CN109321577B - Aptamer group for detecting exosome, lateral flow aptamer biosensor and preparation method thereof - Google Patents
Aptamer group for detecting exosome, lateral flow aptamer biosensor and preparation method thereof Download PDFInfo
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Abstract
The invention provides an aptamer group for detecting exosomes, a lateral-flow aptamer biosensor and a preparation method thereof, and belongs to the technical field of exosome detection. The aptamer group for detecting the exosome comprises a CD63 aptamer and an EpCam aptamer, the CD63 aptamer can be specifically combined with a CD63 protein of the exosome, the EpCam aptamer can be specifically combined with an EPCAM protein of the exosome, and the two aptamer groups are not easily influenced by environmental factors such as pH, temperature and the like and have good stability. The invention also provides a lateral flow aptamer biosensor, which is characterized in that the CD63 aptamer is sprayed on a bonding pad mainly based on the principle of chromatography test paper strips, the Epcam aptamer is sprayed to form a detection line, and a DNA probe which can be matched and bonded with the aptamer marked by the nanogold is sprayed on a quality control line through base complementary pairing. The flow-measuring aptamer biosensor can detect 500000 exosomes, and provides an innovative tool for rapid cancer screening and diagnosis.
Description
Technical Field
The invention belongs to the technical field of exosome detection, and particularly relates to an aptamer group for detecting exosomes, a lateral-flow aptamer biosensor and a preparation method thereof.
Background
Exosomes (exosomes) are vesicular bodies secreted by a variety of living cells, containing various components, such as proteins and RNA. The ubiquitous nanoscale capsule structure in the organism can participate in the exchange of substances and information among cells, and plays an important role in various physiological and pathological processes. Exosomes have high abundance in body fluids such as peripheral blood, urine, saliva, ascites, amniotic fluid and the like, while exosomes from different tissue sources have differences in composition and function, and the differences are dynamically regulated by extracellular matrix and microenvironment. Tumor-derived or tumor-associated exosomes are important mechanisms for regulating and controlling tumorigenesis and development, and analysis and detection of tumor exosomes can assist early diagnosis, curative effect evaluation and prognosis analysis of tumors. Although exosomes are still in the initial stage as biomarkers, with further research, clinical application of exosomes will have good prospects.
At present, the exosome morphology can be directly observed by applying an electron microscope, and the expression quantity of exosome protein can be detected by applying a Western blot method according to transmembrane molecules of markers such as CD9 and CD63 on the surface of the exosome and by combining CD9 and CD63 monoclonal antibodies with corresponding antigens. The analysis of genetic information substances contained in exosomes by using a PCR or sequencing method is a detection method which is widely applied at present, and other identification technologies such as a dynamic light scattering technology, flow cytometry analysis, nanoparticle tracking analysis, electrochemistry, fluorescence, SPR, SPRI, color analysis and the like are available. The above method requires either expensive large-scale instruments or complicated operation steps, which are disadvantageous for wide-screen. The method for detecting the exosome further comprises enzyme-linked immunoassay and immunoblot analysis. Although immunoassay using antigen-antibody can achieve high detection sensitivity and does not require large-scale instruments, the screening of antibodies specifically binding to the exosome surface marker protein is often complicated in steps, and meanwhile, the antibody protein is susceptible to denaturation by environmental factors such as pH, temperature and the like and is expensive to synthesize.
Disclosure of Invention
In view of the above, the present invention provides an aptamer and an aptamer set for detecting exosome with easy synthesis and good stability, and also provides a lateral flow aptamer biosensor with high detection sensitivity, simplicity, convenience, and low cost without complicated and expensive instruments and devices, and a preparation method thereof.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a CD63 aptamer for detecting exosome, wherein the nucleotide sequence of the CD63 aptamer group is shown as SEQ ID No.1 in a sequence table.
The invention provides an aptamer group for detecting exosomes, which comprises a CD63 aptamer and an Epcam aptamer, wherein the nucleotide sequence of the Epcam aptamer is shown as SEQ ID No.2 in a sequence table.
