200μl BG2-FAM溶液室温孵育60min，对照序列染色方法相同，空白不染色；
3)用洗涤缓冲液洗涤三次；
4)干燥，抗淬灭封片剂封片，利用激光共聚焦扫描显微镜观察。
在临床实际标本切片中，可以实现如图6B所示染色，可以看出，BG2可以与结肠癌组织结合。
实施例6、BG2在活动物成像中的应用
1、Alexa Fluor 647荧光分子标记的核酸适配体BG2溶液(BG2-AF647)(3μM)的制备
Alexa Fluor 647标记的核酸适配体BG2为在核酸适配体BG2的5’端偶联Alexa Fluor 647基团、5’端和3’端1-7的核苷酸硫代修饰(序列6)，为了用结合缓冲液溶解BG2-AF647，依据紫外吸收标定浓度(3μM)后，95℃加热5min，冰上放置5min，室温放置15min，得到BG2-AF647溶液。
2、Alexa Fluor 647荧光分子标记的核酸适配体BG2溶液(L45-AF647)(3μM)的制备
Alexa Fluor 647标记的对照核酸序L45为在核酸适配体BG2的5’端偶联Alexa Fluor 647基团、5’端和3’端1-7的核苷酸硫代修饰，用结合缓冲液溶解L45-AF647，依据紫外吸收标定浓度(3μM)后，95℃加热5min，冰上放置5min，室温放置15min，得到L45-AF647溶液。
对照核酸序列L45的核苷酸序列：sNsNsNsNsNsNsNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNsNsNsNsNsNsNsN。(sN为为硫代修饰的A、T、G或C，N为随机的A、T、G或C)
3、荷瘤小鼠模型
1)4-6周的BALB/c nu/nu雄性小鼠购自北京维通利华实验动物技术有限公司。
2)将指数生长期的1×10 7/mL LoVo细胞或PC-3细胞(100μL)用注射器注入右侧腋下皮下。
2)小鼠饲养3-4周，待肿瘤的直径长至0.8–1.2cm。
3)将100μl的BG2-AF647溶液(3μM)或L45-AF647溶液(3μM)通过尾静脉注射至小鼠静脉。
4)注射30min后，麻醉，小鼠在Maestro TM小动物成像系统(美国剑桥科研仪器公司)成像，激发波长586-601nm，用640nm长通滤光片采集发射光，图像用Maestro v2.10.0处理。将小鼠处死后，剥离肿瘤，用相 同的方法成像。
结果如图7所示，可以看出，BG2-AF647可以实现LoVo荷瘤小鼠的活体成像，而且在活体具有很好的特异性，BG2-AF647无法与PC-3荷瘤小鼠结合。
实施例7、核酸适配体BG2对肿瘤细胞的捕获
一、偶联有BG2核酸适配体的磁性微球和纳米颗粒的制备
1、核酸适配体BG2修饰的磁微球制备
生物素标记的核酸适配体BG2(BG2-Bio)为在核酸适配体BG2的5’端偶联生物素基团Bio得到的，用结合缓冲液溶解BG2-Bio，依据紫外吸收标定浓度(200nM)后，95℃加热5min，冰上放置5min，室温放置15min，得到BG2-Bio溶液。
取100μL链霉亲和素修饰的磁球(货号：112.05D，Invitrogen Dynal AS,挪威)溶液，加入1mLPBS，震荡，置于磁力架，洗涤2次，得到磁微球。
然后将上述BG2-Bio溶液和磁微球在室温孵育30min，然后用PBS洗涤2次，得到BG2修饰的磁微球。
2、对照核酸序列L45(L45-Bio)修饰的磁球制备
生物素标记的对照核酸序列L45(L45-Bio)为在对照核酸序列L45的5’端偶联生物素基团Bio得到的，用结合缓冲液溶解L45-Bio，依据紫外吸收标定浓度(200nM)后，95℃加热5min，冰上放置5min，室温放置15min，得到L45-Bio溶液。
