CN107860912B - Detection method of bifunctional aptamer-mediated A549 tumor cells - Google Patents

Abstract

the invention relates to a method for detecting A549 tumor cells mediated by a bifunctional aptamer, which comprises 6 steps of preparing an aptamer-inhibition chain compound, preparing a cell suspension, preparing a hairpin catalysis self-assembly reaction liquid, collecting fluorescence intensity, establishing a standard substance curve equation and calculating cell concentration. The detection method can realize the rapid detection of the tumor cells in a homogeneous system, and has the advantages of high sensitivity, good specificity and capability of being used for rapid screening.

Description

Detection method of bifunctional aptamer-mediated A549 tumor cells

Technical Field

The present invention relates to the field of optical detection, and in particular to the testing or analysis of tumor cells by detecting changes in fluorescence signals.

Background

The traditional tumor cell detection methods mainly comprise immunohistochemistry, flow cytometry, fluorescence in situ hybridization, polymerase chain reaction, second-generation sequencing methods and the like, which solve the problems related to tumor cell detection to different degrees, but a simple, convenient, rapid, high-sensitivity and high-specificity detection method suitable for clinical laboratories is still lacked at present. Flow cytometry is a classical cytometry method that achieves rapid quantification and sorting of cells in a fluidic system by measuring coulter resistance, fluorescence, light scattering, and light absorption. However, the method requires expensive equipment, and has extremely high requirements on the professional performance of the personnel for operation and result analysis, thereby limiting the clinical popularization of the method. Of particular interest in recent years for the detection of circulating tumor cellsThe platform is the only circulating tumor cell detection platform which is approved by the food and drug administration for clinical use at present. The platform utilizes an antibodyAnd enriching the cells by using the body-labeled magnetic beads, fixing the cells, labeling the cells by using a fluorescent dye and a fluorescent antibody, and then identifying the cells. The technology can better protect the morphological structure of the cells, is beneficial to observing the morphology of the tumor cells, provides a new idea for detecting the tumor cells, and has the defects of limited types of markers, high false negative rate, high precision and expense of equipment and the like. Therefore, in order to meet the needs of clinical diagnosis and treatment, especially individualized medical treatment, developing a new method for detecting tumor cells, which is simple, fast, highly sensitive and specific, is an urgent issue to be solved in the field of biosensing research, and is one of the research hotspots in recent years.

in consideration of the characteristics of low abundance, short life and heterogeneity of rare tumor cells, the fluorescence detection technology has attracted wide attention in tumor cell detection due to its characteristics of high sensitivity, simplicity, real-time monitoring and the like. Wherein, the fluorescent dye can be well combined with protein, nucleic acid and nano material to realize the detection of various biomarkers in vivo and in vitro. The developed tumor cell fluorescence detection method is mainly characterized in that the tumor cells are fixed on a detection interface through capture molecules, and then the tumor cells are detected by adopting specific fluorescent molecules. As is commonly used in the method of capturing tumor cells with antibodies, it is necessary to fix the antibodies on the interface, incubate the sample with the antibodies overnight and elute non-targeted cells, and then observe under the mirror by staining with fluorescent dyes. With these methods, tumor cells can be specifically identified, but such methods are time-consuming, require removal of non-target cells by multiple elutions, are complicated in steps, are expensive in reagents and not easy to store, and are not suitable for rapid detection of tumor cells.

