CN109975561B - Ultra-sensitive dopamine detection method based on aptamer - Google Patents
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
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- G01N33/94—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving narcotics or drugs or pharmaceuticals, neurotransmitters or associated receptors
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- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
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- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
- G01N2021/6439—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" with indicators, stains, dyes, tags, labels, marks
Abstract
The invention provides a method for detecting dopamine with ultrasensitive based on aptamer, which comprises the steps of firstly combining ssDNA1 with dopamine aptamer and ssDNA2 by a base complementary pairing principle to form double-stranded DNA1 with two 3 '-protruding ends, wherein the number of bases of ssDNA2 is less than that of ssDNA1 and the ssDNA1 is subjected to complementary pairing, and the dopamine aptamer is subjected to partial complementary pairing with ssDNA1 from the 3' -end of ssDNA 2; then adding a sample to be detected, hairpin DNA, a fluorescent dye SYBR Green I and exonuclease III, wherein the number of bases of the hairpin DNA is less than that of the ssDNA1, and the hairpin DNA and the free ssDNA1 can form double-stranded DNA2 with the 3' -terminal of the hairpin DNA chain sunken through the base complementary pairing principle. The invention recognizes dopamine by dopamine aptamer, so that the aptamer is separated from double-stranded DNA1, thereby continuously initiating enzyme digestion reaction and reducing fluorescence signals.
Description
Technical Field
The invention relates to the field of biomedicine, in particular to an aptamer-based method for detecting dopamine with ultrasensitiveness.
Background
Parkinson's disease is a common neurological disorder disease, mainly affects middle-aged and elderly people, and is the third killer of middle-aged and elderly people after tumor and cardiovascular and cerebrovascular diseases. Surprisingly, approximately 450 million parkinson's disease patients worldwide, nearly half in china. The most important pathological change of the Parkinson disease is degeneration and death of dopaminergic neurons in the midbrain substantia nigra, and the striatum Dopamine (DA) content is obviously reduced to cause diseases. Therefore, detection of neurotransmitters such as dopamine is of great significance for guaranteeing national health and treatment and prevention of diseases.
However, the existing detection method cannot easily realize sensitive detection due to the low content of Dopamine (DA) in blood. On the other hand, various substances are usually present in body fluids, such as ascorbic acid, epinephrine, norepinephrine, etc., which may affect the determination of dopamine. Therefore, it is necessary to develop a recognition probe having good selectivity. Secondly, the sampling problem is also an aspect to be solved. Dopamine was collected by bleeding as well as cerebrospinal fluid. The sampling of the parts has the characteristics of large trauma to the body, poor patient compliance and the like. With the development of ultra-micro detection, non-invasive or minimally invasive detection in the medical field, it is of great significance to develop a new method for minimally invasive detection of Dopamine (DA) with high sensitivity and selectivity.
Currently, main methods for measuring Dopamine (DA) are spectrometry, mass spectrometry, fluorescence, chemiluminescence, capillary electrophoresis, chromatography, and electrochemical methods. For example, Edyta N et al measured dopamine using luminol potassium ferricyanide chemiluminescence system. Andrej K et al performed measurement studies on neurotransmitters in mouse cerebrospinal fluid by a method of liquid chromatography-mass spectrometry. Graphene-modified gold electrodes such as argyongqing were used to simultaneously measure dopamine and uric acid. Although the optical method is convenient and rapid in detection, the detection sensitivity is poor. Although the chromatography has higher sensitivity compared with an optical method, the pretreatment is complicated, the instrument is expensive, the reagent consumption is large, and the operation cost is higher. Although the electrochemical method is simple in equipment, the detection sensitivity is not high, the service life of the electrode is short, and the reproducibility of the detection result is poor.
Disclosure of Invention
The invention aims to provide an aptamer-based ultra-sensitive dopamine detection method, and solves the problems of poor sensitivity, complex operation and large adopted wound of the existing detection method.