对照核酸序列L45的核苷酸序列：TTTNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN。
取100μL链霉亲和素修饰的磁球(货号：112.05D，Invitrogen Dynal AS,挪威)溶液，加入1mLPBS，震荡，置于磁力架，洗涤2次，得到磁微球。
然后将上述L45-Bio溶液和磁微球在室温孵育30min，然后用PBS洗涤2次，得到对照核酸序列L45修饰的磁微球。
3、偶联有BG2核酸适配体的磁性纳米颗粒的制备
取10μL浓度为10mg/mL的链霉亲和素修饰的磁性纳米颗粒(200nm)和10μL 1μM生物素标记的BG2核酸适配体溶液(溶剂为PBS；溶质为BG2-bio)，加入到1mL PBST缓冲液中，室温震荡孵育30min，磁分离1min， PBST缓冲液洗涤2次，磁分离，得到偶联有BG2核酸适配体的磁性纳米颗粒，即为核酸适配体磁性纳米颗粒。
二、核酸适配体功能化的磁性微球捕获细胞的情况
1、核酸适配体磁性微球与细胞孵育：
取1×10 5个指数生长期的LOVO细胞或PC-3细胞，用含5mM EDTA的PBS消化，PBS溶液洗涤2次，分别与BG2修饰的磁微球和对照核酸序列L45修饰的磁微球室温孵育30分钟。
2、磁分离与显微镜观察
然置于磁力架洗涤3次X3分钟，去除没有与磁球结合的细胞，PBS重悬磁球-细胞复合物，重复分散，显微镜下观察并拍照。
结果如图8A所示，BG2修饰的磁性微球球可以捕获表达碱性磷酸酶异源二聚体的LoVo细胞，但不可以捕获碱性磷酸酶异源二聚体阴性表达的PC-3细胞；而对照序列修饰的磁球无法捕获任何一种细胞。这说明BG2核酸适配体可以用于碱性磷酸酶异源二聚体阳性表达细胞的捕获或富集。
三、核酸适配体磁性纳米颗粒捕获及检测靶细胞
1、核酸适配体磁性纳米颗粒与细胞孵育：
取培养的LoVo细胞，EDTA消化后，PBST缓冲液洗涤2次，取不同个数的细胞(25、50、100、200、400、600个)，加入1mL结合缓冲液，加入10μL上述(一)核酸适配体磁性纳米颗粒，4℃震荡孵育30min。
2、磁分离：
将上述孵育后产物置于磁分离架上进行磁分离，以进一步去除非特异性细胞，PBST缓冲液洗涤2次，磁分离后得到所需的肿瘤细胞。
3、靶细胞检测：
在上述2得到的肿瘤细胞中加入10μL显色底物pNPP在工作缓冲液中，37℃静置孵育2h，在405nm用微量分光光度计(NanoDrop 2000)测定吸光度。根据吸光度值与细胞个数建立正相关关系，结果如图8B(a)所示。由图8B(a)可见随着细胞个数增加，吸光度值随之增加，呈正相关关系，证明了该方法的可行性，检测限是5个细胞。
或将上述(一)得到的肿瘤细胞直接置于显微镜下观察，结果如图8B(b)所示。由图8B(b)可见靶细胞LoVo细胞表面被核酸适配体磁性纳米颗 粒包裹，证明了该捕获方法的有效性。
四、核酸适配体磁性纳米颗粒在混合细胞中捕获及检测靶细胞
1、核酸适配体磁性纳米颗粒与细胞孵育：
取1×10 5个指数生长期的LOVO细胞或PC-3细胞，用含5mM EDTA的PBS消化，PBS溶液洗涤2次，分别与BG2修饰的磁微球和对照核酸序列L45修饰的磁微球4℃震荡孵育30min。
取培养的LoVo细胞，EDTA消化后，PBST缓冲液洗涤2次，取不同个数的细胞；取10 6个培养的Jurkat细胞，离心后PBST缓冲液洗涤2次，加入1mL结合缓冲液，将LoVo细胞加入到Jurkat细胞中，加入10μL上述一得到的核酸适配体磁性纳米颗粒，4℃震荡孵育30min。
