CN109470691B - Self-assembled aptamer/protein composite nanoprobe, preparation method, kit and application thereof - Google Patents
Self-assembled aptamer/protein composite nanoprobe, preparation method, kit and application thereof Download PDFInfo
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Abstract
The invention discloses a self-assembly aptamer/protein composite nano probe, a preparation method, a kit and application thereof, wherein three DNA probes are hybridized to prepare the self-assembly aptamer/protein composite nano probe, one of the three DNA probes is a DNA probe containing an aptamer sequence, and the other one is marked with horseradish peroxidase, and magnetic nanoparticles functionalized by the aptamer are used as a magnetic separation solid phase carrier for specific capture of human acute lymphoblastic leukemia T lymphocytes (CCRF-CEM) in blood and improving capture efficiency of a target object High specificity and rapid detection, and has great significance for the auxiliary diagnosis, curative effect evaluation and prognosis judgment of leukemia.
Description
Technical Field
The invention relates to the technical field of molecular biology, in particular to a self-assembled aptamer/protein composite nanoprobe, a preparation method, a kit and application thereof.
Background
Currently, self-assembly, a powerful technique for spontaneously organizing or aggregating functional building blocks into multifunctional materials in a controlled manner, has been successfully used in the fields of sensing and drug delivery in biomedicine. Particularly, the self-assembly based on nucleic acid hybridization regulation has wide application prospects in the aspects of ordered construction of nano materials, detection of nucleic acid, enzyme activity, heavy metal ions and the like due to the advantages of structural and functional diversity, programmable design of sequences and the like of nucleic acids. In recent years, the construction and application of nucleic acid assemblies with aptamers (aptamers) as affinity ligands have also gained much attention. The Aptamer is a single-stranded oligonucleotide screened from an artificially synthesized DNA/RNA library, and can form a specific three-dimensional structure by intramolecular folding, thereby binding with a target substance (e.g., a small molecule, a protein, a cell, etc.) with high affinity and high specificity. The Aptamer serving as an antibody of a chemist has the advantages of small molecular weight, easiness in synthesis and modification, low cost, high stability, programmable sequence design and the like, and plays an important role as a new-generation molecular recognition probe in the fields of biological imaging, detection, treatment and the like. The current research shows that the detection performance of the method on tumor cells can be obviously improved by combining the Aptamer with inorganic nano materials such as quantum dots, gold nanoparticles, graphene and the like. However, tumor cell detection strategies based on the binding of the Aptamer to proteins are rare.
Leukemia is a malignant clonal disease originating from hematopoietic stem cells, also known as "leukemia". The occurrence and development of leukemia are closely related to genetic factors, ionizing radiation, nutritional conditions and the like, and the detection of human acute lymphoblastic leukemia T lymphocytes (CCRF-CEM) in peripheral blood has great significance for the auxiliary diagnosis, curative effect evaluation and prognosis judgment of leukemia.
Disclosure of Invention
In order to overcome the defects of the prior art, one of the objectives of the present invention is to provide a self-assembly aptamer/protein composite nanoprobe, wherein a novel aptamer/protein composite nanoprobe is constructed by a self-assembly method and is used for dual amplification of a specific identification and detection signal of CCRF-CEM, so as to improve the sensitivity and accuracy of detection.
The second purpose of the invention is to provide a preparation method of the self-assembly aptamer/protein composite nanoprobe.
The invention also aims to provide a kit, which uses the self-assembled aptamer/protein composite nano probe to realize the detection of a target object by adopting the chemiluminescence principle and has high detection sensitivity and accuracy.
The fourth purpose of the invention is to provide an application of the kit in detecting the T lymphocytes of the human acute lymphoblastic leukemia.
In order to achieve the above purpose, the technical scheme adopted by the invention is as follows:
a self-assembly nucleic acid aptamer/protein composite nano probe is formed by hybridizing three DNA probes, wherein the first DNA probe is a DNA probe containing a nucleic acid aptamer sequence, is marked as a hybridization probe 1, and has a nucleotide sequence as follows:
5'-ATCTAACTGCTGCGCCGCCGGGAAAATACTGTACGGTTAGAAAAAAAAAAAACTAAAAGGGTCTGAGGG-3'；
the second is a DNA probe for marking horseradish peroxidase, which is marked as a hybridization probe 2, and the nucleotide sequence of the probe is as follows:
5'-HRP-AAAAAAAAAATACTCCCCCAGGTGCCCCTCAGACCCTTTTAGT-3'；
the third DNA probe is marked as a hybridization probe 3, and the nucleotide sequence of the hybridization probe is as follows:
5'-GCACCTGGGGGAGTAAAAACTAAAAGGGTCTGAGGG-3'。
the preparation method of the self-assembly aptamer/protein composite nano probe is completed by taking the hybridization probe 1, the hybridization probe 2 and the hybridization probe 3 to perform vortex hybridization at room temperature, wherein the composite nano probe is marked as SAPPs, and the vortex hybridization time is 1 hour.