The purpose of the invention is realized by the following technical scheme: an ultra-sensitive dopamine detection method based on nucleic acid aptamer comprises the steps of firstly combining ssDNA1 with dopamine aptamer and ssDNA2 by a base complementary pairing principle to form double-stranded DNA1 with two 3 'protruding ends, wherein the number of bases of ssDNA2 is less than that of ssDNA1 and the ssDNA1 is subjected to complementary pairing, and the dopamine aptamer is subjected to partial complementary pairing with ssDNA1 from the 3' end of ssDNA 2; then adding a sample to be detected, hairpin DNA, a fluorescent dye SYBR Green I and exonuclease III, wherein the number of bases of the hairpin DNA is less than that of the ssDNA1, and the hairpin DNA and free ssDNA1 can form double-stranded DNA2 with a sunken 3' tail end of a hairpin DNA chain by a base complementary pairing principle;
dopamine in a sample to be detected competes from the double-stranded DNA1 to obtain a dopamine aptamer, so that the remaining double-stranded DNA1 forms a structure with a concave 3 'end, thereby initiating an enzyme digestion reaction of exonuclease III to form free ssDNA1, the hairpin DNA (hDNA) and the free ssDNA1 form the double-stranded DNA2 with the concave 3' end of the hairpin DNA chain by a base complementary pairing principle, and further initiating the enzyme digestion reaction of the exonuclease III again, the cycle is repeated, so that a fluorescent dye SYBR Green I in the double-stranded DNA structure is continuously released to reduce a fluorescent signal, and the dopamine concentration of the sample to be detected is obtained through a linear relation between the reduction amount of the fluorescent signal and the dopamine concentration.
The method for detecting dopamine with ultrasensitiveness based on the aptamer specifically comprises the following steps:
a. adding equimolar amounts of dopamine aptamer, ssDNA1 and ssDNA2 into a Tris-HCl solution, incubating at room temperature, adding a sample to be detected, and reacting at room temperature; wherein the sequence of the dopamine aptamer is shown as a sequence 1 in a sequence table, the sequence of ssDNA1 is shown as a sequence 2 in the sequence table, and the sequence of ssDNA2 is shown as a sequence 3 in the sequence table;
b. b, adding hairpin DNA and a fluorescent dye SYBR Green I solution into the reaction solution obtained in the step a, after the reaction is completed, adding exonuclease III for enzyme digestion reaction, and cooling to room temperature after the reaction is completed; wherein the hairpin DNA sequence is shown as a sequence 4 in the sequence table;
c. and c, detecting the reaction liquid obtained in the step b by using a fluorescence spectrophotometer to obtain the reduction amount of the fluorescence signal corresponding to the sample to be detected, and obtaining the dopamine concentration of the sample to be detected through a pre-measured standard curve of the reduction amount of the fluorescence signal and the dopamine concentration.
In step a, the incubation time of dopamine aptamer, ssDNA1 and ssDNA2 is 5 hours.
In the step b, the enzyme digestion reaction conditions are as follows: 70U of exonuclease III was added and incubated at 37 ℃ for 1 hour.
In the step c, the excitation wavelength of the fluorescence spectrophotometer is 497nm, the fluorescence emission spectrum is detected at 510-600 nm, the excitation and emission slits are 10.0nm, and the temperature is 25 ℃.
The invention identifies DA through a specific aptamer, constructs a fluorescent detection platform of DA, and simultaneously realizes the ultra-sensitive detection of DA by using a cyclic amplification technology for the first time, and the invention mainly has the following advantages:
(1) the sensitivity is high, the detection sensitivity of the conventional method is mostly hundreds to thousands of nanomolar concentration, and by adopting the method, the sensitivity can reach 0.08 nanomolar level, and the sensitivity is improved by hundreds of thousands of times, so that blood and urine samples with very low dopamine content can be measured;
(2) the sample consumption is small, the sample consumption is mostly hundreds of microliters to several milliliters by methods such as electrochemistry, liquid chromatography and the like, the sample consumption is large, and great wound is often caused to a patient;
(3) the method is simple and convenient to operate and low in cost, and cerebrospinal fluid with high content is often used as a detection sample for obtaining high accuracy clinically. But the cerebrospinal fluid is difficult to sample, and the operation requirement on a sampler is higher, but the invention can utilize blood and urine as samples to carry out determination; in addition, the invention uses the unmarked single-stranded DNA to form double-stranded DNA, and embeds fluorescent dye to perform fluorescence detection, thereby greatly reducing the cost and having excellent sensitivity.
Drawings
FIG. 1 is a schematic diagram of the process of the present invention.
FIG. 2 is a standard curve measured in example 1 of the present invention.
Detailed Description
The technical solution of the present invention will be described in detail with reference to specific examples. The DNA sequences involved in the present invention are synthesized by conventional methods, and the test conditions and procedures not mentioned in the examples of the present invention are performed by conventional methods in the art.
EXAMPLE 1 plotting of Standard Curve
(a) Dopamine standard solutions were prepared at concentrations of 0nM, 0.1nM, 0.3nM, 0.6nM, 0.9nM, 3.0nM, 4.0nM, 5.0nM, and 10.0nM, respectively.