The invention provides a lateral flow aptamer biosensor, which comprises a bottom plate, and a binding pad, a detection pad and an absorption pad which are adhered on the bottom plate and sequentially connected from bottom to top, wherein the detection pad comprises a detection line and a quality control line, the binding pad is sprayed with a CD63 aptamer conjugate marked by nanogold, the detection line is sprayed with an EpCam aptamer conjugate marked by streptavidin-biotin, the quality control line is sprayed with a DNA probe marked by streptavidin-biotin, and the nucleotide sequence of the DNA probe is complementary with the 3' end sequence of the nucleotide sequence of the CD63 aptamer;
the CD63 aptamer in the nanogold-labeled CD63 aptamer conjugate is the CD63 aptamer of the detection exosome;
preferably, the nucleotide sequence of the DNA probe in the streptavidin-biotin labeled DNA probe is shown as SEQ ID No.2 in the sequence table.
Preferably, the nanogold labeled CD63 aptamer conjugate is sprayed at a rate of 3 μ L/cm of the conjugate pad.
Preferably, the spraying times of the streptavidin-biotin labeled Epcam aptamer conjugate are 3 times; the spray volume of the streptavidin-biotin labeled Epcam aptamer conjugate was 3. mu.l/cm detection pad.
The invention provides a preparation method of a lateral flow type aptamer biosensor, which comprises the following steps:
(1) sequentially adding a dATP solution, an SDS solution and a NaCl solution into a nanogold solution under a shaking condition for reaction, mixing the obtained reaction solution with a CD63 aptamer, reacting for 3 hours at 60 ℃, carrying out solid-liquid separation, dissolving the obtained precipitate into a nanoparticle storage solution to obtain a nanogold-labeled CD63 aptamer conjugate solution, and spraying the nanogold-labeled CD63 aptamer conjugate solution on a binding pad to obtain the binding pad sprayed with the nanogold-labeled CD63 aptamer conjugate;
(2) mixing and culturing an Epcam aptamer marked by biotin and streptavidin for 1h, placing the mixture into an ultrafiltration centrifugal tube for ultrafiltration and centrifugation, washing the ultrafiltration centrifugal tube for 2 times, and collecting supernatant as an Epcam aptamer solution marked by streptavidin-biotin;
(3) replacing the Epcam aptamer in the step (2) with a DNA probe, and repeating the operation in the step (3) to obtain a DNA probe solution marked by streptavidin-biotin;
(4) scribing the streptavidin-biotin labeled Epcam aptamer solution in the step (2) and the streptavidin-biotin labeled DNA probe solution in the step (3) on a detection pad in sequence to obtain the detection pad sprayed with a detection line and a quality control line;
(5) cutting the bonding pad sprayed with the CD63 aptamer conjugate marked with the nanogold in the step (1), the detection pad sprayed with the detection line and the quality control line in the step (4) and absorbent paper respectively, then sequentially assembling the pads on a bottom plate from bottom to top, and cutting the pads into strips to obtain the lateral flow aptamer biosensor;
there is no chronological restriction between steps (1) and (2) to (4), and there is no chronological restriction between step (2) and step (3).
Preferably, the volume ratio of the nanogold solution, the dATP solution, the SDS solution and the NaCl solution in the step (1) is 200:2:3: 10; the concentration of the nano gold solution is 15-20 nmol/L, the concentration of the dATP solution is 1mmol/L, the mass concentration of the SDS solution is 1%, and the concentration of the NaCl solution is 0.2 mol/L.
Preferably, the storage solution in the step (1) comprises the following components in percentage by weight: 20mmol/LNa3PO4·12H2O, BSA at 5% mass concentration, Tween20 at 0.25% mass concentration and sucrose at 10% mass concentration.
Preferably, the solid-liquid separation mode in the step (2) comprises centrifugation; the rotating speed of the centrifugation is 6000rpm, the time of the centrifugation is 20min, and the temperature of the centrifugation is 4 ℃.
The invention provides an aptamer group for detecting exosomes, which comprises a CD63 aptamer and an Epcam aptamer, wherein the nucleotide sequence of the CD63 aptamer is shown as SEQ ID No.1 in a sequence table, and the Epcam aptamer is shown as SEQ ID No.2 in the sequence table. The CD63 aptamer can be specifically combined with CD63 protein expressed on the surface of an exosome, the Epcam aptamer can be specifically combined with EPCAM protein expressed on the surface of the exosome, the aptamer group is not easily influenced by environmental factors such as pH and temperature, the stability is good, the aptamer is small in size and easy to synthesize, and the preparation cost is greatly reduced.
