CN117646004B - Label-free biosensor for myrobalan acid aptamer - Google Patents
Label-free biosensor for myrobalan acid aptamer Download PDFInfo
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Abstract
The invention discloses a myrobalan acid aptamer label-free biosensor, which comprises the following components: (1) A sequence of a chebula acid aptamer label-free biosensor; (2) And a signal reporter molecule of the chebula acid aptamer label-free biosensor. The detection principle of the sensor is as follows: when only an aptamer and Thioflavin T (ThT) exist in the system, the ThT can be embedded into a nucleic acid advanced structure, and the fluorescent intensity of the ThT-nucleic acid aptamer complex is higher; when the target myrobalan acid exists in the system, the fluorescence intensity of the THT-nucleic acid aptamer complex is reduced, and the higher the concentration of the myrobalan acid is, the lower the fluorescence intensity of the system is, so that the simple, convenient, quick, low-cost and high-sensitivity detection of the myrobalan acid is realized.
Description
Technical Field
The invention belongs to the field of biosensors, and particularly relates to a myrobalan acid aptamer label-free biosensor.
Background
Chebular acid is a plant extract with various biological activities, such as anti-inflammatory, antioxidant, antitumor, etc. In recent years, the physiological function and medicinal efficacy of myrobalan acid are greatly concerned, and the requirements for detecting the myrobalan acid also relate to different fields and applications, including drug research and development, drug and health care product research, environmental monitoring, quality control and the like.
The aptamer was first screened in 1990 in vitro by the technique of exponential enrichment of ligand system evolution (systematic evolution of ligands by exponential enrichment, SELEX) by Ellington and szostank. In the next thirty years of development, more and more nucleic acid aptamers were isolated that were able to bind target molecules in an affinity, specific manner. In terms of detection, nucleic acid aptamer biosensors have been developed rapidly in recent years, can generate output signals in a target response mode, and are stable in properties and convenient to transport and store. However, some aptamer sensors require modification of the signal output molecule, which can lead to increased costs and affect the folding kinetics of the nucleic acid. Thioflavin T (ThT) is a small fluorescent dye that can be incorporated into a particular nucleic acid conformation to yield higher fluorescence quantum yields, such as G-quadruplex, hairpin, or B-DNA duplex.
The invention provides a myrobalan acid aptamer obtained through SELEX screening, and a myrobalan acid aptamer label-free biosensor is successfully constructed based on the aptamer, so that quick, low-cost and ultrasensitive label-free fluorescence detection of myrobalan acid is finally realized.
Disclosure of Invention
Based on the above, the invention provides a myrobalan acid aptamer, and a myrobalan acid aptamer label-free biosensor is successfully constructed.
In one aspect, the present application provides a chebular acid aptamer.
The myrobalan acid aptamer has the sequence: 5'-CGGGCGGATGGGTCAGGAGCATGGAAGCTTGGGATTGCGGCTAGGGAAGGTGGCA-3', as shown in SEQ ID NO. 1, or a nucleotide sequence having at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 96%, at least 98% or at least 99% homology to the nucleotide sequence shown in SEQ ID NO. 1 and binding chebular acid, e.g. the nucleotide sequence shown in SEQ ID NO. 1 may be deleted for part of the sequence or added for part of the sequence.
In addition, it will be appreciated by those skilled in the art that, as an improvement over the above-described techniques, a modification may be made at a position on the nucleotide sequence of the above-described aptamer, e.g., phosphorylation, methylation, amination, sulfhydrylation, substitution of oxygen with sulfur, substitution of oxygen with selenium, or isotopicization, etc., provided that the thus modified aptamer sequence has desirable properties, e.g., may have an affinity for binding myrobalan acid equal to or greater than the parent aptamer sequence prior to modification, or, although the affinity is not significantly improved, has greater stability.
Thus, in some embodiments, the nucleotide sequence of the nucleic acid aptamer is modified and the modified nucleic acid aptamer binds to myrobalan acid, the modification selected from at least one of phosphorylation, methylation, amination, sulfhydrylation, substitution of oxygen with sulfur, substitution of oxygen with selenium, and isotopicization.
In another aspect, the invention also provides conjugates of chebular acid nucleic acid aptamers.
