CN111500684B - Method for detecting dopamine based on aptamer-G quadruplex nanowire - Google Patents

Method for detecting dopamine based on aptamer-G quadruplex nanowire Download PDF

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CN111500684B
CN111500684B CN202010405160.XA CN202010405160A CN111500684B CN 111500684 B CN111500684 B CN 111500684B CN 202010405160 A CN202010405160 A CN 202010405160A CN 111500684 B CN111500684 B CN 111500684B
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牛凌梅
王艳仙
康维钧
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Hebei Medical University
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Abstract

The invention provides a method for detecting dopamine based on an aptamer-G quadruplex nanowire, which comprises the following steps: (a) Preparing a dopamine aptamer solution, wherein the sequence of the dopamine aptamer is shown as a sequence 1 in a sequence table; (b) Preparing a hairpin DNA solution, wherein the sequence of the hairpin DNA is shown as a sequence 2 in a sequence table; (c) Mixing the dopamine aptamer solution and the hairpin DNA solution in an equimolar amount, and adding a sample to be tested for incubation; (d) After incubation is finished, adding exonuclease III for enzyme digestion reaction, and then adding Mg 2+ And K + Mixing completely and standing the buffer solution; (e) After the reaction was completed, the RRS signal of the reaction solution was detected by a fluorescence spectrophotometer. The invention utilizes the G lead nano structure to amplify RRS signals, and establishes the ultra-sensitive detection method of dopamine. The method has good specificity and high sensitivity, and the detection limit reaches nanomolar level.

