阅读说明：本技术 一种新型无标记的铽(iii)-核酸适体传感器及其制备方法与应用 (Novel unmarked terbium (III) -aptamer sensor and preparation method and application thereof ) 是由 赵永祥 钟莉娉 黄勇 于 2019-08-19 设计创作，主要内容包括：本发明公开了一种新型无标记的铽(III)-适配体传感器及其制备方法与应用。本发明利用核酸适体致敏稀土Tb<Sup>3+</Sup>荧光的特征,设计了一种用于CCRF-CEM细胞的无标记信号敏感荧光检测法。该无标记信号敏感荧光检测法具有选择性高,操作简便,快速的优点,检测限高达5个细胞/毫升。有望为急性淋巴细胞白血病的超灵敏诊断提供一种稳健,简便,廉价的新方法。(The invention discloses a novel unmarked terbium (III) -aptamer sensor and a preparation method and application thereof. The invention uses aptamer to sensitize rare earth Tb 3+ Fluorescence characteristics, a label-free signal sensitive fluorescence detection method for CCRF-CEM cells is designed. The label-free signal sensitive fluorescence detection method has the advantages of high selectivity, simplicity and convenience in operation and rapidness, and the detection limit is up to 5 cells/ml. Is expected to provide a novel method which is steady, simple and convenient and cheap for the ultra-sensitive diagnosis of the acute lymphocytic leukemia.)

1. A probe comprising an aptamer and Tb³⁺；

The aptamer is rich in base G, has a hairpin structure, and targets tumor cells or cancer cells.

2. The probe of claim 1, wherein: the aptamer and the Tb³⁺In a molar ratio of 1: (2000-4000).

3. The probe according to claim 1 or 2, characterized in that: the probe further comprises Mg²⁺；

Or, the aptamer, the Tb³⁺And said Mg²⁺In a molar ratio of 1: (2000-4000): (100-200).

4. A probe according to any of claims 1 to 3, wherein: the aptamer is a aptamer targeting acute lymphocytic leukemia cells;

or, the aptamer targeting acute lymphocytic leukemia cells is an Sgc8 aptamer.

5. A method for preparing the probe according to any one of claims 1 to 4, comprising the steps of: combining the nucleic acid aptamer solution of any one of claims 1-4 with the Tb of any one of claims 1-4³⁺Uniformly mixing the solution to obtain the probe;

the aptamer solution is obtained by uniformly mixing the aptamer and a buffer solution;

tb³⁺The solution is Tb³⁺Mixing with buffer solution.

6. The method of claim 5, wherein:

tb³⁺The solution is prepared by mixing terbium nitrate with Mg²⁺The buffer solution is evenly mixed to obtain the product;

the buffer solution in the aptamer solution is PBS buffer solution;

tb³⁺The buffer in the solution is HEPES buffer.

7. A product comprising a probe according to any one of claims 1 to 4 or prepared according to the method of claim 5 or 6.

8. Use of the probe of any one of claims 1 to 4 or prepared according to the method of claim 5 or 6 or the product of claim 7 in any one of the following a1) -a 8):

a1) detecting or aiding in the detection of tumor cells or cancer cells;

a2) preparing a product for detecting or assisting in detecting tumor cells or cancer cells;

a3) diagnosing a tumor or cancer;

a4) preparing a product for diagnosing tumors or cancers;

a5) detecting or aiding in the detection of acute lymphocytic leukemia cells;

a6) preparing a product for detecting or assisting in detecting acute lymphocytic leukemia cells;

a7) diagnosing acute lymphocytic leukemia;

a8) preparing a product for diagnosing acute lymphocytic leukemia.