Meanwhile, the aptamer group for detecting the exosome provided by the invention is specifically combined with the exosome dual protein, and the visible detection concentration of the exosome detected by the aptamer group provided by the invention is 5 multiplied by 105Has high specificity. Meanwhile, the aptamer group provided by the invention fills the blank of adopting the aptamer to detect the exosome in the prior art, and provides important information for the technology of detecting the exosome by using the aptamer.
The invention provides a lateral flow aptamer biosensor, which is characterized in that a nanogold-labeled aptamer capable of specifically binding with an exosome surface protein is sprayed on a binding pad based on the principle of a chromatography test strip, another aptamer in the scheme is sprayed on a detection line of the detection pad, and a DNA probe which is sprayed on a quality control line and can be bound with the nanogold-labeled aptamer through base complementary pairing. When the sample contains the target exosome, the nanogold-labeled aptamer and the exosome are combined to form a compound which moves upwards in a chromatography mode along with water flow to reach the detection line and is combined with another aptamer on the detection line, meanwhile, the nanogold-labeled aptamer which is not combined with the exosome is combined with the DNA probe on the quality control line, and the detection line and the quality control line turn red. When no exosome exists in the sample or the content of exosome is extremely low, only the quality control line shows red, and the detection line does not show color. The lateral flow aptamer biosensor provided by the inventionCan realize qualitative and quantitative detection. The implementation shows that 500000 exosomes can be detected by the flow-measuring aptamer biosensor provided by the invention. Simultaneously, the concentration of the sample is 0-1 multiplied by 106Within the concentration range, the signal value and the exosome concentration are in a linear relation, and the content of the concentration range indicates that the detection result of the flow-measuring type aptamer biosensor is accurate.
Meanwhile, the lateral flow type aptamer biosensor provided by the invention is simple and convenient to operate, does not depend on large and expensive instruments, and provides an innovative tool for rapid screening and diagnosis of cancers.
The invention also provides a preparation method of the lateral flow aptamer biosensor, and the method has the advantages of good repeatability, simple and quick operation, cheap and easily-obtained raw materials and greatly reduced production cost of the lateral flow aptamer biosensor.
Drawings
FIG. 1 is an assembly structure of a lateral flow aptamer biosensor provided by the present invention;
FIG. 2 is a schematic diagram illustrating the detection principle of the lateral flow aptamer biosensor provided by the present invention;
fig. 3 is a magnetic nanoparticle-aptamer-exosome under Scanning Electron Microscopy (SEM): exosomes captured by aptamers are inside the circle;
FIG. 4 is a standard curve for detection of exosomes.
Detailed Description
The invention provides a CD63 aptamer for detecting exosome, wherein the nucleotide sequence of the CD63 aptamer group is shown as SEQ ID No.1 in a sequence table. The CD63 aptamer can be specifically combined with CD63 protein expressed on the surface of an exosome, and based on the CD63 aptamer, the exosome can be accurately captured.
The invention provides an aptamer group for detecting exosomes, which comprises a CD63 aptamer and an Epcam aptamer in the scheme, wherein the nucleotide sequence of the Epcam aptamer is shown as SEQ ID No.2 in a sequence table.
In the present invention, the sources of the CD63 aptamer and the EpCam aptamer are not particularly limited, and a synthesis method well known in the art may be employed depending on the nucleotide sequence of the aptamer.
Based on the characteristic that the aptamer group provided by the invention can detect exosomes, the invention provides a lateral flow aptamer biosensor, which comprises a bottom plate, and a combination pad, a detection pad and an absorption pad which are adhered to the bottom plate and sequentially connected from bottom to top, wherein the detection pad comprises a detection line and a quality control line, the combination pad is sprayed with a CD63 aptamer conjugate labeled with nanogold, the detection line is sprayed with an EpCam aptamer conjugate labeled with streptavidin-biotin, the quality control line is sprayed with a DNA probe labeled with streptavidin-biotin, and the nucleotide sequence of the DNA probe is complementary with the 3' end sequence of the nucleotide sequence of the CD63 aptamer.