It will be appreciated by those skilled in the art that, as an improvement to the above-described technical scheme, at least one of a fluorescent substance such as FAM, a radioactive substance, a therapeutic substance, biotin, digoxin, a nano luminescent material, a small peptide, siRNA or an enzyme label, etc. may be attached to the nucleotide sequence of the above-described nucleic acid aptamer, provided that the nucleic acid aptamer sequence thus modified has desirable properties, for example, may have an affinity for binding myrobalan acid equal to or higher than that of the parent nucleic acid aptamer sequence before modification, or may have higher stability although the affinity is not significantly improved.
In other words, the above nucleic acid aptamer, whether partially substituted or modified, has substantially the same or similar molecular structure, physicochemical properties and functions as the original nucleic acid aptamer, and can be used for binding with chebulic acid.
Accordingly, the present invention provides a conjugate of a nucleic acid aptamer characterized in that it is a substance for labeling, detecting, diagnosing or treating attached to a nucleotide sequence according to the nucleic acid aptamer (as shown in SEQ ID NO: 1), and the conjugate of the nucleic acid aptamer after the substance is attached is bound to myrobalan acid, the substance being at least one of fluorescent markers such as FAM, radioactive substances, therapeutic substances, biotin, digoxin, nano luminescent materials, small peptides, siRNA and enzyme markers.
On the other hand, the nucleic acid aptamer (shown as SEQ ID NO: 1) or the conjugate thereof is applied to the development of a myrobalan acid detection method, the preparation of a kit, the research and development of medicines or the detection of environment.
In another aspect, the present application provides a label-free biosensor for myrobalan acid aptamer, which is characterized in that the label-free biosensor comprises: (1) A sequence of a chebula acid aptamer label-free biosensor; (2) A signal reporter molecule of the chebula acid aptamer label-free biosensor;
the myrobalan acid aptamer label-free biosensor sequence is as follows: 5'-CGGGCGGATGGGTCAGGAGCATGGAAGCTTGGGATTGCGGCTAGGGAAGGTGGCA-3', as shown in SEQ ID NO. 1;
the signal reporter molecule of the myrobalan acid aptamer label-free biosensor is a ThT fluorescent dye;
the concentration of the THT fluorescent dye in the solution of the myrobalan acid aptamer label-free biosensor is 2.5 mu mol.L -1 ；
The buffer solution of the myrobalan acid aptamer label-free biosensor is as follows: 10 mmol.L -1 Tris-HCl, 10 mmol·L -1 MgCl 2 , 20 mmol·L -1 KCl, 1 mmol·L -1 DTT, pH 7.4；
The detection of the myrobalan acid is realized by changing the intensity of the ThT fluorescent signal based on the addition of the myrobalan acid, and the concentration of the myrobalan acid in the solution and the ThT fluorescent value under specific excitation conditions show gradient change, so that the detection of the myrobalan acid is realized;
the specific standard curve establishment step:
different concentrations of chebular acid and 2.5 mu mol.L -1 ThT is added simultaneously to the mixture containing 1. Mu. Mol.L -1 Buffer solution of aptamer solution (10 mmol.L) -1 Tris-HCl, 10 mmol·L -1 MgCl 2 , 20 mmol·L -1 KCl, 1 mmol·L -1 DTT, pH 7.4) to final concentrations of 0,1,2,5, 10, 15, 20, 30, 50. Mu. Mol.L of chebular acid, respectively -1 Shaking and mixing at room temperature, and incubating for 1 min. Finally, the fluorescence intensity of ThT (excitation wavelength 443 nm, emission wavelength 477 nm) in the solution system was measured by a fluorescence spectrophotometer, and a standard curve was drawn based on the fluorescence value and the concentration of myrobalan acid. The result shows that the myrobalan acid aptamer label-free sensor is 1-50 mu mol.L -1 Has good linear relation (R) in the concentration range of myrobalan acid 2 =0.991), the linear regression equation is y= -1.260X + 248.6, the detection limit is as low as 8.29 nmol·l -1 。
On the other hand, the application of the biosensor in the development of a myrobalan acid detection method or in environmental detection is related.
