CN111289747B - Tumor marker detection kit and preparation method and application thereof - Google Patents

Tumor marker detection kit and preparation method and application thereof Download PDF

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CN111289747B
CN111289747B CN201811492628.2A CN201811492628A CN111289747B CN 111289747 B CN111289747 B CN 111289747B CN 201811492628 A CN201811492628 A CN 201811492628A CN 111289747 B CN111289747 B CN 111289747B
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dna
magnetic beads
cea
anchor
tumor marker
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CN111289747A (en
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孔浩
凡苏
董伟
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Nanjing University of Science and Technology
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57473—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving carcinoembryonic antigen, i.e. CEA
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
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    • G01N33/531—Production of immunochemical test materials
    • G01N33/532—Production of labelled immunochemicals
    • G01N33/533—Production of labelled immunochemicals with fluorescent label
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    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
    • G01N33/54326—Magnetic particles
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label
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    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
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Abstract

The invention discloses a tumor marker detection kit and a preparation method and application thereof. According to the method, the specific combination principle of the Aptamer chain of the tumor marker and the complementary strand thereof is utilized, the anchor-DNA is connected to the surface of the magnetic bead modified with streptavidin, and the other ssDNA with the end connected with a fluorescent group is complementarily coupled to the anchor-DNA through the base complementation principle to form a stable double-chain structure, so that the probe capable of detecting the corresponding tumor marker is obtained. The kit prepared by the invention can be used singly, can be used for detecting single tumor markers, can be used after three probes are mixed in equal quantity, is used for simultaneously detecting three tumor markers, and has the advantages of greatly shortening the detection time, improving the detection efficiency and saving the detection cost.

Description

Tumor marker detection kit and preparation method and application thereof
Technical Field
The invention relates to a tumor marker detection kit and a preparation method and application thereof, and belongs to the technical field of medical detection.
Background
In recent years, with the progress of industrialization and the increasing population, environmental pollution has become more serious, and the incidence of tumors and other diseases has been rising and has been in a trend of younger, particularly the incidence of malignant tumors and mortality have been rising on a large scale. It has been investigated that more than about 80% -90% of the treatments for patients with early cancer are almost curable clinically, so that early diagnosis and early treatment of tumors plays a critical role for survival of the patients. The detection of the tumor markers has great practical value in aspects of tumor screening, diagnosis of tumor recurrence and metastasis, judgment of curative effect, prognosis and the like.
Tumor markers refer to substances which are produced or increased in content by themselves, either by secretory synthesis through intracellular gene expression, or by the body in response to abnormal expression of a tumor, during the production or proliferation of tumor cells. At present, detection of tumor markers clinically is mainly based on immunohistochemical techniques and polymerase chain reaction techniques. The method has the problems of high cost, low detection sensitivity, long time consumption, complex experimental operation and the like. In particular, although the method has high specificity, a monoclonal antibody is needed for detecting a marker, and the immunoassay is complex, time-consuming, labor-consuming and expensive, which is not beneficial to the rapid screening of a large number of tumor markers. The analysis and test means based on the sensor array has the advantages of simultaneously acquiring multidimensional information, rapidly analyzing and identifying complex samples, being simple in instrument and device, convenient and fast to operate and the like, and provides a simpler, efficient and rapid new method for early diagnosis and analysis of tumors.
The domestic Qiu et al successfully achieved visual detection of CEA using godet strands of carcinoembryonic antigen (CEA) and their complementary strands using GOD-CdTe/CdSe QDs, and the detection sensitivity of the probes was very high (anal. Chem.2017, 89, 5152-5160). Qian et al prepared a nanoprobe with high sensitivity to telomerase using a segment of single-stranded DNA as the capture unit of the sensor and nanomaterial mesoporous silica microspheres with good stability and biocompatibility as the carrier (j.am. Chem. Soc.2013, 135, 36, 13282-13285). The detection method of the tumor markers can become an emerging means for rapidly detecting the tumor markers. However, the detection method has the defects of single detection target, complex probe preparation, long later detection time and the like. The invention aims to provide a novel method for simultaneously detecting a plurality of tumor markers, which is rapid, simple and convenient and is not labeled by antibodies.