2、磁分离：
将上述孵育后产物置于磁分离架上进行磁分离，以进一步去除非特异性细胞，PBST缓冲液洗涤2次，磁分离后得到所需的肿瘤细胞。
3、靶细胞检测：
在上述(2)得到的肿瘤细胞中加入10μL显色底物pNPP在工作缓冲液中，37℃静置孵育2h，在405nm用微量分光光度计(NanoDrop 2000)测定吸光度，计算捕获率(捕获率(％)＝捕获后细胞与显色底物反应产生的吸光度值/纯细胞(LoVo细胞)与显色底物反应产生的吸光度值×100)。
结果如图8C所示，BG2核酸适配体捕获的阳性细胞样品(LoVo细胞)的吸光度值较对照核酸适配体序列捕获的阳性细胞样品有显著性差异(P值〈0.01)，而BG2核酸适配体和对照核酸适配体序列捕获的阴性样品(PC3细胞)均无明显吸光度变化。说明该方法可以用以检测待测样品是否含有表达或高表达碱性磷酸酶的肿瘤细胞。
结果如图8D(a)所示，在10 6个非特异性细胞Jurkat细胞中，加入50、100、500、1000个靶细胞LoVo，得到的捕获率均在90％左右。
或将上述(1)得到的肿瘤细胞直接置于显微镜下观察，结果如图8D(b)所示，可以看出，将LoVo细胞(经染料4',6-二脒基-2-苯基吲哚染色后呈蓝色)与PC3细胞(经染料荧光素二乙酸染色后绿色)混合后，可以看到LoVo细胞表面包裹核酸适配体磁性纳米颗粒，而非特异性细胞PC3表面无磁性纳米颗粒。证明了该方法的特异性。
五、核酸适配体磁性纳米颗粒在人全血中捕获及检测靶细胞
1、核酸适配体磁性纳米颗粒与细胞孵育：
取培养的LoVo细胞，EDTA消化后，PBST缓冲液洗涤2次，取不同个数的细胞，将LoVo细胞加入1mL全血中，加入2mL红细胞裂解液，轻轻吹打混匀，室温裂解10min，500g离心5min，弃红色上清，如果发现红细胞裂解不完全，重复1-2次，用PBST缓冲液洗涤2次，保留沉淀为细胞，向沉淀中加入1mL结合缓冲液，加入10μL上述一得到的核酸适配体磁性纳米颗粒，4℃震荡孵育30min。
2、磁分离：
将上述孵育后产物置于磁分离架上进行磁分离，以进一步去除非特异性细胞，PBST缓冲液洗涤2次，磁分离后得到所需的循环肿瘤细胞。
3、靶细胞检测：
在上述(2)得到的循环肿瘤细胞中加入10μL显色底物pNPP在工作缓冲液中，37℃静置孵育2h，在405nm用微量分光光度计(NanoDrop 2000)测定吸光度，计算捕获率(捕获率(％)＝捕获后细胞与显色底物反应产生的吸光度值/纯细胞与显色底物反应产生的吸光度值×100)。
结果如图8E所示，在1mL全血中，加入200、500、1000个靶细胞LoVo，得到的捕获率均在85％以上。证明该方法在人全血样品中也可有效的进行捕获和检测。
六、核酸适配体磁性纳米颗粒在人全血中捕获及镜检靶细胞
下述实施例中BCIP/NBT工作液：BCIP浓度为50mg/mL溶于100％二甲基甲酰胺中，NBT浓度为50mg/mL溶于70％二甲基甲酰胺中，在每1mL工作缓冲液中先加入4μL NBT，混匀后再加入4μL BCIP，再次混匀，该试剂配制好后在1h内使用，当与碱性磷酸酶反应后，产生蓝紫色沉淀。
1、核酸适配体磁性纳米颗粒与细胞孵育：
取培养的LoVo细胞，EDTA消化后，PBST缓冲液洗涤2次，将50个LoVo细胞加入1mL健康个体全血中，加入2mL红细胞裂解液，轻轻吹打混匀，室温裂解10min，500g离心5min，弃红色上清，如果发现红细胞裂解不 完全，重复1-2次，用PBST缓冲液洗涤2次，加入1mL结合缓冲液，加入上述10μL核酸适配体磁性纳米颗粒，4℃震荡孵育30min。
