CN113484387B - DNA biosensor and preparation method thereof - Google Patents

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

A DNA biosensor and its preparation method

Technical field

The invention belongs to the technical field of biosensors, and specifically relates to a DNA biosensor and a preparation method thereof.

Background technique

As an important biomarker, DNA is widely used in the early detection of various diseases. Quartz crystal microbalance is a new biosensor that uses quartz crystal to be sensitive to mass changes. When the crystal surface mass changes, the amount of adsorbed material can be monitored through changes in frequency, and its measurement accuracy can reach the nanogram level. Since the quartz crystal microbalance measures mass changes and does not require labeling, it can simplify the analysis procedures and increase the analysis speed. Therefore, it has the characteristics of fast response, high sensitivity, good specificity, small size and simplicity, and is widely used in the field of biomedical sensing technology. attracted the attention of the industry.

As one of the transition metal chalcogen compounds, molybdenum disulfide nanosheets have a large specific surface area. Therefore, their surface will produce a relatively large electrostatic attraction. At the same time, due to the electron cloud distribution of molybdenum atoms and sulfur atoms, they tend to be in In the middle, one tends to be at both ends, resulting in a certain negative charge on the surface of the molybdenum disulfide nanosheets. For single-stranded nucleic acids, since their bases are exposed, they are easily absorbed by the molybdenum disulfide nanosheets. Adsorption, and for double-stranded nucleic acids, because its negatively charged phosphate backbone is exposed due to the double helix structure, it will produce a greater repulsion to the negatively charged molybdenum disulfide nanosheets on the surface, which is far greater than the electrostatic attraction of adsorption. So it cannot be adsorbed. Taking advantage of the different adsorption properties of molybdenum disulfide nanosheets on single-stranded nucleic acids and double-stranded nucleic acids, a sensing method based on molybdenum disulfide nanosheets for detecting nucleic acids can be constructed.

Currently, most of the sensing methods based on molybdenum disulfide nanosheets are detection methods based on the principle of fluorescence resonance energy. This method requires modifying a fluorescent group at one end of the DNA, which is costly and cumbersome to operate. Therefore, it is of great significance to develop new biosensing methods based on molybdenum disulfide nanosheets and quartz crystal microbalances that can reduce costs and operating procedures.

Contents of the invention

In view of the problems of cumbersome operation and high cost of existing fluorescent sensors for detecting nucleic acids, the present invention provides a method for preparing a new biosensor based on molybdenum disulfide nanosheets and quartz crystal microbalance. The main principle is: when the target DNA does not exist, the single-stranded probe DNA is adsorbed on the surface of the molybdenum disulfide nanosheets, keeping the molybdenum disulfide nanosheets in a dispersed and suspended state; when the target DNA is present, it is adsorbed on the surface of the molybdenum disulfide nanosheets. The single-stranded probe DNA on the surface of the nanosheet will combine with its complementary target DNA to form double-stranded DNA and break away from the surface of the molybdenum disulfide nanosheet. At this time, the mutual repulsion between the molybdenum disulfide nanosheets decreases, and the molybdenum disulfide nanosheets The nanosheets will undergo precipitation and aggregation, the mass of the crystal surface will increase, and the output frequency will decrease.

In order to achieve the above objects, the present invention adopts the following technical solutions:

A method for preparing a DNA biosensor, including the following steps:

(1) Use shear exfoliation method to obtain molybdenum disulfide nanosheet dispersion;

(2) Use Piranha solution (Chinese name: Piranha solution) to clean the gold electrode to the mirror surface, rinse it with deionized water, and blow dry with inert gas, and then assemble the gold electrode and the static cell assembly of the quartz crystal microbalance together;

(3) Add molybdenum disulfide dispersion to the static pool in step (2), then add single-stranded probe DNA, mix evenly, and incubate at room temperature;

(4) Add the soluble metal salt to the suspension obtained in step (3) and mix evenly to obtain the label-free biosensor in the first state;

(5) Add single-stranded target DNA to the suspension obtained in step (4), and react at room temperature to obtain a label-free biosensor in the second state.

Since the surface of molybdenum disulfide nanosheets carries a certain degree of negative charge, there is a certain repulsion between the molybdenum disulfide nanosheets in the dispersion liquid of molybdenum disulfide nanosheets. When metal cations are added, the metal cations are removed due to charge neutralization. Some of them will be free between the sheets, which will weaken the repulsion between the sheets and make the electrostatic adsorption between the molybdenum disulfide nanosheets play a dominant role, making the stacking and deposition of the sheets become relative. It is easier; when there is single-stranded DNA, since the single-stranded DNA is adsorbed on the surface of the molybdenum disulfide nanosheets, it can protect the molybdenum disulfide nanosheets from stacking and deposition at high salt concentrations; when there is a single-stranded probe When the target single-stranded DNA complementary to the DNA exists, the two complement each other to form a double-stranded nucleic acid. Since the double-stranded nucleic acid has a rigid structure, the adsorption force between the double-stranded nucleic acid and the molybdenum disulfide nanosheets is very weak. In an environment with high salt concentration, , stacking and deposition of molybdenum disulfide nanosheets occur. The present invention utilizes this property to construct a label-free biosensor that regulates the sedimentation degree of molybdenum disulfide nanosheets by using single-stranded probe DNA, target single-stranded DNA and metal cations to detect the concentration of target single-stranded DNA.

