CN112251493B - Substance detection method based on fluorescence resonance energy transfer and exonuclease-assisted cyclic amplification strategy - Google Patents

Substance detection method based on fluorescence resonance energy transfer and exonuclease-assisted cyclic amplification strategy Download PDF

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CN112251493B
CN112251493B CN202011139606.5A CN202011139606A CN112251493B CN 112251493 B CN112251493 B CN 112251493B CN 202011139606 A CN202011139606 A CN 202011139606A CN 112251493 B CN112251493 B CN 112251493B
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周楠迪
陈金日
田亚平
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Abstract

The invention discloses a substance detection method based on a fluorescence resonance energy transfer and exonuclease-assisted cyclic amplification strategy, and belongs to the technical field of analytical chemistry. According to the invention, sialic acid is captured by the aptamer, so that a signal probe complementary with the aptamer is released, the signal probe can be combined with a nucleic acid molecular beacon in a fluorescence resonance energy transfer system, meanwhile, the signal probe can also be combined with a hairpin structure nucleic acid probe Hp1, and when exonuclease III is added, the signal probe promotes the nucleic acid molecular beacon to be enzyme-cut with the hairpin structure nucleic acid probe, so that a large number of quantum dot fluorescent signals are recovered, the detection range of the sensor is expanded, and the detection sensitivity is improved. Compared with the traditional sialic acid detection method, the method has the advantages of strong specificity and high sensitivity.

Description

Substance detection method based on fluorescence resonance energy transfer and exonuclease-assisted cyclic amplification strategy
Technical Field
The invention relates to a substance detection method based on a fluorescence resonance energy transfer and exonuclease-assisted cyclic amplification strategy, belonging to the technical field of analytical chemistry.
Background
Sialic acid is also called cubilose acid, is a nine-carbon sugar, has a main component with biological activity in cubilose which is a traditional rare food in China, and provides immunity for the early growth of infants and improves the absorption capacity of intestinal tracts on vitamins and minerals. Sialic acid is neuraminic acid, widely exists in animal tissues and microorganisms, is an important component of cell membrane protein, participates in various physiological functions of cell surfaces, and plays an important role in regulating physiological and biochemical functions of human bodies. Therefore, the development of methods and analytical techniques for detecting sialic acid is of great interest for the detection and diagnosis of sialic acid or sialic acid modified polysaccharides/lipids/proteins. At present, the sialic acid has been studied by high performance liquid chromatography, nuclear magnetic resonance, mass spectrometry, lectin precipitation, lectin affinity chromatography, lectin affinity electrophoresis, lectin enzyme-linked immunosorbent assay, lectin blotting, agglutination, lectin-coated magnetic beads, fluorescence-labeled lectin flow cytometry, and the like. These methods generally have the following disadvantages: before a sample is measured, pretreatment such as separation and purification of the sample is needed, only sugar extracted from cells can be measured, the structure of glycoprotein is easy to change or lose in the extraction process, only a few lectins can be used for detection in a single experiment, if a plurality of lectins are used for detection, multiple reactions are needed, the operation is complicated, sialic acid cannot be analyzed at one time, and the like. In addition, the methods have high requirements on the professional degree of operators, high cost, low accuracy and high detection limit, and influence the detection result. Based on the current research situation, the development of new chemical labeling and analytical detection technologies for sialic acid or sialic acid modified polysaccharide/lipid/protein is urgently needed.
Biosensing analysis has received much attention because of its advantages such as high selectivity and high sensitivity. The biosensor includes a molecular recognition part that recognizes a target and then converts a biological signal into an electrical signal, a fluorescent signal, or other signal, and a signal conversion part. In recent years, with the development of molecular biology and biosensor technologies, various aptamers and Fluorescence Resonance Energy Transfer (FRET) -based biosensors have been designed for detecting biological targets. The adapter has obvious advantages as an identification element: the method is not limited by immune conditions and immunogenicity, has wide target range, can be artificially synthesized in vitro, has mature technology, relatively low cost, reversible denaturation and renaturation, is easy to carry out various chemical modifications, is easy for high-throughput preparation, is easy for long-term storage and the like. These properties make aptamers widely used in the biomedical research field.
Quantum dots are a class of spherical-like nanomaterials, typically composed of semiconductor materials. Generally, the diameter is in the range of 1-100 nm, and the nano-particles are relatively stable nano-particles. The quantum dots have good fluorescence property, long fluorescence life and good stability. Even if the laser is irradiated at high intensity for a long time, the optical characteristics of the quantum dots are not significantly changed. The quantum dots are combined with DNA, so that the constructed biosensor has the advantages of simple test method, good detection selectivity, high sensitivity and the like. However, even when quantum dots are combined with biosensors based on aptamers and fluorescence resonance energy transfer techniques, there still remains a problem of insufficient sensitivity. There is currently no biosensor available that can be used to detect sialic acid with great sensitivity.
