CN113484387A - DNA biosensor and preparation method thereof - Google Patents
DNA biosensor and preparation method thereof Download PDFInfo
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- CN113484387A CN113484387A CN202110710510.8A CN202110710510A CN113484387A CN 113484387 A CN113484387 A CN 113484387A CN 202110710510 A CN202110710510 A CN 202110710510A CN 113484387 A CN113484387 A CN 113484387A
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Abstract
The invention discloses a DNA biosensor and a preparation method thereof, and the preparation method comprises the following steps: (1) obtaining molybdenum disulfide nanosheet dispersion liquid by adopting a shearing and stripping method; (2) cleaning the gold electrode to a mirror surface by using a Piranha solution, washing the gold electrode clean by using deionized water, drying the gold electrode by using inert gas, and then assembling the gold electrode and a static cell component of the quartz crystal microbalance; (3) adding the molybdenum disulfide nanosheet dispersion liquid obtained in the step (1) into the static pool obtained in the step (2), adding the single-chain probe DNA, uniformly mixing, and incubating at room temperature; (4) adding soluble metal salt into the suspension obtained in the step (3), and uniformly mixing to obtain a label-free biosensor in a first state; (5) and (4) adding single-stranded target DNA into the suspension obtained in the step (4), and reacting at room temperature to obtain the label-free biosensor in the second state, namely the DNA biosensor.
Description
Technical Field
The invention belongs to the technical field of biosensors, and particularly relates to a DNA biosensor and a preparation method thereof.
Background
DNA is used as an important biomarker and widely applied to early detection of various diseases. The quartz crystal microbalance is a novel biosensor which utilizes the sensitivity of quartz crystal to mass change, when the surface mass of the crystal changes, the amount of adsorbed substances can be monitored through the change of frequency, and the measurement precision can reach the level of nanogram. The quartz crystal microbalance can be used for measuring mass change without marking, so that the analysis operation procedure can be simplified, and the analysis speed can be increased.
The molybdenum disulfide nanosheet is one of transition metal chalcogen compounds and has a large specific surface area, so that the surface of the molybdenum disulfide nanosheet generates relatively large electrostatic attraction, meanwhile, due to the fact that the molybdenum atom and the sulfur atom are distributed in an electron cloud mode, one electron cloud mode tends to be in the middle, and the other electron cloud mode tends to be at two ends, a certain negative charge is formed on the surface of the molybdenum disulfide nanosheet, for single-stranded nucleic acid, the base of the double-stranded nucleic acid is exposed outside and is easily adsorbed by a molybdenum disulfide nanosheet layer, and for double-stranded nucleic acid, due to the negatively charged phosphate framework of the double-stranded nucleic acid, the negatively charged phosphate framework is exposed outside due to the double-helix structure, the negatively charged molybdenum disulfide nanosheet layer can generate relatively large repulsion to be far larger than the adsorbed electrostatic attraction, and therefore, the negatively charged molybdenum disulfide nanosheet layer cannot be adsorbed. The sensing method for detecting nucleic acid based on the molybdenum disulfide nanosheets can be constructed by utilizing the property that the molybdenum disulfide nanosheets have different adsorption effects on single-stranded nucleic acid and double-stranded nucleic acid.
At present, most of sensing methods based on molybdenum disulfide nanosheets are detection means based on the fluorescence resonance energy principle, and the method needs to modify a fluorescent group at one end of DNA, so that the cost is high and the operation is complicated. Therefore, the development of a novel biosensing method based on the molybdenum disulfide nanosheet and the quartz crystal microbalance can reduce the cost and the operation procedure, and has great significance.
Disclosure of Invention
Aiming at the problems of complex operation, high cost and the like of the existing fluorescence sensor for detecting nucleic acid, the invention provides a method for preparing a novel biosensor based on a molybdenum disulfide nanosheet and a quartz crystal microbalance. The main principle is as follows: when the target DNA does not exist, the single-chain probe DNA is adsorbed on the surface of the molybdenum disulfide nanosheet, so that the molybdenum disulfide nanosheet is kept in a dispersed suspended state; when the target DNA exists, the single-chain probe DNA adsorbed on the surface of the molybdenum disulfide nanosheet can be combined with the complementary target DNA to form double-chain DNA and is separated from the surface of the molybdenum disulfide nanosheet, the mutual repulsion force between the molybdenum disulfide nanosheets is reduced, the molybdenum disulfide nanosheets can be precipitated and aggregated, the quality of the crystal surface is increased, and the output frequency is reduced accordingly.
