CN113834935A - Method for detecting carcinoembryonic antigen - Google Patents
Method for detecting carcinoembryonic antigen Download PDFInfo
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- CN113834935A CN113834935A CN202111110426.9A CN202111110426A CN113834935A CN 113834935 A CN113834935 A CN 113834935A CN 202111110426 A CN202111110426 A CN 202111110426A CN 113834935 A CN113834935 A CN 113834935A
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- 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
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Abstract
The invention discloses a method for detecting carcinoembryonic antigen, which is a method for detecting the carcinoembryonic antigen by a fluorescence biosensor constructed on the basis of magnetic graphene oxide (GO-Fe)3O4) The magnetic material has high adsorption capacity of GO, is easy to obtain, and has the advantages of simple method, low cost and stable property; at the same time, GO-Fe3O4Can effectively improve the water solubility and the dispersibility of the composite material, and can be used for effectively improving the water solubility and the dispersibility of the composite material under the action of an external magnetic field,the interference caused by non-specific adsorption is easy to remove; in addition, in GO-Fe3O4The polypeptide is introduced into the surface, and can remove the non-specific adsorption of the protein on the GO surface, so that the detection sensitivity is improved.
Description
Technical Field
The invention belongs to the technical field of biochemistry, environmental detection and food safety, and relates to a protein detection method, in particular to a method for detecting a carcinoembryonic antigen by a fluorescence biosensor constructed on the basis of magnetic graphene oxide and zwitterionic peptide.
Background
The tumor marker is a substance with carcinogenic effect in normal cell development or different cell development stages[1]It has important reference in the aspects of general investigation, diagnosis, prognosis and prognosis determination of tumor, and therapeutic effect evaluation[2-5]. It is a biological structural substance, most commonly proteins and glycolipids, and also includes DNA, RNA and microRNA (miRNA), etc[6-9]. Carcinoembryonic antigen (CEA) is one of the common tumor markers, first described since 1965[10]CEA has been one of the most widely studied tumor markers.
CEA is a human glycoprotein involved in cell adhesion and expressed during human fetal development, produced by normal fetal intestinal tissue and epithelial tumor cells, and whose serum levels may also be increased in non-malignant diseases such as inflammatory bowel disease[11,12]. After the birth of the fetus, the expression of CEA is largely inhibited, and only lower concentration levels are found in the plasma of healthy adults[13]. However, CEA is overexpressed in many human cancers, including gastric, colorectal (CRC), breast, ovarian, lung, and pancreatic cancers[10,14,15]. CEA is aberrantly expressed in about 95% of CRC, and the correlation between serum CEA (serum CEA, s-CEA) levels and CRC has been extensively studied by a number of investigators[16]. As one of important tumor markers, the detection of CEA has very important significance clinically. CEA is a broad-spectrum tumor marker, and although CEA cannot be a specific index for diagnosing a certain malignant tumor, CEA still has important clinical value in the aspects of differential diagnosis, curative effect evaluation, disease condition monitoring and the like of malignant tumor[17-20]。
Currently, the medical practice for CEA detection is based on immunoassay techniques, including enzyme-linked immunosorbent assays (ELISAs)[21,22]Electrochemical immunoassay[23]Manual radioimmunoassay (radioimmunoassay)say, RIA), immunoradiometric assay (IRMA)[24]And fluorescence immunization[25]And the like. Although immuno-based detection methods have good selectivity, most immunoassays developed rely on specific labels, such as fluorescent molecules, radioactive elements or enzymes, in order to translate antigen-antibody binding information into readable signals[26]. In addition, these techniques require complex and expensive precision instruments, require specialized personnel to operate, and the radioactive elements can cause some harm to the human body, which limits their wide-ranging applications[27-29]. Therefore, the development of a rapid, high-sensitivity, high-selectivity, low-cost and high-benefit CEA detection method has important theoretical significance and application value for human disease detection.
Reference to the literature
[1]Lech G,Slotwinski R,Slodkowski M,et al.Colorectal cancer tumour markers and biomarkers: recent therapeutic advances.World Journal of Gastroenterology,2016,22(5):1745-1755.