The lateral flow type aptamer biosensor provided by the invention comprises a bottom plate. The material of the substrate is not particularly limited, and the substrate for preparing the immune test strip is well known in the field. In the embodiment of the invention, the bottom plate is made of a PVC plate. The PVC sheet of the present invention is not particularly limited in its source, and may be one known in the art. The bottom plate has the functions of bearing the combination pad, the detection pad and the absorption pad, so that the combination pad, the detection pad and the absorption pad form a complete chromatographic test strip.
The lateral flow aptamer biosensor provided by the invention comprises a bonding pad. The conjugate pad was sprayed with a nanogold labeled CD63 aptamer conjugate. The spraying frequency of the streptavidin-biotin labeled DNA probe is preferably 3 times. The bonding pad is made of polyester film or glass fiber. The source of the polyester film or the glass fiber is not particularly limited in the present invention, and polyester films or glass fibers known in the art may be used.
The lateral flow aptamer biosensor provided by the invention comprises a detection pad. The detection pad comprises a detection line and a quality control line. The detection line is sprayed with CD63 aptamer conjugate labeled by nanogold. The amount of the nanogold labeled CD63 aptamer conjugate sprayed is preferably 3 μ L/20cm conjugate pad.
In the invention, a DNA probe marked by streptavidin-biotin is sprayed on the quality control line. The sequence of the DNA probe is not particularly limited, and the nucleotide sequence of the DNA probe is complementary to the 3' -terminal sequence of the nucleotide sequence of the CD63 aptamer. The nucleotide sequence of the DNA probe in the streptavidin-biotin labeled DNA probe is preferably shown as SEQ ID No.2 in the sequence table. The streptavidin-biotin labeled EpCam aptamer conjugate is preferably sprayed 3 times. The control line is used for checking whether the test is effective or not, and when the macroscopic color appears on the control line (the marker is nanogold, and is red), the test strip is indicated to be effective, and then rapid detection can be carried out according to the macroscopic color shade change on the detection line. If the control line is not colored, the test is invalid, and the result on the test line is meaningless.
In the present invention, the detection pad is preferably made of a nitrocellulose membrane (NC membrane). The bonding pad is made of polyester film or glass fiber. The source of the nitrocellulose membrane is not particularly limited in the present invention, and a nitrocellulose membrane known in the art may be used. The NC membrane realizes the stable coating of streptavidin-biotin labeled Epcam aptamer conjugate and streptavidin-biotin labeled DNA probe through the interaction with streptavidin.
The lateral flow aptamer biosensor provided by the invention comprises an absorption pad. The material of the absorption pad is preferably absorbent paper. The absorption pad is used for controlling the redundant absorption amount of the liquid to be detected flowing into the test strip and washing the free labeled compound on the NC membrane, so that background interference can be reduced and the detection sensitivity can be improved.
The invention provides a preparation method of a lateral flow type aptamer biosensor, which comprises the following steps:
(1) sequentially adding a dATP solution, an SDS solution and a NaCl solution into a nanogold solution under a shaking condition for reaction, mixing the obtained reaction solution with a CD63 aptamer, reacting for 3 hours at 60 ℃, carrying out solid-liquid separation, dissolving the obtained precipitate into a nanoparticle storage solution to obtain a nanogold-labeled CD63 aptamer conjugate solution, and spraying the nanogold-labeled CD63 aptamer conjugate solution on a binding pad to obtain the binding pad sprayed with the nanogold-labeled CD63 aptamer conjugate;
(2) mixing and culturing an Epcam aptamer marked by biotin and streptavidin for 1h, placing the mixture into an ultrafiltration centrifugal tube for ultrafiltration and centrifugation, washing the ultrafiltration centrifugal tube for 2 times, and collecting supernatant as an Epcam aptamer solution marked by streptavidin-biotin;
(3) replacing the Epcam aptamer in the step (2) with a DNA probe, and repeating the operation in the step (3) to obtain a DNA probe solution marked by streptavidin-biotin;
(4) scribing the streptavidin-biotin labeled Epcam aptamer solution in the step (2) and the streptavidin-biotin labeled DNA probe solution in the step (3) on a detection pad in sequence to obtain the detection pad sprayed with a detection line and a quality control line;
(5) cutting the bonding pad sprayed with the CD63 aptamer conjugate marked with the nanogold in the step (1), the detection pad sprayed with the detection line and the quality control line in the step (4) and absorbent paper, sequentially assembling the pads on a bottom plate from bottom to top, and cutting the pads into strips to obtain the lateral flow aptamer biosensor;
there is no chronological restriction between steps (1) and (2) to (4), and there is no chronological restriction between step (2) and step (3).
