A kit for detecting AFP-L3% in human serum based on fluorescence resonance energy transfer, its detection method and application

Abstract

The present invention discloses a kit for detecting AFP-L3% in human serum based on fluorescence resonance energy transfer, as well as a detection method and application thereof. The kit includes a magnetic bead reagent, a nucleic acid aptamer reagent, a complementary DNA reagent, a lectin reagent, an AFP standard, an AFP-L3 standard, buffer solution I and buffer solution II. The specific detection method is to react the magnetic bead reagent with biotin-modified complementary DNA, and bind the nucleic acid aptamer reagent to AFP, mix the solutions, magnetically separate to remove the excess nucleic acid aptamer, and measure the fluorescence intensity of the remaining AFP-fluorescent probe-modified nucleic acid aptamer complex as PL1; react the obtained complex with the lectin modified with a fluorescence quencher, and measure the fluorescence intensity as PL2; by comparing the values of PL1 and PL2, AFP-L3% is calculated. The present invention has the advantages of small sample loss, rapid detection, high sensitivity, good selectivity, etc., and can improve the early detection rate of hepatocellular carcinoma.

Description

Technical Field

[0001] The present invention belongs to the technical field of biological detection, and particularly relates to a kit for detecting AFP-L3% in human serum based on fluorescence resonance energy transfer, a detection method thereof and an application thereof. Background Art

[0002] Hepatocellular carcinoma (HCC) is the sixth most common cancer in the world, with approximately 900,000 patients diagnosed each year. At the same time, liver cancer is also the third leading cause of cancer death globally, and only about 7% of advanced patients survive for more than 5 years. The early clinical symptoms of HCC patients are not obvious, and most patients are not discovered until the middle and late stages of hepatocellular carcinoma, resulting in patients missing the best treatment time. Therefore, to improve the curative effect and increase the survival rate of patients depends on improving the early diagnosis rate of HCC, which is also of great significance for the prognosis of patients.

[0003] Traditionally, researchers have detected liver cancer by using the total concentration of the serum biomarker - alpha-fetoprotein (AFP). Studies have shown that the concentration of total AFP in the serum of HCC patients continues to increase. However, this method has certain limitations, such as weak sensitivity and low specificity. AFP also increases to varying degrees in chronic liver diseases (chronic hepatitis, cirrhosis, etc.), and not all HCC patients have elevated AFP. Recent studies have shown that the AFP produced by patients with different liver diseases differs in sugar chains. According to the affinity for reacting with lentil lectin (LCA), AFP has three subtypes, AFP-L1, AFP-L2, and AFP-L3. AFP-L1 mainly comes from normal hepatocytes, AFP-L2 comes from pregnant women, and AFP-L3 mainly comes from cancerous hepatocytes, with a specificity for liver cancer as high as 95%. The US Food and Drug Administration (FDA) has approved the application of AFP-L3 analysis for the early warning of HCC. Taking 10% of the total AFP content as the positive critical value, exceeding this ratio indicates that the incidence of HCC exceeds 95%. Therefore, AFP-L3 can not only be used for the early diagnosis of HCC, but also for the judgment of disease prognosis and recurrence monitoring.

[0004] Currently, the conventional detection methods for AFP-L3 include affinity immunoelectrophoresis, affinity blotting, lectin enzyme-linked immunosorbent assay (lectin-ELISA), etc. Some of these methods have been applied clinically, and some are still in the research stage. However, the basic principle of their detection is mostly to separate AFP-L3 from AFP and then measure it. Since the detection sensitivity and repeatability still need to be further optimized, and the detection cost is relatively high, it is not suitable for large-scale market application. Based on the needs of clinical research and market application, there is an urgent need to develop a simple, rapid, and low-cost detection method for quantitative detection of AFP-L3.