The vortex hybridization process is to mix the hybridization probe 1, the hybridization probe 2 and the hybridization probe 3 according to a certain proportion and carry out vortex hybridization at room temperature. Wherein, the hybridization probe 1 mainly comprises two parts, one part is an aptamer which specifically recognizes CCRF-CEM and is used for recognizing the CCRF-CEM captured by Sgc8 c-MNPs; the other part can be hybridized with the hybridization probe 2 and is used for connecting the hybridization probe 2, and the two parts are connected by a connecting arm with the length of 10A bases. The hybridization probe 1 can be hybridized with the first half part sequence of the hybridization probe 2, the second half part sequence of the hybridization probe 2 can be hybridized with the first half part sequence of the hybridization probe 3, the second half part sequence of the hybridization probe 3 can be hybridized with the first half part of the next hybridization probe 2, and the like, and finally a DNA long chain with a double-helix structure is formed. Each double chain contains at least one HRP, and the formed SAPPs not only have a target recognition function, but also can realize double amplification of detection signals.
Further, the aptamer/protein composite nanoprobe is prepared by hybridizing the hybridization probe 1, the hybridization probe 2 and the hybridization probe 3 with the concentration of 50-250 nmol/L.
Preferably, the aptamer/protein composite nanoprobe is prepared by hybridizing 75nmol/L of hybridization probe 1, 150nmol/L of hybridization probe 2 and 150nmol/L of hybridization probe 3.
A kit comprising a capture probe for capturing a target product, and the self-assembling aptamer/protein composite nanoprobe described above.
The kit also comprises a target capture carrier, a chemiluminescent plate, a washing buffer solution, a binding buffer solution and a chemiluminescent base solution.
The application of the kit is applied to the detection of human acute lymphoblastic leukemia T lymphocytes (CCRF-CEM) in peripheral blood.
Further, the nucleotide sequence of the capture probe is:
5'-ATCTAACTGCTGCGCCGCCGGGAAAATACTGTACGGTTAGAAAAAAAAAAA-3'。
further, the target capture carrier is streptavidin magnetic beads; the capture probes are loaded on streptavidin magnetic beads and are marked as Sgc8 c-MNPs.
The preparation method of the Sgc8c-MNPs comprises the following steps:
a: and (3) cleaning magnetic beads: washing streptavidin magnetic beads by PBS buffer solution;
b: binding of streptavidin magnetic beads to capture probes: adding a capture probe into the washed streptavidin magnetic beads, uniformly mixing, performing vortex reaction at room temperature, performing magnetic separation to obtain magnetic beads, and washing the magnetic beads by adopting PBS (phosphate buffer solution), namely Sgc8 c-MNPs;
c: preserving the magnetic beads: and (4) resuspending the Sgc8c-MNPs prepared in the step B in an immunomagnetic bead preservation solution, and storing at 4 ℃ for later use.
Further, the detection method of the application of the kit comprises the following operation steps:
(1) washing the low-adsorption centrifuge tube: washing the low-adsorption centrifuge tube by using a washing buffer solution;
(2) adding Sgc8 c-MNPs: washing Sgc8c-MNPs3 times by using washing buffer, suspending in the same volume of washing buffer, adding 20 mu L of washing buffer into a low-adsorption centrifuge tube per tube, and washing by using the washing buffer;
(3) adding a sample to be detected: adding a certain volume of sample to be detected into the low-adsorption centrifugal tube treated in the step (2), incubating, carrying out magnetic separation, discarding the supernatant, and washing with a washing buffer solution;
(4) resuspending the sample to be detected obtained in the step (3) and the Sgc8c-MNPs compound in a binding buffer solution, transferring the mixture to a chemiluminescence plate, carrying out magnetic separation, and discarding the supernatant;
(5) adding SAPPs into a chemiluminescence plate, incubating for 20min at 4 ℃, performing magnetic separation, discarding the supernatant, and washing the plate with a washing buffer solution;
(6) adding chemiluminescent base solution into each hole of the chemiluminescent plate, and detecting RLU by using a microporous plate type luminescence detector;
(7) and (3) detecting RLU of CCRF-CEM standard substances with different concentrations by the same method as the steps (1) to (6), drawing a standard curve chart of the relation between the RLU and the CCRF-CEM concentration to obtain a relation between the RLU and the CCRF-CEM concentration, and substituting the RLU of the sample to be detected in the step (6) into the relation to calculate the concentration of the CCRF-CEM in the sample to be detected.