[2]Di Gioia D,Blankenburg I,Nagel D,et al.Tumor markers in the early detection of tumor recurrence in breast cancer patients:CA 125,CYFRA 21-1,HER2 shed antigen,LDH and CRP in combination with CEA and CA 15-3.Clinica Chimica Acta,2016,461:1-7.
[3]Duffy M J,Lamerz R,Haglund C,et al.Tumor markers in colorectal cancer,gastric cancer and gastrointestinal stromal cancers:European group on tumor markers 2014 guidelines update.International Journal of Cancer,2014,134(11):2513-2522.
[4]Shimada H,Noie T,Ohashi M,et al.Clinical significance of serum tumor markers for gastric cancer: a systematic review of literature by the task force of the japanese gastric cancer association.Gastric Cancer, 2014,17(1):26-33.
[5]Lai Y,Wang L,Liu Y,et al.Immunosensors based on nanomaterials for detection of tumor markers[J].Journal of Biomedical Nanotechnology,2018,14(1):44-65.
[6]Wieczorek E,Reszka E.mRNA,microRNA and lncRNA as novel bladder tumor markers.Clinica Chimica Acta,2018,477:141-153.
[7]Han X,Wang J,Sun Y.Circulating tumor DNA as biomarkers for cancer detection.Genomics Proteomics and Bioinformatics,2017,15(2):59-72.
[8]Banin Hirata B K,Oda J M,Losi Guembarovski R,et al.Molecular markers for breast cancer: prediction on tumor behavior.Disease Markers,2014,2014:513158.
[9]Tan M-H,Wang H-Y,Hsieh C-H,et al.Cancers screening in an asymptomatic population by using multiple tumour markers.PLoS One,2016,11(6):e0158285.
[10]Gold P,.S O F.Specific carcinoembryonic antigens of the human digestive system.Journal of Experimental Medicine,1965,122(3):467-481.
[11]Chen C H,Hsieh M C,Lai C C,et al.Lead time of carcinoembryonic antigen elevation in the postoperative follow-up of colorectal cancer did not affect the survival rate after recurrence.International Journal of Colorectal Disease,2010,25(5):567-571.
[12]Chiaravalloti A,Fiorentini A,Palombo E,et al.Evaluation of recurrent disease in the re-staging of colorectal cancer by(18)F-FDG PET/CT:use of CEA and CA 19-9 in patient selection.Oncology Letters,2016, 12(5):4209-4213.
[13]Hensel J A,Khattar V,Ashton R,et al.Recombinant AAV-CEA tumor vaccine in combination with an immune adjuvant breaks tolerance and provides protective immunity.Molecular Therapy-Oncolytics,2019, 12:41-48.
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Disclosure of Invention
In order to overcome the above defects of the prior art, the present invention employs SYBR Green II (SG II) to stain DNA, so that the DNA is fluorescent. SYBR Green II is a highly sensitive nucleic acid staining reagent that dramatically increases the fluorescence of single-stranded DNA in the presence of single-stranded oligonucleotides. After DNA is dyed by SYBR Green II, the DNA is reacted with GO-Fe3SO4And (3) combining, adsorbing the CEA to the tube wall through an external magnetic field, removing the supernatant to achieve the centrifugal effect, further adding a novel antifouling biological material, namely antifouling peptide, into the sensor, and removing non-specific adsorption to improve the detection sensitivity, thereby detecting the CEA.