[0005] The principle of fluorescence resonance energy transfer (FRET) is that when the fluorescence spectrum of the donor molecule partially overlaps with the excitation spectrum of the acceptor molecule, and the distance between the two is less than 10 nm, the excitation energy of the donor molecule induces the acceptor molecule to emit fluorescence, while the fluorescence of the donor molecule itself decays. Due to the high sensitivity, low background, and great application potential of FRET, several FRET proximity ligation-based strategies have been developed for detecting protein-specific glycosylation, such as detecting specific glycoforms of EGFR and MUC1. Currently, the FRET strategy has not been applied to the detection of AFP-L3, probably because lectins that recognize the signature core fucose of AFP-L3 are all large molecular proteins. Due to the large size of the labeling molecule itself, the distance between the donor and acceptor is greater than 10 nm, and FRET cannot occur. Pholiota squarrosa lectin (PhoSL) is a novel lectin with a small molecular weight and good specific recognition ability for core fucose. In addition, it is highly stable over a wide range of pH and temperature and highly soluble in various buffers. These advantages indicate that PhoSL is expected to become a powerful tool for analyzing AFP-L3 and a diagnostic reagent in the near future. Summary of the Invention

[0006] Object of the Invention: Aiming at the problems existing in the prior art, the present invention provides a kit for detecting AFP-L3% in human serum based on fluorescence resonance energy transfer. The kit has the advantages of small sample loss, rapid detection, high sensitivity, good selectivity, etc., and can improve the early detection rate of hepatocellular carcinoma.

[0007] The present invention also provides a detection method and application of a kit for AFP-L3% in human serum based on fluorescence resonance energy transfer.

[0008] Technical Solution: To achieve the above object, a kit for detecting AFP-L3% in human serum based on fluorescence resonance energy transfer according to the present invention includes a magnetic bead reagent, a nucleic acid aptamer reagent, a complementary DNA reagent, a lectin reagent, an AFP standard, an AFP-L3 standard, buffer solution I, and buffer solution II.

[0009] Further, the magnetic bead reagent is a streptavidin-modified magnetic microsphere solution, the size of the magnetic beads is between 0.1 - 10 μm, and the concentration of the magnetic bead solution is 5 - 10 mg / mL.

[0010] Preferably, the size of the magnetic beads is 1 μm.

[0011] Further, the nucleic acid aptamer reagent is an oligonucleotide chain solution labeled with a fluorescent probe and capable of specifically recognizing alpha-fetoprotein AFP, with a concentration of 10 - 100 μM, and the nucleic acid aptamer sequence is as shown in SEQ ID NO.1.

[0012] Furthermore, the complementary DNA reagent is a solution of biotin-modified oligonucleotide chains complementary to the nucleic acid aptamer, with a concentration of 10 - 100 μM, and the complementary DNA sequences are shown as any of SEQ ID NO.2 - SEQ ID NO.6.

[0013] Preferably, the complementary DNA reagent is a solution of biotin-modified oligonucleotide chains complementary to the nucleic acid aptamer, with a concentration of 10 μM, and the complementary DNA sequence is shown as SEQ ID NO.5.

[0014] Furthermore, the lectin reagent is a solution of a novel lectin PhoSL-Dabcyl modified with a fluorescence quencher, with a concentration of 200 - 800 ng / mL.

[0015] Furthermore, the fluorescent probe labeling the nucleic acid aptamer needs to match the fluorescence quencher labeling the lectin, and the fluorescent probe-fluorescence quencher is any one of FAM-BHQ1 or FAM-Dabcyl or Cy3-BHQ2 or Cy5-BHQ3.

[0016] Furthermore, the AFP standard product is a mixed solution of AFP-L1 and AFP-L3 with equal concentrations, with a total concentration of 100 - 300 ng / mL, and the AFP-L3 standard product is a mixed solution of AFP-L1 and AFP-L3 with different concentration ratios, with a total concentration of 5 - 10 ng / mL, and the proportion of AFP-L3 is 0 - 50%.

[0017] Preferably, the AFP standard product is a 1:1 mixed solution of AFP-L1 and AFP-L3, with a total concentration of 200 ng / mL, and the AFP-L3 standard product is a mixed solution of AFP-L1 and AFP-L3 with different concentration ratios, with a total concentration of 8 ng / mL, and the proportion of AFP-L3 is 0 - 50%.

[0018] Furthermore, the components of buffer solution I are 10 - 100 mM Tris-HCl, pH 7.5, 1 - 10 mM EDTA, 1 - 10 M NaCl, 0.01% - 0.1% Tween-20, and the components of buffer solution II are 10 - 100 mM PBS, 5 - 10 g / L glucose, 5 - 10 mmol / L MgCl2, 0.1 - 1 g / L yeast tRNA, 100 - 1000 mL / L fetal bovine serum, 1 - 10 g / L bovine serum albumin.