(b) According to the concentration gradient of the dopamine solution, detection tests are carried out in groups, the operation of each group of tests is the same, and the method specifically comprises the following steps: mu.L of ssDNA1 (5. mu.M), 6. mu.L of ssDNA2 (5. mu.M), 6. mu.L of dopamine aptamer (5. mu.M) was added to 230. mu.L of Tris-HCl (10 mM) and incubated for 5 hours at room temperature; adding 20 μ L of dopamine solution with corresponding concentration, reacting at room temperature for 1 hour, adding 6 μ L of hairpin DNA (15 μ M) and 19 μ L of SYBR Green I (purchased from Biotechnology engineering (Shanghai) GmbH, diluted 1000 times with water), and reacting at room temperature for 45 minutes; adding 70U exonuclease III (Exo-III), incubating for 1 hour at 37 ℃ (the progress of the enzyme digestion reaction is verified by a polyacrylamide gel electrophoresis method), cooling to room temperature, detecting by a fluorescence spectrophotometer, wherein the excitation wavelength is 497nm, the fluorescence emission spectrum is detected at 510-600 nm, the excitation and emission slits are both 10.0nm, and the temperature is 25 ℃.
The sequence of the dopamine aptamer is shown as sequence 1 in a sequence table, the sequence of ssDNA1 is shown as sequence 2 in the sequence table, and the sequence of ssDNA2 is shown as sequence 3 in the sequence table; the hairpin DNA sequence is shown as a sequence 4 in the sequence table; the reaction process is shown in FIG. 1. The ssDNA2 has fewer bases than ssDNA1 and is complementary paired with ssDNA1, and the dopamine aptamer is complementary paired with a portion of ssDNA1 from the 3 'end of ssDNA2, thereby forming double-stranded DNA1 with two 3' overhanging ends. The hairpin DNA has fewer bases than ssDNA1 and can form a double-stranded DNA2 with a dip at the 3' end of the hairpin DNA strand by base complementary pairing with free ssDNA 1.
Dopamine in a sample competes from double-stranded DNA1 to obtain dopamine aptamer, so that the remaining double-stranded DNA1 forms a 3 'end concave structure, thereby initiating an enzyme digestion reaction of exonuclease III to form free ssDNA1, the hairpin DNA and the free ssDNA1 form the double-stranded DNA2 with the 3' end concave structure of the hairpin DNA chain by a base complementary pairing principle, further initiating the enzyme digestion reaction of the exonuclease III again, and the cycle is repeated so that a fluorescent dye SYBR Green I in the double-stranded DNA structure is continuously released to reduce a fluorescent signal, and the dopamine concentration of the sample to be detected is obtained through a linear relation between the reduction amount of the fluorescent signal and the dopamine concentration.
(c) The decrease in fluorescence signal measured for each group was plotted against the corresponding dopamine concentration as a standard curve, as shown in figure 2.
Example 2 detection of dopamine content in mouse brain tissue
The mice were anesthetized by intraperitoneal injection with 1% sodium pentobarbital at a dose of 0.1ml/10g, and after complete anesthesia, the brain tissue was removed at low temperature. The brain tissue was added with a tissue lysate in an amount of 1g:7.5mL, homogenized for 30s, centrifuged at low temperature (14000 r/min,4 ℃ C.) and the supernatant was centrifuged again under the same conditions. The supernatant was subjected to fluorescence detection according to the detection procedure of example 1, and simultaneously to enzyme-linked immunosorbent assay according to a conventional method. The detection results are shown in table 1, and by statistical analysis of P =0.235>0.1, the difference between the two methods is not statistically significant, so that the two methods are considered to have better consistency.
Table 1:
the sensitivity of the detection method of the present invention was compared to other methods of the prior art, as shown in table 2. As can be seen from table 2, the present invention achieves ultrasensitive detection, which is a significant improvement over the prior art.
Table 2:
reference in table 2:
[1] Raj D R, Prasanth S, Vineeshkumar T V, et al. Surface plasmon resonance based fiber optic dopamine sensor using green synthesized silver nanoparticles[J]. Sensors and Actuators B: Chemical, 2016, 224: 600-606.
[2] Liu S, Shi F, Zhao X, et al. 3-Aminophenyl boronic acid-functionalized CuInS2 quantum dots as a near-infrared fluorescence probe for the determination of dopamine[J]. Biosensors and Bioelectronics, 2013, 47: 379-384.
[3] Liu J M, Wang X X, Cui M L, et al. A promising non-aggregation colorimetric sensor of AuNRs–Ag+ for determination of dopamine[J]. Sensors & Actuators B Chemical, 2013, 176(6): 97-102.