Further, the chemiluminescent substrate solution in the step (6) includes a chemiluminescent substrate solution a and a chemiluminescent substrate solution B, wherein the chemiluminescent substrate solution a is luminol and an enhancer (p-hydroxybiphenyl, BIP), and the chemiluminescent substrate solution B is a hydrogen peroxide solution.
Further, the composition of the above washing buffer was 0.01mol/L pH 7.4PBS, 4.5mg/mL glucose, 5mmol/L MgCl2·6H2O, 0.05% Tween-20; the composition of the binding buffer was 0.01mol/L pH 7.4PBS, 4.5mg/mL glucose, 5mmol/L MgCl2·6H2O，0.1mg/mL yeast tRNA，0.05％Tween-20。
Compared with the prior art, the invention has the beneficial effects that: the invention makes hybridization between three DNA probes, one of which is the DNA probe containing the sequence of the aptamer and the other is marked with horseradish peroxidase, to prepare the self-assembly aptamer/protein composite nano probe, and the partial sequences of the three DNA probes are combined with each other in the hybridization process to realize circular hybridization, and finally form a DNA long chain with a double-spiral structure. Each double chain contains at least one HRP, and the formed self-assembly aptamer/protein composite nano probe not only has a target recognition function, but also can realize double amplification of detection signals. The magnetic nanoparticle with the functionalized aptamer is used as a magnetic separation solid phase carrier, is used for specific capture of CCRF-CEM in blood, improves capture efficiency of a target object, is matched with the self-assembly aptamer/protein composite nanoprobe for use, has wide linear range, low detection limit, high precision and high accuracy for detecting the CCRF-CEM, realizes high sensitivity, high specificity and rapid detection of the CCRF-CEM in peripheral blood samples, and has great significance for auxiliary diagnosis, curative effect evaluation and prognosis judgment of leukemia.
Drawings
FIG. 1 is a schematic diagram showing the hybridization of ApDNA (hybridization probe 1), H1 DNA (hybridization probe 2), and H2 DNA (hybridization probe 3) to form SAPPs;
FIG. 2 is a graph showing the results of agarose gel electrophoresis of hybridized substances of ApDNA (hybridization probe 1), H1 DNA (hybridization probe 2) and H2 DNA (hybridization probe 3);
FIG. 3 is a graph of the capture efficiency of Sgc8c-MNPs against CCRF-CEM at various concentrations;
FIG. 4 is an imaging diagram of Sgc8 recognition of target cells captured by the MNPs 8 c;
FIG. 5 is a schematic diagram of the detection principle of CCRF-CEM using the self-assembled aptamer/protein composite nanoprobe of the present invention;
FIG. 6 is a graph of the statistical effect of Tween-20 in wash buffer on chemiluminescence intensity;
FIG. 7 is a graph of the statistics of the effect of Tween-20 in binding buffer on the intensity of chemiluminescence;
FIG. 8 is a standard graph of detection of CCRF-CEM;
FIG. 9 is a statistic of the results of the effect of H1 DNA (hybridization probe 2) concentration on chemiluminescence intensity;
FIG. 10 is a graph showing the results of an experiment for detecting CCRT-CEM cells in human peripheral blood by the method of the present invention.
Detailed Description
The present invention will be further described with reference to the accompanying drawings and the detailed description, and it should be noted that any combination of the embodiments or technical features described below can be used to form a new embodiment without conflict.
The target cell line used in the experiment is human acute lymphoblastic leukemia T lymphocyte (CCRF-CEM), and the control cell is human acute lymphoblastic leukemia B lymphocyte (Ramos). The culture medium is RPMI 1640 (supplemented with 10% fetal calf serum), the cell culture temperature is 37 deg.C, and CO is added2The content is 5%.
In the present invention, the equipment, materials and the like used are commercially available or commonly used in the art, and the methods in the following examples are conventional in the art unless otherwise specified.
The nucleotide sequences used in the present invention are shown in table 1:
TABLE 1
Example 1
Preparing a self-assembled aptamer/protein composite nanoprobe: mixing 75nmol/L hybridization probe 1, 150nmol/L hybridization probe 2 and 150nmol/L hybridization probe 3, adding into a vortex mixing instrument, and performing vortex hybridization at room temperature for 1h to obtain the self-assembled aptamer/protein composite nanoprobe, wherein the preparation process is shown in figure 1.