1mL结肠癌个体全血，加入2mL红细胞裂解液，轻轻吹打混匀，室温裂解10min，500g离心5min，弃红色上清，如果发现红细胞裂解不完全，重复1-2次，用PBST缓冲液洗涤2次，加入1mL结合缓冲液，加入上述10μL核酸适配体磁性纳米颗粒，4℃震荡孵育30min。
2、磁分离：
将上述孵育后产物置于磁分离架上进行磁分离，以进一步去除非特异性细胞，PBST缓冲液洗涤2次，磁分离后得到所需的循环肿瘤细胞。
3、靶细胞检测：
在上述(一)得到的肿瘤细胞中加入10μL BCIP/NBT工作液，室温静置孵育30min后，直接置于显微镜下观察。
结果如图8F所示，图8F(a)为外加肿瘤的全血中捕获所得循环肿瘤细胞，图8F(b)为实际癌症患者全血中捕获的循环肿瘤细胞，可以看出碱性磷酸酶高表达的循环肿瘤细胞与BCIP/NBT反应后，在细胞表面产生蓝紫色沉淀，证明该方法有望可以临床样本的镜检。因此，这种高效、高选择性、快速捕获的方法有望用于临床样本中循环肿瘤细胞的检测，从而有助于癌症的早期诊断和预后评价。
上述结果表明，核酸适配体和磁性纳米颗粒可用于检测待测样品中是否含有循环肿瘤细胞，检测所用的试剂盒包括如下物质：
链霉亲和素修饰的磁性纳米颗粒、生物素标记的BG2核酸适配体，其中，BG2核酸适配体的核苷酸为序列表中序列1；
还包括用于循环肿瘤细胞捕获的其他物质，如PBST缓冲液、结合缓冲液、红细胞裂解液和磁分离架；
还包括用于循环肿瘤细胞检测的其他物质，如用于能结合碱性磷酸酶的染色物(如显色底物pNPP或BCIP/NBT)、荧光底物、分光光度计或显微镜。
检测的方法具体步骤如下：
1)将上述链霉亲和素修饰的磁性纳米颗粒和上述生物素标记的BG2核酸适配体偶联，得到核酸适配体磁性纳米颗粒；
2)将待测样品与所述核酸适配体磁性纳米颗粒结合后再进行磁分离，去除非特异性细胞，得到含有循环肿瘤细胞的产物；
3)检测含有循环肿瘤细胞的产物，实现捕获和/或检测待测样品中循环肿瘤细胞。
上述检测含有循环肿瘤细胞的产物的方法为如下A或B：
A、将含有循环肿瘤细胞的产物用pNPP染色显色，然后检测吸光度；以对照核酸适配体序列作为对照，若与对照核酸适配体序列捕获后吸光度值有显著差异，则待测样品含有或候选含有循环肿瘤细胞；若与对照序列捕获后吸光度值无显著差异，则待测样品不含有或候选不含有循环肿瘤细胞；
B、将含有循环肿瘤细胞的产物用BCIP/NBT染色后显微镜观察，若观察到表面带有蓝紫色沉淀的细胞，则待测样品含有或候选含有循环肿瘤细胞；若无表面蓝紫色细胞，则待测样品不含有或候选不含有循环肿瘤细胞。
对照核酸适配体L45，生物素标记在该对照核酸适配体适配体的5'端。
实施例8、核酸适配体磁性纳米颗粒对外泌体的捕获及检测
(一)外泌体的捕获：
(1)链霉亲和素修饰的磁性纳米颗粒与识别BG2核酸适配体偶联：
取1μL浓度为10mg/mL的链霉亲和素修饰的200nm磁性纳米颗粒和10μL 1μM生物素标记的BG2核酸适配体，在1mL PBST缓冲液中，室温震荡孵育30min，磁分离1min，PBST缓冲液洗涤2次，得核酸适配体磁性纳米颗粒。
(2)外泌体的制备
外泌体通过常用的超高速离心法制备，首先LoVo细胞在正常含有血清的培养基中培养，细胞密度达到70-80％后，去除原有培养基，换为无血清培养基，继续培养48h后，收集细胞上清，800×g，4℃离心10min，小心吸取上清，2000×g，4℃离心20min，收集上清，10000×g，4℃离心30min，收集上清，确保细胞或细胞碎片去除干净，将上清120000×g，4℃离心120min，弃上清，用PBS缓冲液重悬沉淀，120000×g，4℃离心120min，用200μL PBS缓冲液分散所得外泌体。