Aptamers are oligonucleotides which can interact with target specificity and are obtained by repeatedly screening random oligonucleotide sequence libraries which are artificially synthesized in vitro through an exponential enrichment ligand phylogenetic technology, and aptamers which can be specifically combined with proteins, living cells, metals, bacteria, small molecules and the like are screened at present. The aptamer has the advantages of high affinity, high specificity, small volume, easy synthesis, stable property, easy structure control and the like. In the study of tumor cells, aptamers have been screened from various cell lines, such as non-small cell lung cancer, liver cancer, glioma, adenocarcinoma of lung, colon cancer, etc. In recent years, many methods for detecting tumor cells have been established using aptamers, and the principles of action of these methods can be roughly classified into three categories. The method established by the earliest using aptamer is mainly to label single/double fluorescent groups or some new luminescent materials on the aptamer to form a probe structure, the aptamer is specifically combined with tumor cells to generate conformational transition so as to release a fluorescent signal, the method is simple to operate and mature in fluorescent labeling technology, but the signal and the target of the method are in one-to-one relationship, and the sensitivity is poor. Many nanomaterials are introduced into tumor cell detection because of their strong biocompatibility, small size, and controllable color and luminescence. When the tumor cells exist, the aptamer is combined with the target to cause the change of the existing form of the nanometer material, so that different signals are collected for tumor cell analysis. If researchers mark nucleic acids which are respectively complementary with two ends of the MCF-7 cell aptamer on the gold nanoparticles, when MCF-7 cells exist, the aptamer is combined with target cells and is not complementary with oligonucleotides on the gold nanoparticles, so that the gold nanoparticles are changed into a dispersion state from an aggregation state, and the solution is also correspondingly changed into red from purple. The method provides a new idea for detecting tumor cells, but has the defects of difficult preparation of nano materials, high price and the like. The polymerase chain reaction carries out signal amplification through exponential amplification to bring milestone changes to biomolecule detection, and other signal amplification modes are developed concomitantly to realize trace detection of nucleic acid. Based on the nucleic acid properties of aptamers, some researchers have attempted to apply these signal amplification methods to tumor cell detection in an attempt to achieve highly sensitive detection of tumor cells. The sequences which are complementary with the aptamer are designed and are correspondingly amplified after being replaced, the sensitivity of the method is improved to a certain extent by the method, but the replaced sequences are polydisperse and exist in the supernatant and are difficult to concentrate, so that the real signal amplification reaction is limited to a certain extent.

the catalytic hairpin self-assembly reaction is developed from a DNA nano structure, the reaction is realized by two stem-loop nucleic acids and one chain nucleic acid which are well designed, and the structure is changed by utilizing the base complementary pairing principle and the topological reaction kinetic effect of naked nucleic acid of which the nuclein forming area on the stem-loop structure can be complemented by sequence, so that the self-assembly and de-assembly processes between the nucleic acids are completed. This reaction is of great interest because of its fast and efficient signal amplification properties. In biomolecule detection, a target nucleic acid molecule generally plays a role in starting a signal amplification reaction by a chain nucleic acid, and therefore, the reaction is mainly applied and developed in a nucleic acid molecule detection process. Due to the nature of nucleic acid, small volume, specificity recognition target and the like, the aptamer realizes the homogeneous detection of various biomolecules, and avoids complicated operation. In order to realize homogeneous detection of tumor cells quickly, the invention introduces the aptamer to carry out specific recognition of target cells. The existing tumor cell detection method based on the aptamer still has the defects of complex operation, difficult preparation, high price, poor sensitivity and the like.

Disclosure of Invention

In view of the defects of the prior art, the technical problem to be solved by the invention is to provide a method for detecting the A549 tumor cells mediated by the bifunctional aptamer, which has the characteristics of high sensitivity, quick reaction, simple and convenient operation and the like.

The technical scheme for solving the problems is as follows:

A method for detecting A549 tumor cells mediated by bifunctional aptamer, which comprises the following steps:

(1) The bifunctional aptamer and the inhibitory strand were dissolved and diluted to 2. mu. mol L with TE buffer, respectively^-1and 4. mu. molL^-1Then mixing the two in equal volume proportion, reacting at 95 ℃ for 5min, and slowly cooling to room temperature to obtain an aptamer-inhibition chain compound; wherein the content of the first and second substances,

The nucleic acid sequence of the bifunctional aptamer is as follows:

5'-TGAGGTAGTAGGTTGTGTGGTTGCAGTTGATCCTTTGGATACCCTGG-3'(SEQ ID NO.1)；

The sequence of the inhibition chain is as follows:

5'-TCAACTGCAACCACACAACCT-3'(SEQ ID NO.2)；

(2) Taking cell suspension to be detected, adding 10 mu L of aptamer-inhibition chain compound obtained in the step (1), dissolving to 1mL by using a binding buffer solution, placing in a hybridization instrument, hybridizing at a medium speed of 37 ℃ for 1h, centrifuging, removing supernatant, and re-suspending precipitate by using 15 mu L of sterile PBS buffer solution to obtain cell suspension; wherein the pH of the binding buffer solution is 7.5, and the binding buffer solution is prepared from Tris-HCl, NaCl and MgCl₂and deionized water, wherein the Tris-HCl, NaCl, MgCl₂The final concentrations of (A) are in order: 10 mmoleL of^-1、500mmolL^-1、1mmolL^-1；

(3) Taking the cell suspension obtained in the step (2), and adding 5 mu molL of the cell suspension^-1H1-F2. mu.L, concentration 5. mu. molL^-1Placing H22 mu L and TNaK buffer solution 1 mu L in a PCR instrument, and incubating for 45min at 37 ℃ in an isothermal mode to obtain hairpin catalytic self-assembly reaction solution; wherein the pH value of the TNaK buffer solution is 7.5, and the TNaK buffer solution consists of Tris-HCl, NaCl, KCl and deionized water,

The final concentrations of Tris-HCl, NaCl and KCl are as follows in sequence: 10 mmoleL of^-1、125mmolL^-1、20mmolL^-1；

The sequence of H1-F is as follows:

5'-FAM-AACCACACAACCTACTACCTCAAGAAGAAGGTGTTGAGGTAGTAGGTT-BHQ1-3'(SEQ ID NO.3)；

The sequence of H2 is:

5'-TACCTCAACACCTTCTTCTTGAGGTAGTAGGTTAGAAGAAGGTGTTTAAGTA-3'(SEQ ID NO.4)；

(4) Diluting the hairpin catalytic self-assembly reaction liquid obtained in the step (3) to 60 mu L by DEPC water, and collecting fluorescence intensity with the wavelength of 515nm by a fluorescence spectrophotometer;

(5) Firstly, the A549 tumor cells with the concentration of 10 cells per mL-10 are prepared⁵Repeating the steps (3) and (4) for each mL of a series of standard cell sap, drawing a curve of the corresponding relation between the fluorescence intensity and the cell concentration in a rectangular coordinate system by taking the fluorescence intensity as a vertical coordinate and the cell concentration of the standard cell sap as a horizontal coordinate, and performing linear regression to obtain a standard product curve equation;

(6) And (5) substituting the fluorescence intensity obtained in the step (4) into the standard substance curve equation established in the step (5) to obtain the concentration of the A549 tumor cells in the cell suspension to be detected.

In the above method, the first 22 bases in the nucleic acid sequence of the bifunctional aptamer are the nucleic acid sequence triggering the hairpin structure consisting of H1-F and H2, and the last 25 bases are the nucleic acid sequence specifically recognizing mucin 1(MUC-1) on A549 tumor cells.

The detection principle of the method of the invention is as follows: firstly, designing a double-hairpin structure and a trigger sequence in a hairpin catalysis self-assembly reaction system, efficiently and specifically amplifying signals, integrating the trigger sequence to an aptamer for specifically recognizing mucin 1 on the surface of a target cell A549, designing an inhibition chain to effectively inhibit the bifunctional aptamer from triggering a non-specific hairpin catalysis self-assembly reaction, and ensuring the recognition effectiveness of the aptamer. During detection, firstly, tumor cells are specifically identified through the aptamer, a trigger sequence is exposed, and then a hairpin catalysis self-assembly reaction is started to amplify signals, so that fluorescent signals are collected, and the number of detected cells can be analyzed (see fig. 1).