And (3) hybridization result characterization: ApDNA, H1 DNA and H2 DNA are respectively diluted to the concentration of 2 mu mol/L, then 10 mu L of the above solution, 10 mu L of mixed solution of 1 mu mol/L ApDNA, 2 mu mol/L H1 and 2 mu mol/L H2 are respectively subjected to 2% agarose gel electrophoresis under the conditions of voltage 100V and time 1H, and finally gel imaging is carried out, as shown in FIG. 2, wherein the gel imaging is carried out in lane 1 in FIG. 2: a primer; lane 2: a binding buffer; lane 3: 2 mu mol/L ApDNA; lane 4: 2. mu. mol/L H1 DNA; lane 5: 2 μmol/L H2; lane 6: mixed solutions of ApDNA, H1, and H2 at concentrations of 1. mu. mol/L, 2. mu. mol/L, and 2. mu. mol/L, respectively. As can be seen from fig. 2: after the ApDNA, H1 and H2 are vortexed at room temperature for 1H, a series of hybrid bands with different lengths can be seen in an electrophoretogram, and no obvious single bands of ApDNA, H1 and H2 are observed. The results indicate that ApDNA, H1 and H2 hybridize under room temperature vortexing, forming self-assembling probes SAPPs.
Example 2
Sgc 8A method for preparing 8c-MNPs, which comprises the following steps:
a: and (3) cleaning magnetic beads: mu.L of 2mg/mL magnetic beads (MNPs) were placed in a 1.5mL low-adsorption centrifuge tube, magnetically separated, and the supernatant was discarded. Then 300. mu.L PBS buffer solution was added, mixed well, separated magnetically, the supernatant was discarded, and washed 3 times in total.
B: binding of MNPs to Sgc8 c: 500 μ L of 400nmol/L Sgc8c (another aliquot of Sgc8c at the same concentration, labeled as pre) was added to the low adsorption centrifuge tube and mixed well. Performing vortex reaction at room temperature for 30min, performing magnetic separation, collecting supernatant, marking as post, washing magnetic beads with PBS for 2 times, each time 500 μ L, collecting supernatant, respectively marking as wash1 and wash 2;
c: the Sgc8c modified MNPs (Sgc8c-MNPs) are resuspended in 600. mu.L of immunomagnetic bead preservation solution and stored at 4 ℃ for later use.
Characterization of the above fixed results: the absorption of the solutions pre, post, wash1 and wash2 at 260nm is detected by utilizing the characteristic that nucleic acid has a specific absorption peak at 260nmThe luminosity, the binding efficiency was calculated according to the following calculation formula for binding efficiency,
the results are shown in Table 2. Sgc8c and MNPs have binding efficiency ranging from 70.85% to 75.31%, which indicates that Sgc8c-MNPs have been successfully prepared.
TABLE 2
A nanometer particle size and Zeta potential analyzer is used for detecting the hydrated particle size and the Zeta potential of aptamer-streptavidin magnetic beads (Sgc8c-MNPs), whether Sgc8c is successfully fixed on the surfaces of the MNPs is identified, the hydrated particle size of the MNPs is 313.2nm, the hydrated particle size is increased to 532.1nm after Sgc8c is modified on the surfaces of the MNPs, and is increased by 218.9nm in total compared with the hydrated particle size before modification, which indicates that Sgc8c is successfully modified on the surfaces of the MNPs, and Sgc8c-MNPs complex is formed. Meanwhile, the results of the Zeta potential measurements showed that the Zeta potentials of MNPs, Sgc8c, and Sgc8c-MNPs were-2.31 mV, -20.7mV, and-11.1 mV, respectively. Sgc8 Zeta potential of 8c-MNPs is biased towards Sgc8c potential, indicating that Sgc8c-MNPs have been successfully prepared.
Example 3
Sgc8 Capture of target cells in single CCRF-CEM suspensions by 8 c-MNPs:
(1) and (3) washing the cells: CCRF-CEM was washed 3 times with binding buffer and then resuspended to 106cells/mL of cell suspension;
(2) cell staining: add 5. mu.L of 2mmol/L DiD dye per ml of CCRF-CEM cell suspension and incubate at 37 ℃ for 5 min. Cells were washed 3 times with binding buffer;
(3) sgc8c-MNPs capture CCRF-CEM: the stained cells were diluted to five different concentrations: 500. 1000, 2500, 5000 and 10000 cells/mL. 1mL of CCRF-CEM with different concentrations was mixed with Sgc8c-MNPs (Sgc8c-MNPs in 20. mu.L of stock solution), incubated at 4 ℃ for 20min, subjected to magnetic separation, and the supernatant was collected. Centrifuging at 1000r/min for 5min, and dispersing the cell precipitate in 100 μ L binding buffer;
(4) cell counting: the CCRF-CEM in the supernatant was subjected to fluorescence counting under a confocal laser microscope, and the Capture Efficiency (CE) was calculated according to the following formula, and the result is shown in fig. 3, and can be seen from fig. 3: when the CCRF-CEM concentration is in the range of 500cells/mL to 10000cells/mL, the capture efficiency of Sgc8c-MNPs is about 92 percent.