Preferably, in step (1), the molybdenum disulfide nanosheet dispersion can be prepared by shear peeling method; the specific method is as follows:

(1-1) Add 2g of sodium cholate powder and 10g of molybdenum disulfide powder to 200mL of deionized water, and stir intermittently at a speed of 10,000 to 15,000 rpm for 80 to 90 minutes;

(1-2) After stirring is completed, centrifuge at 1500 rpm for 90 minutes and take the supernatant;

(1-3) Centrifuge the supernatant in step (1-2) for 20 minutes at a speed of 10,000 to 15,000 rpm, take the precipitate, and add deionized water to the precipitate to dissolve the precipitate;

(1-4) Repeat step (1-3) three times in total, take the precipitate, add deionized water to the precipitate and sonicate for 5 minutes to obtain a uniformly dispersed dispersion of molybdenum disulfide nanosheets.

Preferably, in step (2), freshly prepared Piranha solution (prepared by mixing concentrated sulfuric acid and 30% mass percent hydrogen peroxide at a volume ratio of 3:1) is dropped onto the surface of the gold electrode.

Preferably, in step (2), the reaction time of the Piranha solution on the gold electrode surface is 15 to 30 s, and the deionized water rinsing time is 30 to 60 s.

Preferably, in step (2), the inert gas is nitrogen.

Preferably, in step (3), single-stranded probe DNA is added to the molybdenum disulfide nanosheet dispersion, and the reaction is carried out at room temperature for 20 to 60 minutes.

Preferably, the single-stranded probe DNA and single-stranded target DNA in steps (3) and (5) are both dissolved in Tris-Hcl buffer, with a concentration of 25 nM and pH=7.4.

Preferably, in step (3), the incubation time between molybdenum disulfide and 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 between the suspension and the single-stranded target DNA is 6 to 8 hours.

The invention also provides a DNA biosensor prepared by the above preparation method.

The beneficial effects of the present invention are:

(1) The DNA biosensor of the present invention has a wide range of applications and can be used in specific cancer screening, genetic engineering, food safety, etc.

(2) The present invention has simple operation process, low cost, does not require expensive detection devices, and has good biocompatibility.

(3) The present invention is simple to prepare, does not require cumbersome pretreatment, is small in size, and can achieve efficient real-time detection.

Description of the drawings

Figure 1 is a schematic diagram of the detection principle of DNA biosensor.

Detailed ways

The preferred embodiments of the present invention will be described in detail below.

This embodiment is a method for preparing a DNA biosensor, including the following steps:

Step (1), use shear peeling method to obtain molybdenum disulfide nanosheet dispersion; the operation of this step is as follows:

(1-1) Add 2g of sodium cholate powder and 10g of molybdenum disulfide powder to 200mL of deionized water, and stir intermittently at a speed of 10,000 to 15,000 rpm for 80 to 90 minutes;

(1-2) After stirring is completed, centrifuge at 1500 rpm for 90 minutes and take the supernatant;

(1-3) Centrifuge the supernatant in step (1-2) for 20 minutes at a speed of 10,000 to 15,000 rpm, take the precipitate, and add deionized water to the precipitate to dissolve the precipitate;

(1-4) Repeat step (1-3) three times, take the precipitate, add deionized water to the precipitate and sonicate for 5 minutes to obtain a uniformly dispersed dispersion of molybdenum disulfide nanosheets.

Step (2): Clean the gold electrode with Piranha solution to the mirror surface, rinse with deionized water, and blow dry with nitrogen. Then assemble the gold electrode and the static cell assembly of the quartz crystal microbalance together.

In this step (2), freshly prepared Piranha solution (concentrated sulfuric acid and 30% hydrogen peroxide by mass percentage are mixed and prepared at a volume ratio of 3:1) is dropped onto the surface of the gold electrode. The reaction time of Piranha solution on the gold electrode surface is 15 to 30 s, and the deionized water rinsing time is 30 to 60 s.

In step (3), first add molybdenum disulfide dispersion to the static pool in step (2), then add single-stranded probe DNA, mix evenly, incubate at room temperature, and react at room temperature for 20 to 60 minutes.

Step (4), add soluble metal salt NaCl to the suspension obtained in step (3), and mix evenly to obtain a label-free biosensor in the first state.

Step (5): Add single-stranded target DNA to the suspension obtained in step (4), and react at room temperature for 6 to 8 hours to obtain a label-free biosensor in the second state.

In this example, the single-stranded probe DNA and single-stranded target DNA in steps (3) and (5) were dissolved in Tris-Hcl buffer (Chinese name: tris-hydroxymethylaminomethane hydrochloride, purchased from (Beijing Solebao Technology Co., Ltd., product number: T1090), its concentration is 25nM, pH=7.4. The present invention constructs a molybdenum disulfide nanosheet based on the different affinities of molybdenum disulfide nanosheets to single-stranded DNA and double-stranded DNA and the sensitivity of quartz crystal microbalance (QCM) to mass changes. The label-free biosensor detects the mass changes on the gold electrode surface caused by different states of molybdenum disulfide nanosheets in the static pool of a quartz crystal microbalance, and obtains the corresponding frequency changes, and quickly detects specific DNA through this frequency change. . The method of the invention is simple to operate, easy to implement and low in cost.

The preferred embodiments and principles of the present invention have been described in detail above. For those of ordinary skill in the art, there will be changes in the specific implementation methods based on the ideas provided by the present invention, and these changes should also be regarded as the present invention. scope of protection.

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.