The invention has the beneficial effects that:

(1) The method can effectively distinguish the tumor cells from the normal cells, can generate signals for different numbers of tumor cells in various clinical specimens, has a result similar to that in a buffer solution, and has a detection limit of 10 cells/mL.

(2) In the method, the fluorescent group rapidly releases signals along with the progress of hairpin catalyzed self-assembly reaction, and the number of tumor cells can be accurately quantified.

(3) The method converts the tumor cell detection into nucleic acid detection, simply and conveniently realizes homogeneous phase detection of rare tumor cells in clinical blood body fluid specimens, reduces complicated separation and elution operations, and is easy to realize the on-site detection of the tumor cells.

(4) The method can well distinguish the tumor cells from normal cells and common interfering cells, can be applied to various clinical specimens, is expected to realize the rapid screening of the rare tumor cells in the fields of tumor liquid biopsy, individualized medical treatment and the like, and has potential application value.

Drawings

FIG. 1 is a schematic diagram of the method of the present invention.

FIG. 2 is a diagram of analysis of the quantitative detection effect of the method of the present invention, in which the diagram A is an electrophoresis diagram of reaction products of different hairpin catalyzed self-assembly systems, and the diagram B is a fluorescence spectrum diagram of the reaction products of different hairpin catalyzed self-assembly systems.

FIG. 3 is a graph of fluorescence spectra of different numbers of target cells.

Fig. 4 is a histogram of a statistical analysis of signal-to-noise ratio.

FIG. 5 is a graph of signal intensity versus reaction time.

FIG. 6 is a histogram of statistical analysis of signal-to-noise ratios at different temperatures.

FIG. 7 is a graph showing the relationship between the fluorescence intensity of the cell concentration in the cell suspension.

fig. 8 is a linear fit of a standard curve.

FIG. 9 histogram of statistical analysis of specific effects.

FIG. 10 is a histogram of statistical analysis of clinical effects according to the method of the present invention.

Detailed Description

Example 1 (bifunctional aptamer-mediated A549 tumor cell detection method)

1. materials and methods

1.1 materials

Non-small cell lung cancer A549 cell line, purchased from cell bank of Chinese academy of sciences.

Hairpin H1-F, having the sequence shown in SEQ ID NO.3, synthesized by Shanghai bioengineering, Inc., dissolved in TE buffer and diluted to 5. mu. mol L^-1。

hairpin H2, sequence shown in SEQ ID NO.4, synthesized by Shanghai bioengineering, Inc., dissolved in TE buffer and diluted to 5. mu. mol L^-1。

Bifunctional aptamers (Apt)_S2.2) Synthesized by Shanghai bioengineering Co., Ltd according to the sequence shown in SEQ ID NO.1, dissolved in TE buffer and diluted to 2. mu. mol L^-1。

Suppression ofChain (Inh), having the sequence shown in SEQ ID NO.2, was synthesized by Shanghai bioengineering, Inc., dissolved in TE buffer and diluted to 4. mu. mol L^-1。

DEPC water was purchased from Shanghai Biotech engineering, Inc.

RPMI 1640 medium, fetal bovine serum, Phosphate Buffered Saline (PBS), pancreatin and penicillin-streptomycin were purchased from sermer flyer.

Chemical reagents NaCl, KCl and NH₄Cl、KHCO₃And EDTANA₂All purchased from Sigma.

TE buffer was purchased from Biosharp company.