In order to achieve the purpose, the invention provides the following technical scheme: a method for detecting carcinoembryonic antigen, comprising the steps of:
s1 preparation of GO-Fe3O4(ii) a 0.4g of GO was added to 60mL of ethylene glycol and the resulting mixture was sonicated until solidUntil the solution is homogeneous, 0.65g of FeCl is added3Added to the solution and further sonicated for 10 min. Adding 2.6g of sodium acetate, stirring vigorously for 20min, transferring the mixture into an autoclave with polytetrafluoroethylene as an inner liner, heating at 180 ℃ for 10h, cooling to room temperature, and adding black product GO-Fe3O4Washed with ethanol and dried with a vacuum freeze dryer. To a solution containing 1mL NHS (50mM) and 1mL EDC (500mM) was added 4mg GO-Fe3O4Activating carboxyl group, activated GO-Fe3O4The concentration of (A) is 2 mg/mL;
s2, modifying amino NH at the end of DNA sequence2Dissolving the modified DNA sequence and polypeptide in Tris-HCl solution with pH value in the range of 5-9;
s3, dyeing the single-stranded DNA with SG II, and mixing the DNA with GO-Fe3O4In combination, the concentration range of SG II is set to (0.4X, 0.6X, 0.8X, 1.0X, 1.2X and 1.5X), GO-Fe3O4The concentration ranges of (5, 10, 15, 20 and 25. mu.g/mL);
s4, mixing GO-Fe in S33O4Introducing polypeptide on the surface to remove non-specific adsorption; the concentration range of the polypeptide is set as (0, 25, 50, 75, 100, 125, 150. mu.g/mL);
s5, combining the GO-Fe with DNA and polypeptide in S4 under the action of external magnetic field3O4Adsorbing to the bottom or the tube wall, eluting to remove SG II not bound with DNA and GO-Fe not bound3O4The polypeptide of (1);
s6, after fluorescence is quenched by FRET, CEA is added to the sensor prepared in S5; binding of CEA to the aptamer induces a conformational change in the aptamer, resulting in fluorescence recovery;
s7, obtaining the linear relation between CEA and its nucleic acid aptamer; adding a series of CEA solutions with known concentrations into the solution obtained in S6, reacting at normal temperature, and specifically binding DNA in the solution obtained by the aptamer S6 and CEA to induce conformation change, so that the DNA part with CEA fluorescence is far away from GO-Fe3O4The fluorescence is recovered to a certain degree, after the reaction is finished, the fluorescence value is measured, and a corresponding linear relation graph is prepared according to the measured fluorescence value and CEA;
above, the CEA solution concentration was set to (0.001, 0.01, 0.05, 0.1, 0.5, 0.8, 1 and 5 ng/mL);
s8, CEA detection; adding a certain amount of CEA with unknown concentration into the solution obtained in S6, reacting at normal temperature, measuring the fluorescence recovery value after the reaction is finished, and obtaining the concentration of the CEA according to the linear relation obtained in S7;
preferably, the pH value of the Tris-HCl solution is selected to be 7.5;
the sequence of the DNA is: DNA: 5' -ATA CCA GCT TAT TCA ATT TTT TTT TTT T-NH2-3’；
The polypeptide is: EKEKEKEPPPPC, respectively;
the final concentration of the modified DNA sequence was 50nM in Tris-HCl buffer.
Preferably, in S3, SYBR II is 1X SYBR II, with fluorescent DNA and GO-Fe3O4The binding time was 30min, the concentration range of SG II was set to 1.0 ×, GO-Fe3O4The concentration of (2) was set to 15. mu.g/mL.
Preferably, in S4, the concentration of the introduced polypeptide is set to 50. mu.g/mL, the total volume after the reaction is 500. mu.L, and the reaction conditions are 4 ℃ overnight.
Preferably, in S5, the elution is performed by eluting with Tris-HCl to remove unbound substances and measuring the fluorescence by pipetting 200. mu.L.
Preferably, in S6, the incubation time is 30min and the reaction temperature is 4 ℃.
Preferably, in S7, the reaction time at room temperature is 30min, and the pH of the CEA solution is set to 7.5.