(3)核酸适配体磁性纳米颗粒与外泌体孵育：取不同浓度(0.25、0.5、 1、2、4、8μg/mL)的上述(2)所得外泌体，加入1mL结合缓冲液，加入上述(1)核酸适配体磁性纳米颗粒，4℃震荡孵育30min。
(4)磁分离：
将上述孵育后产物置于磁分离架上进行磁分离，PBS缓冲液洗涤2次，磁分离后得到所需的外泌体。
(二)外泌体检测：
在上述(一)得到的外泌体中加入10μL显色底物pNPP在工作缓冲液中，37℃静置孵育2h，在405nm用微量分光光度计(NanoDrop 2000)测定吸光度，根据吸光度值与外泌体浓度建立正相关关系，结果如图9所示。由图9可见随着外泌体浓度增加，吸光度值随之增加，呈正相关关系，证明了该方法的可行性，检测限为0.03μg/mL。
实施例9、肿瘤患者血浆/血清样品的自由蛋白检测
(一)可溶性碱性磷酸酶的捕获：
(1)链霉亲和素修饰的磁性纳米颗粒与识别BG2核酸适配体偶联：
取1μL浓度为10mg/mL的链霉亲和素修饰的200nm磁性纳米颗粒(结合生物素标记的寡核苷酸300pmol/mg)和10μL 1μM生物素标记的BG2核酸适配体，在1mL PBST缓冲液中，室温震荡孵育30min，磁分离1min，PBST缓冲液洗涤2次，得核酸适配体磁性纳米颗粒。
(2)核酸适配体磁性纳米颗粒与人血清/血浆样品孵育：
将不同量的碱性磷酸酶(4、8、16、30、60、120、240微活力单位),加入1mL结合缓冲液，加入上述(1)核酸适配体磁性纳米颗粒，4℃震荡孵育30min。
在10μL血清/血浆加入1mL结合缓冲液，加入上述(1)核酸适配体磁性纳米颗粒，4℃震荡孵育30min。
(4)磁分离：
将上述孵育后产物置于磁分离架上进行磁分离，以进一步去除非特异性蛋白结合，PBST缓冲液洗涤2次，磁分离后得到所需的细胞可溶性碱性磷酸酶。
(二)可溶性碱性磷酸酶的检测：
在上述(一)得到的可溶性碱性磷酸酶中加入10μL显色底物pNPP在工作缓冲液中，37℃静置孵育2h，在405nm用微量分光光度计(NanoDrop2000)测定吸光度，根据吸光度值与可溶性碱性磷酸酶的浓度建立正相关关系，结果如图10A所示。由图10A可见随着可溶性碱性磷酸酶浓度增加，吸光度值随之增加，呈正相关关系，证明了该方法的可行性。
将10μL血清样品所测的吸光度值带入上述标准曲线，得血清中可溶性碱性磷酸酶的浓度为20U/L。
实施例10、细胞培养基中的自由碱性磷酸酶异源二聚体蛋白检测
(1)分别取1mL指数生长期的LoVo细胞的培养基和1mL指数生长期的PC-3细胞的培养液，1000rpm离心5min去掉细胞碎片，分别与10nM生物素标记的BG2核酸适配体BG2-bio溶液或生物素标记的对照核酸序列L45-bio溶液(4℃)震荡孵育30分钟。
(2)然后分别加入10μL链霉亲和素修饰的琼脂糖微球(GE公司，货号：17-5113-01)在4℃震荡孵育60分钟。
(3)2000rpm离心去除上清，PBST缓冲液洗涤2次。
(4)在上述得到的可溶性碱性磷酸酶中加入10μL显色底物pNPP在工作缓冲液中，37℃静置孵育2h，在405nm用微量分光光度计(NanoDrop2000)测定吸光度，结果如图10B所示。
或，在(3)得到产物中，加入100μL的10μM荧光素二磷酸在工作缓冲液中，37℃静置孵育1h，在用酶标仪(SpectraMax M5)在488nm激发，测定530nm发射，结果如图10C所示。
