CN113484387B - DNA biosensor and preparation method thereof - Google Patents
DNA biosensor and preparation method thereof Download PDFInfo
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- CN113484387B CN113484387B CN202110710510.8A CN202110710510A CN113484387B CN 113484387 B CN113484387 B CN 113484387B CN 202110710510 A CN202110710510 A CN 202110710510A CN 113484387 B CN113484387 B CN 113484387B
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Abstract
The invention discloses a DNA biosensor and a preparation method thereof, wherein the preparation method comprises the following steps: (1) A shearing stripping method is adopted to obtain molybdenum disulfide nanosheet dispersion liquid; (2) Cleaning a gold electrode to a mirror surface by using Piranha solution, washing the mirror surface by using deionized water, drying the mirror surface by using inert gas, and then assembling the gold electrode and a static cell assembly 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 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 (3) adding single-stranded target DNA into the suspension obtained in the step (4), and reacting at room temperature to obtain the second-state label-free biosensor, 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 quartz crystals to be sensitive to quality change, when the quality of the surface of the crystal changes, the quantity of adsorbed substances can be monitored through the change of frequency, and the measurement precision can reach the nanogram level. The quartz crystal microbalance is used for measuring the mass change without marking, so that the analysis operation procedure can be simplified, and the analysis speed can be improved, therefore, the quartz crystal microbalance has the characteristics of quick response, high sensitivity, good specificity, small size, simplicity and the like, and attracts the attention of the industry in the technical field of biomedical sensing.
The molybdenum disulfide nanosheets are used as one of transition metal chalcogenides and have larger specific surface area, so that relatively larger electrostatic attraction force is generated on the surfaces of the nanosheets, meanwhile, molybdenum atoms and sulfur atom electron clouds are distributed in the middle, and one of the nanosheets tends to be arranged at two ends, so that the surfaces of the nanosheets are charged with certain negative charges, for single-stranded nucleic acids, the bases of the single-stranded nucleic acids are exposed to be easily adsorbed by the nanosheets, and for double-stranded nucleic acids, the negative phosphate frameworks are exposed to the outside due to the double-helix structure, larger repulsion is generated on the surface of the negatively charged nanosheets, and the nanosheets are far more than the electrostatic attraction force of adsorption, so that the nanosheets cannot be adsorbed. By utilizing the property that the molybdenum disulfide nanosheets have different adsorption effects on single-stranded nucleic acid and double-stranded nucleic acid, a sensing method for detecting nucleic acid based on the molybdenum disulfide nanosheets can be constructed.
At present, most of sensing methods based on molybdenum disulfide nanosheets are detection means based on a fluorescence resonance energy principle, and fluorescent groups need to be modified at one end of DNA, so that the method is high in cost and complex in operation. Therefore, the development of a novel biosensing method based on molybdenum disulfide nanosheets and quartz crystal microbalances can reduce cost and operation procedures, and has great significance.
Disclosure of Invention
Aiming at the problems of complicated 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 molybdenum disulfide nanosheets and quartz crystal microbalances. The main principle is as follows: when the target DNA does not exist, the single-stranded probe DNA is adsorbed on the surface of the molybdenum disulfide nanosheets, so that the molybdenum disulfide nanosheets are kept in a dispersed and suspended state; when target DNA exists, the single-stranded probe DNA adsorbed on the surface of the molybdenum disulfide nanosheets is combined with the complementary target DNA to form double-stranded DNA and is separated from the surface of the molybdenum disulfide nanosheets, at the moment, the repulsive force between the molybdenum disulfide nanosheets is reduced, the phenomenon of precipitation aggregation of the molybdenum disulfide nanosheets occurs, the quality of the crystal surface is increased, and the output frequency is reduced.