The invention has the technical effects and advantages that:
magnetic graphene oxide (GO-Fe) in the invention3O4) The magnetic GO is easy to obtain due to the high adsorption capacity and the magnetic material of GO, and the method is simple, low in cost and stable in property;
at the same time, GO-Fe3O4The water solubility and the dispersibility of the composite material can be effectively improved, and the interference caused by non-specific adsorption can be easily removed under the action of an external magnetic field;
at GO-Fe3O4The polypeptide is introduced into the surface, and can remove the non-specific adsorption of the protein on the GO surface, so that the detection sensitivity is improved;
drawings
FIG. 1 is a technical scheme for the detection of CEA according to the present invention;
FIG. 2 shows GO-Fe3O4A TEM image of (a);
FIG. 3 is a graph showing the selection of the optimum concentration of SG II;
FIG. 4 is GO-Fe3O4An optimal concentration selection map;
FIG. 5 is a selection graph of pH optimum concentration of Tris-HCl;
FIG. 6 is a graph of the sensitivity of aptamers;
FIG. 7 is a selectivity diagram of aptamers;
FIG. 8 is a graph showing the change in fluorescence intensity after addition of different proteins;
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1
A method for detecting carcinoembryonic antigen comprising the steps of:
s1 preparation of GO-Fe3O4(ii) a 0.4g of GO was added to 60mL of ethylene glycol, the resulting mixture was sonicated until the solution was homogeneous, then 0.65g of FeCl was added3Added to the solution and further sonicated for 10 min. 2.6g of sodium acetate are added and the mixture is stirred vigorously for 20min, and the mixture is transferred to a high pressure with polytetrafluoroethylene liningHeating at 180 deg.C for 10 hr in kettle, cooling to room temperature, and adding black product GO-Fe3O4Washed with ethanol and dried with a vacuum freeze dryer. To a solution containing 1mL NHS (50mM) and 1mL EDC (500mM) was added 4mg GO-Fe3O4Activating carboxyl group, activated GO-Fe3O4The concentration of (A) is 2 mg/mL;
s2, modifying amino NH at the end of DNA sequence2Dissolving the modified DNA sequence and the polypeptide in a Tris-HCl solution with the ph value of 7.5; the sequence of the DNA is: DNA: 5' -ATA CCA GCT TAT TCA ATT TTT TTT TTT T-NH2-3'; the polypeptide is: EKEKEKEPPPPC, respectively; the final concentration of the modified DNA sequence was 50nM in Tris-HCl buffer.
S3, staining the single-stranded DNA with SG II with the concentration of 1.0 multiplied by the X, and combining the stained DNA with GO-Fe3O4Combined for 30min, GO-Fe3O4The concentration range of (2) is set to 15 mug/mL; SYBR II is 1X SYBR II.
S4, mixing GO-Fe in S33O4Introducing polypeptide with the concentration of 50 mug/mL on the surface to remove nonspecific adsorption, wherein the total volume after reaction is 500 mug L, and the reaction condition is overnight at 4 ℃;
s5, combining the GO-Fe with DNA and polypeptide in S4 under the action of external magnetic field3O4Adsorbing to the bottom or tube wall, eluting with Tris-HCl to remove SG II unbound with DNA and GO-Fe unbound3O4And aspirating 200. mu.L of the polypeptide of (4) to measure the fluorescence thereof;
s6, after fluorescence is quenched through FRET, CEA is added into the sensor prepared in S5, the incubation time is 30min, the reaction temperature is 4 ℃, and the binding of the CEA and the aptamer induces the conformation change of the aptamer, thereby leading to the recovery of fluorescence;
s7, obtaining the linear relation between CEA and its nucleic acid aptamer; adding CEA solution with concentration of 0.01, 0.05, 0.1, 0.5, 0.8, 1 and 5ng/mL into the solution obtained in S6, respectively, reacting at room temperature to obtain DNA in the solution obtained from aptamer S6, which specifically binds to CEA to induce conformation change, and collecting the DNAWhile keeping the DNA part with CEA fluorescence away from GO-Fe3O4The fluorescence is recovered to a certain degree, after the reaction is finished, the fluorescence value is measured, and a corresponding linear relation graph is prepared according to the measured fluorescence value and CEA;
s8, CEA detection; and adding a certain amount of CEA with unknown concentration into the solution obtained in S6, reacting at normal temperature, measuring the fluorescence recovery value after the reaction is finished, and obtaining the concentration of the CEA according to the linear relation obtained in S7.