Disclosure of Invention
Aiming at the problems of the detection of the common tumor markers at present, the invention provides a tumor marker detection kit which utilizes the specific binding principle of the tumor markers and the corresponding Aptamer chains and a simple and rapid magnetic separation mode to realize the simultaneous detection of any one or more of CEA, PSA and AFP.
The technical scheme of the invention is as follows:
a tumor marker detection kit comprising at least one of streptavidin magnetic beads of anchored DNA (Anchor-CEA) of Biotin (Biotin) modified carcinoembryonic antigen (CEA), streptavidin magnetic beads of anchored DNA (Anchor-PSA) of Biotin modified Prostate Specific Antigen (PSA) and streptavidin magnetic beads of anchored DNA (Anchor-AFP) of Biotin modified Alpha Fetoprotein (AFP) attached to the surface, and fluorescent dye modified DNA (FL-DNA) complementary to the anchored DNA; the anchoring DNA of the CEA is 5'-biotin-TTTTTTATACCAGCTTATTCAATT-3', and the complementary strand is 5'-FAM-AATTGAATAA-3'; the anchoring DNA of the PSA is 5'-biotin-TTTTTTATTAAAGCTCGCCATCAAATAGCTTT-3', and the complementary strand is 5'-ROX-AAAGCTATTTGATGGC-3'; the anchor DNA of AFP is 5'-biotin-TTTTTTTCAGGTGCAGTTCTCGACTCGGTCTTGATGTGGGT-3' and the complementary strand is 5'-Cy5.5-ACCCACATCAAGACCGAGTC-3'.
Preferably, in the detection kit, the concentration of the streptavidin magnetic beads is 5-10 mg/mL.
The invention also provides a preparation method of the tumor marker detection kit, which comprises the following specific steps:
step 1, uniformly dispersing the washed streptavidin magnetic beads in a connection Buffer solution (Binding Buffer), adding anchoring DNA for reaction, after the reaction is finished, magnetically separating, washing off the anchoring DNA which is not connected to the surface of the magnetic beads, and then re-dispersing the anchoring DNA into a phosphate Buffer solution (PB Buffer solution) to obtain streptavidin magnetic beads (anchor-magnetic beads) with the surface connected with the anchoring DNA;
and 2, adding fluorescent dye modified DNA (FL-DNA) complementary to the anchored DNA into streptavidin magnetic beads with the surface connected with the anchored DNA, after the reaction, magnetically separating, washing the unconjugated FL-DNA, and then re-dispersing into PB buffer solution to obtain the tumor marker detection kit (anchor-FL-magnetic beads).
Preferably, in the step 1, the concentration of the streptavidin magnetic beads is 5-10 mg/mL.
Preferably, in the step 1, the molar ratio of the mass of the streptavidin to the anchored DNA is 1-2: 10 -3 ，mg：μmol。
Preferably, in step 1, the ligation buffer is 20mM Tris HCl,1.0M NaCl,1mM EDTA,0.02%Triton X-100, pH7.4.
Preferably, in steps 1 and 2, the pH of the phosphate buffer is 7.0-7.4 and the concentration is 10mM.
Preferably, in steps 1 and 2, the reaction temperature is 37 ℃.
Preferably, in step 1, the reaction time is 15 to 60 minutes.
Preferably, in the step 2, the reaction time is 1-2 h.
Further, the invention provides application of the tumor marker detection kit in tumor marker detection, and the specific application method comprises the following steps: adding the tumor marker detection kit into serum to be detected for reaction, magnetically separating after the reaction is finished, taking supernatant to measure the fluorescence intensity of a corresponding wave band, and calculating to obtain the concentration of the tumor marker in the serum to be detected according to the linear relation between the fluorescence intensity and the concentration of the tumor marker.
Preferably, the volume ratio of the tumor marker detection kit to serum is 2-5:25.
Preferably, the reaction time is 1 to 2 hours.
Preferably, the optimal excitation/emission wavelength for CEA detection is 492/518nm; the optimal excitation/emission wavelength for detecting PSA is 585/605nm; the optimal excitation/emission wavelength for detecting AFP is 682/696nm.