TNaK buffer as hybridization buffer: the pH value is 7.5, and the reagent is composed of Tris-HCl, NaCl, KCl and deionized water, wherein the final concentrations of the Tris-HCl, the NaCl and the KCl are as follows in sequence: 10 mmoleL of^-1、125mmolL^-1、20mmolL^-1。

Binding solution of target cells and aptamers: pH 7.5, Tris-HCl, NaCl, MgCl₂And deionized water, wherein the Tris-HCl, NaCl, MgCl₂The final concentrations of (A) are in order: 10 mmoleL of^-1、500mmolL^-1、1mmolL^-1。

Erythrocyte lysate: pH of 7.2 to 7.4, from NH₄Cl、KHCO₃、EDTANa₂and deionized water, wherein NH₄Cl、KHCO₃、EDTANa₂in the order of 1.5mol L^-1、100mmolL^-1、1mmolL^-1。

1.2 detection Instrument

Fluorescence/phosphorescence/spectrophotometer LS55 is a PerkinElmer company, uk.

2. Preparation method

(1) the bifunctional aptamer and the inhibitory strand were dissolved and diluted to 2. mu. mol L with TE buffer^-1And 4. mu. molL^-1Mixing the two solutions in equal volume ratio, reacting at 95 deg.C for 5min, and slowly cooling to room temperature to obtain 1 μmolL^-1the aptamer-inhibitory strand complex of (a);

(2) Taking cell suspension to be detected, adding 10 mu L of aptamer-inhibition chain compound in the step (1), adding a binding buffer solution to dissolve the mixture to 1mL, then placing the reaction mixed solution into a hybridization instrument, hybridizing at a medium speed of 37 ℃ for 1h, placing the mixture into a centrifuge, centrifuging at 1000rpm for 15min, discarding supernatant, and re-suspending the precipitate by using 15 mu L of sterile PBS buffer solution to obtain cell suspension;

(3) Taking 15 mu L of the cell suspension prepared in the step (2), and adding 5 mu molL of the cell suspension^-1H1-F2. mu.L, concentration 5. mu. molL^-1Putting 1 mu L of H22 mu L, TNaK buffer solution into a PCR instrument, and incubating for 45min at 37 ℃ in an isothermal mode to obtain hairpin catalytic self-assembly reaction solution;

(4) Diluting the hairpin catalytic self-assembly reaction liquid prepared in the step (3) to 60 mu L by DEPC water, and collecting the fluorescence intensity with the wavelength of 515nm by using a fluorescence spectrophotometer LS 55.

(5) Culturing non-small cell lung cancer A549 cell line, digesting with pancreatin when the cells are in logarithmic growth phase, centrifuging at 1000rpm for 3min to collect cells, resuspending with sterile PBS buffer solution, and counting to obtain 10, 10²、10³、5×10³、10⁴、5×10⁴、10⁵And (3) repeating the steps (3) and (4) again for each milliliter of standard cell sap, drawing a curve of the corresponding relation between the fluorescence intensity and the cell concentration in a rectangular coordinate system by taking the fluorescence intensity as a vertical coordinate and the cell concentration of the standard cell sap as a horizontal coordinate, and performing linear regression to obtain a standard curve equation. 74.47+7.399log F₁₀C (F is fluorescence intensity, C is the concentration of A549 tumor cells, and the correlation coefficient is 0.9791), and a standard curve corresponding to the equation is shown in FIG. 8;

(6) and (5) substituting the fluorescence intensity obtained in the step (4) into the standard substance curve equation established in the step (5) to obtain the concentration of the A549 tumor cells in the cell suspension to be detected.

example 2 (verification of quantitative determination Effect)

Hairpin catalysis self-assembly reaction analysis and verification

1. Experimental methods

To verify the effectiveness of the A549 tumor cell detection method, we first analyzed the hairpin-catalyzed self-assembly reactionand evaluating. Control groups containing only 500nM H1, only 500nM H2, 500nM H1+500nM H2, and different concentrations of trigger sequence (10 nmolL) were added^-1And 1. mu. molL^-1) The reaction was carried out under the same conditions (37 ℃ C., 60 min). Finally, the product was analyzed by 12% polyacrylamide gel electrophoresis and the fluorescence response of the reaction was observed.