Disclosure of Invention
In order to solve the problems, the invention provides a substance detection method based on a fluorescence resonance energy transfer and exonuclease-assisted cyclic amplification strategy, which has the advantages of high sensitivity, high specificity, accurate measurement and the like.
The first purpose of the invention is to provide a substance detection method based on a fluorescence resonance energy transfer and exonuclease-assisted cycle amplification strategy, which comprises the following steps:
s1, selecting an aptamer matched with a target substance, and constructing an aptamer-based nucleic acid molecule hybridization system, wherein the nucleic acid molecule hybridization system comprises a capture probe and a signal probe which form a complementary structure with the aptamer;
s2, adding a sample to be detected into the nucleic acid molecule hybridization system in the step S1, releasing a signal probe, and separating a free signal probe;
s3, constructing a fluorescence resonance energy transfer system based on quantum dots and a quencher, wherein the fluorescence resonance energy transfer system based on the quantum dots and the quencher is obtained by respectively incubating three nucleic acid molecule beacons capDNA1, capDNA2 and capDNA3 containing the quencher with the quantum dots, and one nucleic acid molecule beacon capDNA1 can be complementarily hybridized with a signal probe;
s4, adding the signal probe separated in the step S2 into the fluorescence resonance energy transfer system in the step S3, adding two hairpin structure nucleic acid probes Hp1 and Hp2, adding exonuclease III for enzyme digestion reaction, and starting multi-stage fluorescence signal amplification; the single-stranded ssDNA1 can be complementarily hybridized with a nucleic acid molecular beacon capDNA2 and complementarily hybridized with another hairpin structure nucleic acid probe Hp2, the single-stranded ssDNA2 can be complementarily hybridized with a nucleic acid molecular beacon capDNA3, and the single-stranded ssDNA2 can be complementarily hybridized with a nucleic acid molecular beacon capDNA 3;
and S5, calculating the content of the target substance in the sample to be detected by detecting the intensity of the fluorescence signal.
Furthermore, the quantum dot is a water-soluble quantum dot modified by streptavidin, carboxyl or amino.
Furthermore, the water-soluble quantum dots are CdSe/ZnS quantum dots, znCdSe/ZnS quantum dots, cdSe/ZnS/CdSe quantum dots, cdSe/CdS/ZnS quantum dots or CdTe/Cd quantum dots.
Further, the quenching agent is a black hole quenching agent BHQ0, BHQ1, BHQ2, BHQ3 or BHQX.
Further, the aptamer-based nucleic acid molecule hybridization system is obtained by performing denaturation and renaturation treatment on the aptamer, the capture probe and the signal probe respectively, incubating the treated sequences, and separating magnetic beads. Specifically, the aptamer is denatured at the high temperature of 75 ℃ for 5min and is subjected to ice bath for 10min, and meanwhile, the capture probe and the signal probe are denatured at the high temperature of 95 ℃ for 5min and are subjected to ice bath for 10min; and (3) incubating the renaturation treated sequence at 37 ℃, adding streptavidin modified magnetic beads, and slightly shaking for reaction for a period of time to form an aptamer-based sandwich structure nucleic acid molecule hybridization system.
Further, the target substance is sialic acid, sialic acid modified polysaccharide, sialic acid modified lipid or sialic acid modified protein.
Further, the nucleotide sequence of the aptamer is shown as SEQ ID NO. 1.
Further, the nucleotide sequence of the capture probe is shown as SEQ ID NO. 2.
Further, the nucleotide sequence of the signal probe is shown as SEQ ID NO. 3.
Further, the nucleotide sequences of the nucleic acid molecular beacons capDNA1, capDNA2 and capDNA3 are shown as SEQ ID NO.4, SEQ ID NO.5 and SEQ ID NO.6, respectively.
Further, the nucleotide sequences of the hairpin structure nucleic acid probes Hp1 and Hp2 are shown as SEQ ID NO.7 and SEQ ID NO.8, respectively.