According to the invention, dATP solution, SDS solution and NaCl solution are sequentially added into nanogold solution under a shaking condition for reaction, the obtained reaction solution is mixed with CD63 aptamer, the reaction is carried out for 3h at 60 ℃, solid-liquid separation is carried out, the obtained precipitate is dissolved in a nanoparticle storage solution, the nanogold labeled CD63 aptamer conjugate solution is obtained, and the nanogold labeled CD63 aptamer conjugate solution is sprayed on a binding pad, so that the binding pad sprayed with the nanogold labeled CD63 aptamer conjugate is obtained; the CD63 aptamer is a CD63 aptamer with the nucleotide sequence shown in SEQ ID No.1 in the sequence table in the aptamer group of the scheme.
In the present invention, the volume ratio of the nanogold solution, the dATP solution, the SDS solution, and the NaCl solution is preferably 200:2:3: 10. The concentration of the nano gold solution is preferably 15-20 nmol/L, and more preferably 18 nmol/L. The concentration of the dATP solution is preferably 1mmol/L, the mass concentration of the SDS solution is preferably 1%, and the concentration of the NaCl solution is preferably 0.2 mol/L. The preferred shaking time for adding the dATP solution is 20 min. The shaking time for adding the SDS solution is preferably 10 min. The shaking time for adding the NaCl solution is preferably 10 min. The adding speed of the NaCl solution is controlled to be 2 mu l every 2-3 min.
In the present invention, the volume ratio of the nanogold solution to the CD63 aptamer solution is preferably 100: 1. The CD63 aptamer was at a concentration of 1 OD. In the present invention, the rotation speed of the centrifugation is preferably 12,000 rpm. The time for the centrifugation is preferably 10 min. The type of the centrifuge is not particularly limited, and a centrifuge known to those skilled in the art may be used.
In the present invention, the resulting precipitate is preferably washed and centrifuged before being dissolved in the nanoparticle storage solution. The washing solution was 0.01mol/LPBS solution. The number of washing and centrifugation is preferably 2-3. After washing and centrifugation, the present invention dissolves the centrifuged precipitate into the nanoparticle stock solution.
In the present invention, the storage liquid preferably comprises the following components in percentage by weight: 20mmol/LNa3PO4·12H2O, BSA at 5% mass concentration, Tween20 at 0.25% mass concentration and sucrose at 10% mass concentration. The nanoparticle stock solution with the nanogold-CD 63 aptamer conjugate dissolved therein is preferably stored at 4 ℃.
In the present invention, the nano gold labeled CD63 aptamer conjugate solution spraying method is preferably performed by using a gold spraying instrument. The spraying speed of the nanogold-labeled CD63 aptamer conjugate solution is preferably 50 mm/s. The amount of the nanogold labeled CD63 aptamer conjugate solution sprayed was 3. mu.L/cm of the conjugate pad. After spraying, the present invention preferably dries the conjugate pad sprayed with the nanogold labeled CD63 aptamer conjugate. The temperature of the drying is preferably 37 ℃, and the time of the drying is preferably 30 min.
And (2) performing mixed culture on the Epcam aptamer marked by the biotin and streptavidin, performing solid-liquid separation, collecting supernatant, washing and performing solid-liquid separation, and collecting the supernatant as a streptavidin-biotin marked Epcam aptamer solution.
The source of the biotin-labeled Epcam aptamer was synthesized by the manufacturer. The volume ratio of the biotin-labeled Epcam aptamer to the streptavidin solution is 1: 4. The concentration of the biotin-labeled EpCam aptamer solution is preferably 50 nmol/L. The concentration of the streptavidin solution is preferably 2.5 mg/ml. The mixed culture is preferably performed in a PBS buffer. The concentration of the PBS buffer is preferably 0.01 mol/L. The time of the mixed culture is preferably 55-70 min, and more preferably 60 min.