CE(％)＝NC/N0×100％
In the formula: CE is the capture efficiency; n is a radical ofCThe captured target number; n is a radical of0Is the original target number.
Sgc8 Capture of target cells in CCRF-CEM and Ramos mixtures by 8 c-MNPs:
(1) and (3) washing the cells: CCRF-CEM, Ramos were washed 3 times with binding buffer and then resuspended to 10%6cells/mL of cell suspension.
(2) Cell staining: 5. mu.L of 2mmol/L DiD per ml of CCRF-CEM cell suspension and 5. mu.L of 2mmol/L DiO per ml of Ramos cell suspension were added and incubated at 37 ℃ for 5 min. Cells were washed 3 times with binding buffer.
(3) Sgc8 incubation of 8c-MNPs with cells: the preparation contains CCRF-CEM and Ramos with the concentration of 10000cells/mL and 10 respectively5cells/mL of mixed cell suspension. Mixing 1mL of cell suspension with Sgc8c-MNPs (Sgc8c-MNPs in 20 μ L of stock solution), incubating at 4 deg.C for 20min, magnetically separating, and collecting supernatant. Another 1mL of cell suspension (as control) was centrifuged simultaneously with the supernatant, and the cells were collected and resuspended in 100 μ L of binding buffer, respectively. The centrifugation condition is 1000r/min of rotation speed and 5min of time.
(4) Fluorescence imaging: cell imaging was performed under a laser confocal fluorescence microscope.
Sgc8c-MNPs are respectively mixed with CCRF-CEM and Ramos, and after incubation, imaging is carried out by a laser confocal fluorescence microscope, and the result is shown in figure 4, wherein figure 4A shows that Sgc8c-MNPs are mixed with CCRF-CEM for incubation and then imaged, figure 4B shows that Sgc8c-MNPs are mixed with Ramos for incubation and then imaged, and figure 4A shows that after Sgc8c-MNPs are mixed with CCRF-CEM for incubation, obvious black particles, namely Sgc8c-MNPs, are attached to the surface of CCRF-CEM; as can be seen in FIG. 4B, when Sgc8c-MNPs were incubated in combination with Ramos, no Sgc8c-MNPs were clearly attached to the surface of Ramos. Sgc 8-8 c-MNPs are shown to be capable of specifically recognizing CCRF-CEM, but have no obvious recognition and capture to Ramos, and the efficiency of capturing the CCRF-CEM in mixed cell suspension by Sgc 8-8 c-MNPs is about 90%.
Example 4
A kit comprises a low chemiluminescence plate, magnetic beads, capture probes, self-assembled aptamer/protein composite nano probes, chemiluminescence base solution, binding buffer solution and washing buffer solution; the chemiluminescence base solution comprises solution A and solution B, wherein the solution A is luminol + reinforcing agent, and the solution B is hydrogen peroxide solution; the magnetic beads are Sgc8c-MNPs magnetic beads prepared according to the method described in example 2; the self-assembled aptamer/protein composite nanoprobe is prepared according to the method described in example 1.
The kit is applied to the detection of CCRF-CEM, and the specific detection method comprises the following steps:
(1) washing the low-adsorption centrifuge tube: 1.5mL of low-adsorption centrifuge tube is taken and washed for 1 time by 1.5mL of washing buffer solution, so that the non-specific adsorption of the low-adsorption centrifuge tube to the reactant is reduced;
(2) adding Sgc8 c-MNPs: washing Sgc8c-MNPs3 times with washing buffer, suspending in washing buffer with the same volume, adding 20 microliter of washing buffer into a low-adsorption centrifuge tube, and washing with washing buffer for 1 time;
(3) adding CCRF-CEM: CCRF-CEM was diluted to different concentrations with binding buffer, 1mL per tube was added to a low adsorption centrifuge tube, 4 replicates per concentration, incubated at 4 ℃ for 20min, magnetically separated, the supernatant discarded, and washed 1 time with wash buffer.