The invention firstly carries out high-temperature denaturation on an aptamer, a capture probe and a signal probe sequence, and the aptamer is placed in a water bath kettle at 37 ℃ for incubation after renaturation treatment to form a complementary sequence with a sandwich structure. In the presence of sialic acid, the aptamer moiety in the complementary sequence of the sandwich binds to sialic acid, altering the secondary structure of the complementary sequence, resulting in release of the signaling probe from the complementary sequence of the sandwich. Secondly, three nucleic acid molecular beacons (capDNA 1, capDNA2 and capDNA 3) containing the black hole quencher (BHQ 2) are respectively incubated with CdSe/ZnS Quantum Dots (QDs), and three fluorescence resonance energy transfer systems based on the quantum dots and the black hole quencher are respectively BHQ2-capDNA1-QDs, BHQ2-capDNA2-QDs and BHQ2-capDNA3-QDs. When the signal probe released from the complementary sequence of the sandwich structure is added into the fluorescence resonance energy transfer system, the signal probe can carry out sequence complementary pairing with a nucleic acid molecule beacon capDNA1 in the fluorescence resonance energy transfer system BHQ2-capDNA 1-QDs. After exonuclease III is added, the 3 'end of the nucleic acid molecular beacon capDNA1 is completely complementary to the combination of the signal probe, so that the nucleic acid molecular beacon capDNA1 is digested by the exonuclease III, and the black hole quencher BHQ2 modified by the 3' end of the nucleic acid molecular beacon capDNA1 is released, so that the fluorescence of the quantum dot is recovered. When the molecular beacon capDNA1 is cut by enzyme, the signal probe complementary with the molecular beacon capDNA1 is released, and the released signal probe can be subjected to complementary hybridization with other molecular beacon capDNA1 again, so that a new round of fluorescence resonance energy transfer system is stopped, a large amount of black hole quenchers combined on the quantum dots are separated, and the fluorescence intensity of the quantum dots is further recovered; in addition, in order to further amplify the fluorescent signal, a double-hairpin structure nucleic acid probe is added into the reaction system, the signal probe can be combined with the hairpin structure nucleic acid probe Hp1 to induce the Hp1 to be cut by exonuclease III to form a large amount of single-stranded ssDNA1, the ssDNA1 can be further used as the signal probe to carry out sequence complementary pairing with a nucleic acid molecule beacon capDNA2 in a fluorescence resonance energy transfer system BHQ2-capDNA2-QDs, and the nucleic acid molecule beacon capDNA2 is induced to be cut by exonuclease III. When the molecular beacon capDNA2 is cut by enzyme, the single-chain ssDNA1 complementary to the molecular beacon capDNA2 is released, and the released single-chain ssDNA1 can be subjected to complementary hybridization with other molecular beacon capDNA2 again, so that a new round of fluorescence resonance energy transfer system is terminated, the black hole quenchers combined on the quantum dots are separated in a large amount, and the fluorescence intensity of the quantum dots is enhanced again; meanwhile, the ssDNA1 sequence can be combined with the hairpin structure nucleic acid probe Hp2 to induce the Hp2 to be cut by exonuclease III to form a large amount of single-chain ssDNA2, the ssDNA2 can be further used as a signal probe to carry out sequence complementary pairing with the nucleic acid molecule beacon capDNA3 in the fluorescence resonance energy transfer system BHQ2-capDNA3-QDs, and the nucleic acid molecule beacon capDNA3 is induced to be cut by the exonuclease III to recover a large amount of quantum dot fluorescence. When the molecular beacon capDNA3 is cut by enzyme, the single-chain ssDNA2 which is complementary to the molecular beacon capDNA3 is released, the released single-chain ssDNA2 can be subjected to complementary hybridization with other molecular beacon capDNA3 again, so that a new round of fluorescence resonance energy transfer system is stopped again, the black hole quenchers combined on the quantum dots are separated in a large amount, and the fluorescence intensity of the quantum dots is further enhanced again; through the above multiple rounds of cyclic reactions, the number of the enzyme-digested molecular beacons capDNA1, capDNA2 and capDNA3 is increased, so that the fluorescence signal is enhanced, and the sensitivity of the biosensor is improved (fig. 1). And finally, establishing a linear relation between the fluorescence signal intensity and the sialic acid quantity, and calculating the sialic acid content in the sample by using the standard curve.
The invention has the beneficial effects that:
according to the invention, sialic acid is captured by the aptamer, so that a signal probe complementary with the aptamer is released, the signal probe can be combined with a nucleic acid molecular beacon in a fluorescence resonance energy transfer system, meanwhile, the signal probe can also be combined with a hairpin structure nucleic acid probe Hp1, and when exonuclease III is added, the signal probe promotes the nucleic acid molecular beacon to be enzyme-cut with the hairpin structure nucleic acid probe, so that a large number of quantum dot fluorescent signals are recovered, the detection range of the sensor is expanded, and the detection sensitivity is improved. Compared with the traditional sialic acid detection method, the method has the advantages of strong specificity and high sensitivity.