In the present invention, the solid-liquid separation is preferably centrifugation. The tube for centrifugation is preferably an ultrafiltration centrifuge tube. The dialysis tubing preferably has a molecular weight cut-off of 30000. In the embodiment of the invention, the ultrafiltration centrifugal tube is an ultrafiltration dialysis tube produced by Millipore corporation. The rotation speed of the centrifugation is preferably 6000 rpm. The time for the centrifugation is preferably 20 min. The temperature of the centrifugation is preferably 4 ℃. The purpose of the centrifugation is to remove unbound aptamer probes. And (3) centrifuging to obtain supernatant, namely streptavidin-biotin Epcam aptamer, adding PBS buffer solution for washing, repeating the steps twice, and finally collecting centrifuged solution to be constant volume of 600 mu l.
According to the invention, the DNA probe marked by biotin is used for replacing the Epcam aptamer marked by biotin, and the operation of the step (3) is repeated to obtain a DNA probe solution marked by streptavidin-biotin. The biotin-labeled DNA probe was synthesized by Biotech Ltd.
And obtaining a streptavidin-biotin labeled Epcam aptamer solution and a streptavidin-biotin labeled DNA probe solution.
The method of spraying is not particularly limited in the present invention, and a spraying method known to those skilled in the art may be used. The spraying speed is preferably 50 mm/s. The number of spraying is preferably independently 3. The spraying volume of the streptavidin-biotin labeled Epcam aptamer conjugate or streptavidin-biotin labeled DNA probe solution is 3 mul/cm of the detection pad.
The invention cuts the binding pad sprayed with the CD63 aptamer conjugate with the nanogold mark, the detection pad sprayed with the detection line and the quality control line and the water absorption pad, assembles the combination pad on a bottom plate from bottom to top, and cuts the combination pad into strips to obtain the lateral flow aptamer biosensor.
In the present invention, the overlapping length of the conjugate pad or absorbent pad and the detection pad at the time of assembly is preferably 2 mm. During assembly, the detection pad is firstly pasted, and then the combination pad or the water absorption pad is pasted so as to ensure that the solution to be detected can smoothly move on the test strip.
In the present invention, the slitting is preferably performed using a slitter. The width of the cutting strip is preferably 2-4 mm.
The present invention preferably provides a method for detecting exosomes based on the lateral flow aptamer biosensor prepared as described above, preferably comprising the steps of:
slowly dripping 100 mu l of sample liquid containing exosomes on a bonding pad of a lateral flow aptamer biosensor, adding 20 mu l of buffer solution for washing after 5min, observing the color shade change of a detection line according to different exosome concentrations in the sample by naked eyes, finally placing the test strip in a strip reading machine to read signals, and calculating the content of exosomes in the sample according to a standard curve.
In the present invention, the buffer is a Tris-HCl solution containing BSA at a mass concentration of 1%. The standard curve is a linear relationship between the signal value and the exosome concentration, and the standard curve can be drawn by a conventional method well known in the art. The standard equation of the standard curve is that y is 0.0007x +6.3364, R20.981, wherein y represents signal intensity and x represents the concentration of exosomes.
The aptamer set for detecting exosomes, the lateral flow aptamer biosensor and the preparation method thereof provided by the invention are described in detail below with reference to the examples, but they should not be construed as limiting the scope of the invention.
Example 1
Mixing 10 μ L of streptavidin labeled magnetic nanoparticles (Shanghai Producer No. NO. D110557) and 10 μ L and 100 μmol/L of biotin modified CD63 aptamer (Shanghai Producer) in 0.01mol/LPBS buffer solution for 1h, washing twice with 0.01mol/LPBS buffer solution after magnet separation, adding 20 μ L of plasma (containing exosomes in the plasma), culturing for 2h, and separating with magnet. The magnetic nanoparticle-aptamer-exosome conjugate was observed under SEM scanning electron microscope (carl zeiss EVO-18) (fig. 3), and the circled portion was found to be exosome captured by the aptamer. This demonstrates that the CD63 aptamers provided herein bind specifically to exosomes.