2. Analysis and conclusions

FIG. 2A shows the electrophoresis of the reaction products of different hairpin catalyzed self-assembled systems:

Lane 1 is hairpin structure H1 alone;

lane 2 is the isolated hair scaffolding structure H2;

Lane 3 is a mixture of two hairpin structures H1, H2;

Lane 4 shows two hairpin structures H1, H2 plus 10nmolL^-1A trigger sequence;

lane 5 shows two hairpin structures H1, H2 plus 1. mu. molL^-1A trigger sequence.

When only the hairpin catalyzed self-assembly substrates H1 and H2 were present in the system, the corresponding lanes had only one distinct band (FIG. 2A-lanes 1 and 2), indicating that the secondary structure formed by the two substrates used in the system was good and no other interfering structures were present. The existence of two independent clear bands in the lane corresponding to the positions of H1 and H2 and the absence of other bands in the lane when the hairpin-catalyzed self-assembly substrates H1 and H2 coexist further proves that the hairpin-catalyzed self-assembly substrates used in the method have no obvious non-specific reaction, and further proves that the designed hairpins H1 and H2 are suitable for the reaction. When a trigger sequence is added into the system, a bright band with larger molecular weight is added in the lane; and the brightness of the strip increases with the increase of the concentration of the trigger sequence, and when the concentration of the trigger sequence is 1 mu molL^-1In the time, the bands at the corresponding positions of the two hairpin structures in the lane are not visible to the naked eye (fig. 2A-lanes 4 and 5), which shows that the hairpin catalyzed self-assembly reaction can be smoothly performed, the reaction is efficient and rapid, and the rapid amplification of the signal can be realized.

FIG. 2B shows fluorescence spectra of reaction products of different hairpin-catalyzed self-assembly systems, which sequentially correspond to lanes 1, 3, 4, and 5 in FIG. 2A from bottom to top:

Purple curve a is H1-F alone;

Curve b is a coexisting system of H1-F, H2;

Curve c is H1-F, H2 plus 10nmolL^-1a trigger sequence;

Curve d is H1-F, H2 plus 100nmolL^-1A trigger sequence.

The fluorescence response of this system is consistent with the results in FIG. 2A. Only very weak fluorescence background signals were collected when only H1-F or H1-F, H2 coexisted in the reaction system (FIG. 2B-curves a and B). When a trigger sequence was added to the system, the fluorescence signal increased dramatically and increased with increasing trigger sequence concentration (FIG. 2B-curves c and d). This result is also illustrated: the designed nucleic acid sequence has good secondary structure formation, high-efficiency and quick reaction and high specificity, and can effectively analyze data through fluorescence signal output.

Secondly, verifying the detection effect of tumor cells

1. Experimental methods

Based on the hairpin catalyzed self-assembly reaction with excellent reaction, the feasibility of the A549 tumor cell detection method is further verified. In one aspect, control groups and tumor cells containing different concentrations of A549 were designed (10)³、5×10⁴、10⁵one/mL) of the groups, according to the operations of the steps (1) to (4) of the example 1, the reaction system is placed at 37 ℃ for reaction for 45min, and then the fluorescence signals of different reaction products are detected; on the other hand, we performed real-time fluorescence monitoring of these sets of experiments at 2.5min intervals for 45min total.

2. analysis and conclusions

As shown in fig. 3, fluorescence spectrograms acquired by adding different numbers of target cells a549 in the bifunctional aptamer-mediated a549 tumor cell detection method are from bottom to top:

Curve a is a blank control with no cells added;

Curve b is the addition of 10³one/mL target cell;

curve c is plus5X 10⁴one/mL target cell;

Curve d is addition of 10⁵one/mL target cell.

it is obvious from the acquired fluorescence spectrogram that the fluorescence signal shows a growing trend along with the increase of the number of cells and is obviously distinguished from a blank control; the fluorescence dynamic analysis chart also shows that the signal shows a trend of gradually increasing and stabilizing in a certain time. The result can preliminarily confirm that the bifunctional aptamer-mediated A549 tumor cell detection method can be used for detecting tumor cells.