In another aspect, the invention provides a kit, which is characterized in that the kit comprises a nucleic acid aptamer as shown in SEQ ID NO. 1 or a conjugate thereof.
Compared with the prior art, the invention has the beneficial effects that:
1. the invention provides a chebula acid aptamer obtained by SELEX screening, which has better affinity and an affinity constant of 2.770 mu mol.L -1 ；
2. The myrobalan acid aptamer label-free biosensor provided by the invention can realize quick, low-cost, stable and ultrasensitive myrobalan acid label-free detection, and has certain universality and industrialization potential;
3. the myrobalan acid aptamer label-free biosensor is 1-50 mu mol.L -1 Has good linear relation (R) in the concentration range of myrobalan acid 2 =0.991), the linear regression equation is y= -1.260X + 248.6, the detection limit is as low as 8.29 nmol·l -1 。
Drawings
FIG. 1 shows the secondary structure of chebula acid aptamer.
Fig. 2 is a graph of qualitative results of AuNPs colorimetric experiments.
Fig. 3 is a graph of the quantitative results of AuNPs colorimetric experiments.
FIG. 4 is a graph showing the results of determining the affinity constant of the myrobalan acid aptamer using an AuNPs colorimetric assay.
FIG. 5 shows the fluorescence signal of ThT at different myrobalan acid concentrations.
FIG. 6 is a standard graph of detection of chebular acid by an aptamer label-free sensor.
Detailed Description
The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Example 1 SELEX screening of chebula acid aptamer
The invention provides a method for screening myrobalan acid aptamer, which comprises the following steps:
(1) Double-stranded complex preparation
Mixing the random single-stranded DNA library with the fixed sequence, and putting the mixture into a PCR instrument for annealing (cooling from 95 ℃ to 25 ℃) to obtain a nucleic acid double-stranded complex;
(2) Target incubation
Adding target myrobalan acid into the double-chain complex system, and incubating to enable single-chain DNA specifically combined with the myrobalan acid to be dissociated from the double-chain complex to form a single-chain DNA-target complex;
(3) Endonuclease cleaves double strand
Adding endonuclease into the system after target incubation to cut the double-stranded complex, wherein the double-stranded complex cannot be amplified after cutting;
(4) PCR amplification
Performing PCR amplification by using the single-stranded DNA bound to the target as a template;
(5) Preparation of single strands
Recovering and purifying the amplified product of the step (4) and preparing single-stranded DNA (deoxyribonucleic acid) to obtain a secondary library;
(6) Multiple round screening
Replacing the random single-stranded DNA library in the step (1) with the secondary library obtained in the step (5), and repeatedly screening for a plurality of rounds according to the steps (1) to (5);
(7) High throughput sequencing
And after screening, carrying out high-throughput sequencing analysis on the obtained latest secondary library to obtain the alternative chebula acid aptamer to be tested.
Example 2 Structure prediction and affinity determination of chebula acid aptamer
Firstly, carrying out structure prediction on the chebula acid aptamer obtained by sequencing (shown as SEQ ID NO: 1). The secondary structure of the aptamer was predicted using RNAfold WebServer, and the results are shown in fig. 1, and it was found that the chebula acid aptamer obtained by screening contained a stem-loop structure.
Subsequently, the affinity of the myrobalan acid aptamer (shown as SEQ ID NO: 1) was verified by using a gold nanoparticle (AuNPs) colorimetric experiment. The AuNPs colorimetric experiment can verify the affinity of the DNA aptamer, and the principle is as follows: under the condition of high salt (NaCl), auNPs are agglomerated by the action of salt, and the color of the solution is changed from red to blue. The nucleic acid can be adsorbed on the surface of AuNPs through electrostatic action, so that the AuNPs are protected, and the AuNPs can not be aggregated in a high-salt solution to be red. Based on the above, the myrobalan acid aptamer can be adsorbed on AuNPs, and the AuNPs are stabilized under the high-salt condition; when the myrobalan acid exists in the system, the myrobalan acid can be specifically combined with the aptamer, so that the aptamer is separated from the surface of the AuNPs, and the free AuNPs aggregate to be blue when meeting salt. The affinity of the aptamer for the target was judged by measuring the absorbance at solution 525/626 nm.