Example 2
1. Preparation of nanogold-CD 63 aptamer conjugate
Taking 1mL of ten-fold concentrated nanogold (15-20 nm) solution, adding 10 microliters of 1mM dATP, shaking for 20min at room temperature by using a shaker, then adding 15 mul of SDS with the mass concentration of 1% for shaking culture for ten minutes, adding 50 mul of 0.2mol/L NaCl (the speed is controlled to be 2 mul added every 2-3 min), then adding 10 mul of 1OD CD63 aptamer probe, reacting for 3h at 60 ℃, centrifuging by using a centrifuge (the rotating speed is 12,000rpm and 10min), removing supernatant, washing for 3 times by using PBS buffer solution (pH7.2-7.4), and finally dissolving the precipitate in 1mL of nanoparticle storage solution (20 mmol/LNa/20 mmol3PO4·12H2O, BSA with the mass concentration of 5%, Tween20 with the volume concentration of 0.25% and sucralose with the mass concentration of 10%), and storing the conjugate solution in a refrigerator at 4 ℃ for later use.
2. Binding of Biotin-labeled Epcam aptamer to streptavidin
Labeling Epcam aptamer with 50nmol/L biotin
After incubation of (5 '-Bio-CACTACAGAGGTTGCGTCTGTCCCACGTTGTCATGGGGGGTTGGCCTG-3' Shanghai Biotech) with 200. mu.l of 2.5mg/ml streptavidin (purchased from Shanghai Biotech) for 1h in 0.01mol/LPBS, the mixture was transferred to a dialysis tube (molecular weight cut-off 30000) and centrifuged (6000rpm, 20min, 4 ℃) in a refrigerated centrifuge to remove unbound aptamer probes. Adding 0.01mol/LPBS buffer solution, repeating the steps twice, and finally collecting the centrifuged solution to make the volume of the solution reach 600 mu l. And finally, spraying the mixture solution combined by the Epcam aptamer and the streptavidin marked by the biotin three times on the detection line of the test strip.
Streptavidin-biotin labeled DNA probes were prepared as described above. And spraying the prepared streptavidin-biotin labeled DNA probe on a detection line of the test strip for three times.
3. Assembly of aptamer-labeled nanogold test strip
And spraying the mixture of the nano gold and the CD63 aptamer on the bonding pad by using a gold spraying instrument, and spraying the Epcam aptamer marked by streptavidin and biotin on a detection line of the NC membrane by using the gold spraying instrument. And finally, assembling all parts of the test strip on the bottom plate according to a certain sequence, wherein each part is overlapped by 2mm so as to ensure that the solution to be tested can smoothly move on the test strip. And (4) cutting the assembled bottom plate into test strips with proper width by using a strip cutting machine, and storing the test strips in a refrigerator at 4 ℃ for later use.
Example 3
Drawing 0-1 × 10 according to the detection method of exosomes in example 26Standard curves in the concentration range, the study signal values and exosome concentrations are in a linear relationship. The results are shown in FIG. 4. The detection sensitivity is 5X 105。
The exosome detection method of example 2 was performed on different samples, including tumor patient samples, healthy person samples and blank controls. The test paper for detecting the tumor patient sample generates two red lines through visual observation, and the test paper for detecting the healthy patient sample and the test paper for detecting the blank control sample generate one red line on the quality control line.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
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Claims (7)
1. A lateral flow aptamer biosensor comprises a bottom plate, and a binding pad, a detection pad and an absorption pad which are adhered to the bottom plate and sequentially connected from bottom to top, wherein the detection pad comprises a detection line and a quality control line, the lateral flow aptamer biosensor is characterized in that the binding pad is sprayed with a CD63 aptamer conjugate marked by nanogold, the detection line is sprayed with an EpCam aptamer conjugate marked by streptavidin-biotin, the quality control line is sprayed with a DNA probe marked by streptavidin-biotin, and the nucleotide sequence of the DNA probe is complementary with the 3' end sequence of the nucleotide sequence of the CD63 aptamer;
the nucleotide sequence of the CD63 aptamer in the nanogold-labeled CD63 aptamer conjugate is shown as SEQ ID No.1 in a sequence table;
the nucleotide sequence of the Epcam aptamer in the streptavidin-biotin labeled Epcam aptamer conjugate is shown as SEQ ID No.2 in a sequence table.
2. The lateral flow aptamer biosensor as claimed in claim 1, wherein the nucleotide sequence of the DNA probe in the streptavidin-biotin labeled DNA probe is shown as SEQ ID No.3 in the sequence table.