9. A method of detecting or aiding in the detection of a tumor cell or cancer cell comprising the steps of:

(b1) drawing a standard curve

Preparing tumor cell or cancer cell solutions with a series of concentrations, mixing the obtained several tumor cell or cancer cell solutions with the probe according to any one of claims 1 to 4 or the probe prepared according to the method of claim 5 or 6, respectively, to obtain detection solutions; incubating the detection solution in the dark, centrifuging, and collecting the supernatant; detecting the fluorescence intensity of the supernatant by using a fluorescence spectrophotometer; taking the concentration or logarithm value of the tumor cells or the cancer cell solution as a horizontal coordinate, taking the fluorescence intensity as a vertical coordinate, and drawing a standard curve graph to obtain a standard curve equation;

(b2) detection of a sample to be tested

Mixing a sample to be tested with the probe according to any one of claims 1 to 4 or the probe prepared according to the method of claim 5 or 6 to obtain a detection solution; incubating the detection solution in the dark, centrifuging, and collecting the supernatant; detecting the fluorescence intensity of the supernatant by using a fluorescence spectrophotometer; substituting the fluorescence intensity into the standard curve equation obtained in the step b1), and calculating to obtain the concentration value of the tumor cells or the cancer cells in the sample to be detected.

10. The probe according to any one of claims 1 to 4 or the product according to claim 7 or the use according to claim 8 or the method according to claim 9, wherein: the tumor or cancer is acute lymphocytic leukemia; the tumor cells or cancer cells are acute lymphocytic leukemia cells.

Technical Field

The invention belongs to the technical field of biomedicine, and particularly relates to a novel unmarked terbium (III) -aptamer sensor, a preparation method and application thereof, in particular to application of the novel unmarked terbium (III) -aptamer sensor in preparation of a product for diagnosing acute lymphocytic leukemia.

Background

Acute lymphocytic leukemia is a common and fatal cancer, usually beginning in the bone marrow, characterized by the presence of large numbers of immature leukocytes in children and adolescents. This disease not only requires a large number of health and medical resources, but also has a negative impact on the living standard and social welfare of people. Acute lymphoblastic leukemia can lead to abnormal proliferation and accumulation of primary cells in the bone marrow and inhibit hematopoietic development, resulting in anemia, thrombocytopenia and neutropenia. In addition, primary cells can also infiltrate the liver, spleen and other external tissues, causing corresponding lesions.

At present, the methods for clinical detection of leukemia mainly include fluorescence labeling analysis, flow cytometry analysis, immunohistochemical analysis and the like. However, these methods are not suitable for simple and rapid medical analysis, since they are not only expensive, time consuming, complex and labor intensive, but also require complex instruments, have low sensitivity and require multi-step processing, etc.

Aptamers (aptamers) are a class of single-stranded DNA, RNA, peptide nucleic acids or chemically modified nucleic acid sequences, usually consisting of 15-80 nucleotides, that specifically interact with a target substance. The aptamer can act on inorganic ions, small molecules and biological macromolecules, can be combined with supramolecular substances such as cells, viruses and pathological tissue sections, has the characteristics of high affinity, good specificity, no immunogenicity, easy synthesis, modification and modification, good biochemical stability, reversible denaturation and renaturation and the like, and is called as a chemical antibody.

Rare earth terbium ion (Tb)³⁺) Is a promising non-labeled fluorescent probe, which is of great interest due to its unique optical properties, such as long fluorescence lifetime, large stokes shift and narrow emission band.

Disclosure of Invention

The invention aims to provide a novel unmarked terbium (III) -aptamer sensor and application thereof in preparing a product for diagnosing acute lymphocytic leukemia.

In order to achieve the above object, the present invention first provides a probe.

The name of the probe provided by the invention is Tb³⁺-Apt probe or terbium (III) -aptamer sensor comprising an aptamer and Tb³⁺；

The aptamer is rich in base G, has a hairpin structure, and targets tumor cells or cancer cells.

Tb mentioned above³⁺-Apt probe, wherein said base G rich is at least 20% of base G content.