Description

Method for detecting dopamine based on aptamer-G quadruplex nanowire
Technical Field
The invention relates to the field of biomedicine, in particular to a method for detecting dopamine based on an aptamer-G quadruplex nanowire.
Background
Dopamine (DA), a catecholamine neurotransmitter, plays an important role in maintaining normal physiological functions of the body and in systems such as the central nervous system, endocrine system, kidney system and cardiovascular system. Excessive and excessive levels of dopamine in humans can cause many diseases. For example, the major neurochemical disturbance of Parkinson's disease is associated with a significant reduction in dopamine levels in the striatum. Symptoms such as motor dysfunction in aids patients are associated with a deficiency of dopamine in the striatum. Huntington's disease, attention deficit hyperactivity disorder, schizophrenia, paragangliomas and pheochromocytomas are also associated with abnormal levels of dopamine. Due to the low dopamine content and the high amount of interfering substances in body fluids, a highly selective and sensitive detection method is urgently needed. Therefore, detection of neurotransmitters such as dopamine is of great significance for guaranteeing national health and treatment and prevention of diseases. However, because the content of the substance in blood is low, the existing detection method is not easy to realize sensitive detection. On the other hand, various substances, such as ascorbic acid, epinephrine, serotonin, etc., are usually present in body fluids, and may affect the measurement 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 taking blood 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 with high sensitivity and high selectivity.
Currently, the main methods for measuring Dopamine (DA) include spectrophotometry, chemiluminescence, fluorescence, high performance liquid chromatography, capillary electrophoresis, and electrochemical methods. 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. Despite decades of research, these methods still suffer from drawbacks such as complex pretreatment process, poor selectivity, low sensitivity, and the need for specialized equipment and trained personnel.
Disclosure of Invention
The invention aims to provide a method for detecting dopamine based on an aptamer-G quadruplex nanowire, and the method is used for solving the problems of poor sensitivity, complex operation and large wound of the existing detection method.
The purpose of the invention is realized by the following technical scheme: a method for detecting dopamine based on aptamer-G quadruplex nanowires, comprising the following steps:
(a) Preparing a dopamine aptamer solution, wherein the sequence of the dopamine aptamer is shown as a sequence 1 in a sequence table;
(b) Preparing a hairpin DNA solution, wherein the hairpin DNA comprises a G-quadruplex forming sequence, the base of the 5' -end part of the hairpin DNA is complementarily paired with the dopamine aptamer, and the sequence of the hairpin DNA is shown as a sequence 2 in a sequence table;
(c) Mixing the dopamine aptamer solution and the hairpin DNA solution in an equimolar amount, and adding a sample to be tested for incubation;
(d) After incubation is complete, nucleic acid is addedCarrying out enzyme digestion reaction by using exonuclease III, and then adding Mg 2+ And K + The buffer solution is mixed completely and then stands to form a G quadruplex nano wire in a reaction system;
(e) And after the reaction is finished, detecting the RRS signal of the reaction solution by using a fluorescence spectrophotometer, and obtaining the content of the dopamine in the sample to be detected according to a pre-made standard curve.
In step (c), the incubation conditions were 2 hours at 4 ℃.
In the step (d), the enzyme digestion reaction conditions are as follows: 60U of exonuclease III was added and incubated at 4 ℃ for 1 hour.
In step (d), the buffer solution is 150mM KCl and 200mM MgCl 2 And a pH of 7.4.
In step (e), the resonant Rayleigh scattering spectra are set to wavelengths for simultaneous scanning of λ ex = λ em The synchronous fluorescence wavelength scanning range is 220nm-650nm, the excitation and emission slit is 10.0nm, and the temperature is 4 ℃.
The invention designs the appropriate binding capacity of the dopamine aptamer and the hairpin DNA, so that the dopamine aptamer can be timely detached from the hairpin DNA in the presence of dopamine. At the same time, hairpin DNA sequences capable of forming G-quadruplexes are designed to achieve the effect on Mg 2+ G-wire nanostructures are formed in the presence. It mainly has the following advantages:
(1) The nucleic acid aptamer is used for identifying dopamine, and an RRS detection platform of the dopamine is constructed;
(2) The G-quadruplex nano-wire structure is used for signal amplification for the first time, and a new method for ultra-sensitive detection of dopamine is established;
(3) The RRS signal is enhanced by utilizing the G-wire nano structure, and the detection is carried out without a fluorescent marker, so that the cost is greatly reduced, and the sensitivity is excellent; the detection sensitivity of the conventional method is mostly hundreds to thousands of nanomolar concentration, and the sensitivity can reach 0.01 nanomolar level by adopting the method. The sensitivity is improved by hundreds of thousands of times, and the measurement of blood and urine samples with very low dopamine content is facilitated.
(4) 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 the heavy burden is often caused to a patient. The method has the advantages that the sample amount is very small, the measurement requirement can be met only by a few microliters, the burden of a patient is relieved, and the possibility of future home service is created;
(5) The sampling is simple, and cerebrospinal fluid with higher content is often used as a detection sample for obtaining higher accuracy clinically. But cerebrospinal fluid sampling is difficult, patients are traumatized greatly, and compliance is poor. The method has the advantages of simple and convenient sampling, and can adopt blood and urine samples with very low contents due to the ultrahigh sensitivity.
Drawings