由图10B和10C可见在碱性磷酸酶表达的细胞系(LoVo)培养液中可以捕获到碱性磷酸酶，而对阴性表达细胞(PC3)的培养液中没有。这证明了该方法可以用于实际样品中自由碱性磷酸酶异源二聚体的捕获和检测。
工业应用
本发明首次发现核酸适配体BG2可以特异性识别并结合碱性磷酸酶异 源二聚体，本发明的核酸适配体BG2具有亲和力高、特异性强、无免疫原性和无毒性等特点，基于核酸适配体BG2建立的检测碱性磷酸酶异源二聚体的方法可用于碱性磷酸酶异源二聚体表达的检测和相关疾病的诊断的产品。本发明的基于核酸适配体磁性颗粒技术用于外周血中高表达碱性磷酸酶的循环肿瘤细胞捕获和检测的方法，可以实现对目标细胞高选择性的捕获及检测。磁性颗粒既可起到尺寸放大作用，用于富集循环肿瘤细胞，还可用于磁分离实现高效率捕获。通过利用碱性磷酸酶自身与显色底物的酶反应，对分离得到的循环肿瘤细胞进行可视化检测，实现了信号放大，提高了灵敏度。本发明的捕获及检测方法，无需复杂的修饰及操作过程，步骤简单，高效快速且成本低廉，可用于临床样品的检测。捕获的肿瘤细胞可以用于进一步的培养或基因检测等。

Claims (20)

  1. 一种核酸适配体或其衍生物，为如下1)-7)中任一种：
    1)序列1所示的单链DNA分子；
    2)将1)所示的核酸适配体删除或增加一个或几个核苷酸，得到与所述核酸适配体具有相同功能的核酸适配体的衍生物；
    3)将1)所示的核酸适配体进行核苷酸取代或修饰，得到与所述核酸适配体具有相同功能的核酸适配体的衍生物；
    4)将1)所示的核酸适配体的骨架改造为硫代磷酸脂骨架，得到与所述核酸适配体具有相同功能的核酸适配体的衍生物；
    5)由1)所示的核酸适配体编码的RNA分子，得到与所述核酸适配体具有相同功能的核酸适配体衍生物；
    6)由1)所示的核酸适配体编码的肽核酸，得到与所述核酸适配体具有相同功能的核酸适配体的衍生物；
    7)将1)-6)任一所示的核酸适配体的一端或中间接上信号分子和/或活性分子和/或功能基团和/放射性核素，得到与所述核酸适配体具有相同功能的核酸适配体的衍生物。
  2. 根据权利要求1所述的核酸适配体或其衍生物，其特征在于：
    所述核酸适配体的衍生物为去除或改变序列1所示的核酸适配体核苷酸序列自5’端第1个核苷酸起包括5’端第一个核苷酸残基的1-7个核苷酸；和/或，去除序列1所示的核酸适配体的核苷酸序列自3’端第1个核苷酸起包括3’端第一个核苷酸残基的1-7个核苷酸；
    或所述核酸适配体的衍生物为将序列1所示的核酸适配体核苷酸序列5’端或3’端增加若干个核苷酸或修饰基团且不影响序列1第10-36位形成的结构，保留的核苷酸残基组成的核酸适配体。
  3. 根据权利要求1或2所述的核酸适配体或其衍生物，其特征在于：
    所述核酸适配体的衍生物为如下1)-6)中任一种：
    1)序列2所示的单链DNA分子；
    2)序列3所示的单链DNA分子；
    3)序列4所示的单链DNA分子；
    4)序列5所示的单链DNA分子；
    5)序列6所示的单链DNA分子；
    6)序列7所示的单链DNA分子。
  4. 根据权利要求1-3中任一所述的核酸适配体或其衍生物，其特征在于：
    7)所示的核酸适配体衍生物为在1)-6)任一所示的核酸适配体的5’端或3’端标记荧光基团、生物素基团或放射性核素。
  5. 权利要1-4中任一所述的核酸适配体或其衍生物在如1)-4)中至少一种中的应用：
    1)检测或诊断碱性磷酸酶；
    2)制备检测或诊断碱性磷酸酶产品；
    3)提取或捕获碱性磷酸酶；
    4)制备提取或捕获碱性磷酸酶产品；