[0019] The method for the kit of the present invention to detect the AFP-L3% in human serum specifically includes the following steps:

[0020] (1) Add buffer solution I to the magnetic bead reagent (streptavidin-modified magnetic beads, MB-SA), wash and separate, discard the supernatant, add the complementary DNA reagent (biotinylated complementary DNA (C-DNA-Biotin)), shake and suspend, separate, discard the supernatant, to form a complementary DNA magnetic bead (C-DNA-MB complex). After magnetic separation and washing with buffer solution I, a pure C-DNA-MB complex is obtained;

[0021] (2) Add the aptamer reagent (Aptamer-FL) to the AFP standard and buffer solution II to obtain an AFP-Aptamer-FL complex solution. In addition, the solution also contains an excess of free Aptamer-FL;

[0022] (3) Mix all the solids and solutions obtained in steps (1) and (2). The free Aptamer-FL and C-DNA-MB are synthesized into an Aptamer-FL-C-DNA-MB complex. After magnetic separation, the Aptamer-FL-C-DNA-MB complex is removed to obtain a solution containing only the AFP-Aptamer-FL complex;

[0023] (4) Perform fluorescence spectroscopy on the solution obtained in step (3), and the measured fluorescence intensity is PL1;

[0024] (5) Add the lectin reagent (PhoSL-FQ) to the solution obtained in step (3);

[0025] (6) Perform fluorescence spectroscopy on the solution obtained in step (5), and the measured fluorescence intensity is PL2;

[0026] (7) By comparing the PL1 and PL2 values, calculate that the AFP-L3% in the serum to be tested = (PL1 - PL2) / PL1.

[0027] Among them, the magnetic bead reagent is a streptavidin-modified magnetic bead solution, the aptamer reagent is an oligonucleotide chain solution specifically recognizing alpha-fetoprotein AFP labeled with a fluorescent probe, the complementary DNA reagent is an oligonucleotide chain solution modified with biotin and complementary to the aptamer, and the lectin reagent is a novel lectin PhoSL solution modified with a fluorescence quencher.

[0028] When using this kit to detect AFP-L3%, first react streptavidin-modified magnetic beads with biotin-modified complementary DNA, and bind the aptamer reagent to AFP in the serum to be tested. Mix the above two solutions, and use the principle of nucleic acid complementary pairing and magnetic separation to remove the excess aptamer. The fluorescence intensity of the remaining AFP-fluorescent probe-modified aptamer complex is measured as PL1. Further, react the obtained complex with the lectin modified with a fluorescence quencher to cause fluorescence resonance energy transfer, and measure the fluorescence intensity as PL2. By comparing the values of PL1 and PL2, AFP-L3% = (PL1 - PL2) / PL1 is calculated.

[0029] Application of the kit for detecting AFP-L3% in human serum based on fluorescence resonance energy transfer in the early detection of hepatocellular carcinoma.

[0030] Application of the detection method of the detection kit of the present invention in the early detection of hepatocellular carcinoma.

[0031] Application of the detection kit of the present invention in the preparation of tools or reagents for detecting AFP-L3% in human serum or liver cancer cells.

[0032] Currently, the conventional detection methods for AFP-L3 include affinity immunoelectrophoresis, affinity blotting, lectin enzyme-linked immunosorbent assay (lectin-ELISA), etc. Some of these methods have been applied clinically, and some are still in the research stage. However, the fundamental principle of their detection is mostly to separate AFP-L3 from AFP and then measure it. Since the detection sensitivity and repeatability still need to be further optimized, and at the same time the detection cost is relatively high, it is not suitable for large-scale market application. Based on the needs of clinical research and market application, there is an urgent need to develop a simple, rapid, and low-cost detection method for quantitative detection of AFP-L3.