FIG. 1 is a schematic diagram of the process of the present invention. In the figure, the ab curve is a comparison of the results of the response with and without the presence of the target.
FIG. 2 is a resonance Rayleigh spectrum of dopamine of different concentrations in example 1 of the present invention. In the figure, (a) 0.0nM, (b) 0.05nM, (c) 0.07nM, (d) 0.09nM, (e) 0.2nM, (f) 0.4nM, (g) 0.6nM, (h) 0.6nM, (i) 0.8nM, (j) 1.0nM, (k) 3.0nM, (l) 5.0nM, (m) 8.0nM, (n) 10.0nM.
FIG. 3 is a standard curve measured in example 1 of the present invention.
FIG. 4 is a gel electrophoresis image of G-wire nanostructures. In the figure, (A) RRS spectrum of aptamer sensor, in which (a) hairpin DNA + aptamer + Exo III + Mg 2+ (b) hairpin DNA + aptamer + DA + Exo III + Mg 2+ (ii) a (B) Polyacrylamide gel electrophoresis analysis, wherein (1) DNA marker, (2) hairpin DNA (3.0. Mu.M), (3) aptamer (3.0. Mu.M), (4) hairpin DNA (3.0. Mu.M) + aptamer (3.0. Mu.M) + DA (10.0 nM) + Exo III (6.0U/. Mu.L) + Mg 2+ (200.0 mM). (DA, hairpin DNA, aptamer, exo and Mg 2+ Are 10.0nM, 3.0. Mu.M, 6.0U/. Mu.L, 200.0 mM), respectively).
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 preparation of Standard Curve
(a) Dopamine standard solutions were prepared at concentrations of 0.0nM, 0.05nM, 0.07nM, 0.09nM, 0.2nM, 0.4nM, 0.6nM, 0.8nM, 1.0nM, 3.0nM, 5.0nM, 8.0nM, 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: the prepared hairpin DNA is first heated to 95 ℃ for 5 minutes and then slowly cooled to room temperature for further use. The hybridization reaction was performed with dopamine aptamer (3. Mu.M, 20. Mu.L), hairpin DNA (3. Mu.M, 20. Mu.L), and 20. Mu.L of dopamine solutions of different concentrations in Tris-HCl A (20mM Tris, pH 7.4) buffer, and the mixture was incubated at 4 ℃ for 2 hours, and the number of complementary bases between the hairpin DNA and the aptamer was 3. Followed by addition of 10. Mu.L of Exo-III (6U/. Mu.L) and incubation at 4 ℃ for 1 hour. Followed by Tris-HCl B buffer (150mM KCl,200mM MgCl) 2 pH 7.4) to 300. Mu.L, and left to stand at 4 ℃ for 2 hours to promote the formation of G-nanowires. The RRS spectral intensity of the solution was measured by a spectrofluorometer, and the results are shown in FIG. 2. A low-temperature control thermostatic bath is arranged, and the detection temperature is controlled to be 4 ℃. The wavelength setting of the resonant Rayleigh scattering spectrum is a synchronous scan λ ex = λ em (Δ λ =0 nm). The synchronous fluorescence wavelength scanning range is 220nm-650nm, and the slit (EX/EM) is 10/10 nm. In the experiment, the voltage of the photomultiplier was selected to be 400V. The varying RRS intensity (Δ I) is defined as Δ I = I 1 -I 0 ，I 1 And I 0 Representing the RRS intensity in the presence and absence of dopamine, respectively. The amount of change in the fluorescence signal measured for each group was plotted against the corresponding dopamine concentration as a standard curve, as shown in fig. 3.
The dopamine aptamer sequence is shown as sequence 1 in the sequence table, and the aptamer is a fragment which is screened from an oligonucleotide library in vitro through a systematic evolution technology (SELEX) for exponentially enriching ligands and synthesized in vitro and is used for specific recognition of a target object. The hairpin DNA comprises a G-quadruplex forming sequence, the 5' -end part base of the hairpin DNA is complementarily matched with the dopamine aptamer, and the sequence of the hairpin DNA is shown as a sequence 2 in a sequence table.
The method of the present invention is shown in FIG. 1, and dopamine aptamer is combined with an equimolar amount of hairpin DNA by the base complementary pairing principle to form a stable double-stranded DNA. In the presence of dopamine, the aptamer binds to it, thereby releasing the aptamer from the hairpin DNA, which in turn activates digestion of Exo III to form a G-quadruplex structure, in Mg 2+ And K + Under the induction of (3), forming a long-thread-shaped G-wire nano structure. The G-wire has significant RRS enhancement characteristics. The higher the dopamine concentration, the more G-wire is formed per unit time, and the stronger the RRS detection signal. The RRS signal of the above solution was detected by a fluorescence spectrophotometer. On the basis, the invention considers the reaction time of the hairpin DNA and the aptamer, the number of complementary pairing bases of the hairpin DNA and the aptamer, and Mg 2+ Concentration, exo-III Activity and Mg 2+ The influence of the reaction time on the construction of the aptamer sensor finds the optimal construction condition of the RRS aptamer sensor. Formation of the G-wire nanostructure was verified by polyacrylamide gel electrophoresis, which is shown in fig. 3.
Example 2 detection of dopamine content in mouse brain tissue
This example randomly divided mice (25-28 g) into 4 groups: a high-fat feed potassium perfluorooctane sulfonate administration group, a common feed potassium perfluorooctane sulfonate administration group, a high-fat feed sodium carboxymethyl cellulose control group and a common feed sodium carboxymethyl cellulose control group. Continuously perfusing the stomach for 8 weeks, killing the mice, taking brain tissue, adding 7.5mL/g of tissue lysate, homogenizing 30 s, then centrifuging at 14000 r/min for 15min at 4 ℃, taking supernatant, adding perchloric acid precipitator according to the ratio of 2: 1, centrifuging at 14000 r/min for 15min at 4 ℃, taking supernatant, and performing RRS analysis and ELISA experiment. The detection results are shown in table 1, and the two methods have better consistency.
Table 1:
Figure 373760DEST_PATH_IMAGE001
the sensitivity of the detection method of the present invention was compared to other methods of the prior art, as shown in table 2.
Table 2:
Figure DEST_PATH_IMAGE002
as can be seen from table 2, the present invention achieves ultra-sensitive detection, which is a significant advance over the prior art.
Sequence listing
<120> method for detecting dopamine based on aptamer-G quadruplex nanowire
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 58
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
gtctctgtgt gcgccagaga acactggggc agatatgggc cagcacagaa tgaggccc 58
<210> 2
<211> 28
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
gacagggtgg ggagggtggg gccaccct 28