In order to achieve the above purpose, the present invention adopts the following technical scheme:
a method for preparing a DNA biosensor, comprising the steps of:
(1) A shearing stripping method is adopted to obtain molybdenum disulfide nanosheet dispersion liquid;
(2) Cleaning a gold electrode to a mirror surface by using Piranha solution (Chinese name: piranha solution), washing with deionized water, drying by using inert gas, and then assembling the gold electrode and a static cell component of the quartz crystal microbalance together;
(3) Adding molybdenum disulfide dispersion liquid into the static pool in the step (2), then 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 (3) 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 nanosheets has negative charges to a certain extent, in the dispersion liquid of the molybdenum disulfide nanosheets, certain repulsive interaction exists between the sheets, when metal cations are added, the metal cations can be partially dissociated between the sheets due to the charge neutralization effect, so that the repulsive interaction between the sheets can be weakened, the electrostatic adsorption between the molybdenum disulfide nanosheets plays a leading role, and the stacking and deposition of the sheets are relatively easy; when single-stranded DNA exists, the single-stranded DNA is adsorbed on the surface of the molybdenum disulfide nanosheets, so that the molybdenum disulfide nanosheets can be protected from stacking and depositing under high salt concentration; when target single-stranded DNA complementary to the single-stranded probe DNA exists, the target single-stranded DNA and the target single-stranded probe DNA are complementary and paired to form 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 nanosheets is weak, and the molybdenum disulfide nanosheets can be stacked and deposited in a high salt concentration environment. The invention utilizes the property to construct the label-free biosensor which adjusts the sedimentation degree of the molybdenum disulfide nanosheets by the single-stranded probe DNA, the target single-stranded DNA and the metal cations, thereby realizing 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 shearing and 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 for 80-90 min at a rotating speed of 10000-15000 rpm;
(1-2) centrifuging at 1500rpm for 90min after the completion of stirring, and collecting the supernatant;
(1-3) centrifuging the supernatant in the step (1-2) at a rotating speed of 10000-15000 rpm for 20min, taking a 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 carrying out ultrasonic treatment for 5min to obtain the uniformly dispersed molybdenum disulfide nanosheet dispersion liquid.
Preferably, in the step (2), freshly prepared Piranha solution (prepared by mixing concentrated sulfuric acid and 30% hydrogen peroxide by mass percent according to 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 deionized water flushing time is 30-60 s.
Preferably, in step (2), the inert gas is nitrogen.
Preferably, in the step (3), single-stranded probe DNA is added into the molybdenum disulfide nanosheet dispersion liquid, and the reaction is carried out for 20-60 min at room temperature.
Preferably, the single-stranded probe DNA and the single-stranded target DNA in step (3) and step (5) are both dissolved in Tris-HCl buffer at a concentration of 25nM and pH=7.4.
Preferably, in the step (3), the incubation time of the molybdenum disulfide and the single-stranded probe DNA is 20 to 60 minutes.
Preferably, in step (4), the soluble metal salt is NaCl.
Preferably, in step (5), the reaction time of the suspension with the single-stranded target DNA is 6 to 8 hours.
The invention also provides a DNA biosensor prepared by the preparation method.
The beneficial effects of the invention are as follows:
(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 device and good biocompatibility.
(3) The invention has simple preparation, no need of complex pretreatment, small size and high-efficiency real-time detection.
Drawings
FIG. 1 is a schematic diagram of the detection principle of a DNA biosensor.
Detailed Description
The preferred embodiments of the present invention will be described in detail.
The preparation method of the DNA biosensor comprises the following steps:
step (1), a shearing stripping method is adopted to obtain molybdenum disulfide nanosheet dispersion liquid; the 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 for 80-90 min at a rotating speed of 10000-15000 rpm;
(1-2) centrifuging at 1500rpm for 90min after the completion of stirring, and collecting the supernatant;
(1-3) centrifuging the supernatant in the step (1-2) at a rotating speed of 10000-15000 rpm for 20min, taking a precipitate, and adding deionized water into the precipitate to dissolve the precipitate;
(1-4) repeating the step (1-3) for 3 times, taking a precipitate, adding deionized water into the precipitate, and carrying out ultrasonic treatment for 5min to obtain the uniformly dispersed molybdenum disulfide nanosheet dispersion liquid.