Example 3 (comparison of Effect)

The double-functional aptamer developed by the invention can specifically recognize target cells and can also specifically trigger hairpin catalytic self-assembly signal amplification reaction, thereby realizing the rapid and sensitive detection of A549 cells. In this regard, we have designed experiments and observed the difference between the detection effects of the bifunctional aptamer and the traditional aptamer on A549 cells. The traditional aptamer is an oligonucleotide sequence which is screened by an exponential enrichment ligand phylogeny technology and can be specifically combined with mucin 1 on the surface of a tumor cell, and the specific sequence is as follows:

5'-GCAGTTGATCCTTTGGATACCCTGG-3'(SEQ ID NO.5)

In the experiment, the bifunctional aptamer and the traditional aptamer are respectively added into a detection system, and the same quantity of A549 cells (0 and 10) are respectively added into two groups⁵One) for detection. The fluorescence signal was measured and the signal-to-noise ratio under the two aptamer conditions was analyzed, as shown in fig. 4, the signal-to-noise ratio of the conventional aptamer group was 2.350, while the signal-to-noise ratio of the bifunctional aptamer group was significantly improved to 5.353. This result confirms that the bifunctional aptamers can indeed trigger a signal amplification reaction on the basis of specific recognition of a549 tumor cells.

Example 4 (optimization of reaction conditions)

the important reaction conditions involved in the present invention mainly include reaction temperature and reaction time. Therefore, we all design a series of reaction conditions to observe the detection effect of the detection method, so as to grope the optimal conditions to achieve the optimal reaction performance.

The temperature can change the structure, reaction rate and the like of the reaction substrate, thereby influencing the sensitivity and specificity of the reaction. Therefore, we first investigated the effect of different reaction temperatures on the reaction results. As can be seen in FIG. 5, blank control (no target cells added) and experimental group (10 added) were set⁵Individual target cells), the reaction is respectively placed in five environments of 4 ℃, 25 ℃, 37 ℃, 42 ℃ and 55 ℃, the fluorescent signal is collected after the reaction is finished, and the signal-to-noise ratios at different reaction temperatures are respectively calculated to be 2.44, 3.44, 4.17, 2.70 and 1.20, so that the optimal reaction temperature of the detection method is selected to be 37 ℃.

Similarly, to examine the effect of different reaction times on the assay, we set blank control (no target cells added) and experimental group (10 added)⁵Individual target cells) and collecting fluorescence signal values of different points at intervals of 15min to 75min, and counting results show that: the signal value of the blank group has no obvious change from 15min to 75min, while the fluorescence signal value of the experimental group gradually rises within 0-45 min and tends to be stable after 45min (FIG. 6). Therefore, the optimum reaction time for the process was selected to be 45 min.

Example 5 (sensitivity analysis)

In order to evaluate the performance of the double-function aptamer mediated A549 tumor cell detection method, the method is used for detecting the quantity of A549 cells with different concentrations, under the optimal reaction condition, in the logarithmic growth phase of the A549 cells, cell suspension is digested and prepared, the cells are counted and subjected to gradient dilution to obtain 10 and 10²、10³、5×10³、10⁴、5×10⁴、10⁵(pieces/ml) of cell suspension, and these different concentrations of cell suspension (3 sets of parallel assays) were analyzed by this assay.

As can be seen from the collected spectrograms, the fluorescence spectrogram gradually rose as the number of cells increased (FIG. 7). The fluorescence signals were statistically analyzed to obtain FIG. 8: the number of A549 cells is 10-10³In the range of each cell/mL, the fluorescence signal is in direct proportion to the number of tumor cells; the mathematical model can be obtained by linear fittingthe linear equation is F ═ 74.47+7.399log₁₀C (F is fluorescence intensity, C is concentration of a549 cells), correlation coefficient is 0.9791; the detection limit was estimated from the signal value corresponding to the blank signal plus 3 times the standard deviation, and calculated to be 10 cells/mL.