Example 2
The detection method comprises the following steps:
s1 preparation of GO-Fe3O4(ii) a 0.4g of GO was added to 60mL of ethylene glycol, the resulting mixture was sonicated until the solution was homogeneous, then 0.65g of FeCl was added3Added to the solution and further sonicated for 10 min. Adding 2.6g of sodium acetate, stirring vigorously for 20min, transferring the mixture into an autoclave with polytetrafluoroethylene as an inner liner, heating at 180 ℃ for 10h, cooling to room temperature, and adding black product GO-Fe3O4Washed with ethanol and dried with a vacuum freeze dryer. To a solution containing 1mL NHS (50mM) and 1mL EDC (500mM) was added 4mg GO-Fe3O4Activating carboxyl group, activated GO-Fe3O4The concentration of (A) is 2 mg/mL;
s2, modifying amino NH at the end of DNA sequence2Dissolving the modified DNA sequence and the modified polypeptide in a Tris-HCl solution with the ph value of 9; the sequence of the DNA is: DNA: 5' -ATA CCA GCT TAT TCA ATT TTT TTT TTT T-NH2-3'; the polypeptide is: EKEKEKEPPPPC, respectively; the final concentration of the modified DNA sequence was 50nM in Tris-HCl buffer.
S3, staining the single-stranded DNA with SG II with the concentration of 1.5 times, and mixing the stained DNA with GO-Fe3O4Combined for 30min, GO-Fe3O4The concentration range of (2) is set to 25. mu.g/mL; SYBR II is 1X SYBR II.
S4, mixing GO-Fe in S33O4Introducing 150 microgram/mL polypeptide to the surface to remove non-specific adsorption, and reactingThe total volume of the reaction solution is 500 mu L, and the reaction condition is overnight at the temperature of 4 ℃;
s5, combining the GO-Fe with DNA and polypeptide in S4 under the action of external magnetic field3O4Adsorbing to the bottom or tube wall, eluting with Tris-HCl to remove SG II unbound with DNA and GO-Fe unbound3O4And aspirating 200. mu.L of the polypeptide of (4) to measure the fluorescence thereof;
s6, after fluorescence is quenched through FRET, CEA is added into the sensor prepared in S5, the incubation time is 30min, the reaction temperature is 4 ℃, and the binding of the CEA and the aptamer induces the conformation change of the aptamer, thereby leading to the recovery of fluorescence;
s7, obtaining the linear relation between CEA and its nucleic acid aptamer; adding CEA solutions with the concentrations of 0.01, 0.05, 0.1, 0.5, 0.8, 1 and 5ng/mL into the solution obtained in S6, respectively, reacting at normal temperature, and allowing DNA in the solution obtained from the aptamer S6 to specifically bind with CEA to induce conformational change, so that the DNA part with CEA fluorescence is far away from GO-Fe3O4The fluorescence is recovered to a certain degree, after the reaction is finished, the fluorescence value is measured, and a corresponding linear relation graph is prepared according to the measured fluorescence value and CEA;
s8, CEA detection; and adding a certain amount of CEA with unknown concentration into the solution obtained in S6, reacting at normal temperature, measuring the fluorescence recovery value after the reaction is finished, and obtaining the concentration of the CEA according to the linear relation obtained in S7.
Example 3
The detection method comprises the following steps:
s1 preparation of GO-Fe3O4(ii) a 0.4g of GO was added to 60mL of ethylene glycol, the resulting mixture was sonicated until the solution was homogeneous, then 0.65g of FeCl was added3Added to the solution and further sonicated for 10 min. Adding 2.6g of sodium acetate, stirring vigorously for 20min, transferring the mixture into an autoclave with polytetrafluoroethylene as an inner liner, heating at 180 ℃ for 10h, cooling to room temperature, and adding black product GO-Fe3O4Washed with ethanol and dried with a vacuum freeze dryer. In a container containing 1mLNHS (50mM) and 1mL EDC (500mM) with 4mg GO-Fe3O4Activating carboxyl group, activated GO-Fe3O4The concentration of (A) is 2 mg/mL;
s2, modifying amino NH at the end of DNA sequence2Dissolving the modified DNA sequence and the modified polypeptide in a Tris-HCl solution with the ph value of 5; the sequence of the DNA is: DNA: 5' -ATA CCA GCT TAT TCA ATT TTT TTT TTT T-NH2-3'; the polypeptide is: EKEKEKEPPPPC, respectively; the final concentration of the modified DNA sequence was 50nM in Tris-HCl buffer.
S3, staining the single-stranded DNA with SG II with the concentration of 0.4 multiplied by the X-ray fluorescence, and reacting the stained DNA with GO-Fe3O4Combined for 30min, GO-Fe3O4The concentration range of (2) is set to 5 mug/mL; SYBR II is 1X SYBR II.