And (2) cleaning the gold electrode to a mirror surface by using Piranha solution, washing the mirror surface by using deionized water, drying the mirror surface by using nitrogen, and then assembling the gold electrode and a static cell assembly of the quartz crystal microbalance together.
In the step (2), freshly prepared Piranha solution (prepared by mixing concentrated sulfuric acid and 30% hydrogen peroxide by mass percent according to a volume ratio of 3:1) is dropwise added to the surface of the gold electrode. The reaction time of Piranha solution on the surface of the gold electrode is 15-30 s, and the deionized water flushing time is 30-60 s.
And (3) adding molybdenum disulfide dispersion liquid into the static pool in the step (2), then adding 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 hours 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 step (3) and step (5) were dissolved in Tris-HCl buffer (Chinese name: tris hydrochloride, commercially available from Beijing Soy Corp., product No. T1090) at a concentration of 25nM and pH=7.4. The invention constructs a label-free biosensor based on molybdenum disulfide nanosheets based on the different affinities of the molybdenum disulfide nanosheets for single-stranded DNA and double-stranded DNA and the characteristic that quartz crystal microbalances (Quartz Crystal Microbalance, QCM) are sensitive to quality change, obtains corresponding frequency change by detecting the quality change of gold electrode surfaces caused by different states of the molybdenum disulfide nanosheets in a static cell of the quartz crystal microbalances, and rapidly detects specific DNA through the frequency change. The method of the invention has simple operation, easy realization and low cost.
While the foregoing has been with reference to the preferred embodiments and principles of the present invention, it will be apparent to those skilled in the art that changes in this embodiment may be made without departing from the principles of the invention.
Claims (8)
1. A method for preparing a DNA biosensor, comprising the steps of:
(1) A shearing stripping method is adopted to obtain molybdenum disulfide nanosheet dispersion liquid;
(2) Cleaning a gold electrode to a mirror surface by using Piranha solution, washing the mirror surface by using deionized water, drying the mirror surface by using inert gas, and then assembling the gold electrode and a static cell assembly 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 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) Adding single-stranded target DNA into the suspension obtained in the step (4), and reacting at room temperature to obtain a second-state label-free biosensor, namely the DNA biosensor;
in the step (4), the soluble metal salt is NaCl;
the single-stranded target DNA in step (5) and the single-stranded probe DNA in step (3) are complementary DNAs.
2. The method for preparing a DNA biosensor according to claim 1, wherein: in the step (1), the shear 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 for 80-90 min at a rotating speed of 10000-15000 rpm;
(1-2) centrifuging at 1500rpm for 90min after the completion of stirring, and collecting the supernatant;
(1-3) centrifuging the supernatant in the step (1-2) at a rotating speed of 10000-15000 rpm for 20min, taking a 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 carrying out ultrasonic treatment for 5min to obtain the uniformly dispersed molybdenum disulfide nanosheet dispersion liquid.
3. The method for preparing a 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 with 30% hydrogen peroxide; the reaction time of the Piranha solution on the surface of the gold electrode is 15-30 s; the deionized water flushing time is 30-60 s.
4. A method for producing a DNA biosensor according to any one of claims 1 to 3, wherein in the step (2), the inert gas is nitrogen.
5. The method for preparing a DNA biosensor according to claim 1, wherein in the step (3), the molybdenum disulfide nanosheet dispersion liquid is incubated with the single-stranded probe DNA at room temperature for 20 to 60 minutes.
6. The method for producing a DNA biosensor according to any one of claims 1 to 3 or 5, wherein in step (5), the reaction time of the single-stranded target DNA with the suspension is 6 to 8 hours.
7. The method for preparing a DNA biosensor according to 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 dissolved in Tris-HCl buffer at a concentration of 25nM and pH=7.4.
8. A DNA biosensor produced by the production method according to any one of claims 1 to 7.
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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 |
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| Development of an effective electrochemical platform for highly sensitive DNA detection using MoS2 - polyaniline nanocomposites;Shibsankar Dutta 等;《Biochemical Engineering Journal》;第140卷;130-139 * |
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