Compared with the prior art, the invention has the following advantages:
the invention prepares the kit capable of successfully detecting the corresponding tumor marker through the combination application of the magnetic beads and the Aptamer chains corresponding to the tumor marker. The kit is nontoxic and harmless, and the detection waste does not pollute the environment; the kit has the characteristics of simple preparation process, short preparation time, good stability, easy storage, high detection sensitivity, convenient and quick detection process and the like. The kit prepared by the invention can be used singly, can be used for detecting single tumor markers, can be used after three probes are mixed in equal quantity, is used for simultaneously detecting three tumor markers, and has the advantages of greatly shortening the detection time, improving the detection efficiency and saving the detection cost.
Drawings
Fig. 1 is a schematic diagram of the detection principle of the present invention.
FIG. 2 is a graph showing fluorescence intensity versus CEA concentration of a probe prepared from FL-CEA-2 and Anchor-CEA sequences in example 1, as measured by detection of CEA and the corresponding full complement.
FIG. 3 is a graph showing fluorescence intensity versus CEA concentration of a probe prepared from FL-CEA-1 sequence and Anchor-CEA sequence in example 1, and detected CEA and corresponding full complement.
FIG. 4 is a graph showing fluorescence intensity versus CEA concentration of a probe prepared from FL-CEA-3 sequence and Anchor-CEA sequence in example 1, and the detection of CEA and the corresponding full complement.
FIG. 5 is a graph of CEA detection concentration versus fluorescence signal intensity for example 2.
FIG. 6 is a graph of fluorescence signal spectra corresponding to CEA at various concentrations in example 2.
FIG. 7 is a chart showing fluorescence signal spectra obtained by detecting an analyte using a probe prepared from FL-PSA-2 sequence and Anchor-PSA sequence at different concentrations of PSA in example 3.
FIG. 8 is a spectrum of fluorescent signals obtained by detecting the PSA and corresponding full complement of the analyte using the FL-PSA-1 sequence and Anchor-PSA sequence prepared in example 3.
FIG. 9 is a chart showing fluorescence signal spectra obtained by detecting the PSA and corresponding full complement of the analyte using the FL-PSA-3 sequence and Anchor-PSA sequence prepared in example 3.
FIG. 10 is a graph showing the results of detection of a series of samples under excitation at 492nm after equal amounts of the three probes in example 4.
FIG. 11 is a graph showing the detection results of a series of samples under excitation at 585nm wavelength after mixing the same amounts of the three probes in example 4.
FIG. 12 is a graph showing the detection results of a series of samples under test under 695nm excitation after mixing the three probes in equal amounts in example 4.
FIG. 13 is a graph showing the detection result of carcinoembryonic antigen CEA by using a probe prepared by using a carboxyl-modified magnetic bead and an amino-modified DNA in comparative example 1.
FIG. 14 is a graph showing the detection result of carcinoembryonic antigen CEA by using a probe prepared from a silver nanocluster synthesized using a carboxyl group-modified magnetic bead and an amino group-modified DNA in comparative example 2.
Detailed Description
The present invention will be described more fully hereinafter with reference to the accompanying drawings, in which preferred embodiments are shown, for the purpose of illustrating the invention, but the scope of the invention is not limited to the specific embodiments shown.
All terms of art used hereinafter are defined as commonly understood by one skilled in the art unless otherwise defined. The terminology used herein is for the purpose of describing particular examples only and is not intended to be limiting of the scope of the present invention.
Unless specifically indicated otherwise, reagents, materials, equipment and the like used in the present invention are commercially available or may be prepared by existing methods.
Streptavidin magnetic beads were purchased from bordeaux biotechnology limited.
DNA molecules were synthesized by Shanghai Biotechnology Inc.
TABLE 1 DNA sequences
Figure BDA0001896055760000041
Figure BDA0001896055760000051
Example 1
(1) Three groups of 200. Mu.l streptavidin magnetic beads were taken, washed three times with 200. Mu.l Binding Buffer, then re-uniformly dispersed in 200. Mu.l Binding Buffer, then added with corresponding 20. Mu.l (100. Mu.M) Anchor-DNA (Anchor-CEA), reacted under gentle shaking for 30min, then magnetically separated, washed off the Anchor-DNA not attached to the surface of the magnetic beads, and re-dispersed in 200. Mu.l PB Buffer to obtain Anchor-magnetic beads.