Tb mentioned above³⁺-Apt probe, said aptamer and said Tb³⁺May be 1: (2000-4000). In one embodiment of the inventionIn examples, the aptamer is conjugated to the Tb³⁺In a molar weight ratio of 5X 10^-3：20(1：4000)。

Further, the probe may further include Mg²⁺(ii) a The Mg²⁺For stabilizing oligonucleotide (aptamer) conformation and reducing non-specific binding between other metal ions and the oligonucleotide (aptamer).

The aptamer, Tb³⁺And said Mg²⁺May be 1: (2000-4000): (100-200). In a specific embodiment of the present invention, said aptamer, said Tb³⁺And said Mg²⁺In a molar weight ratio of 5X 10^-3：20：1(1：4000：200)。

Further, the probe may further include a buffer; the buffer can be specifically PBS buffer and HEPES buffer.

Tb mentioned above³⁺In the Apt probe, the aptamer may be an aptamer targeting acute lymphoblastic leukemia cells, such as Sgc8 aptamer targeting human acute lymphoblastic leukemia T-lymphocyte CCRF-CEM. The Sgc8 aptamer is a nucleic acid aptamer that specifically recognizes CCRF-CEM cells. Sgc8 aptamer has a base G content of 26.8% and has a hairpin structure (the DNA molecules shown at positions 2-7 of the Sgc8 aptamer pair complementarily with the DNA molecules shown at positions 35-41, forming a hairpin structure).

In one embodiment of the invention, the probe consists of Sgc8 aptamer, terbium nitrate, magnesium nitrate, PBS buffer and HEPES buffer. Wherein the final concentration of the Sgc8 aptamer in the probe is 25 mu M, Tb³⁺The final concentration in the probe was 100mM, Mg²⁺The final concentration in the probe was 5 mM.

The probe can be used for detecting or assisting in detecting tumor cells or cancer cells (such as acute lymphocytic leukemia cells), and further realizes diagnosis of tumors or cancers (such as acute lymphocytic leukemia). In practical application, a sample solution to be tested (such as blood sample or tumor cell suspension or cancer cell suspension) can be mixed with Tb³⁺Uniformly mixing an-Apt probe according to the volume ratio of 4:1 to obtain Tb³⁺-Apt working fluid. Aptamers (e.g. Sgc8 aptamer) at Tb³⁺Final concentration in Apt working solution 5. mu.M, Tb³⁺At Tb³⁺Final concentration of 20mM, Mg in Apt working solution²⁺At Tb³⁺Final concentration in Apt working solution 1 mM.

In order to achieve the purpose, the invention also provides the Tb³⁺-a method for preparing an Apt probe.

Tb provided by the invention³⁺The preparation method of the Apt probe comprises the following steps: mixing aptamer solution and Tb³⁺Uniformly mixing the solution to obtain the probe;

the aptamer solution is obtained by uniformly mixing the aptamer and a buffer solution;

tb³⁺The solution is Tb³⁺Mixing with buffer solution.

Further, the buffer solution in the aptamer solution is PBS buffer solution.

Tb³⁺The solution is prepared by mixing terbium nitrate with Mg²⁺The buffer solution is mixed evenly. Said containing Mg²⁺The buffer of (1) is prepared by mixing magnesium nitrate and HEPES buffer.

Further, in the aptamer solution, the concentration of the aptamer may be 50 μ M.

Tb³⁺In solution, the Tb³⁺The concentration may be 200mM, said Mg²⁺The concentration may be 10 mM.

The aptamer solution and the Tb³⁺The solution was mixed in equal volume.

In the method, the aptamer is Sgc8 aptamer targeting human acute lymphoblastic leukemia T lymphocyte CCRF-CEM. The final concentration of the Sgc8 aptamer in the probe was 25 μ M, the Tb³⁺The final concentration in the probe was 100mM, the Mg²⁺The final concentration in the probe was 5 mM.

In order to achieve the above object, the present invention further provides a product.