S4, mixing GO-Fe in S33O4Introducing polypeptide with the concentration of 25 mug/mL on the surface to remove nonspecific adsorption, wherein the total volume after reaction is 500 mug L, and the reaction condition is overnight at 4 ℃;
s5, combining the GO-Fe with DNA and polypeptide in S4 under the action of external magnetic field3O4Adsorbing to the bottom or tube wall, eluting with Tris-HCl to remove SG II unbound with DNA and GO-Fe unbound3O4And aspirating 200. mu.L of the polypeptide of (4) to measure the fluorescence thereof;
s6, after fluorescence is quenched through FRET, CEA is added into the sensor prepared in S5, the incubation time is 30min, the reaction temperature is 4 ℃, and the binding of the CEA and the aptamer induces the conformation change of the aptamer, thereby leading to the recovery of fluorescence;
s7, obtaining the linear relation between CEA and its nucleic acid aptamer; adding CEA solutions with the concentrations of 0.01, 0.05, 0.1, 0.5, 0.8, 1 and 5ng/mL into the solution obtained in S6, respectively, reacting at normal temperature, and allowing DNA in the solution obtained from the aptamer S6 to specifically bind with CEA to induce conformational change, so that the DNA part with CEA fluorescence is far away from GO-Fe3O4Resulting in a certain recovery of fluorescence, measuring the fluorescence value after the reaction is finished, and determining the fluorescence value according to the measurementPreparing a corresponding linear relation graph by the determined fluorescence value and the CEA;
s8, CEA detection; and adding a certain amount of CEA with unknown concentration into the solution obtained in S6, reacting at normal temperature, measuring the fluorescence recovery value after the reaction is finished, and obtaining the concentration of the CEA according to the linear relation obtained in S7.
With reference to the attached figures 1-8, the three embodiments described above make it possible to obtain:
from fig. 3, it can be seen that the optimum concentration of SG ii is 1.0 ×: SG II (0.4X, 0.6X, 0.8X, 1.0X, 1.2X and 1.5X) was added to Tris-HCl buffer containing 50nM DNA at various concentrations, and the fluorescence intensity was measured after reaction at room temperature for 30min, and the results are shown in FIG. 3. The fluorescence intensity also increased with the increase of SG II concentration, and the fluorescence increase relatively tended to be gentle after the concentration reached 1.0X. By further increasing the concentration of SG II, there is no significant increase in fluorescence, and excessive SG II may interfere with later experiments. Therefore, 1.0 × SG II was selected as the optimum concentration for the experiment.
From FIG. 4, it can be derived, GO-Fe3O4The optimal concentration is 15 mug/mL: to Tris-HCl buffer containing 50nM DNA and 1.0 XSXII was added GO-Fe at different concentrations3O4(5, 10, 15, 20 and 25. mu.g/mL) were prepared, and then 0.8ng/mL of CEA was added, and the fluorescence intensity thereof was measured and compared for changes in the fluorescence intensity. In the range of 5-25 μ g/mL with GO-Fe3O4Increase in concentration, F/F0-1 is also increasing and reaches a maximum at 15. mu.g/mL, and F/F when the concentration exceeds 15. mu.g/mL0-1 is continuously decreased. Therefore, 15 μ g/mL was chosen as the best GO-Fe for this experiment3O4And (4) concentration.
From FIG. 5, it can be seen that the optimal pH of the Tris-HCl solution is 7.5: due to the sensitivity of SG II to pH, the influence of different pH conditions on the experimental results is explored. We prepared Tris-HCl in the pH range of 5 to 9 and recorded the effect on SG II and DNA binding at different pH (FIG. 5A) and the effect on the change in fluorescence intensity after CEA (0.8ng/mL) (FIG. 5B). Too high or too low a pH is detrimental to SG IIThe DNA staining is not beneficial to the DNA and GO-Fe3O4The combination of (1). The results in FIG. 5 show that the sensor works best at pH 7.5. Therefore, the pH 7.5 was chosen as the optimum pH for the sensor.