(2) Adding 20 microliters of FL-DNA (FL-CEA-1, FL-CEA-2 and FL-CEA-3) into the Anchor-magnetic beads obtained in the step (1), reacting for 2 hours under mild vibration, then magnetically separating, washing off unconnected FL-DNA, and then redispersing into 200 microliters of PB buffer solution to obtain the Anchor-FL-magnetic beads, namely the probes for detecting the corresponding tumor markers.
(3) Three groups of to-be-detected solutions with the volume of 275 microliters are prepared by taking CEA, the final concentration is 100ng/mL, and the complete complementary strand of CEA Aptamer strand is additionally taken to prepare the complete complementary strand to be-detected solution with the concentration consistent with that of FL-DNA on the probe.
(4) And (3) taking 25 microliters of the probes prepared in the step (2), respectively adding the probes into the liquid to be tested prepared in the step (3), and reacting for 2 hours under mild vibration.
(5) And (3) taking the reaction systems of which the reactions are finished, respectively carrying out magnetic separation, and taking the supernatant to detect the intensity of fluorescent signals under the same excitation wavelength.
Fig. 1 is a schematic diagram of the detection principle of the present invention. When a tumor marker exists, double chains can be opened to release single-chain DNA carrying dye, so that fluorescence signals can be detected; in the absence of tumor markers, the double strand could not be opened, so that no fluorescent signal could be detected in the supernatant. When the Anchor-CEA sequence and the FL-CEA-2 sequence are used simultaneously, the test result shown in FIG. 2 is obtained, and the existence of CEA can be obviously detected; when the Anchor-CEA sequence is used together with the FL-CEA-1 sequence or the FL-CEA-3 sequence, the detection results are as shown in FIGS. 3 and 4, and the detection of CEA as a test substance is hardly detected.
Example 2
(1) 200 microliter of streptavidin magnetic beads are taken, washed three times by 200 microliter of Binding Buffer, then uniformly dispersed in 200 microliter of Binding Buffer, 20 microliter (100 microliter) of Anchor-DNA (Anchor-CEA) is added, the mixture is reacted for 30 minutes under mild vibration, then magnetic separation is carried out, the Anchor-DNA which is not connected to the surface of the magnetic beads is washed off, and then the mixture is redispersed in 200 microliter of PB to obtain the Anchor-magnetic beads.
(2) Adding 20 microliters of FL-CEA-2 into the Anchor-magnetic beads obtained in the step (1), reacting for 2 hours under mild vibration, then magnetically separating, washing off the unconnected FL-CEA-2, and then redispersing the unconnected FL-CEA-2 in 200 microliters of PB buffer solution to obtain the Anchor-FL-magnetic beads, namely the corresponding probes for tumor marker detection.
(3) Taking CEA to-be-tested liquid with a volume of 275 microlitres, wherein the final concentrations of the CEA to-be-tested liquid are respectively as follows: 0. 5, 50, 100, 200 (ng/mL), and a full complement of the CEAAptamer strand was prepared to give a full complement of the test solution corresponding to the concentration of FL-DNA on the probe.
(4) And (3) taking 25 microliters of the probes prepared in the step (2), respectively adding the probes into the liquid to be tested prepared in the step (3), and reacting for 2 hours under mild vibration.
(5) And (3) taking the reaction systems of which the reactions are finished, respectively carrying out magnetic separation, and taking the supernatant to detect the intensity of fluorescent signals under the same excitation wavelength.
The detection results are shown in fig. 5 and 6. FIG. 5 is a graph showing the linear relationship between CEA detection concentration and fluorescence signal intensity, i.e. the fluorescence signal intensity detected at the same excitation wavelength in the presence of CEA at different concentrations, and it can be seen that the fluorescence signal intensity shows a certain linear trend with increasing CEA concentration, and the detection effect is good. Performing linear fitting to obtain a fluorescence intensity-marker concentration change standard curve, wherein the linear formula is as follows:
y=0.97x+4, where y is fluorescence intensity and x is tumor marker concentration. FIG. 6 shows fluorescence signal spectra corresponding to CEA at different concentrations, and fluorescence signal spectra detected at the same excitation wavelength at different CEA concentrations, so that it can be seen that the optimal excitation wavelength is almost the same, and the detection effect is good.