Drawings
FIG. 1 is a schematic diagram of sialic acid high-sensitivity detection based on fluorescence resonance energy transfer and exonuclease assisted cycle amplification strategies;
FIG. 2 is a sialic acid fluorescence detection standard curve.
Detailed Description
The present invention is further described below in conjunction with specific examples to enable those skilled in the art to better understand the present invention and to practice it, but the examples are not intended to limit the present invention.
The aptamers described in the examples below were purchased from Bei Xin Biotech, suzhou, and complementary nucleic acids and nucleic acid molecular beacons, hairpin nucleic acids were purchased from Biotechnology engineering (Shanghai) Inc.
The sequences of the aptamers, capture probes, signaling probes, nucleic acid molecular beacons, and hairpin nucleic acid probes used in the examples of the invention are shown in Table 1.
TABLE 1 sequence listing
Figure BDA0002737858110000061
Example 1: drawing of standard curve of sialic acid concentration
(1) Preparing target recognition magnetic beads: the nucleic acid sequence is shown in Table 1, including sialic acid aptamer sequence, and signal probe sequence and capture probe sequence complementary to the aptamer, denaturing the aptamer at 75 ℃ for 5min, ice-bathing for 10min, at the same time, denaturing the signal probe and the sequence modified with biotin at 95 ℃ for 5min, ice-bathing for 10min; incubating the renaturation treated sequence at 37 ℃, adding streptavidin modified magnetic beads, slightly shaking for reaction for a period of time, forming an aptamer-based sandwich structure nucleic acid molecule hybridization system, carrying out magnetic separation on the solution by using a magnet, discarding supernatant, washing the precipitate for 3 times by using PBS buffer solution, and then suspending the precipitate in the PBS buffer solution;
(2) Target recognition and signaling probe acquisition: adding 50 mu L of sialic acid containing different concentrations into the PBS buffer solution, uniformly mixing, and incubating for 1h at room temperature; carrying out magnetic separation by using a magnet to obtain supernatant, wherein the supernatant contains the signal probe;
(3) Constructing a fluorescence resonance energy transfer system: 50 μ L of 1 μmol/L each -1 Nucleic acid molecular beacons (capDNA 1, capDNA2, capDNA 3) containing Black hole quencher (BHQ 2) and 50. Mu.L of 1. Mu. Mol. L -1 Mixing CdSe/ZnS fluorescent Quantum Dots (QDs) at room temperatureSlightly shaking the solution, incubating for 25min, and constructing three fluorescence resonance energy transfer systems based on quantum dots and black hole quenchers, namely BHQ2-capDNA1-QDs, BHQ2-capDNA2-QDs and BHQ2-capDNA3-QDs; the nucleic acid molecule beacon sequence is shown in table 1;
(4) Enzyme digestion multistage circulation amplification and signal amplification: adding 50 mu L of the solution obtained in the step (2) into the constructed fluorescence resonance energy transfer system, simultaneously adding 50 mu L of hairpin structure nucleic acid probes Hp1 and Hp2 respectively, and then adding 150U of exonuclease III for enzyme digestion reaction for 1.5h, so that the molecular beacon modified on the quantum dot is cut off by the exonuclease III, and the black hole quencher BHQ2 is separated from the quantum dot, the fluorescence resonance energy transfer system disappears, and the fluorescence is enhanced; the sequences of the hairpin structure nucleic acid probes Hp1 and Hp2 are shown in Table 1;
(5) Fluorescence signal detection and standard curve drawing: and (3) detecting a product obtained after the enzyme digestion reaction for 1.5h in the step (4) by using a fluorescence spectrophotometer, comparing the product with a blank sample, reading the change of a fluorescence value, adopting an excitation wavelength of 380nm, measuring a fluorescence signal with an emission wavelength of 605 +/-5 nm, diluting the concentration of sialic acid by times, and drawing a corresponding linear relation curve according to the relation between the measured fluorescence value and the concentration of the added sialic acid.
As shown in FIG. 2, the fluorescence intensity increased with increasing sialic acid concentration, and the linear regression equation was y =0.096log c Neu5Ac +0.893(R 2 = 0.995), wherein y denotes the relative fluorescence intensity, c Neu5Ac Indicates the concentration pmolL of sialic acid -1 The detection limit of the method is 78.13fmolL -1 。
Example 2: determination of sialic acid content in real samples
In order to further verify the accuracy of the method in determining the sialic acid content in the actual sample, a non-pretreated milk product and a human serum sample were selected.