The product provided by the invention comprises the Tb³⁺-Apt probeOr according to the above Tb³⁺-preparing an Apt probe by a method for preparing an Apt probe;

the product has the functions of any one of a1) -a 8):

a1) detecting or aiding in the detection of tumor cells or cancer cells;

a2) preparing a product for detecting or assisting in detecting tumor cells or cancer cells;

a3) diagnosing a tumor or cancer;

a4) preparing a product for diagnosing tumors or cancers;

a5) detecting or aiding in the detection of acute lymphocytic leukemia cells;

a6) preparing a product for detecting or assisting in detecting acute lymphocytic leukemia cells;

a7) diagnosing acute lymphocytic leukemia;

a8) preparing a product for diagnosing acute lymphocytic leukemia.

Further, the product may further comprise a culture medium for culturing tumor cells or cancer cells, a buffer for washing tumor cells or cancer cells, and a fluorescence spectrophotometer.

Further, the medium is a medium 1640 containing 10% fetal bovine serum and 100U/mL penicillin-streptomycin; the buffer solution is PBS buffer solution; the fluorescence spectrophotometer is an F-7000 fluorescence spectrophotometer.

In order to achieve the purpose, the invention also provides the Tb³⁺-Apt probe or Tb according to above³⁺-Apt probe preparation method and new use of the prepared probe or the product.

The invention provides the Tb³⁺-Apt probe or Tb according to above³⁺-Apt Probe preparation method the probe prepared or the use of the above product in any of the following a1) -a 8):

a1) detecting or aiding in the detection of tumor cells or cancer cells;

a2) preparing a product for detecting or assisting in detecting tumor cells or cancer cells;

a3) diagnosing a tumor or cancer;

a4) preparing a product for diagnosing tumors or cancers;

a5) detecting or aiding in the detection of acute lymphocytic leukemia cells;

a6) preparing a product for detecting or assisting in detecting acute lymphocytic leukemia cells;

a7) diagnosing acute lymphocytic leukemia;

a8) preparing a product for diagnosing acute lymphocytic leukemia.

In order to achieve the above object, the present invention finally provides a method for detecting or aiding in the detection of tumor cells or cancer cells.

The method for detecting or assisting in detecting the tumor cells or the cancer cells comprises the following steps:

(b1) drawing a standard curve

Preparing tumor cell or cancer cell solution with series concentration, mixing the obtained tumor cell or cancer cell solution with Tb³⁺-Apt probe or Tb according to above³⁺-mixing the probes prepared by the method for preparing the Apt probe to respectively obtain detection solutions; incubating the detection solution in the dark, centrifuging, and collecting the supernatant; detecting the fluorescence intensity of the supernatant by using a fluorescence spectrophotometer; taking the concentration or logarithm value of the tumor cells or the cancer cell solution as a horizontal coordinate, taking the fluorescence intensity as a vertical coordinate, and drawing a standard curve graph to obtain a standard curve equation;

(b2) detection of a sample to be tested

Mixing the sample to be tested with Tb³⁺-Apt probe or Tb according to above³⁺-mixing the probes prepared by the method for preparing an Apt probe to obtain a detection solution; incubating the detection solution in the dark, centrifuging, and collecting the supernatant; detecting the fluorescence intensity of the supernatant by using a fluorescence spectrophotometer; substituting the fluorescence intensity into the standard curve equation obtained in the step b1), and calculating to obtain the concentration value of the tumor cells or the cancer cells in the sample to be detected.

In the above method, the tumor cell or cancer cell solution or sample solution to be tested and the Tb are mixed³⁺-Apt probe or Tb according to above³⁺-Apt Probe volumeThe ratio may be 4: 1. The final concentration of the aptamer in the detection solution is 5 mu M, Tb³⁺The final concentration in the test solution was 20mM, the Mg²⁺The final concentration in the test solution was 1 mM.

In the above method, the incubation is carried out at 4 ℃ in the dark for 10 min.

The fluorescence intensity of the supernatant was measured with a fluorescence spectrophotometer at an excitation wavelength of 290 nm. The method also comprises the step of adjusting the pH value of the supernatant to 9.6 before detecting the fluorescence intensity of the supernatant.