Example 3
(1) Three groups of 200. Mu.l streptavidin magnetic beads were taken, washed three times with 200. Mu.l Binding Buffer, then dispersed again in 200. Mu.l Binding Buffer, then the corresponding 20. Mu.l (100. Mu.M) Anchor-DNA (Anchor-PSA sequence) was added, reacted under gentle shaking for 30min, then magnetically separated, the Anchor-DNA not attached to the surface of the magnetic beads was washed off, and then dispersed again in 200. Mu.l PB Buffer to obtain Anchor-magnetic beads.
(2) And (3) adding 20 microliters of FL-DNA (FL-PSA-1, FL-PSA-2 and FL-PSA-3) into the Anchor-DNA-magnetic beads obtained in the step (1), reacting for 2 hours under mild vibration, then magnetically separating, washing the unconnected FL-DNA, and then re-dispersing into 200 microliters of PB buffer solution to obtain the Anchor-FL-magnetic beads, namely the probes for detecting the corresponding tumor markers.
(3) Three groups of test solutions with volumes of 275 microliters were prepared from PSA, with final concentrations of 1, 10, 100, 200ng/mL, respectively.
(4) And (3) taking 25 microliters of the probes prepared in the step (2), respectively adding the probes into the liquid to be tested prepared in the step (3), and reacting for 2 hours under mild vibration.
(5) And (3) taking the reaction systems of which the reactions are finished, respectively carrying out magnetic separation, and taking the supernatant to detect the intensity of fluorescent signals under the same excitation wavelength.
As a result of the detection, when the Anchor-PSA sequence and the FL-PSA-2 sequence are used simultaneously, the detection result as shown in fig. 7 is obtained, the detection result is obvious, and the existence of the PSA to be detected can be successfully detected; when the Anchor-PSA sequence is used together with the FL-PSA-1 sequence or the FL-PSA-3 sequence, the detection results are shown in FIGS. 8 and 9, and the probe has a weak response to the full complement of the Aptamer strand of PSA, but it cannot respond to the PSA of the analyte.
Example 4
(1) Three groups of 200. Mu.L streptavidin magnetic beads were taken, washed three times with 200. Mu.L Binding Buffer, then dispersed again in 200. Mu.L Binding Buffer, 20. Mu.L (100. Mu.M) of Anchor-DNA (Anchor-CEA, anchor-PSA, anchor-AFP) was added, respectively, reacted under gentle shaking for 30min, then magnetically separated, the Anchor-DNA not attached to the surface of the magnetic beads was washed off, and then dispersed again into 200. Mu.L PB Buffer to obtain the respective Anchor-magnetic beads.
(2) 20 microliters of corresponding FL-DNA (FL-CEA-2, FL-PSA-2, FL-AFP) are added into the Anchor-DNA-magnetic beads obtained in the step (1), the reaction is carried out for 2 hours under mild vibration, then the unconnected FL-DNA is removed by magnetic separation, and the unconnected FL-DNA is redispersed in 100 microliters of PB buffer solution, so that the respective Anchor-FL-magnetic beads, namely the probes for detecting the corresponding tumor markers, are obtained.
(3) Mixing the probes of the three markers prepared in the step (2) together, fully and uniformly mixing, and taking CEA, PSA, AFP to-be-detected liquid with the volume of 275 microlitres respectively, wherein the composition and the final concentration of each to-be-detected liquid are respectively as follows: (1) 100cea+100psa+100afp; (2) 100cea+100psa; (3) 100cea+100afp; (4) 100psa+100afp; (5) 100CEA; (6) 100PSA; (7) 100AFP (ng/mL).
(4) And (3) taking 25 microliters of probes uniformly mixed in the step (3), respectively adding the probes into the liquid to be tested prepared in the step (3), and reacting for 2 hours under mild vibration.
