CN110873745A - Novel method for detecting acute myelogenous leukemia marker Siglec-5 - Google Patents
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Abstract
A novel method for detecting an acute myelogenous leukemia marker Siglec-5. The principle is as follows: in the work of the invention, a novel electrochemical biosensor is constructed based on a DNA hairpin structure and a nucleic acid aptamer and is used for detecting an acute myelogenous leukemia marker Siglec-5. Modifying sulfydryl-SH at the 5' -end of the designed DNA hairpin structure, so that the hairpin structure can be modified on the surface of a gold electrode through an Au-S bond; meanwhile, the 3 '-end is modified with methylene blue MB, and the MB at the 3' -end is close to the surface of the electrode due to the characteristic of the hairpin structure, so that a larger MB signal can be detected. In the experimental process, the Siglec-5 aptamer is firstly combined with a DNA hairpin structure connected to the surface of a gold electrode, the hairpin structure is opened, MB is far away from the surface of the electrode, and the obtained MB signal is weakened; however, when Siglec-5 exists in the detection system, the Siglec-5 aptamer is combined with the Siglec-5 due to the difference of affinity, DNA modified on the surface of the electrode re-forms a hairpin structure, the distance between the MB and the electrode is pulled, and the MB signal is changed from weak to strong. Therefore, we achieved efficient, sensitive and specific detection of Siglec-5 by variation of MB signal using a simple electrochemical workstation.
Description
Technical Field
The invention belongs to the field of analytical chemistry, and particularly relates to detection of an acute myelogenous leukemia marker and research on application of the marker.
Background
Acute leukemia is a malignant tumor disease that seriously harms human health. The cause of leukemia is not well defined at present, and studies have shown that it may be associated with various factors. Leukemia can be classified into two major categories, Acute leukemia (Acute leukemia) and Chronic leukemia (Chronic leukemia), according to the state of leukemia cells, wherein Acute leukemia is further classified into Acute myelogenous leukemia (ALL) and Acute lymphatic leukemia (AML). Sialic acid (SIA) binding immunoglobulin class (Ig) lectins (Siglecs) are a family of type I transmembrane proteins, each containing an N-terminal Sialic acid binding V region and a distinct intracellular region and showing selective expression in hematopoietic lineages, where Siglec-5 can play an important role in inhibiting leukocyte receptors using SHP-1. Related studies have shown that Siglec-5 can be used as a marker for diagnosing acute myeloid leukemia.
The development of a rapid, sensitive and simple detection method is one of the current research directions for early diagnosis of tumors. Proteins are important functional units of the human body, and in tumor diagnosis, protein tumor markers are usually produced by tumor cells and are present in blood and urine of patients. Studies have shown that the detection of these proteins can be used to diagnose tumors or indicate prognosis of tumor therapy. Meanwhile, the electrochemical biosensor realizes the cross development of electrochemical technology and biology, and has the advantages of sensitivity, rapidness and low cost in detection and analysis, so that a great deal of research and development work is devoted to researching and developing the electrochemical biosensor for detecting protein tumor markers at present.
With the mature development of SELEX technology, aptamers have gradually become widely used in scientific research. In tumor diagnosis, antibodies are currently the most commonly used biological recognition elements. Compared with antibodies, the nucleic acid aptamer has the advantages of small volume, stable chemical property, low immunogenicity, simple production mode, low price, multiple identifiable targets and the like, and along with the deep development of scientific research, the nucleic acid aptamer becomes a substitute biological identification element with unique performance.
In the work of the invention, a novel electrochemical biosensor is constructed based on a DNA hairpin structure and a nucleic acid aptamer and is used for detecting an acute myelogenous leukemia marker Siglec-5. The 5' -end of the DNA hairpin structure is modified with sulfhydryl-SH and is used for being combined with a gold electrode through an Au-S bond; the 3' -end is modified with MB for generating electrochemical signals. Thus, with a simple electrochemical workstation, we achieved efficient, sensitive and specific detection of Siglec-5.