[0033] In the prior art, there is no detection method for AFP-L3% based on the principle of fluorescence resonance energy transfer. The main reason is that the lectins used in the conventional methods mainly include LCA, ConA, etc. These lectins are all macromolecular proteins with relatively large sizes, which cannot meet the requirements of fluorescence resonance energy transfer for the distance between the fluorescence donor and the receptor (<10 nm). In addition, the relatively large recognition molecules also bring relatively large steric hindrance, resulting in a decrease in the reaction and detection efficiency. In the present invention, PhoSL lectin is innovatively used. This lectin only contains 40 amino acids and has a size of about 3 - 4 nm, which meets the requirements of fluorescence resonance energy transfer and has relatively small steric hindrance, and can effectively improve the detection efficiency.

[0034] Principle of the invention: This application provides a method for detecting AFP-L3 based on the principle of fluorescence resonance energy transfer. The donor molecule (fluorescent probe) is linked to the target protein AFP, and the acceptor molecule (fluorescent quencher) is linked to the sequence of α-linked mannose residues on the alpha-fetoprotein isoform. By detecting the change in fluorescence intensity, AFP-L3 is quantitatively detected. Magnetic beads and complementary strand DNA are linked through the interaction between streptavidin and biotin.

[0035] An aptamer is selected to recognize AFP. Compared with antibodies, aptamers have the following advantages: (1) High specificity, with an affinity for the target molecule equivalent to or even higher than that of antibodies; (2) Aptamers are easier to obtain than antibodies and can be synthesized in vitro in large quantities and quickly, and the preparation method is simpler; (3) They can be screened for different types of target molecules, broadening their application scope; (4) They have better stability than antibodies, which is beneficial for storage. (5) Small steric hindrance, which is more conducive to fluorescence resonance energy transfer. A lectin is selected to recognize the sequence of α-linked mannose residues on the alpha-fetoprotein isoform, and the type of lectin is controlled so that the lectin can effectively capture AFP-L3 and improve the accuracy of subsequent detection.

[0036] This method relies on the competitive reaction between complementary strand DNA-aptamer and aptamer-target. By designing complementary strands of different lengths, the complementary strands cannot compete with the aptamers that bind to the target. An excessive amount of aptamer is first mixed with the target, and then an appropriate amount of complementary strand is added. After screening, the complementary strand will bind to the free aptamer and form a precipitate together with the magnetic beads. The concentration of aptamer-fluorescent probe in the solution is equivalent to the concentration of the target. Finally, a lectin modified with a quenching group is added to specifically recognize AFP-L3. After sufficient reaction, the fluorescence intensity of the solution is significantly quenched, and finally the AFP-L3% is calculated.

[0037] Beneficial effects: Compared with the prior art, the present invention has the following advantages:

[0038] (1) The present invention first proposes to use a fluorescence analysis method based on fluorescence resonance energy transfer to detect AFP-L3% in human serum, and creatively uses PhoSL lectin. This lectin contains only 40 amino acids and has a size of about 3-4 nm, meeting the requirements of fluorescence resonance energy transfer and having a small steric hindrance, which can effectively improve the detection efficiency.

[0039] (2) The present invention has the advantages of less sample consumption, simple detection, good selectivity, high sensitivity and high specificity.

[0040] (3) The present invention uses products such as aptamers and lectins to recognize the target, and has the advantages of high specificity and good selectivity.

[0041] (4) The present invention can be directly used to detect AFP-L3% in serum. Without separating AFP and AFP-L3, the detection of AFP-L3% can be completed. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 It is a method for detecting AFP-L3% in human serum by the kit of the present invention;

[0043] Figure 2 It is an AFP standard curve;

[0044] Figure 3 It is an AFP-L3% standard curve;

[0045] Figure 4 In 4A, it is the correlation curve of detecting AFP-L3% in human serum by the detection method established by the present invention and detecting AFP-L3% by ELISA of the kit; in 4B, it is the detection result chart of AFP-L3% in the sera of normal people, patients with liver cirrhosis, and patients with liver cancer;

[0046] Figure 5 Fluorescence reaction comparison chart of complementary sequences. DETAILED DESCRIPTION OF THE INVENTION

[0047] Drugs and reagents: All DNAs used in the experiment were synthesized by Sangon Biotech (Shanghai). The magnetic bead reagent MB-SA was purchased from Shanghai Aladdin S8040-A1μm-1EA; the lectin reagent was purchased from Guoping Pharmaceutical; both AFP-L1 and AFP-L3 were purchased from Shanghai Enzyme-linked Biotechnology, with the numbers ml023557 and ml058717 respectively; yeast tRNA was purchased from Shanghai Merck Sigma, with the number 9014-25-9.