The specific AuNPs colorimetric experiment steps are as follows:
5. mu L aptamer (1 mu mol. L) -1 ) And 5 mu L of myrobalan acid solution with different concentrations, incubating for 40 min, addingIncubate with 100 μl citrate coated AuNPs for 40 min. To the system was added 8. Mu.L of 0.5 mol.L -1 Sodium chloride solution, incubated for 15 min, photographed and absorbance was recorded simultaneously at 525 nm and 626 nm using a microplate reader. The data processing uses the ratio of absorbance at 626 and 525 nm to indicate the affinity of the chebular acid aptamer, with a high A525 value and a low A626 value in the dispersed state, i.e., a low A626/A525 value.
As shown in figures 2-3, auNPs colorimetric experiment results show that the chebular acid aptamer obtained through screening (shown as SEQ ID NO: 1) has good affinity with a target, and the A626/A525 value is increased along with the increase of the chebular acid concentration. Nonlinear fitting is carried out on the results of the AuNPs colorimetric experiment, and the affinity constant of the myrobalan acid aptamer (shown as SEQ ID NO: 1) is 2.770 mu mol.L -1 (FIG. 4).
Example 3 myrobalan acid aptamer label-free sensor sensitivity assessment
Because the G base content of the myrobalan acid aptamer (shown as SEQ ID NO: 1) exceeds 49 percent and has a hairpin and other advanced structures (shown in figure 1), thT is introduced as a fluorescent small molecule to construct the myrobalan acid aptamer label-free sensor. The specific detection principle is as follows: when only the aptamer and the ThT exist in the system, the ThT can be embedded into a nucleic acid advanced structure, and the fluorescent intensity of the ThT-nucleic acid aptamer complex is higher; when the target myrobalan acid exists in the system, the fluorescence intensity of the THT-nucleic acid aptamer complex is reduced, and the higher the concentration of the myrobalan acid is, the lower the fluorescence intensity of the system is.
And detecting the chebular acid with known concentration by using chebular acid nucleic acid aptamer (shown as SEQ ID NO: 1), and preparing a standard curve according to the change of the THT fluorescence value in the solution. Different concentrations of chebular acid and 2.5 mu mol.L -1 ThT is added simultaneously to the mixture containing 1. Mu. Mol.L -1 Buffer solution of aptamer solution (10 mmol.L) -1 Tris-HCl, 10 mmol·L - 1 MgCl 2 , 20 mmol·L -1 KCl, 1 mmol·L -1 DTT, pH 7.4) to final concentrations of 0,1,2,5, 10, 15, 20, 30, 50. Mu. Mol.L of chebular acid, respectively -1 Shaking and mixing at room temperature, and incubating for 1 min. Finally, fluorescence is utilizedThe spectrophotometer measures the fluorescence intensity of ThT (excitation wavelength 443 nm, emission wavelength 477 nm) in the solution system, and a standard curve is drawn based on the fluorescence value and the concentration of myrobalan acid.
As shown in FIG. 5, the myrobalan acid aptamer label-free sensor is 1-50 mu mol.L -1 In the range of the concentration of the myrobalan acid, the fluorescence signal of the myrobalan acid is reduced along with the increase of the concentration of the myrobalan acid, and the myrobalan acid has good detection trend. And the myrobalan acid aptamer label-free sensor is 1-50 mu mol.L -1 Has good linear relation (R) in the concentration range of myrobalan acid 2 =0.991), the linear regression equation is y= -1.260X + 248.6, the detection limit is as low as 8.29 nmol·l -1 (FIG. 6).
Claims (10)
1. A myrobalan acid aptamer, characterized in that the aptamer sequence is: 5'-CGGGCGGATGGGTCAGGAGCATGGAAGCTTGGGATTGCGGCTAGGGAAGGTGGCA-3', as shown in SEQ ID NO. 1.
2. A conjugate of a chebular acid aptamer, characterized in that the conjugate is a substance for labelling, detection linked to the nucleotide sequence of the nucleic acid aptamer according to claim 1, and the conjugate of the nucleic acid aptamer after linking the substance binds to chebular acid, the substance being a fluorescent label.
3. The conjugate of claim 2, wherein the fluorescent label is at least one of FAM, biotin, digoxigenin, a nano luminescent material, and/or an enzyme label.