Milk preparations and human serum samples were diluted 50-fold with PBS buffer. Then adding sialic acid with different concentrations and mixing evenly. 5 mu mol. L -1 The aptamer sequence and the complementary nucleic acid sequence are subjected to ice bath for 10min after 5min of high-temperature denaturation, and then are incubated for 1h at 37 ℃,then, the supernatant was removed by magnetic separation, and the pellet was washed four times with PBS buffer and resuspended, mixed with 50. Mu.L of milk products containing sialic acid at different concentrations and human serum samples, and incubated at room temperature with gentle shaking for 30min. And then supernatant is obtained by magnetic separation. Taking 50 mu L of supernatant, adding 150 mu L of supernatant into the constructed fluorescence resonance energy transfer system, mixing uniformly, and then respectively adding 50 mu L of 1 mu mol.L -1 Adding exonuclease 150U into the hairpin structure probes Hp1 and Hp2 at 37 ℃ to react for 1.5h, finally, placing the enzyme digestion reaction solution in a fluorescence spectrophotometer to detect fluorescence, and substituting the fluorescence into a standard curve to calculate the concentration of sialic acid.
Specific samples and test results are shown in table 2.
TABLE 2
Figure BDA0002737858110000081
The above-mentioned embodiments are merely preferred embodiments for fully illustrating the present invention, and the scope of the present invention is not limited thereto. The equivalent substitution or change made by the technical personnel in the technical field on the basis of the invention is all within the protection scope of the invention. The protection scope of the invention is subject to the claims.
Sequence listing
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Claims (3)

1. A substance detection method based on fluorescence resonance energy transfer and exonuclease assisted cycle amplification strategies is characterized by comprising the following steps:
s1, selecting an aptamer matched with a target substance, and constructing an aptamer-based nucleic acid molecule hybridization system, wherein the nucleic acid molecule hybridization system comprises a capture probe and a signal probe which form a complementary structure with the aptamer; the aptamer-based nucleic acid molecule hybridization system is obtained by performing denaturation and renaturation treatment on an aptamer, a capture probe and a signal probe respectively, incubating the treated sequences and separating magnetic beads; the target substance is sialic acid, sialic acid modified polysaccharide, sialic acid modified lipid or sialic acid modified protein;
s2, adding a sample to be detected into the nucleic acid molecule hybridization system in the step S1, releasing a signal probe, and separating a free signal probe;
s3, constructing a fluorescence resonance energy transfer system based on quantum dots and a quencher, wherein the fluorescence resonance energy transfer system based on the quantum dots and the quencher is obtained by respectively incubating three nucleic acid molecule beacons capDNA1, capDNA2 and capDNA3 containing the quencher with the quantum dots, and one nucleic acid molecule beacon capDNA1 can be complementarily hybridized with a signal probe;
s4, adding the signal probe separated in the step S2 into the fluorescence resonance energy transfer system in the step S3, adding two hairpin structure nucleic acid probes Hp1 and Hp2, adding exonuclease III for enzyme digestion reaction, and starting multi-stage fluorescence signal amplification; the single-stranded ssDNA1 can be complementarily hybridized with a nucleic acid molecular beacon capDNA2 and complementarily hybridized with another hairpin structure nucleic acid probe Hp2, the single-stranded ssDNA2 can be complementarily hybridized with a nucleic acid molecular beacon capDNA3, and the single-stranded ssDNA2 can be complementarily hybridized with a nucleic acid molecular beacon capDNA 3;
s5, calculating the content of the target substance in the sample to be detected by detecting the intensity of the fluorescence signal;
wherein, the nucleotide sequence of the aptamer is shown as SEQ ID NO.1, the nucleotide sequence of the capture probe is shown as SEQ ID NO.2, the nucleotide sequence of the signal probe is shown as SEQ ID NO.3, the nucleotide sequences of the nucleic acid molecular beacons capDNA1, capDNA2 and capDNA3 are respectively shown as SEQ ID NO.4, SEQ ID NO.5 and SEQ ID NO.6, and the nucleotide sequences of the hairpin structure nucleic acid probes Hp1 and Hp2 are respectively shown as SEQ ID NO.7 and SEQ ID NO. 8.
2. The substance detection method according to claim 1, wherein the quantum dot is a streptavidin-modified, carboxyl-modified, or amino-modified water-soluble quantum dot.
3. The method for detecting a substance according to claim 1, wherein the quencher is a black hole quencher BHQ0, BHQ1, BHQ2, BHQ3 or BHQX.
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