Example 6 (specificity analysis)

The differentiation of tumor cells from normal cells and leukocytes is of great importance for the understanding of free tumor cells in tumor tissues and body fluids. In order to explore the detection specificity of the bifunctional aptamer-mediated A549 tumor cell detection method, human bronchial epithelial cell 16HBE cell line and white blood cells are used as control cells for analysis. The results are shown in FIG. 9 for 10⁵And detecting the leucocytes, the 16HBE cells and the A549 cells, wherein the detection method only detects the fluorescence signal value close to that of a blank group for the leucocytes and the 16HBE cells, and detects a remarkably enhanced fluorescence signal for the A549 cells. At 10⁵When equal amount of white blood cells and 16HBE cells are respectively mixed in each A549 cell, the signal detected by the method is close to that of the A549 cell alone. The result shows that the prepared method for detecting the A549 tumor cells mediated by the bifunctional aptamer can effectively distinguish the tumor cells from leukocytes and 16HBE cells, can effectively avoid the interference of the two cells on the detection of the leukocytes and has good specificity.

Example 7 (evaluation of clinical Properties)

In order to evaluate the clinical application value of the bifunctional aptamer mediated A549 tumor cell detection method, tumor cells (200, 10) with different orders of magnitude are used⁴、10⁵Individual cells/mL) into four kinds of body fluid specimens of serum, urine, cerebrospinal fluid and hydrothorax and ascites, detecting the number of tumor cells in the specimens by an established method, and comparing the result with the detection result of the tumor cells with the same order of magnitude in PBS cell suspension. The results show that the detection method detects different signals for different numbers of tumor cells in the four body fluid samples respectively, and the obtained signals are close to the results in the PBS buffer (FIG. 10). The result proves that the bifunctional aptamer-mediated A549 tumor cell detection method can distinguish multiple bodiesThe number of the tumor cells in the liquid sample is close to the detection result of the tumor cells in the PBS according to the analysis result of the tumor cells in the liquid sample, and the method has certain potential in detecting the number of the tumor cells in the clinical sample.

Sequence listing

<110> southern hospital of southern medical university

<120> A549 tumor cell detection method mediated by bifunctional aptamer

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Claims (1)

1. an agent for detecting a549 tumor cells, the agent comprising:

Reagent A: the reagent is formed by mixing TE buffer solution and bifunctional aptamers, and the concentration of the bifunctional aptamers in the TE buffer solution is 2 mu mol/L; wherein the nucleic acid sequence of the bifunctional aptamer is:

5'-TGAGGTAGTAGGTTGTGTGGTTGCAGTTGATCCTTTGGATACCCTGG-3'（SEQ ID NO.1）；

And (3) reagent B: the reagent consists of TE buffer solution and inhibitory chains, and the concentration of the inhibitory chains in the TE buffer solution is 4 mu mol/L; wherein the sequence of the inhibitory strand is:

5'-TCAACTGCAACCACACAACCT-3'（SEQ ID NO.2）；

And (3) reagent C: the reagent consists of TE buffer solution and hairpin H1-F, and the concentration of the hairpin H1-F in the TE buffer solution is 5 mu mol/L; wherein the sequence of hairpin H1-F is:

5'-FAM-AACCACACAACCTACTACCTCAAGAAGAAGGTGTTGAGGTAGTAGGTT -BHQ1-3'（SEQ ID NO.3）；

and (3) reagent D: the reagent consists of TE buffer solution and hairpin H2, and the concentration of the hairpin H2 in the TE buffer solution is 5 mu mol/L; wherein the sequence of hairpin H2 is:

5'-TACCTCAACACCTTCTTCTTGAGGTAGTAGGTTAGAAGAAGGTGTTTAAGTA-3'（SEQ ID NO.4）。