4. A label-free biosensor of myrobalan acid aptamer, characterized in that the label-free biosensor comprises: (1) A sequence of a chebula acid aptamer label-free biosensor; (2) A signal reporter molecule of the chebula acid aptamer label-free biosensor;
the myrobalan acid aptamer label-free biosensor sequence is as follows: 5'-CGGGCGGATGGGTCAGGAGCATGGAAGCTTGGGATTGCGGCTAGGGAAGGTGGCA-3', as shown in SEQ ID NO. 1.
5. The myrobalan acid aptamer label-free biosensor according to claim 4, wherein the signal reporter molecule of the myrobalan acid aptamer label-free biosensor is ThT fluorescent dye; the concentration of the ThT fluorescent dye in the solution of the biosensor is 2.5 mu mol.L -1 The method comprises the steps of carrying out a first treatment on the surface of the The buffer solution of the biosensor is as follows: 10 mmol.L -1 Tris-HCl, 10 mmol·L -1 MgCl 2 , 20 mmol·L -1 KCl, 1 mmol·L -1 DTT, pH 7.4。
6. The method for quantitatively detecting chebular acid by using the chebular acid aptamer label-free biosensor according to claim 4, wherein a standard curve is established:
different concentrations of chebular acid and 2.5 mu mol.L -1 ThT is added simultaneously to the mixture containing 1. Mu. Mol.L -1 In the buffer solution of the aptamer solution, the final concentration of the myrobalan acid is respectively 0,1,2,5, 10, 15, 20, 30 and 50 mu mol.L -1 Shaking and mixing at room temperature, and incubating for 1 min; finally, the fluorescence intensity of the ThT in the solution system is measured by using a fluorescence spectrophotometer, wherein the excitation wavelength is 443 nm, the emission wavelength is 477 nm, and a standard curve is drawn based on the fluorescence value and the concentration of the myrobalan acid.
7. A kit comprising the nucleic acid aptamer of claim 1 or the conjugate of any one of claims 2 or 3.
8. Use of the aptamer of claim 1 or the conjugate of any one of claims 2 or 3 in the development of a myrobalan acid detection method, kit preparation, drug development or environmental detection.
9. Use of the biosensor of any one of claims 4 or 5 or the method of claim 6 in the development of a method for detecting myrobalan acid or in environmental detection.
10. The use of the kit of claim 7 in the development of a method for detecting myrobalan acid or in environmental detection.
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| WO2022054100A1 (en) * | 2020-09-12 | 2022-03-17 | Council Of Scientific And Industrial Research | Chebulinic acid and its enriched fraction from the fruits of terminalia chebula for treatment of benign prostatic hyperplasia (bph) and its preparation thereof |
| CN114414701A (en) * | 2022-01-28 | 2022-04-29 | 辽宁中医药大学 | Preparation method and content determination method of myrobalamin acid in myrobalan meat |
| CN114414702A (en) * | 2022-01-28 | 2022-04-29 | 辽宁中医药大学 | Preparation method and content determination method of chebulagic acid in chebula meat |
| CN114965751A (en) * | 2022-05-05 | 2022-08-30 | 天津中医药大学 | Method for measuring chemical component content in myrobalan |
| CN115627270A (en) * | 2022-12-22 | 2023-01-20 | 中国农业大学 | Label-free ratio type malachite green luminescence aptamer biosensor |
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| WO2022054100A1 (en) * | 2020-09-12 | 2022-03-17 | Council Of Scientific And Industrial Research | Chebulinic acid and its enriched fraction from the fruits of terminalia chebula for treatment of benign prostatic hyperplasia (bph) and its preparation thereof |
| CN114414701A (en) * | 2022-01-28 | 2022-04-29 | 辽宁中医药大学 | Preparation method and content determination method of myrobalamin acid in myrobalan meat |
| CN114414702A (en) * | 2022-01-28 | 2022-04-29 | 辽宁中医药大学 | Preparation method and content determination method of chebulagic acid in chebula meat |
| CN114965751A (en) * | 2022-05-05 | 2022-08-30 | 天津中医药大学 | Method for measuring chemical component content in myrobalan |
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