(4) Resuspending the CCRF-CEM and Sgc8c-MNPs complex obtained in the step (3) in 100 mu L of binding buffer, transferring the complex to a chemiluminescence plate, carrying out magnetic separation, and discarding the supernatant;
(5) SAPPs were added to the chemiluminescent plate at 100. mu.L per well and incubated at 4 ℃ for 20 min. Magnetic separation, discarding supernatant, washing the plate with washing buffer solution for 3 times;
(6) adding 100 mu L of chemiluminescent substrate solution A and 100 mu L of chemiluminescent substrate solution B into each hole, and detecting RLU by using a microporous plate type luminescence detector;
(7) detecting RLU of CCRF-CEM standard substances with different concentrations by the same method as the steps (1) to (5), drawing a standard curve graph of the relation between the RLU and the CCRF-CEM concentration to obtain a relation between the RLU and the CCRF-CEM concentration, and substituting the RLU of the sample to be detected in the step (5) into the relation to calculate the concentration of the CCRF-CEM in the sample to be detected;
the establishment process of the standard curve in the step (7) is as follows: CCRF-CEM was diluted with binding buffer to different concentrations of cell suspension: 150. 200, 500, 750, 1000, 2500, 5000, 7500, 10000 cells/mL. CCRF-CEM was detected in cell suspensions in parallel 4 times at each concentration using the method described above. And (3) carrying out linear fitting on the experimental result, establishing a standard curve for detecting CCRF-CEM, and obtaining a corresponding relation between the concentration and the RLU, wherein the corresponding relation is as follows: Y-0.27615X-0.49613 (wherein X is lgC)CCRF-CEMY is lg (RLU/RLU)0And r ═ 0.9918), as shown in fig. 8.
The detection principle of the detection method is as follows: as shown in fig. 5: first, biotinylated Sgc8c was immobilized to the surface of MNPs using a strong specific interaction between streptavidin and biotin, forming Sgc8 c-MNPs. When the target CCRF-CEM exists, Sgc8c-MNPs can specifically recognize tyrosine kinase 7 on the surface of CCRF-CEM so as to capture the target CCRF-CEM (incubation for 20 min). After magnetic separation and washing, adding the self-assembled aptamer/protein composite nano-probe, incubating for 20min again, wherein the self-assembled composite nano-probe can specifically recognize and bind with the CCRF-CEM captured by Sgc8-MNPs, and removing unbound probe by magnetic separation and washing. And finally, adding a chemiluminescence base solution, wherein the nucleic acid aptamer/protein composite nano probe can catalyze a chemiluminescence substrate and generate chemiluminescence. The higher the concentration of the CCRF-CEM is, the stronger the chemiluminescence intensity is, and therefore, the effective capture and high-sensitivity rapid detection of the CCRF-CEM can be realized.
Example 5
In order to improve the accuracy of CCRF-CEM detection, each condition in the CCRF-CEM detection process is optimized, and the specific optimization process is as follows:
1. effect of Tween-20 in washing buffer on chemiluminescence intensity
FIG. 6A is the effect of the addition or absence of Tween-20 in the wash buffer on solvent-blank RLU; b is the effect of the addition or non-addition of Tween-20 in the washing buffer on the chemiluminescence intensity, and the buffer composition of group 1 is 0.01mol/L of pH 7.4PBS, 4.5mg/mL of glucose, and 5mmol/L of MgCl2·6H2O; the composition of the buffer in group 2 was 0.01mol/L pH 7.4PBS, 4.5mg/mL glucose, 5mmol/L MgCl2·6H2O, 0.05% Tween-20, as can be seen in FIG. 6: the solvent blank signal values for the Tween-20 group (group 2) added to the wash buffer were low, indicating that Tween-20 reduced non-specific adsorption. As seen in FIG. 6B, RLU/RLU of group 2 when the cell concentration was the same0Above group 1, it is believed that the addition of Tween-20 to the wash buffer had no significant effect on the binding of CCRF-CEM to Sgc8 c. Therefore, the present invention selects a solution containing 0.05% Tween-20 as the washing buffer.
2. Selection of binding buffer
As shown in fig. 7: a: solvent blank RLU corresponding to different kinds of binding buffer; b: effect of binding buffer on chemiluminescence intensity. Group 1 consists of: 0.01mol/L pH 7.4PBS, 4.5mg/mL glucose, 5mmol/L MgCl2·6H2O, 0.1mg/mL yeast tRNA, 1mg/mL BSA; group 2 consists of: 0.01mol/L pH 7.4PBS, 4.5mg/mL glucose, 5mmol/L MgCl2·6H2O, 0.1mg/mL yeast tRNA, 0.05% Tween-20; group 3 consists of: 0.01mol/L pH 7.4PBS, 4.5mg/mL glucose, 5mmol/L MgCl2·6H2O, 0.1mg/mL yeast tRNA, as can be seen in FIG. 7: the solvent blank signal value for the added Tween-20 group (group 2) was lower than for the other two groups, and RLU/RLU0 was significantly higher than for the other two groups, because Tween-20 reduced non-specific adsorption of the chemiluminescent plate to the reactants, the blank signal value was reduced, and the signal to background ratio was increased. Therefore, the present inventionA solution with 0.05% Tween-20 was selected as the binding buffer.
3. Selection of the amounts of reactants
(1) Sgc8 dosage of 8 c-MNPs: sgc8, the dosage of 8c-MNPs has direct influence on the experimental result, the dosage is too small, and the capture efficiency of the MNPs on target cells is low; if the dosage is too much, the experiment cost is increased, and the waste is caused, and the experiment result shows that the dosage of Sgc8c-MNPs is 20 mu L of stock solution per tube, RLU/RLU0To a maximum.