    所述检测或诊断或提取或捕获的靶标为碱性磷酸酶本身、碱性磷酸酶异源二聚体、含有碱性磷酸酶或其异源二聚体的细胞、含有碱性磷酸酶或其异源二聚体的外泌体、含有碱性磷酸酶或其异源二聚体的组织切片、含有碱性磷酸酶或异源二聚体的动物活体。
  6. 根据权利要求5所述的应用，其特征在于：所述检测或诊断的样本为全血、血清、培养液、唾液、组织切片或活体；
  7. 根据权利要求5或6所述的应用，其特征在于：所述检测或诊断方式为荧光成像，比如细胞的荧光成像、组织切片的荧光成像、活体的荧光成像，进而镜检。
  8. 权利要1-4中任一所述的核酸适配体或其衍生物在如a1-a26至少一种中的应用：
    a1)富集提取碱性磷酸酶；
    a2)识别并结合或辅助识别并结合碱性磷酸酶；
    a3)识别并结合或辅助识别并结合表达碱性磷酸酶的细胞；
    a4)检测待测样品中碱性磷酸酶含量或活性；
    a5)检测待测样品中是否含有碱性磷酸酶；
    a6)检测与抗碱性磷酸酶的抗体结合的物质；
    a7)检测与碱性磷酸酶相互作用的蛋白；
    a8)制备富集提取碱性磷酸酶产品；
    a9)制备识别并结合或辅助识别并结合碱性磷酸酶产品；
    a10)制备检测待测样品中碱性磷酸酶含量或活性产品；
    a11)制备检测待测样品中是否含有碱性磷酸酶产品；
    a12)制备检测与抗碱性磷酸酶的抗体结合的物质产品；
    a13)制备检测与碱性磷酸酶相互作用的蛋白产品；
    a14)制备诊断和/或治疗与碱性磷酸酶相关疾病的产品；
    a15)捕获和/或检测表达或高表达碱性磷酸酶表达细胞或外泌体；
    a16)制备捕获和/或检测表达或高表达碱性磷酸酶细胞或外泌体的产品；
    a17)制备用于靶向碱性磷酸酶的动物成像的探针；
    a18)制备用于靶向碱性磷酸酶治疗的产品；
    a19)检测或捕获待测样品中表达或高表达碱性磷酸酶的肿瘤或肿瘤细胞；
    a20)检测或捕获待测样品中表达或高表达碱性磷酸酶的循环肿瘤细胞；
    a21)检测或捕获待测样品本中表达或高表达碱性磷酸酶的外泌体；
    a22)检测或捕获待测样品中的可溶性碱性磷酸酶；
    a23)制备检测或捕获待测样品中表达或高表达碱性磷酸酶的肿瘤或肿瘤细胞产品；
    a24)制备检测或捕获待测样品中表达或高表达碱性磷酸酶的循环肿瘤细胞产品；
    a25)制备检测或捕获待测样品中表达或高表达碱性磷酸酶的外泌体产品；
    a26)制备检测或捕获待测样品中的可溶性碱性磷酸酶产品。
  9. 一种试剂盒，包括权利要1-4中任一所述的核酸适配体或其衍生物，以及，固定或偶联所述核酸适配体或其衍生物的载体。
  10. 根据权利要求9所述的试剂盒，其特征在于：
    所述试剂盒具有如下b1-b11中至少一种功能：
    b1)富集提取碱性磷酸酶；
    b2)识别并结合或辅助识别并结合碱性磷酸酶；
    b3)识别并结合或辅助识别并结合表达碱性磷酸酶的细胞；
    b4)检测待测样品中碱性磷酸酶含量或活性；
    b5)检测待测样品中是否含有碱性磷酸酶；
    b6)检测与抗碱性磷酸酶的抗体结合的物质；
    b7)检测与碱性磷酸酶相互作用的蛋白；
    b8)检测或捕获待测样品中表达或高表达碱性磷酸酶的肿瘤或肿瘤细胞；
    b9)检测或捕获待测样品中表达或高表达碱性磷酸酶的循环肿瘤细胞；
    b10)检测或捕获待测样品中表达或高表达碱性磷酸酶的外泌体；
    b11)检测或捕获待测样品中的可溶性碱性磷酸酶.