In the above method, the sample to be tested may be a blood sample or a tumor cell suspension or a cancer cell suspension of a patient to be tested. In one embodiment of the present invention, the sample to be tested is a CCRF-CEM cell suspension.

In the above method, the tumor cell or cancer cell may be acute lymphocytic leukemia cell, specifically human acute lymphocytic leukemia T-lymphocyte CCRF-CEM.

In the above method, the standard linear equation may be F-751.67 log^c+5435.7, where F is the fluorescence intensity and C is the CCRF-CEM cell concentration (number of cells per ml).

Terbium (III) -based aptamers (Tb) according to the invention³⁺Apt) the principle of detection of leukemia cells by a label-free signal fluorescent aptamer sensor is as follows: tb³⁺In the Apt probe, the aptamer can make Tb³⁺The fluorescence of the T lymphocyte is sensitive and forms strong fluorescence, and the CCRF-CEM of the T lymphocyte of the human acute lymphoblastic leukemia as a target cell can react with Tb³⁺-Apt probes combined to form Tb³⁺-Apt-CEM complex, which upon centrifugation forms a supernatant, the fluorescence signal of which can be detected with a fluorescence spectrophotometer. The concentration range is 5-5 × 10⁶The cell/mL CCRF-CEM cell concentration has a good linear relationship between the logarithm value and the fluorescence signal (R)²0.9881), therefore, the method can realize the detection of CCRF-CEM cells in a sample to be detected, and further realize the diagnosis of acute lymphocytic leukemia.

The invention uses aptamer to sensitize rare earth Tb³⁺Fluorescent featureIn the characterization, a label-free signal sensitive fluorescence detection method for CCRF-CEM cells is designed. The detection limit of the detection method of the invention is up to 5 cells/ml. The label-free signal sensitive fluorescence detection method has the advantages of high selectivity, simplicity and convenience in operation and rapidness, and is expected to provide a novel method which is stable, simple and convenient and low in cost for the ultra-sensitive diagnosis of acute lymphocytic leukemia.

Drawings

FIG. 1 shows Tb for detection of CCRF-CEM cells³⁺Schematic representation of Apt fluorescent aptamer sensor.

FIG. 2 shows the fluorescence intensity measurements of different detection systems. FIG. 2A shows Tb under different conditions³⁺Fluorescence emission spectra of Apt and of cellular CCRF-CEM: a. tb³⁺；b、Apt；c、Tb³⁺+Apt；d、Tb³⁺+Apt+CEM；e、Tb³⁺+ Apt + Romas; f. CEM; g. romos. FIG. 2B shows Tb under different conditions³⁺Quantitative analysis of fluorescence emission spectra of Apt and CCRF-CEM. NS, no statistical significance,. P<0.05, has statistical significance. FIG. 2C is a fluorescence emission spectrum of aptamers with different metal ions. FIG. 2D is a statistical analysis of fluorescence emission spectra of aptamers with different metal ions, showing Tb³⁺And other ions.

FIG. 3 is Tb³⁺And (4) optimizing concentration and detection conditions. FIG. 3A shows Tb at various concentrations³⁺(20mM) fluorescence intensity measurement. FIG. 3B shows the fluorescence intensity measurements of the detection systems with different pH values. FIG. 3C shows the fluorescence intensity measurements of the detection system incubated for different periods of time. FIG. 3D shows the fluorescence intensity measurements of the detection systems incubated at different temperatures.