(2) ApDNA concentration: the ApDNA mainly consists of two parts, wherein one part is an aptamer which specifically recognizes CCRF-CEM and is used for recognizing the CCRF-CEM captured by Sgc8 c-MNPs; the other part was able to hybridize with H1 for ligation with H1. The two parts are connected by a linker arm of 10A bases in length. The results of optimizing the ApDNA concentration during the formation of SAPPs indicate that RLU/RLU increases with the ApDNA concentration0Increasing first and then decreasing. RLU/RLU when the concentration of ApDNA is 75nmol/L0At the maximum, the optimal concentration of ApDNA is therefore 75 nmol/L.
(3) H1 concentration: the concentration of H1 during formation of the self-assembled probe was optimized, and when the concentration of H1 was increased from 50nmol/L to 150nmol/L, RLU/RLU was used0Gradually increasing and reaching a maximum, and continuing to increase the concentration of H1, RLU/RLU0Gradually decreases. Therefore, the optimum concentration of H1 was 150nmol/L, as shown in FIG. 9.
4. Selection of standard curves
Using RLU/RLU, respectively0-C、RLU/RLU0-lgC、lg(RLU/RLU0) C and lg (RLU/RLU)0) And linear fitting is carried out on the experimental data in four modes of-lgC, and the obtained linear equation and the correlation coefficient r are shown in table 3. The fitting way of the maximum correlation coefficient r, i.e. in lg (RLU/RLU), is chosen0) And (4) fitting an equation in an lgC mode to obtain a final linear equation, wherein a standard curve corresponding to the equation is shown in FIG. 9. Lg (RLU/RLU) when the concentration of the CCRF-CEM suspension is in the range of 150cells/mL to 10000cells/mL0) And lgC, and the linear equation is Y-0.27615X-0.49613 (wherein X is lgC)CCRF-CEMY is lg (RLU/RLU)0And r ═ 0.9918), as shown in fig. 8.
TABLE 3 CCRF-CEM detection linear equation obtained by different fitting modes
Example 6
Evaluation of the detection method described in example 3:
1. examination of detection limits: measuring 4 solvent blanks and calculating to obtain
RLU75 when the CCRF-CEM concentration is 75cells/mL>8261.76, therefore, the detection limit of the established CCRF-CEM detection method is 75 cells/mL.
2. Precision investigation: the CCRF-CEM standard solutions with the concentrations of 5000cells/mL, 1000cells/mL and 200cells/mL are respectively prepared by using the binding buffer solution, and the detection is carried out by adopting the method disclosed by the invention, so that the precision range in the plate is 2.08-21.83%, and the precision range between plates is 8.48-26.86%. When the concentration of the target cells is higher, the precision is better; when the cell concentration is low, precision is slightly poor.
3. Examination of the recovery rate by adding standard: the test results of the standard recovery rate show that the method provided by the invention is used for detecting CCRF-CEM standard solutions with the concentrations of 5000cells/mL, 1000cells/mL and 200cells/mL respectively, the standard recovery rates are 94.61%, 116.93% and 97.47%, and the experimental results show that the method has high accuracy in detecting the target object.
4. Investigation of specificity: for the CCRF-CEM group, RLU/RLU with increasing cell concentration0Gradually increasing; whereas for the Ramos group, the signal value RLU/RLU increased with increasing cell concentration0Is substantially unchanged. The method is proved to have high specificity.
Example 7
CCRF-CEM cells with different concentrations are respectively added into peripheral blood of a normal person to obtain samples with final concentrations of 200cells/mL, 500cells/mL and 1000cells/mL respectively, then erythrocyte lysate is added for processing, and after a precipitate part is centrifugally collected, cells are resuspended and detected by the method provided by the invention. As shown in FIG. 10, the experimental results show that the method can realize high-sensitivity detection of CCRF-CEM in whole blood (the detection limit is 200cells/ml) within 1 hour.
The invention combines the magnetic separation technology with the double signal amplification function of the self-assembly aptamer/protein composite nano probe and the chemiluminescence detection technology, establishes a method with high sensitivity, strong specificity and simple and convenient operation, and is successfully applied to the detection method of CCRF-CEM in peripheral blood. The standard curve of the method is Y-0.27615X-0.49613 (wherein X is lgC)CCRF-CEMY is lg (RLU/RLU)0R ═ 0.9918) Y is the corresponding RLU-RLU0) The actually measured detection limit in the buffer obtained by gradually reducing the concentration of the target substance is 75cells/mL, and the detection limit of CCRF-CEM in whole blood is 200 cells/mL; the precision (RSD) range of the plate is 2.08-21.83 percent, and the precision (RSD) range of the plate is 8.48-26.86 percent; the range of the recovery rate of the added standard is 94.61-116.93%, and the detection result of CCRF-CEM in peripheral blood shows that the method can be effectively used for detecting CCRF-CEM in peripheral blood, and has great significance for the auxiliary diagnosis, curative effect evaluation and prognosis judgment of leukemia.