Disclosure of Invention
The invention aims to provide detection of an acute myelogenous leukemia marker and research on application of the marker.
The technical scheme is as follows:
in the work of the invention, a novel electrochemical biosensor is constructed based on a DNA hairpin structure and a nucleic acid aptamer and is used for detecting an acute myelogenous leukemia marker Siglec-5. As shown in FIG. 1, we modified thiol-SH at the 5' -end of the designed DNA hairpin structure, the sequence shown in the following table, so that the hairpin structure can be modified on the surface of gold electrode through Au-S bond; meanwhile, the 3 '-end is modified with methylene blue MB, and the MB at the 3' -end is close to the surface of the electrode due to the characteristic of the hairpin structure, so that a larger MB signal can be detected. In the experimental process, the Siglec-5 aptamer is firstly combined with a DNA hairpin structure connected to the surface of a gold electrode, the hairpin structure is opened, MB is far away from the surface of the electrode, and the obtained MB signal is weakened; however, when Siglec-5 exists in the detection system, due to the difference of affinity, the Siglec-5 aptamer is combined with the Siglec-5, DNA modified on the surface of the electrode re-forms a hairpin structure, the distance between the MB and the electrode is pulled, and the MB signal is changed from weak to strong. Therefore, we achieved efficient, sensitive and specific detection of Siglec-5 by variation of MB signal using a simple electrochemical workstation.
TABLE 1 nucleic acid sequences used in the experiments (MB-modified DNA hairpin probe, Siglec-5 from Shanghai Biotech Ltd.).
In the work of the invention, a novel electrochemical biosensor is constructed based on a DNA hairpin structure and a nucleic acid aptamer and is used for detecting an acute myelogenous leukemia marker Siglec-5. In the scheme of the invention, an MB-DNA hairpin structure is designed, a 5 '-terminal of the hairpin structure is modified to be used for-SH connected with a gold electrode, a 3' -terminal of the hairpin structure is modified to be used for generating MB of an electrochemical signal, and the signal output is realized by utilizing a simple electrochemical workstation, so that the Siglec-5 is efficiently, sensitively and specifically detected.
Drawings
FIG. 1: schematic diagram of a novel method for detecting an acute myelogenous leukemia marker Siglec-5.
FIG. 2: and (3) adopting EIS to represent the modification condition of each substance on the surface of the electrode, and gradually increasing the impedance value of the surface of the electrode along with the gradual modification of MB-DNA and Siglec-5 aptamers on the surface of the electrode.
FIG. 3: the feasibility of the experimental method was verified using DPV. After the MB-DNA hairpin structure modified on the surface of the electrode is opened, the Siglec-5 added in the step (a) is combined with the Siglec-5 aptamer, so that the MB signal is changed; (b) CEA added in the process can not be combined with Siglec-5 aptamer, and MB signals have no obvious change.
FIG. 4: the concentration condition of the Siglec-5 aptamer combined with the MB-DNA is optimized.
FIG. 5: the time condition for binding of Siglec-5 to the aptamer is optimized.
FIG. 6: and (4) carrying out quantitative analysis on Siglec-5 by adopting a DPV detection method. (a) The MB signal increases with increasing concentration of Siglec-5; (b) there is a linear relationship between the current peaks recorded by DPV and the log of Siglec-5 concentration.
FIG. 7: the specificity of the detection method for protein selection was verified in 1% serum, and the current peaks recorded by DPV showed that larger peak currents could be detected only when Siglec-5 was present in the system.