3. The lateral flow aptamer biosensor of claim 1 or 2, wherein the nanogold labeled CD63 is sprayed in a volume of 3 μ Ι/cm of conjugate pad.
4. The lateral flow aptamer biosensor of claim 1, wherein the streptavidin-biotin labeled Epcam aptamer conjugate is sprayed 3 times in a volume of 3 μ l/cm of the detection pad.
5. The method for preparing the lateral-flow aptamer biosensor as claimed in any one of claims 1 to 4, comprising the steps of:
(1) sequentially adding dATP solution, SDS solution and NaCl solution into the nano-gold solution under the condition of oscillation for reaction,
mixing the obtained reaction solution with CD63 aptamer, reacting for 3h at 60 ℃, performing solid-liquid separation, dissolving the obtained precipitate in a nanoparticle storage solution to obtain a nano-gold labeled CD63 aptamer conjugate solution, and spraying the nano-gold labeled CD63 aptamer conjugate solution on a bonding pad to obtain the bonding pad sprayed with the nano-gold labeled CD63 aptamer conjugate;
the nano particle storage solution comprises the following components in percentage by weight: 20mmol/L Na3PO4·12H2O, BSA with the mass concentration of 5%, Tween20 with the mass concentration of 0.25% and sucrose with the mass concentration of 10%;
(2) mixing and culturing an Epcam aptamer marked by biotin and streptavidin for 1h, placing the mixture into an ultrafiltration centrifugal tube for ultrafiltration and centrifugation, washing the ultrafiltration centrifugal tube for 2 times, and collecting supernatant as an Epcam aptamer solution marked by streptavidin-biotin;
(3) replacing the Epcam aptamer in the step (2) with a DNA probe, and repeating the operation in the step (3) to obtain a DNA probe solution marked by streptavidin-biotin;
(4) scribing the streptavidin-biotin labeled Epcam aptamer solution in the step (2) and the streptavidin-biotin labeled DNA probe solution in the step (3) on a detection pad in sequence to obtain the detection pad sprayed with a detection line and a quality control line;
(5) cutting the bonding pad sprayed with the CD63 aptamer conjugate marked with the nanogold in the step (1), the detection pad sprayed with the detection line and the quality control line in the step (4) and absorbent paper respectively, then sequentially assembling the pads on a bottom plate from bottom to top, and cutting the pads into strips to obtain the lateral flow aptamer biosensor;
there is no chronological restriction between steps (1) and (2) to (4), and there is no chronological restriction between step (2) and step (3).
6. The preparation method according to claim 5, wherein the volume ratio of the nanogold solution, the dATP solution, the SDS solution and the NaCl solution in the step (1) is 200:2:3: 10; the concentration of the nano gold solution is 15-20 nmol/L, the concentration of the dATP solution is 1mmol/L, the mass concentration of the SDS solution is 1%, and the concentration of the NaCl solution is 0.2 mol/L.
7. The production method according to claim 5 or 6, wherein the means for solid-liquid separation in the step (2) comprises centrifugation; the rotating speed of the centrifugation is 6000rpm, the time of the centrifugation is 20min, and the temperature of the centrifugation is 4 ℃.
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| CN111157504B (en) * | 2020-01-14 | 2022-06-14 | 东南大学 | Exosome multivariate detection method based on photonic crystal |
| CN111458506B (en) * | 2020-03-08 | 2023-01-06 | 复旦大学 | Colorectal cancer exosome detection method and system based on TdT signal amplification |
| CN111398587B (en) * | 2020-04-02 | 2023-02-28 | 安徽科技学院 | Colloidal gold lateral chromatography test strip for detecting cervical cancer and preparation method thereof |
| CN111521785A (en) * | 2020-04-28 | 2020-08-11 | 中国海洋大学 | Kit for quickly detecting cancer cell exosomes and preparation method and application thereof |
| CN111965366B (en) * | 2020-07-09 | 2022-11-29 | 何凤屏 | Method and kit for rapidly detecting exosome troponin by saliva |
| CN113567668A (en) * | 2021-08-02 | 2021-10-29 | 瑞太生物科技(沈阳)有限公司 | Preparation and application of fluorescent immunochromatographic test strip for exosome quantification |
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