The above embodiments are only preferred embodiments of the present invention, and the protection scope of the present invention is not limited thereby, and any insubstantial changes and substitutions made by those skilled in the art based on the present invention are within the protection scope of the present invention.
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Claims (9)
1. A self-assembly aptamer/protein composite nano probe is characterized by being formed by hybridizing three DNA probes, wherein the first DNA probe is a DNA probe containing an aptamer sequence, is marked as a hybridization probe 1, and has a nucleotide sequence as follows:
5'-ATCTAACTGCTGCGCCGCCGGGAAAATACTGTACGGTTAGAAAAAAAAAAAACTAAAAGGGTCTGAGGG-3'；
the second is a DNA probe for marking horseradish peroxidase, which is marked as a hybridization probe 2, and the nucleotide sequence of the probe is as follows:
5'-HRP-AAAAAAAAAATACTCCCCCAGGTGCCCCTCAGACCCTTTTAGT-3'；
the third DNA probe is marked as a hybridization probe 3, and the nucleotide sequence of the hybridization probe is as follows:
5'-GCACCTGGGGGAGTAAAAACTAAAAGGGTCTGAGGG-3'。
2. the method for preparing the self-assembly aptamer/protein composite nanoprobe as claimed in claim 1, wherein the hybridization of the hybridization probe 1, the hybridization probe 2 and the hybridization probe 3 is performed by vortex hybridization at room temperature, and the composite nanoprobe is marked as SAPPs.
3. A kit comprising a capture probe for capturing a target product and the self-assembling aptamer/protein complex nanoprobe of claim 1.
4. The kit of claim 3, further comprising a target capture support, a chemiluminescent plate, a chemiluminescent substrate, a wash buffer and a binding buffer.
5. Use of a kit according to claim 4 for the detection of human acute lymphoblastic leukemia T lymphocytes in peripheral blood.
6. The use of a kit according to claim 5, wherein the capture probe has the nucleotide sequence:
5'-ATCTAACTGCTGCGCCGCCGGGAAAATACTGTACGGTTAGAAAAAAAAAAA-3'。
7. the use of the kit according to claim 6, wherein the target capture carrier is streptavidin magnetic beads; the capture probes are loaded on streptavidin magnetic beads and are marked as Sgc8 c-MNPs.
8. The use of the kit according to claim 6, wherein the detection method comprises the following steps:
(1) washing the low-adsorption centrifuge tube: washing the low-adsorption centrifuge tube by using a washing buffer solution;
(2) adding Sgc8 c-MNPs: washing Sgc8c-MNPs3 times by using washing buffer, suspending in the same volume of washing buffer, adding 20 mu L of washing buffer into a low-adsorption centrifuge tube per tube, and washing by using the washing buffer;
(3) adding a sample to be detected: adding a certain volume of sample to be detected into the low-adsorption centrifugal tube treated in the step (2), incubating, carrying out magnetic separation, discarding the supernatant, and washing with a washing buffer solution;
(4) resuspending the sample to be detected obtained in the step (3) and the Sgc8c-MNPs compound in a binding buffer solution, transferring the mixture to a chemiluminescence plate, carrying out magnetic separation, and discarding the supernatant;
(5) adding SAPPs into a chemiluminescence plate, incubating for 20min at 4 ℃, performing magnetic separation, discarding the supernatant, and washing the plate with a washing buffer solution;
(6) adding chemiluminescent base solution into each hole of the chemiluminescent plate, and detecting RLU by using a microporous plate type luminescence detector;
(7) and (3) detecting RLU of the human acute lymphoblastic leukemia T lymphocyte standard substance with different concentrations according to the same method of the steps (1) to (6), drawing a standard curve graph of the relation between the RLU and the human acute lymphoblastic leukemia T lymphocyte concentration to obtain a relation between the RLU and the human acute lymphoblastic leukemia T lymphocyte concentration, and substituting the RLU of the sample to be detected in the step (6) into the relation to calculate the concentration of the human acute lymphoblastic leukemia T lymphocyte in the sample to be detected.
9. The use of the kit according to claim 8, wherein the chemiluminescent substrate solution of step (6) comprises a chemiluminescent substrate solution A and a chemiluminescent substrate solution B, wherein the chemiluminescent substrate solution A is luminol and an enhancer, and the chemiluminescent substrate solution B is a hydrogen peroxide solution.
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