[0048] Buffer solution I and buffer solution II were prepared in the laboratory.

[0049] Example 1

[0050] A kit for determining AFP-L3% in human serum, the kit includes a magnetic bead reagent, a nucleic acid aptamer reagent, a complementary DNA reagent, a lectin reagent, an AFP standard, an AFP-L3 standard, buffer solution I and buffer solution II.

[0051] The magnetic bead reagent is a streptavidin-modified magnetic bead solution, the magnetic bead size is 1μm, and the concentration of the magnetic bead solution is 10mg / mL;

[0052] The nucleic acid aptamer reagent is an oligonucleotide chain solution specifically recognizing alpha-fetoprotein AFP labeled with a fluorescent probe, wherein the nucleic acid aptamer sequence is shown in Table SEQ ID NO.1, and the concentration of the nucleic acid aptamer is 10μM;

[0053] The complementary DNA reagent is a solution of biotinylated oligonucleotide chains complementary to the nucleic acid aptamer. The concentration of the complementary DNA reagent is 10 μM, and the complementary DNA sequence is as shown in SEQ ID NO.5. Other complementary DNA sequences SEQ ID NO.2 - SEQ ID NO.4 and SEQ ID NO.6 can also be used to construct the complementary DNA reagent according to the above method.

[0054] The lectin reagent is a solution of a novel lectin PhoSL modified with a fluorescence quencher, with a concentration of 200 ng / mL. The fluorescent probe labeled with the nucleic acid aptamer needs to match the fluorescence quencher labeled with the lectin. That is to say, the fluorescence emission wavelength range of the fluorescent probe needs to be within the quenching wavelength range of the fluorescence quencher. Among them, the fluorescence probe - fluorescence quencher FAM - Dabcyl combination is selected, that is, FAM labels the nucleic acid aptamer, and Dabcyl labels the lectin PhoSL. The sequence of Dabcyl - labeled lectin PhoSL is as shown in SEQ ID NO.7. Other fluorescence probe - fluorescence quencher pairs such as FAM - BHQ1, FAM - Dabcyl, Cy3 - BHQ2, and Cy5 - BHQ3 can also be used to construct the lectin reagent according to the above method.

[0055] The AFP standard is a mixed solution of AFP - L1 and AFP - L3 with a concentration ratio of 1:1, and its initial concentration is 200 ng / mL (subsequently diluted to different concentrations for use); the AFP - L3% standard is a mixed solution of AFP - L1 and AFP - L3 with different concentration ratios. The total concentration of AFP - L1 and AFP - L3 is 8 ng / mL, and the concentration ratio of AFP - L3 ranges from 0 - 50% (i.e., 0 - 4 ng / mL); the buffer solution I component is 10 mM Tris - HCl, pH 7.5, 1 mM EDTA, 1 M NaCl, 0.01% - 0.1% Tween - 20; the buffer solution II component is 10 mM PBS, 4.5 g / L glucose, 5 mM MgCl2, 0.1 g / L yeast tRNA, 100 mL / L fetal bovine serum, 1 g / L bovine serum albumin.

[0056] Table 1 Nucleic Acid Sequence Table

[0057]

[0058] Example 2

[0059] A method for detecting AFP - L3% in human serum based on the said kit, which is composed of the kit in Example 1.

[0060] As Figure 1 shown, in the first step, the preparation of the complementary DNA magnetic bead (C - DNA - MB) complex:

[0061] Take 10 μL of streptavidin-modified magnetic beads (magnetic bead size is 1 μm, magnetic bead solution concentration is 10 mg / mL) MB-SA and fully suspend it. It can be vortexed on a mixer for 20 s. Transfer the resuspended liquid MB-SA to a new EP tube, place it on a magnetic stand, perform magnetic separation, and discard the supernatant. Add 200 μL of buffer solution I (10 mM Tris-HCl, pH 7.5, 1 mM EDTA, 1 M NaCl, 0.01%-0.1% Tween-20), fully wash the magnetic beads, perform magnetic separation, and discard the supernatant. Add 10 μL of 10 μM complementary DNA reagent (the sequence is as shown in SEQ ID NO.5, biotinylated complementary strand DNA (C-DNA-Biotin)), fully shake and suspend it, shake at room temperature for 3 h, perform magnetic separation, and discard the supernatant to form a complementary DNA magnetic bead (C-DNA-MB) complex. After three magnetic separations and washing with buffer solution I of Example 1, a pure C-DNA-MB complex solid is obtained;

[0062] Step 2, fluorescent probe-modified nucleic acid aptamer labels the target alpha-fetoprotein AFP:

[0063] Take 5 μL of nucleic acid aptamer reagent (Aptamer-FL) (10 μM) solution, add the AFP standard product (10 μL) prepared in Example 1 and 185 μL of buffer solution II (10 mM PBS, 4.5 g / L glucose, 5 mmol / L MgCl2, 0.1 g / L yeast tRNA, 100 mL / L fetal bovine serum, 1 g / L bovine serum albumin), incubate at 4°C for 3 h. Utilize the specific recognition between Aptamer-FL and AFP to obtain an AFP-Aptamer-FL complex solution, and the complex solution also contains excessive free Aptamer-FL;

[0064] Step 3, competitive reaction between the aptamer and the target and between it and the complementary strand

[0065] Mix all the solids and solutions obtained in Steps 1 and 2, resuspend, incubate at 4°C for 2 h. The free Aptamer-FL prepared in Step 2 and the C-DNA-MB prepared in Step 1 form double-stranded DNA through base complementary pairing. After magnetic separation, the precipitated Aptamer-FL-C-DNA-MB complex is removed, and the supernatant is retained. After adding 50 μL of buffer solution II, a solution containing only the AFP-Aptamer-FL complex is obtained.

[0066] Step 4, fluorescence detection

[0067] Perform fluorescence spectroscopy on the solution obtained in Step 3. The excitation wavelength is 495 nm, and the fluorescence intensity at 520 nm is measured as PL1.

[0068] Step 5: Lectin specifically recognizes core fucose on AFP

[0069] Add 50 μL of lectin reagent PhoSL-DabcyL solution (200 ng / mL) to the solution (200 μL) obtained in Step 3, mix well, incubate at 37 °C for 1 h. The lectin PhoSL specifically binds to the core fucose on AFP-L3, and the fluorescence probe bound to AFP-L3 undergoes fluorescence resonance energy transfer with the fluorescence quencher, resulting in fluorescence quenching.

[0070] Step 6: Fluorescence detection

[0071] Perform fluorescence spectroscopy on the solution obtained in Step 5. The excitation wavelength is 495 nm, and the fluorescence intensity at 520 nm is measured as PL2.

[0072] Step 7: Calculation of AFP or AFP-L3%

[0073] By comparing the values of PL1 and PL2, calculate that AFP-L3% in the serum to be tested = (PL1 - PL2) / PL1.

[0074] Step 8: Establishment of AFP standard curve

[0075] Dilute the AFP standard solution prepared in Example 1 in different proportions so that the final concentrations in the system of Step 2 are 0.2, 10, 40, 80, 100, 150 ng / mL. Use the above Steps 1 to 4 to measure the corresponding PL1 values for each concentration. Prepare 3 replicate samples in parallel for each proportion solution and perform measurements. With the concentration as the abscissa and the PL1 value as the ordinate, draw a standard curve according to the fluorescence results of the gradient AFP standard solution, as Figure 2 shown. The detection range is 0.1 - 100 ng / mL, and the detection limit is 0.66 ng / mL.

[0076] Step 8: Establishment of AFP-L3% standard curve

[0077] Keep the total AFP concentration unchanged, change the concentration ratio of AFP-L1 and AFP-L3 (the proportion of AFP-L3% concentration is 3.13%, 6.25%, 12.5%, 25%, 50% respectively). Use the above Steps 1 to 7 to measure the corresponding PL1 and PL2 values for each proportion solution. Prepare 3 replicate samples in parallel for each concentration and perform measurements. With AFP-L3% as the abscissa and (PL1 - PL2) / PL1 value as the ordinate, draw a standard curve according to the fluorescence results of the gradient AFP-L3% standard solution. As Figure 3 shown, and then calculate AFP-L3% in the serum sample to be tested according to the standard curve.