FIG. 4 shows Tb for detection of CCRF-CEM cells³⁺Determination of the sensitivity and specificity of Apt fluorescent aptamer sensors. FIG. 4A shows Tb in the presence of different concentrations of CCRF-CEM cells³⁺Fluorescence emission spectra of Apt fluorescent aptamer sensors, a, 10¹Cell/200. mu.L, d, 10²Cell/200. mu.L, e, 10³Cell/200. mu.L, f, 10⁴Cells/200. mu.L, g, 10⁵Cell/200. mu.L, h, 10⁶Cells/200. mu.L. FIG. 4B isLinear relationship between fluorescence intensity and CCRF-CEM cell concentration. FIG. 4C is Tb for detection of CCRF-CEM cells³⁺Specificity of Apt fluorescent aptamer sensor. FIG. 4D shows a schematic representation of a scheme with Tb³⁺Fluorescence micrographs of six different cells after Apt mixing. Nuclei were stained with DAPI. The scale bar represents 50 μm.

Detailed Description

The following examples are given to facilitate a better understanding of the invention, but do not limit the invention. The experimental procedures in the following examples are conventional unless otherwise specified. The test materials used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified. The quantitative tests in the following examples, all set up three replicates and the results averaged.

Having 5' -A in the examples which followTCTAACTGCTGCGCCGCCGGGAAAATACTGTACGGTTAGAThe unlabeled Sgc8 aptamer (Apt) of the-3' sequence is Shanghai Sangon Biological engineering technology&Products of services (shanghai, china). Sgc8 aptamers are nucleic acid aptamers that specifically recognize CCRF-CEM cells. Sgc8 aptamer has a base G content of 26.8% and has a hairpin structure (the DNA molecules shown at positions 2-7 of the Sgc8 aptamer pair complementarily with the DNA molecules shown at positions 35-41, forming a hairpin structure).

4- (2-hydroxy-ethyl) piperazine-1-ethanesulfonic acid (HEPES, pH 7.2-7.4,>99.5%) nitrate of a metal salt(s) ((ii)>99%) including potassium (K)⁺) Sodium, sodium (Na)⁺) Silver (Ag)⁺) Magnesium (Mg)²⁺) Calcium (Ca)²⁺) Copper (Cu)²⁺) Iron (Fe)³⁺) And terbium (Tb)³⁺) Are all products of Sigma-Aldrich, Inc.

The PBS buffer (10mM, pH7.2) in the following examples is a product of Beijing Solebao Tech Co.

All solutions in the following examples were prepared with 18.2 M.OMEGA.ultra pure water purified from a Milli-Q purification system (Milli-Pore, Bedford, Mass.).

The following examples of the human acute lymphoblastic leukemia T-lymphocyte cell line CCRF-CEM, the human Burkitt lymphoma cell line Ramos, the chronic myelogenous leukemia cell line K562, the human promyelocytic leukemia cell line HL-60, the human peripheral blood lymphocytes PBL, the human macrophage cell line Thp-1 and the human histiocytic lymphoma cell line U937 are all products of the cell bank of the Chinese academy of sciences (Shanghai, China).

The following examples illustrate the culture of CCRF-CEM, Ramos, K562, HL-60, Thp-1 and U937 cell lines: the culture was carried out at 37 ℃ in 5% carbon dioxide (95% oxygen) in a medium 1640 containing 10% fetal bovine serum (FBS, Gibco) and 100U/mL penicillin-streptomycin (Gibco, Grand Island, NY, USA).

All fluorescence measurements in the following examples were carried out in 350. mu.L quartz cuvettes, with an excitation wavelength of 290nm and emission spectra recorded with an excitation and emission slit of 5nm using an F-7000 spectrofluorometer (Hitachi Company, Tokyo, Japan). The peak intensity was obtained at 545nm and the sampling range was recorded at 460nm to 560 nm.

All atomic force microscopy imaging in the examples below was performed by fluorescence microscopy (Nikon DS-Ri 1; Nikon, Tokyo, Japan).

The cell count in the following examples was carried out using cell count kit-8, and cell count kit-8 was a product of Dojindo, Japan.

Statistical analysis methods in the following examples: each experiment was performed in triplicate. Data are presented as mean ± SD or median (range). All statistical analyses were performed using GraphPad Prism 6.02(GraphPad Software, San Diego, CA, USA). P <0.05 was considered statistically significant.