From FIG. 6, it can be concluded that the optimal polypeptide concentration is 50. mu.g/mL: in the presence of 50nM DNA, 1.0 XSSG II, 15. mu.g/mL GO-Fe3O4Tris-HCl buffer (g) was added to the polypeptide at different concentrations and the change in fluorescence intensity after addition of 0.8ng/mL CEA was compared. As can be seen from FIG. 6, 50. mu.g/mL of the polypeptide was most effective in the range of 0-150. mu.g/mL, and thus 50. mu.g/mL of the polypeptide was selected as the optimal concentration for this experiment.
As shown in fig. 7: the change of fluorescence intensity after adding different kinds of proteins into the aptamer solution. The change of fluorescence intensity and fluorescence recovery rate (F/F0-1) after adding CEA of different concentrations into the aptamer solution. The concentration of AFP added was gradually increased from 0 to a final concentration of 150. mu.g/mL.
As shown in fig. 8: the fluorescence intensity changes after addition of different proteins. And (3) the change of fluorescence intensity after different proteins are added into the aptamer solution, wherein the final concentration of CEA is 5ng/mL, and the final concentration of other proteins is 50 ng/mL.
In the above embodiment, the method further comprises the following steps: in GO-Fe containing 50nM DNA, 15. mu.g/mL3O4CEA (0.001, 0.01, 0.05, 0.1, 0.5, 0.8, 1 and 5ng/mL) with different concentrations is added into Tris-HCl of 1.0 times SG II and 50 mu g/mL polypeptide and reacted for 30min at room temperature, and the fluorescence spectrum is measured and recorded, and the recovery condition of the fluorescence is checked;
in addition, the method also comprises the following steps of selective detection: several different substances (CEA, BSA, IgG, VEGF, Thrombin, Uric acid and Glucose) were added to the sensor for interference experiments and reacted at room temperature for 30 min. Under optimal conditions, CEA (5ng/mL) and the same concentration of BSA, IgG, Lysozyme, VEGF, Thrombin, Uric acid, Glucose (50ng/mL) were added to the sensor, the fluorescence intensity was measured, and F/F0-1 (526nm) was compared, and the results are shown in FIG. 8. Even if the concentration of the interferent is 10 times of the concentration of the CEA, the fluorescence intensity cannot be obviously increased due to the addition of other substances, the change of the fluorescence intensity caused by the CEA with low concentration can still be distinguished from other substances, and the result shows that the detection of the CEA by the sensor can be distinguished from the detection of other substances, and the sensor has good specificity.
The technical features of the above embodiments can be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the above embodiments are not described, however, as long as there is no contradiction between the combinations of the technical features, the scope of the present description should be considered as being described in the present specification.
In addition, the above examples only express three embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the present invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.
And finally: the above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that are within the spirit and principle of the present invention are intended to be included in the scope of the present invention.
Claims (8)
1. A method for detecting carcinoembryonic antigen, comprising: the method comprises the following steps:
s1 preparation of GO-Fe3O4；
S2, modifying amino NH at the end of DNA sequence2Dissolving the modified DNA sequence and the modified polypeptide in Tris-HCl;
s3, dyeing the single-stranded DNA with SG II, and mixing the DNA with GO-Fe3O4Combining;
s4, mixing GO-Fe in S33O4Introducing polypeptide on the surface to remove non-specific adsorption;
s5, combining the GO-Fe with DNA and polypeptide in S4 under the action of external magnetic field3O4AdsorptionTo the bottom or the tube wall, eluting to remove the SG II not bound with DNA and the GO-Fe not bound3O4The polypeptide of (1);
s6, after fluorescence is quenched by FRET, CEA is added to the sensor prepared in S5;
s7, obtaining the linear relation between CEA and its nucleic acid aptamer;
s8, CEA detection;