[0078] Example 3

[0079] Application of the Kit and AFP-L3% Testing Method in the Detection of Liver Diseases

[0080] Serum samples of liver cancer patients, liver cirrhosis patients and normal people were collected. Numbers 1-10 respectively represent: numbers 1-4 represent normal people (Healthy), numbers 5-7 (BLD) represent liver cirrhosis patients, and numbers 8-10 (HCC) represent liver cancer patients. The AFP-L3% of each serum solution was measured by the AFP-L3% detection method in Example 2 and the standard ELISA method respectively, and the measurement results were compared. The obtained results are as Figure 4 shown, and the linear curve is as Figure 4 shown in A. The results measured by both show a high degree of consistency, indicating the authenticity and reliability of the present invention. By comparing the AFP-L3% test results of normal people (Healthy), liver cirrhosis patients (BLD), and liver cancer patients (HCC), it was found that the AFP-L3% of liver cancer patients exceeded the critical value of 10%, and the AFP-L3% of liver cirrhosis patients and healthy people were both lower than 10%, as Figure 4 shown in B, indicating the authenticity and reliability of the present invention. The test results of the present invention show a high degree of consistency with the test results of ELISA kits on the market, indicating the accuracy of the present invention. By comparing the AFP-L3% test results of serum samples of normal people, liver cirrhosis patients, and liver cancer patients, it was found that the AFP-L3% of liver cancer patients exceeded the critical value of 10%, and the AFP-L3% of liver cirrhosis patients and healthy people were both lower than 10%

[0081] The ELISA kit can only detect the concentration of AFP-L1 or AFP-L3 individually, and finally the AFP-L3% can be obtained by comparison. As long as the linear curve is known in the method of the present invention, the AFP-L3% can be known. In summary, the kit of the present invention can be used to accurately measure the AFP-L3% in human serum, providing an effective reference for the early detection rate of liver cancer.

[0082] Example 4

[0083] Optimization of Complementary DNA Sequences SEQ ID NO.2-SEQ ID NO.6

[0084] Form double-stranded complexes of Aptamer-FAM and C-DNA of different lengths by molecular hybridization. Using the method of Example 2, different complementary DNA reagents with sequences shown in SEQ ID NO.2 - SEQ ID NO.6 were used to prepare different solid C-DNA-MB complexes. Take 5 μL of 50 pmol nucleic acid aptamer reagent Aptamer-FL and 2 ng / mL AFP standard (10 μL) and mix them in 185 μL of Buffer II, with a volume of 200 μL. Then carry out a competitive reaction with C-DNA-MB in Step 3. After incubating at room temperature for 60 minutes, perform magnetic separation and take the supernatant to measure the fluorescence intensity of the reaction system supernatant. The excitation wavelength is 495 nm, and the emission wavelength range is from 500 nm to 650 nm. The positive control is an equal amount of Aptamer-FL, showing a significantly high fluorescence signal. Theoretically, there will be two situations after mixing with magnetic beads. One is F Aptamer-C-DNA <F Aptamer-AFP , in which the complementary strand only binds to the free Aptamer, forming a precipitate. The other is F Aptamer-C-DNA >F Aptamer-AFP , so C-DNA binds to both the free Aptamer and competes for the Aptamer of AFP-Aptamer-FL, and only AFP is in the supernatant, with weak or no fluorescence. From the fluorescence results as Figure 5 shown, the complementary DNA sequences SEQ ID NO.5, SEQ ID NO.6 (C-DNA-11, 12) are consistent with the first situation above, and SEQ ID NO.2 - SEQ ID NO.4 (C-DNA-13, C-DNA-14, C-DNA-15) are consistent with the second situation above. This indicates that SEQ ID NO.5 (C-DNA-12) can not only maintain a stable double-stranded complex but also is the minimum value insufficient to trigger a competitive reaction.