[4] Yan Y, Liu Q, Du X, et al. Visible light photoelectrochemical sensor for ultrasensitive determination of dopamine based on synergistic effect of graphene quantum dots and TiO2 nanoparticles[J]. Analytica chimica acta, 2015, 853: 258-264.
[5] Mir T A, Akhtar M H, Gurudatt N G, et al. An amperometric nanobiosensor for the selective detection of K+-induced dopamine released from living cells[J]. Biosensors and Bioelectronics, 2015, 68: 421-428.
[6] Azadbakht A, Roushani M, Abbasi A R, et al. Design and characterization of electrochemical dopamine–aptamer as convenient and integrated sensing platform[J]. Analytical biochemistry, 2016, 507: 47-57.
[7] Zhou X, Ma P, Wang A, et al. Dopamine fluorescent sensors based on polypyrrole/graphene quantum dots core/shell hybrids[J]. Biosensors & Bioelectronics, 2015, 64: 404-410.
SEQUENCE LISTING
<110> Hebei university of medical science
<120> ultra-sensitive dopamine detection method based on aptamer
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Claims (4)
1. An aptamer-based method for detecting dopamine with ultra sensitivity is characterized in that ssDNA1 is firstly combined with dopamine aptamer and ssDNA2 by a base complementary pairing principle to form double-stranded DNA1 with two 3 '-protruding ends, wherein the number of bases of ssDNA2 is less than that of ssDNA1 and is complementarily paired with ssDNA1, and the dopamine aptamer is complementarily paired with ssDNA1 part from the 3' -end of ssDNA 2; then adding a sample to be detected, hairpin DNA, a fluorescent dye SYBR Green I and exonuclease III, wherein the number of bases of the hairpin DNA is less than that of the ssDNA1, and the hairpin DNA and free ssDNA1 can form double-stranded DNA2 with a sunken 3' tail end of a hairpin DNA chain by a base complementary pairing principle;
dopamine in a sample to be detected competes from double-stranded DNA1 to obtain a dopamine aptamer, so that the remaining double-stranded DNA1 forms a 3 'end concave structure, thereby initiating an enzyme digestion reaction of exonuclease III to form free ssDNA1, the hairpin DNA and the free ssDNA1 form the double-stranded DNA2 with the 3' end concave of the hairpin DNA chain by a base complementary pairing principle, and further initiating the enzyme digestion reaction of the exonuclease III again, the cycle is repeated, so that a fluorescent dye SYBR Green I in the double-stranded DNA structure is continuously released, a fluorescent signal is reduced, and the dopamine concentration of the sample to be detected is obtained through a linear relation between the reduction amount of the fluorescent signal and the dopamine concentration; the method specifically comprises the following steps:
a. adding equimolar amounts of dopamine aptamer, ssDNA1 and ssDNA2 into a Tris-HCl solution, incubating at room temperature, adding a sample to be detected, and reacting at room temperature; wherein the sequence of the dopamine aptamer is shown as a sequence 1 in a sequence table, the sequence of ssDNA1 is shown as a sequence 2 in the sequence table, and the sequence of ssDNA2 is shown as a sequence 3 in the sequence table;
b. b, adding hairpin DNA and a fluorescent dye SYBR Green I solution into the reaction solution obtained in the step a, after the reaction is completed, adding exonuclease III for enzyme digestion reaction, and cooling to room temperature after the reaction is completed; wherein the hairpin DNA sequence is shown as a sequence 4 in the sequence table;
c. and c, detecting the reaction liquid obtained in the step b by using a fluorescence spectrophotometer to obtain the reduction amount of the fluorescence signal corresponding to the sample to be detected, and obtaining the dopamine concentration of the sample to be detected through a pre-measured standard curve of the reduction amount of the fluorescence signal and the dopamine concentration.
2. The method for detecting dopamine with ultrasensitive property based on aptamer according to claim 1, wherein the incubation time of dopamine aptamer, ssDNA1 and ssDNA2 in step a is 5 hours.
3. The method for detecting dopamine with ultrasensitive sensitivity based on aptamer according to claim 1, wherein in the step b, the enzyme digestion reaction conditions are as follows: 70U of exonuclease III was added and incubated at 37 ℃ for 1 hour.
4. The method for the ultrasensitive detection of dopamine according to claim 1, wherein in step c, the excitation wavelength of a fluorescence spectrophotometer is 497nm, the fluorescence emission spectrum is 510-600 nm, the excitation and emission slit is 10.0nm, and the temperature is 25 ℃.
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