In order to achieve the purpose, the invention adopts the following technical scheme:
a preparation method of a DNA biosensor comprises the following steps:
(1) obtaining molybdenum disulfide nanosheet dispersion liquid by adopting a shearing and stripping method;
(2) cleaning the gold electrode with Piranha solution (Chinese name: Piranha solution) to a mirror surface, washing with deionized water, blow-drying with inert gas, and assembling the gold electrode and a static cell assembly of a quartz crystal microbalance;
(3) adding molybdenum disulfide dispersion liquid into the static pool in the step (2), adding single-stranded probe DNA, uniformly mixing, and incubating at room temperature;
(4) adding soluble metal salt into the suspension obtained in the step (3), and uniformly mixing to obtain a label-free biosensor in a first state;
(5) and (4) adding single-stranded target DNA into the suspension obtained in the step (4), and reacting at room temperature to obtain the label-free biosensor in the second state.
Because the surface of the molybdenum disulfide nanosheet has a certain degree of negative charge, a certain repulsion action exists between the molybdenum disulfide nanosheets in the dispersion liquid of the molybdenum disulfide nanosheets, when metal cations are added, the metal cations can be partially dissociated between the molybdenum disulfide nanosheets due to the charge neutralization action, so that the repulsion action between the molybdenum disulfide nanosheets can be weakened, the electrostatic adsorption between the molybdenum disulfide nanosheets can play a leading role, and the stacking and deposition of the molybdenum disulfide nanosheets can be relatively easy; when single-stranded DNA exists, the single-stranded DNA is adsorbed on the surface of the molybdenum disulfide nanosheet, so that the molybdenum disulfide nanosheet can be protected from being stacked and deposited under high salt concentration; when a target single-stranded DNA complementary with the single-stranded probe DNA exists, the target single-stranded DNA and the target single-stranded DNA are complementarily paired to form the double-stranded nucleic acid, and the double-stranded nucleic acid has a rigid structure, so that the adsorption force between the double-stranded nucleic acid and the molybdenum disulfide nanosheet is very weak, and the molybdenum disulfide nanosheet can be stacked and deposited in an environment with high salt concentration. By utilizing the property, the invention constructs the label-free biosensor which adjusts the sedimentation degree of the molybdenum disulfide nanosheet by the single-stranded probe DNA, the target single-stranded DNA and the metal cation, and realizes the detection of the concentration of the target single-stranded DNA.
Preferably, in the step (1), the molybdenum disulfide nanosheet dispersion liquid can be prepared by a shear stripping method; the specific method comprises the following steps:
(1-1) adding 2g of sodium cholate powder and 10g of molybdenum disulfide powder into 200mL of deionized water, and intermittently stirring at the rotating speed of 10000-15000 rpm for 80-90 min;
(1-2) after stirring, centrifuging for 90min at the rotating speed of 1500rpm, and taking supernatant;
(1-3) centrifuging the supernatant obtained in the step (1-2) at the rotating speed of 10000-15000 rpm for 20min, taking the precipitate, and adding deionized water into the precipitate to dissolve the precipitate;
and (1-4) repeating the step (1-3) for three times, taking the precipitate, adding deionized water into the precipitate, and performing ultrasonic treatment for 5min to obtain the uniformly dispersed molybdenum disulfide nanosheet dispersion liquid.
Preferably, in the step (2), a freshly prepared Piranha solution (prepared by mixing concentrated sulfuric acid and 30% hydrogen peroxide in percentage by mass in a volume ratio of 3: 1) is dropwise added to the surface of the gold electrode.
Preferably, in the step (2), the reaction time of the Piranha solution on the surface of the gold electrode is 15-30 s, and the washing time of deionized water is 30-60 s.
Preferably, in the step (2), the inert gas is nitrogen.
Preferably, in the step (3), adding the single-stranded probe DNA into the molybdenum disulfide nanosheet dispersion, and reacting for 20-60 min at room temperature.
Preferably, the single-stranded probe DNA and the single-stranded target DNA in steps (3) and (5) are each dissolved in Tris-Hcl buffer at a concentration of 25nM and a pH of 7.4.
Preferably, in the step (3), the incubation time of the molybdenum disulfide and the single-stranded probe DNA is 20-60 min.
Preferably, in the step (4), the soluble metal salt is NaCl.
Preferably, in the step (5), the reaction time of the suspension and the single-stranded target DNA is 6-8 h.