(5) And (3) taking the reaction systems of which the reactions are finished, respectively carrying out magnetic separation, and taking the supernatant to detect the intensity of fluorescent signals under the same excitation wavelength.
As shown in fig. 10, 11 and 12, the detection results are evident, and when three markers are present at the same time, the fluorescence signal intensity reaches the maximum.
Comparative example 1
(1) 200 microliters of carboxyl modified magnetic beads are taken, washed three times by 200 microliters of phosphate buffer (PBS, 10mM,pH7.4, 136.7mM NaCl,2.7mM KCl) respectively, then dispersed in 200 microliters of PBS again uniformly, and 50mg/mL of freshly prepared EDC and NHS are added into the mixture respectively, so that the final concentration of EDC and NHS is 10mg/mL, and the carboxyl on the surface of the magnetic beads is activated as much as possible by mild shaking reaction for 1 hour at room temperature.
(2) After the activation of the step (1), 20 microliter (100 mu M) of amino-modified Anchor-DNA (the base sequence is consistent with that of Anchor-CEA) is added, the reaction is carried out for 6 hours under mild vibration, the reaction is finished, the magnetic separation is carried out, the Anchor-DNA which is not connected to the surface of the magnetic bead is washed by using PB buffer solution, and then the solution is redispersed in 200 microliter PB buffer solution, so that the Anchor-magnetic bead is obtained.
(3) Adding 20 microliters of corresponding FL-DNA (FL-CEA-2) into the Anchor-magnetic beads obtained in the step (2), reacting for 2 hours under mild oscillation, after the reaction is finished, magnetically separating, washing the unconnected FL-DNA by using PB buffer solution, and then re-dispersing into 200 microliters of PB buffer solution to obtain the Anchor-FL-magnetic beads, namely the probes for detecting the corresponding tumor markers.
(4) A test solution with a CEA preparation volume of 275 microliters and a final concentration of 100ng/mL was prepared, and a full-complement test solution with the complete complementary strand of the CEA Aptamer strand consistent with the concentration of FL-DNA on the probe was prepared.
(5) And (3) taking 25 microliters of the probes prepared in the step (2), respectively adding the probes into the liquid to be tested prepared in the step (3), and reacting for 2 hours under mild vibration.
(6) And (3) taking the reaction systems of which the reactions are finished, respectively carrying out magnetic separation, and taking the supernatant to detect the intensity of fluorescent signals under the same excitation wavelength.
As shown in FIG. 13, it was found that the probe prepared from the carboxyl group magnetic beads and the amino DNA had a weak fluorescence signal because the anchored DNA was hardly attached to the surface of the magnetic beads.
Comparative example 2
(1) 200 microliters of carboxyl modified magnetic beads are taken, washed three times by 200 microliters of phosphate buffer (PBS, 10mM,pH7.4, 136.7mM NaCl,2.7mM KCl) respectively, then dispersed in 200 microliters of PBS again uniformly, and 50mg/mL of freshly prepared EDC and NHS are added into the mixture respectively, so that the final concentration of EDC and NHS is 10mg/mL, and the carboxyl on the surface of the magnetic beads is activated as much as possible by mild shaking reaction for 1 hour at room temperature.
(2) 20 microliters of CEA-AgNC sequence DNA was taken, 176.8 microliters of PB buffer (pH 7.0,20 mM) was added, and 1.6 microliters of 7.5mM AgNO was added 3 Shaking strongly for 1min, storing in dark for 30min, and adding 1.6 microlitres of freshly prepared NaBH 4 After strong shaking for 2min, the mixture is preserved for 4 hours at room temperature in a dark place and then is placed in a refrigerator for refrigeration at 2-8 ℃ for overnight.
(3) After the activation of the step (1), 20 microliter (100 mu M) of amino-modified Anchor-DNA (the base sequence is consistent with that of Anchor-CEA) is added, the reaction is carried out for 6 hours under mild vibration, the reaction is finished, the magnetic separation is carried out, the Anchor-DNA which is not connected to the surface of the magnetic bead is washed by using PB buffer solution, and then the solution is redispersed in 200 microliter PB buffer solution, so that the Anchor-magnetic bead is obtained.