Detailed Description
The preparation process of the invention is as follows:
example I treatment and modification of gold electrode surface
Firstly, a gold electrode is soaked in a Tiger solution (Piranha solution: 70% concentrated sulfuric acid and 30% hydrogen peroxide) for 5-10min, and then the surface of the electrode is polished by aluminum powder with the particle size of 1.0, 0.3 and 0.05 mu m respectively. Then, the polished electrode was set at 50%Soaking in concentrated nitric acid for 30min, and performing ultrasonic treatment in ethanol and pure water for 5 min. Finally, with N2And drying the surface of the electrode for later use. MB-DNA was dissolved in TE buffer, denatured at 90 ℃ for 5min in a metal bath, and then cooled to room temperature for further use. Subsequently, a DNA fixative and TCEP (1mM Tris (2-carboxyethyl) phosphine) were added to the above solution, and at this time, the concentration of MB-DNA was 1.5. mu.M. The prepared MB-DNA solution is dripped on the surface of a dried electrode and incubated for 3 hours at the temperature of 25 ℃. Finally, the electrode modified with MB-DNA was soaked in 1mM MCH (2-Mercaptoethanol) solution and allowed to stand in a fume hood for 1h to remove the adsorption of non-specific hairpin structures and occupy the remaining non-specific binding sites, thereby obtaining an ordered self-assembled hairpin monolayer. Thus, an electrochemical sensing probe MB-DNA was prepared. The Siglec-5 aptamer DNA was dissolved in TE buffer, and a DNA hybridization solution was added to the solution to prepare a solution concentration of 1.5. mu.M. Subsequently, the solution was dropwise added to the electrode surface modified with the MB-DNA probe, and incubated at 25 ℃ for 3 hours. As shown in FIG. 2, the impedance diagram of the bare gold electrode is similar to a straight line, when the MB-DNA hairpin structure and the Siglec-5 aptamer are gradually modified on the surface of the electrode, the impedance value of the surface of the electrode is sequentially increased, the semicircular radius is gradually increased in the graph recorded by EIS, and the process that the MB-DNA hairpin structure and the Siglec-5 aptamer are gradually modified on the surface of the electrode by the bare electrode is verified.
Example two, experimental feasibility
To further explore the feasibility of the experimental approach, we chose CEA for experimental analysis. Electrochemical measurements were performed on the electrode surfaces using the CHI660D electrochemical workstation and a conventional three-electrode system (working, reference, counter). Electrochemical Impedance Spectroscopy (EIS) was recorded using a 1M potassium nitrate solution containing 5mM potassium ferricyanide/potassium ferrocyanide, experimental parameters: the offset potential is 0.224V, the amplitude is 5mV, and the frequency range is 0.1 Hz-10 kHz. Differential Pulse Voltammetry (DPV) spectra were recorded using PBS solution with scans ranging from-0.5V to 0.3V. First, the surface of the gold electrode was modified with an MB-DNA hairpin structure, as shown by the black line in FIGS. 3a and 3 b. A fixed amount of Siglec-5 aptamer was added dropwise to the surface of the gold electrode modified with the MB-DNA hairpin structure, and incubated at 25 ℃ for 60min, at which time the MB-DNA hairpin structure was opened and the MB at the 3' -end was away from the electrode surface, so that a reduced MB electrochemical signal was detected, as shown by the red line in FIGS. 3a and 3 b. Subsequently, Siglec-5 and CEA were dropped onto the electrode surfaces, respectively. When Siglec-5 was added to the system, Siglec-5 bound to the aptamer in the system, allowing part of the MB-DNA to reform into a hairpin structure, so that the MB electrochemical signal was increased more than before as shown by the green line in fig. 3 a; when CEA was added to the system, there was no significant change in MB electrochemical signal as shown by the blue line in FIG. 3b, since CEA did not bind to the Siglec-5 aptamer in the system. Thus, we verified the feasibility of the experimental approach.