in the above S1, 0.2-0.6g of GO is added into 40-80mL of ethylene glycol, the mixture is treated by ultrasonic wave until the solution is uniform, and then 0.5-0.8g of FeCl is added3Added to the solution and further sonicated for 8-12 min. Adding 2.2-3.0g of sodium acetate, stirring vigorously for 15-25min, transferring the mixture to an autoclave with polytetrafluoroethylene as a lining, heating at 160-200 ℃ for 8-12h, cooling to room temperature, and adding black product GO-Fe3O4Washed with ethanol and dried with a vacuum freeze dryer. To a solution containing 1mL NHS (50mM) and 1mL EDC (500mM) was added 2-6mg GO-Fe3O4Activating a carboxyl group;
in the above S2, the pH value range of Tris-HCl is set to 5-9;
in the above S3, the concentration range of SG ii is set to (0.4 ×, 0.6 ×, 0.8 ×, 1.0 ×, 1.2 ×, and 1.5 ×);
in addition, GO-Fe3O4The concentration ranges of (5, 10, 15, 20 and 25. mu.g/mL);
in the above S4, the concentration range of the polypeptide is set to (0, 25, 50, 75, 100, 125, 150. mu.g/mL);
in S6 above, the binding of CEA to the aptamer induces a conformational change in the aptamer, resulting in the restoration of fluorescence;
in the step S7, a series of CEA solutions with known concentrations are added into the solution obtained in the step S6, reaction is carried out at normal temperature, DNA in the solution obtained by the aptamer S6 is subjected to specific binding with CEA to induce conformational change, and therefore the DNA part with CEA fluorescence is far away from GO-Fe3O4To cause a certain degree of recovery of fluorescence, measuring the fluorescence value after the reaction is finished, and determining the fluorescence value based on the measured fluorescence valuePreparing a corresponding linear relation graph with the CEA;
above, the CEA solution concentration was set to (0.001, 0.01, 0.05, 0.1, 0.5, 0.8, 1 and 5 ng/mL);
and in the step S8, adding a certain amount of CEA with unknown concentration into the solution obtained in the step S6, reacting at normal temperature, measuring the fluorescence recovery value after the reaction is finished, and obtaining the concentration of the CEA according to the linear relation obtained in the step S7.
2. The method of claim 1, wherein the detection of carcinoembryonic antigen comprises: in S1, the initial GO amount is 0.4g, the ethylene glycol amount is 60mL, and FeCl is selected30.65g, 10min of ultrasonic treatment, 2.6g of sodium acetate, 20min of vigorous stirring after adding the sodium acetate, heating the reaction mixture in an autoclave with polytetrafluoroethylene as a lining at 180 ℃ for 10h, and subsequently adding GO-Fe3O4The amount of GO-Fe is 4mg after reaction3O4The concentration of (2) was 2 mg/mL.
3. The method of claim 1, wherein the detection of carcinoembryonic antigen comprises: in S2:
the sequence of the DNA is as follows: DNA: 5' -ATA CCA GCT TAT TCA ATT TTT TTT TTT T-NH2-3’；
The polypeptide is: EKEKEKEPPPPC, respectively;
the final concentration of the modified DNA sequence was 50nM in Tris-HCl buffer.
4. The method of claim 1, wherein the detection of carcinoembryonic antigen comprises: in S3, SYBR II is 1X SYBR II, DNA with fluorescence and GO-Fe3O4The bonding time is 30min, GO-Fe3O4The concentration of (2) was set to 15. mu.g/mL.
5. The method of claim 1, wherein the detection of carcinoembryonic antigen comprises: in S4, the concentration of the introduced polypeptide was set to 50. mu.g/mL, the total volume after the reaction was 500. mu.L, and the reaction conditions were overnight at 4 ℃.
6. The method of claim 1, wherein the detection of carcinoembryonic antigen comprises: in S5, the elution is performed by eluting with Tris-HCl to remove unbound substances, and measuring the fluorescence by pipetting 200. mu.L.
7. The method of claim 1, wherein the detection of carcinoembryonic antigen comprises: in S6, the incubation time was 30min and the reaction temperature was 4 ℃.
8. The method of claim 1, wherein the detection of carcinoembryonic antigen comprises: in S7, the reaction time was 30min at room temperature, and the pH of the CEA solution was set to 7.5.
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| CN117723748A (en) * | 2024-02-07 | 2024-03-19 | 首都医科大学附属北京天坛医院 | Immunomagnetic bead for enhancing target protein signal for targeted detection, and preparation method and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN117723748A (en) * | 2024-02-07 | 2024-03-19 | 首都医科大学附属北京天坛医院 | Immunomagnetic bead for enhancing target protein signal for targeted detection, and preparation method and application thereof |
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