(4) Adding all silver nanoclusters prepared in the step (2) into the Anchor-magnetic beads prepared in the step (3), reacting for 2 hours under mild vibration, magnetically separating, washing off unconnected CEA-AgNC by using PB buffer solution, and then redispersing into 200 microliters of PB buffer solution to obtain the Anchor-FL-magnetic beads, namely the corresponding probes for detecting tumor markers.
(5) A test solution with a CEA preparation volume of 275 microliters and a final concentration of 100ng/mL was prepared, and a full-complement test solution with the complete complementary strand of the CEA Aptamer strand consistent with the concentration of FL-DNA on the probe was prepared.
(6) And (3) taking 25 microliters of the probes prepared in the step (4), respectively adding the probes into the liquid to be tested prepared in the step (5), and reacting for 2 hours under mild vibration.
(7) And (3) taking the reaction systems of which the reactions are finished, respectively carrying out magnetic separation, and taking the supernatant to detect the intensity of fluorescent signals under the same excitation wavelength.
As a result of the detection, as shown in FIG. 14, the signal of the silver nanocluster itself was extremely weak, and the coupling effect of the carboxyl magnetic beads and the amino DNA was poor, so that the detection effect was poor.
Sequence listing
<110> university of Nanjing's science
<120> tumor marker detection kit and preparation method and application thereof
<141> 2018-12-07
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Claims (7)

1. A tumor marker detection kit comprising at least one of streptavidin magnetic beads with surface-linked anchored DNA of biotin-modified CEA, streptavidin magnetic beads with surface-linked anchored DNA of biotin-modified PSA, and streptavidin magnetic beads with surface-linked anchored DNA of biotin-modified AFP, and fluorescent dye-modified DNA complementary to the anchored DNA; the anchoring DNA of the CEA is 5'-biotin-TTTTTTATACCAGCTTATTCAATT-3', and the complementary strand is 5'-FAM-AATTGAATAA-3'; the anchoring DNA of the PSA is 5'-biotin-TTTTTTATTAAAGCTCGCCATCAAATAGCTTT-3', and the complementary strand is 5'-ROX-AAAGCTATTTGATGGC-3'; the anchor DNA of AFP is 5'-biotin-TTTTTTTCAGGTGCAGTTCTCGACTCGGTCTTGATGTGGGT-3' and the complementary strand is 5'-Cy5.5-ACCCACATCAAGACCGAGTC-3'.
2. The tumor marker detection kit according to claim 1, wherein the concentration of streptavidin magnetic beads is 5-10 mg/mL.
3. The method for preparing a tumor marker detection kit according to claim 1 or 2, characterized by comprising the following specific steps:
step 1, uniformly dispersing the washed streptavidin magnetic beads in a connection buffer solution, adding anchoring DNA for reaction, magnetically separating after the reaction is finished, washing off the anchoring DNA which is not connected to the surface of the magnetic beads, and then re-dispersing the anchoring DNA into a phosphate buffer solution to obtain the streptavidin magnetic beads with the surface connected with the anchoring DNA;
and 2, adding fluorescent dye modified DNA complementary to the anchored DNA into streptavidin magnetic beads with the surface connected with the anchored DNA, magnetically separating after the reaction is finished, washing the unconjugated FL-DNA, and then re-dispersing into PB buffer solution to obtain the tumor marker detection kit.
4. The method of claim 3, wherein in step 1, the molar ratio of streptavidin to anchored DNA is 1-2: 10 -3 ，mg：μmol。
5. The method of claim 3, wherein in step 1, the ligation buffer is 20mM Tris ∙ HCl,1.0M NaCl,1mM EDTA,0.02%Triton X-100, pH7.4; in the steps 1 and 2, the pH of the phosphate buffer solution is 7.0-7.4, and the concentration is 10mM; in steps 1 and 2, the reaction temperature was 37 ℃.
6. The preparation method according to claim 3, wherein in the step 1, the reaction time is 15-60 min.
7. The method according to claim 3, wherein in the step 2, the reaction time is 1 to 2 hours.
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