Example three, optimization of experimental conditions
In order to control the reaction conditions, MB-DNA solution with the concentration of 1.5 μ M is selected to be dripped on the surface of the gold electrode when the surface of the gold electrode is modified. When the modification is complete, a larger electrochemical signal can now be detected. Subsequently, the concentration gradient of the added dropwise Siglec-5 aptamer was set to 0.5, 0.7, 0.9, 1.1, 1.3, 1.5, 1.7, 1.9 μ M, the incubation was performed at 25 ℃, the MB signal of the electrode surface was recorded using DPV, and as a result, as shown in fig. 4, the concentration of the added dropwise Siglec-5 aptamer at the electrode surface was selected to be 1.5 μ M. Through the optimization of the above conditions, experiments show that when the MB-DNA solution with the concentration of 1.5 mu M is selected to be dripped on the surface of the gold electrode for modification, the optimal concentration of the Siglec-5 aptamer is subsequently added to be 1.5 mu M. Based on the above, we optimize the time for Siglec-5 to bind to the aptamer. The MB signal from the electrode surface was recorded every 20min using DPV and the results are shown in FIG. 5, where the signal reached a plateau after 60min, so we took 60min as the time for incubation of the Siglec-5 aptamer with the MB-DNA modified gold electrode.
Example four, Siglec-5 quantitative assay
We used DPV recording experiments for quantitative analysis of Siglec-5. Dropwise adding MB-DNA with the concentration of 1.5 uM on the surface of the electrode for modification, and then dropwise adding 1.5 mu M of Siglec-5 aptamer to continuously modify the surface of the electrode, wherein no Siglec-5 exists in the detection system, and the Siglec-5 aptamer is combined with the MB-DNA hairpin structure to generateThe clamp structure was opened and the MB at the' end of MB-DNA3 was far from the electrode surface, and as a result, the electrochemical signal recorded by DPV was low as shown in FIG. 6 a. As the concentration of Siglec-5 in the detection system increases, due to the difference of affinity, Siglec-5 is combined with the Siglec-5 aptamer, so that MB-DNA modified on the surface of the electrode re-forms a hairpin structure, the distance between MB and the electrode is pulled, and the electrochemical signal recorded by DPV is continuously increased. Thus, the relation between the DPV peak current variation value and the Siglec-5 concentration is established. Fig. 6b shows that there is a linear relationship between the MB current signal peak recorded by DPV and the log of Siglec-5 concentration (y ═ 0.0003x-5.8183, R)2＝0.96)。
Example five detection System selection specificity analysis
To further explore the selectivity and specificity of the detection system for Siglec-5, we selected CEA and AFP for experimental analysis. After modifying fixed amounts of MB-DNA and Siglec-5 on the surface of the electrode, the Siglec-5, GP73 and CEA are mixed in 1% serum separately or simultaneously, and incubated with the electrode, the peak value of the DPV current is detected as shown in FIG. 7, and the larger peak current can be detected only when Siglec-5 exists in the system. This demonstrates that the experimental system we designed is selective for Siglec-5.
Claims (3)
1. Claim 1 claims the idea of the inventive concept: the novel electrochemical biosensor is constructed based on a DNA hairpin structure and a nucleic acid aptamer and used for detecting an acute myelogenous leukemia marker Siglec-5. in the invention scheme, an MB-DNA hairpin structure is designed, the 5 '-end of the hairpin structure is modified to be used for-SH connected with a gold electrode, the 3' -end of the hairpin structure is modified to be used for generating MB of an electrochemical signal, and a simple electrochemical workstation is utilized to realize signal output, so that the Siglec-5 is efficiently, sensitively and specifically detected.
2. Claim 2 claims a new method for detecting the acute myeloid leukemia marker Siglec-5 comprising the steps of: 1. processing and modifying the surface of the gold electrode 2, optimizing experimental conditions 3 and quantitatively detecting conditions Siglec-5.
3. Claim 3 claims the design of the sequence nucleotides in this patent: specifically, the two strands include MB-DNA hairpin and Siglec-5 aptamer.
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| CN113122575A (en) * | 2021-05-07 | 2021-07-16 | 华中科技大学同济医学院附属梨园医院 | Application of siglec-5, gene expression antagonist or protein activity antagonist |
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|---|---|---|---|---|
| CN101825597A (en) * | 2010-04-20 | 2010-09-08 | 上海大学 | DNA aptamer modified bioelectrochemical sensor and preparation method thereof |
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