  11. 根据权利要求9或10所述的试剂盒，其特征在于：
    所述固定或偶联核酸适配体或其衍生物的载体为纳米颗粒或微米颗粒或芯片。
  12. 根据权利要求11所述的试剂盒，其特征在于：
    所述纳米颗粒为修饰物修饰的纳米/微米颗粒；
    或，所述纳米颗粒为磁性纳米颗粒；
    或，所述修饰物为链霉亲和素、生物素、羧基、氨基或巯基。
  13. 根据权利要求9-12中任一所述的试剂盒，其特征在于：
    所述试剂盒还包括与碱性磷酸酶反应的显色底物；
    所述显色底物为荧光底物分子、化学发光底物分子或发可见光底物分子。
  14. 根据权利要求5-8任一所述的应用或权利要求9-13中任一所述的试剂盒，其特征在于：
    所述表达或高表达碱性磷酸酶表达细胞为表达或高表达碱性磷酸酶的肿瘤细胞；
    和/或，所述表达或高表达碱性磷酸酶的肿瘤细胞为人宫颈癌细胞、人乳腺癌细胞、人结肠癌细胞或人肝细胞癌细胞；
    或，所述表达或高表达碱性磷酸酶的循环肿瘤细胞为人宫颈癌循环肿瘤细胞、人乳腺癌循环肿瘤细胞、人结肠癌循环肿瘤细胞或人肝细胞癌循环肿瘤细胞。
  15. 根据权利要求5-8任一所述的应用或权利要求9-13中任一所述的试剂盒，其特征在于：所述碱性磷酸酶为异源二聚体碱性磷酸酶；
    和/或，所述碱性磷酸酶为PALP、IAP、GCAP或任意的异源二聚体。
  16. 权利要1-4中任一所述的核酸适配体或其衍生物，以及，固定或偶联所述核酸适配体或其衍生物的载体在制备具有如下b1-b11至少一种功能的产品中的应用：
    b1)富集提取碱性磷酸酶；
    b2)识别并结合或辅助识别并结合碱性磷酸酶；
    b3)识别并结合或辅助识别并结合表达碱性磷酸酶的细胞；
    b4)检测待测样品中碱性磷酸酶含量或活性；
    b5)检测待测样品中是否含有碱性磷酸酶；
    b6)检测与抗碱性磷酸酶的抗体结合的物质；
    b7)检测与碱性磷酸酶相互作用的蛋白；
    b8)检测或捕获待测样品中表达或高表达碱性磷酸酶的肿瘤或肿瘤细胞；
    b9)检测或捕获待测样品中表达或高表达碱性磷酸酶的循环肿瘤细胞；
    b10)检测或捕获待测样品中表达或高表达碱性磷酸酶的外泌体；
    b11)检测或捕获待测样品中的可溶性碱性磷酸酶。
  17. 根据权利要求5-8任一所述的应用或权利要求9-13中任一所述的试剂盒或权利要求16所述的应用，其特征在于：
    所述待测样品为外周血全血、血清、血浆、细胞培养液或唾液。
  18. 一种捕获和/或检测待测样品是否含有表达或高表达碱性磷酸酶的肿瘤细胞的方法，包括如下步骤：
    1)制备核酸适配体磁性纳米颗粒和去除待测样品的红细胞；
    所述制备核酸适配体磁性纳米颗粒为将磁性纳米颗粒和碱性磷酸酶核酸适配体连接，得到核酸适配体磁性纳米颗粒；
    2)将所述去除红细胞外周血全血样本与所述核酸适配体磁性纳米颗粒结合后再进行磁分离，去除非特异性细胞，得到含有循环肿瘤细胞的产物；
    3)检测含有循环肿瘤细胞的产物，根据吸光度或细胞表面是否生成沉淀判断待测样品是否含有表达或高表达碱性磷酸酶的肿瘤细胞。
  19. 一种捕获和/或检测待测样品是否含有表达或高表达碱性磷酸酶的外泌体的方法，包括如下步骤：
    1)制备核酸适配体磁性纳米颗粒和收集待测样品的肿瘤细胞外泌体；
    所述制备核酸适配体磁性纳米颗粒为将磁性纳米颗粒和碱性磷酸酶核酸适配体连接，得到核酸适配体磁性纳米颗粒；
    所述收集待测样品的肿瘤细胞外泌体为从待测样品的血清或血浆中收集肿瘤细胞外泌体；
    2)将所述外泌体与所述核酸适配体磁性纳米颗粒结合后再进行磁分离，去除非特异性结合，得到含有外泌体产物；
    3)检测含有外泌体产物，通过检测含有外泌体产物的吸光度或产物表面是否生成沉淀，判断待测样品是否含有表达或高表达碱性磷酸酶的肿瘤细胞。
  20. 一种捕获和/或检测待测样品的游离蛋白中是否含有碱性磷酸酶的方法，包括如下步骤：
    1)制备核酸适配体磁性纳米颗粒和收集待测样品的游离蛋白；
    所述制备核酸适配体磁性纳米颗粒为将磁性纳米颗粒和碱性磷酸酶核酸适配体连接，得到核酸适配体磁性纳米颗粒；
    所述收集待测样品的游离蛋白为从待测样品的血清或血浆中收集游离蛋白；
    2)将所述游离蛋白与所述核酸适配体磁性纳米颗粒结合后再进行磁分离，去除非特异性蛋白，得到含有游离蛋白产物；
    3)检测含有游离蛋白产物，通过检测含有游离蛋白产物的吸光度或游离蛋白是否能够荧光染色判断待测样品的游离蛋白中是否含有碱性磷酸酶。
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