Claims (5)

1. A method for detecting dopamine based on an aptamer-G quadruplex nanowire is characterized by comprising the following steps:
(a) Preparing a dopamine aptamer solution, wherein the sequence of the dopamine aptamer is shown as a sequence 1 in a sequence table;
(b) Preparing a hairpin DNA solution, wherein the hairpin DNA comprises a G-quadruplex forming sequence, the base of the 5' end part of the hairpin DNA is complementarily paired with the dopamine aptamer, and the sequence of the hairpin DNA is shown as a sequence 2 in a sequence table;
(c) Mixing the dopamine aptamer solution and the hairpin DNA solution in an equimolar amount, and adding a sample to be tested for incubation;
(d) After incubation is finished, adding exonuclease III for enzyme digestion reaction, and then adding Mg 2+ And K + The buffer solution is mixed completely and then stands to form a G quadruplex nano wire in a reaction system;
(e) And after the reaction is finished, detecting the RRS signal of the reaction solution by using a fluorescence spectrophotometer, and obtaining the content of the dopamine in the sample to be detected according to a pre-made standard curve.
2. The method for detecting dopamine according to claim 1, wherein the incubation is performed at 4 ℃ for 2 hours in step (c).
3. The method for detecting dopamine based on aptamer-G quadruplex nanowires as claimed in claim 1, wherein in the step (d), the enzyme digestion reaction conditions are as follows: 60U of exonuclease III was added and incubated at 4 ℃ for 1 hour.
4. The method for detecting dopamine according to claim 1, wherein the buffer solution is 150mM KCl and 200mM MgCl in step (d) 2 And a pH of 7.4.
5. The method for detecting dopamine according to claim 1, wherein the wavelength setting of the resonant rayleigh scattering spectrum in step (e) is synchronous scanning λ ex = λ em The synchronous fluorescence wavelength scanning range is 220nm-650nm, the excitation and emission slit is 10.0nm, and the temperature is 4 ℃.
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CN104569424A (en) * 2014-12-19 2015-04-29 汕头大学 Hairpin DNA probe and method for quantitatively detecting thrombin

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CN104569424A (en) * 2014-12-19 2015-04-29 汕头大学 Hairpin DNA probe and method for quantitatively detecting thrombin

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Quadruplex integrated DNA(QuID) nanosensors for monitoring dopamine;Jennifer M.Morales;《Sensors(Basel)》;20150831;第15卷(第8期);第19912-19924页 *
光电化学传感器的构建及研究进展;郝旭峰等;《化学研究与应用》;20191130;第31卷(第11期);第1858-1868页 *

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