The invention also provides a DNA biosensor prepared by the preparation method.
The invention has the beneficial effects that:
(1) the DNA biosensor has wide application range, and can be used for specific cancer screening, genetic engineering, food safety and other aspects.
(2) The invention has simple operation process, low cost, no need of expensive detection devices and good biocompatibility.
(3) The invention has simple preparation, small size and can realize high-efficiency real-time detection without fussy pretreatment.
Drawings
FIG. 1 is a schematic view showing the detection principle of a DNA biosensor.
Detailed Description
The following describes in detail preferred embodiments of the present invention.
The present embodiment is a method for preparing a DNA biosensor, comprising the following steps:
step (1), obtaining a molybdenum disulfide nanosheet dispersion liquid by a shearing and stripping method; the operation of the step is as follows:
(1-1) adding 2g of sodium cholate powder and 10g of molybdenum disulfide powder into 200mL of deionized water, and intermittently stirring at the rotating speed of 10000-15000 rpm for 80-90 min;
(1-2) after stirring, centrifuging for 90min at the rotating speed of 1500rpm, and taking supernatant;
(1-3) centrifuging the supernatant obtained in the step (1-2) at the rotating speed of 10000-15000 rpm for 20min, taking the precipitate, and adding deionized water into the precipitate to dissolve the precipitate;
and (1-4) repeating the step (1-3) for 3 times, taking the precipitate, adding deionized water into the precipitate, and performing ultrasonic treatment for 5min to obtain uniformly dispersed molybdenum disulfide nanosheet dispersion liquid.
And (2) cleaning the gold electrode to a mirror surface by using a Piranha solution, washing the gold electrode by using deionized water, drying the gold electrode by using nitrogen, and assembling the gold electrode and a static cell assembly of the quartz crystal microbalance.
In the step (2), a freshly prepared Piranha solution (prepared by mixing concentrated sulfuric acid and 30% hydrogen peroxide in percentage by mass according to a volume ratio of 3: 1) is dropwise added to the surface of the gold electrode. The reaction time of the Piranha solution on the surface of the gold electrode is 15-30 s, and the washing time of deionized water is 30-60 s.
And (3) adding the molybdenum disulfide dispersion solution into the static pool in the step (2), adding the single-stranded probe DNA, uniformly mixing, incubating at room temperature, and reacting for 20-60 min at room temperature.
And (4) adding soluble metal salt NaCl into the suspension obtained in the step (3), and uniformly mixing to obtain the label-free biosensor in the first state.
And (5) adding single-stranded target DNA into the suspension obtained in the step (4), and reacting at room temperature for 6-8 h to obtain the label-free biosensor in the second state.
In this example, the single-stranded probe DNA and the single-stranded target DNA in steps (3) and (5) were dissolved in Tris-HCl buffer (a Chinese name: Tris HCl, available from Beijing Sorboard technologies, Ltd., product number: T1090) at a concentration of 25nM and a pH of 7.4. The invention constructs a label-free biosensor based on molybdenum disulfide nanosheets based on different affinities of the molybdenum disulfide nanosheets to single-stranded DNA and double-stranded DNA and the characteristic that Quartz Crystal Microbalance (QCM) is sensitive to mass change, obtains corresponding frequency change by detecting mass change of the surface of a gold electrode caused by different states of the molybdenum disulfide nanosheets in a static pool of the Quartz Crystal Microbalance, and rapidly detects specific DNA through the frequency change. The method is simple to operate, easy to realize and low in cost.
While the preferred embodiments and principles of this invention have been described in detail, it will be apparent to those skilled in the art that variations may be made in the embodiments based on the teachings of the invention and such variations are considered to be within the scope of the invention.
Claims (10)
1. A preparation method of a DNA biosensor is characterized by comprising the following steps:
(1) obtaining molybdenum disulfide nanosheet dispersion liquid by adopting a shearing and stripping method;
(2) cleaning the gold electrode to a mirror surface by using a Piranha solution, washing the gold electrode clean by using deionized water, drying the gold electrode by using inert gas, and then assembling the gold electrode and a static cell component of the quartz crystal microbalance;
(3) adding the molybdenum disulfide nanosheet dispersion liquid obtained in the step (1) into the static pool obtained in the step (2), adding the single-chain probe DNA, uniformly mixing, and incubating at room temperature;
(4) adding soluble metal salt into the suspension obtained in the step (3), and uniformly mixing to obtain a label-free biosensor in a first state;
(5) and (4) adding single-stranded target DNA into the suspension obtained in the step (4), and reacting at room temperature to obtain the label-free biosensor in the second state, namely the DNA biosensor.