Claims

1. A kit for detecting AFP-L3% in human serum based on fluorescence resonance energy transfer, characterized in that, It includes magnetic bead reagent, nucleic acid aptamer reagent, complementary DNA reagent, lectin reagent, AFP standard, AFP-L3 standard, buffer solution I and buffer solution II; The nucleic acid aptamer reagent is a solution of oligonucleotide chains specifically recognizing alpha-fetoprotein AFP labeled with a fluorescent probe, with a concentration of 10 - 100 μM, and the nucleic acid aptamer sequence is as shown in SEQ ID NO.1; The complementary DNA reagent is a solution of oligonucleotide chains complementary to the nucleic acid aptamer modified with biotin, with a concentration of 10 - 100 μM, and the complementary DNA sequence is any one of SEQ ID NO.2 - SEQ ID NO.6; The lectin reagent is a solution of a novel lectin PhoSL-Dabcyl modified with a fluorescence quencher, with a concentration of 200 - 800 ng / mL.

2. The kit for detecting human serum AFP-L3% based on fluorescence resonance energy transfer according to claim 1, wherein, The magnetic bead reagent is a solution of magnetic microbeads modified with streptavidin, the size of the magnetic beads is between 0.1 - 10 μm, and the concentration of the magnetic bead solution is 5 - 10 mg / mL.

3. The kit for detecting AFP-L3% in human serum based on fluorescence resonance energy transfer according to claim 1, wherein The fluorescent probe labeling the nucleic acid aptamer reagent needs to match the fluorescence quencher labeling the lectin, and the fluorescent probe-fluorescence quencher is any one of FAM-BHQ1, FAM-Dabcyl, Cy3-BHQ2 or Cy5-BHQ3.

4. The kit for detecting AFP-L3% in human serum based on fluorescence resonance energy transfer according to claim 1, wherein The AFP standard is an equiconcentration mixed solution of AFP-L1 and AFP-L3, with a total concentration of 100 - 300 ng / mL, and the AFP-L3 standard is a mixed solution of AFP-L1 and AFP-L3 with different concentration ratios, with a total concentration of 5 - 10 ng / mL, and the proportion of AFP-L3 is 0 - 50%.

5. The kit for detecting AFP-L3% in human serum based on fluorescence resonance energy transfer according to claim 1, wherein The components of buffer solution I are 10 - 100 mM Tris-HCl, pH 7.5, 1 - 10 mM EDTA, 1 - 10 M NaCl, 0.01% - 0.1% Tween-20, and the components of buffer solution II are 10 - 100 mM PBS, 5 - 10 g / L glucose, 5 - 10 mmol / L MgCl2, 0.1 - 1 g / L yeast tRNA, 100 - 1000 mL / L fetal bovine serum, 1 - 10 g / L bovine serum albumin.

6. A method for detecting AFP-L3% in human serum using the kit according to claim 1, characterized in that, Specifically, it includes the following steps: (1) Add buffer solution I to the magnetic bead reagent (MB-SA), wash and separate, discard the supernatant, add the complementary DNA reagent (C-DNA-Biotin), shake and suspend, separate, discard the supernatant, to form a C-DNA-MB complex, and after magnetic separation and washing with buffer solution I, obtain a pure C-DNA-MB complex; (2) Add the nucleic acid aptamer reagent (Aptamer-FL) to the AFP standard and buffer solution II to obtain an AFP-Aptamer-FL complex solution. In addition, the solution also contains an excess of free Aptamer-FL; (3) Mix all the solids and solutions obtained in steps (1) and (2). The free Aptamer-FL and C-DNA-MB synthesize the Aptamer-FL-C-DNA-MB complex. After magnetic separation to remove the Aptamer-FL-C-DNA-MB complex, a solution containing only the AFP-Aptamer-FL complex is obtained. (4) Perform fluorescence spectroscopy on the solution obtained in step (3), and the measured fluorescence intensity is PL1. (5) Add a lectin reagent to the solution obtained in step (3). (6) Perform fluorescence spectroscopy on the solution obtained in step (5), and the measured fluorescence intensity is PL2. (7) By comparing the values of PL1 and PL2, calculate that the AFP-L3% in the serum to be tested is (PL1 - PL2) / PL1.

7. Use of the kit according to any one of claims 1-5 in the preparation of a reagent for early detection of hepatocellular carcinoma.

Images