2. The method for preparing a DNA biosensor according to claim 1, wherein: in the step (1), the shearing and stripping method comprises the following steps:
(1-1) adding 2g of sodium cholate powder and 10g of molybdenum disulfide powder into 200mL of deionized water, and intermittently stirring at the rotating speed of 10000-15000 rpm for 80-90 min;
(1-2) after stirring, centrifuging for 90min at the rotating speed of 1500rpm, and taking supernatant;
(1-3) centrifuging the supernatant obtained in the step (1-2) at the rotating speed of 10000-15000 rpm for 20min, taking the precipitate, and adding deionized water into the precipitate to dissolve the precipitate;
(1-4) repeating the step (1-3) for three times, taking the precipitate, adding deionized water into the precipitate, and performing ultrasonic treatment for 5min to obtain the uniformly dispersed molybdenum disulfide nanosheet dispersion liquid.
3. The preparation method of the DNA biosensor according to claim 1, wherein in the step (2), the Piranha solution is prepared by mixing concentrated sulfuric acid with a volume ratio of 3:1 and 30% hydrogen peroxide by mass percent; the reaction time of the Piranha solution on the surface of the gold electrode is 15-30 s; the deionized water washing time is 30-60 s.
4. The method for preparing a DNA biosensor in accordance with any one of claims 1 to 3, wherein the inert gas in step (2) is nitrogen.
5. The method for preparing the DNA biosensor according to claim 1, wherein in the step (3), the molybdenum disulfide nanosheet dispersion and the single-stranded probe DNA are incubated at room temperature for 20-60 min.
6. The method for preparing a DNA biosensor in accordance with any one of claims 1 to 3 or 5, wherein the single-stranded target DNA in step (5) and the single-stranded probe DNA in step (3) are complementary DNAs.
7. The method of preparing a DNA biosensor in accordance with any one of claims 1 to 3 or 5, wherein in the step (5), the reaction time of the single-stranded target DNA with the suspension is 6 to 8 hours.
8. The method of producing a DNA biosensor in accordance with any one of claims 1 to 3 or 5, wherein the single-stranded probe DNA in step (3) and the single-stranded target DNA in step (5) are each dissolved in Tris-HCl buffer at a concentration of 25nM and a pH of 7.4.
9. The method for preparing a DNA biosensor in accordance with any one of claims 1 to 3 or 5, wherein in the step (4), the soluble metal salt is NaCl.
10. A DNA biosensor produced by the production method according to any one of claims 1 to 9.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102183433A (en) * | 2011-02-25 | 2011-09-14 | 中国科学院化学研究所 | Method for detecting mercury ion concentration of water sample |
| CN104502219A (en) * | 2014-12-18 | 2015-04-08 | 江苏大学 | Amyloid-polypeptide aggregation inhibitor as well as inhibition effect evaluation and verification method |
| CN105004775A (en) * | 2015-07-08 | 2015-10-28 | 青岛大学 | Preparation method of disulfide dot/nanosheet compound DNA electrochemical probe |
| CN108535207A (en) * | 2018-03-07 | 2018-09-14 | 浙江大学 | Label-free biosensor based on tungsten disulfide nano slices and its preparation method and application |
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- 2021-06-25 CN CN202110710510.8A patent/CN113484387B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102183433A (en) * | 2011-02-25 | 2011-09-14 | 中国科学院化学研究所 | Method for detecting mercury ion concentration of water sample |
| CN104502219A (en) * | 2014-12-18 | 2015-04-08 | 江苏大学 | Amyloid-polypeptide aggregation inhibitor as well as inhibition effect evaluation and verification method |
| CN105004775A (en) * | 2015-07-08 | 2015-10-28 | 青岛大学 | Preparation method of disulfide dot/nanosheet compound DNA electrochemical probe |
| CN108535207A (en) * | 2018-03-07 | 2018-09-14 | 浙江大学 | Label-free biosensor based on tungsten disulfide nano slices and its preparation method and application |
Non-Patent Citations (1)
| Title |
|---|
| SHIBSANKAR DUTTA 等: "Development of an effective electrochemical platform for highly sensitive DNA detection using MoS2 - polyaniline nanocomposites", 《BIOCHEMICAL ENGINEERING JOURNAL》, vol. 140, pages 130 - 139, XP085530946, DOI: 10.1016/j.bej.2018.09.016 * |
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