CN114354696A

CN114354696A - DNA biosensor driven by friction nano generator and application thereof

Info

Publication number: CN114354696A
Application number: CN202111413513.1A
Authority: CN
Inventors: 王鹏; 曲晓琳
Original assignee: Institute of Oceanology of CAS
Current assignee: Institute of Oceanology of CAS
Priority date: 2021-11-25
Filing date: 2021-11-25
Publication date: 2022-04-15
Anticipated expiration: 2041-11-25
Also published as: CN114354696B

Abstract

The invention belongs to the technical field of biological detection, and particularly relates to a DNA biosensor driven by a friction nano generator and application thereof. A tribo-nanogenerator driven DNA biosensor comprising: the friction nano generator is used for converting external mechanical energy into electric energy and outputting the electric energy; the resistance-type biosensor is electrically connected with the friction nano generator and is driven by electric energy output by the friction nano generator to capture target DNA; and a carbon nanotube for binding the target DNA and causing the resistive biosensor to generate a sensing signal; the surface of the resistance biosensor is modified with a DNA capture probe; the surfaces of the carbon nano tubes are all modified with DNA signal probes. The DNA sensor has a simple structure, is driven by the friction nano generator, does not need an external power supply, and improves the portability of the sensor; the DNA probe technology is adopted, the pM-level DNA concentration detection can be realized, and the sensitivity and the specificity are high.

Description

DNA biosensor driven by friction nano generator and application thereof

Technical Field

The invention belongs to the technical field of biological detection, and particularly relates to a DNA biosensor driven by a friction nano generator and application thereof.

Background

The current "gold standard" for the detection and identification of bacteria is the use of culture-based methods. Although traditional culture-based techniques are reliable and accurate and relatively inexpensive, the method is time-consuming and laborious, may take days or even weeks to obtain results, and is limited to culturable bacteria. In addition, there are some important bacteria that are difficult or impossible to culture and cannot be detected by the culture method. Molecular techniques are particularly useful for detecting a wide variety of substances or low template amounts. The detection of specific DNA sequences is becoming increasingly important in bacterial detection. The analysis of bacterial DNA is a common bacterial analysis approach and is of great importance in molecular diagnostics of bacteria, rapid classification and phenotyping of bacterial species. Many detection methods have been proposed, including fluorescence, electrochemiluminescence, enzymatic, surface plasmon resonance, Polymerase Chain Reaction (PCR) related diagnostic methods for detection of 16S ribosomal RNA (16S rRNA) fragments. However, these methods have the disadvantages of harsh experimental conditions, complicated operation, environmental pollution, unstable activity when applied to complicated samples, and false positive, which makes them an important bottleneck in molecular diagnosis. Therefore, a method for detecting bacterial DNA fragments which is faster, more convenient and easier to operate is needed.

Among the existing methods, electrochemical DNA sensors have become an interesting choice because of their simplicity, portability and low cost. But it has limited application due to the additional tag and power required. Since 2012, triboelectric nanogenerators proposed based on the coupling of triboelectric charging and electrostatic induction effects have been an efficient means and method for converting various mechanical energies (e.g., human walking, wind energy, water wave energy, etc.) into electrical energy. Meanwhile, a tribo nano-generator (TENG) is also used to prepare various self-driven sensors. The method does not need a power supply and has high sensitivity, so that the development of a novel self-driven DNA sensor based on TENG is a promising idea.

Disclosure of Invention

The invention aims to provide a DNA biosensor driven by a friction nano generator aiming at the problems in the existing biological detection technology, the DNA biosensor utilizes the friction nano generator as a driving power supply to detect the DNA of a biological sample, and has the advantages of high sensitivity, quick detection, no need of an external power supply and the like.

In order to achieve the purpose, the invention adopts the technical scheme that: a tribo-nanogenerator driven DNA biosensor comprising:

the friction nano generator is used for converting external mechanical energy into electric energy and outputting the electric energy;

the resistance-type biosensor is electrically connected with the friction nano generator and is driven by electric energy output by the friction nano generator to capture target DNA; and the number of the first and second groups,

the carbon nano tube is used for combining the target DNA and causing the resistance type biosensor to generate a sensing signal;

the surface of the resistance biosensor is modified with a DNA capture probe;

and the surface of the carbon nano tube is modified with a DNA signal probe.

Furthermore, the friction nano generator comprises two conductive layers, wherein a friction layer is arranged on the surface of one conductive layer; the friction layer is used as a negative electrode of the power generation layer, the other conducting layer is used as a positive electrode of the power generation layer, and the positive electrode of the power generation layer and the negative electrode of the power generation layer generate power through friction under the action of external force.

Further, the friction layer is a perfluoroethylene propylene copolymer film with the thickness of 50 microns.

Further, the conductive layer is an aluminum thin film, and the thickness of the conductive layer is 50 micrometers.

Furthermore, a supporting substrate is arranged on one surface of the conducting layer opposite to the friction surface of the conducting layer; an elastic element is connected between the two supporting substrates.

Furthermore, the resistance type sensor is made of etched ITO conductive glass, a gap with the width of 50 microns is etched in the middle of the ITO conductive glass, and the etching depth is equal to the thickness of the ITO film layer; and silane is modified on the surface of the etched ITO conductive glass.

Further, the sensing system further comprises a variable resistor connected in series between the friction nanogenerator and the resistive biosensor.

Furthermore, two ends of the variable resistor are connected with an alarm device in parallel.

The invention also provides application of the DNA biosensor driven by the friction nano generator, and the sensor can be applied to detecting bacterial DNA.

The invention also provides a DNA detection method driven by the friction nano generator, which comprises the following steps:

(1) soaking the resistance-type biosensor in a DNA solution to be tested, and incubating for 45min at 37 ℃;

(2) taking out the sensor, soaking the sensor in a carbon nanotube solution modified with a DNA signal probe, and incubating for 45min at 37 ℃;

(3) taking out the sensor, slightly washing out the unbound carbon nanotubes by deionized water, and naturally drying;

(4) driving by a friction nano generator, and testing voltage values at two ends of the resistance-type biosensor;

(5) and calculating the concentration of the target DNA in the solution to be detected by using a pre-fitted voltage-DNA concentration curve equation.

According to the invention, a capture probe and a signal probe which can be specifically and complementarily combined with target DNA are adopted to form a sandwich structure with the target DNA, so that the carbon nanotube for modifying the signal probe is combined on the resistance-type biosensor to cause resistance change, the voltage at two ends of the resistance is further changed, and the concentration of the target DNA can be inverted according to the voltage.

The DNA biosensor driven by the friction nano generator provided by the invention has the following beneficial effects:

1. in the invention, the sensor is driven by the friction nano generator, mechanical energy can be converted into electric energy, an external power supply is not required, and the portability of the sensor is improved;

2. the DNA sensor of the invention adopts the DNA probe technology, can realize the detection of the DNA concentration of pM level, and has very high sensitivity;

3. the base sequences of different bacterial DNAs are different, so that the types of bacteria can be simply identified through voltage signals;

4. the detection method is simple and quick, the result can be obtained only in a few hours, and compared with the traditional detection method, the efficiency is greatly improved;

5. the DNA sensor has the advantages of simple structure, working at normal temperature and normal pressure, no need of external power supply, contribution to industrial popularization and potential application value in developing a self-driven DNA sensing network oriented to wireless sensing.

Drawings

FIG. 1 is a schematic structural diagram of a DNA biosensor driven by a friction nanogenerator according to an embodiment of the invention;

FIG. 2 is a schematic structural diagram of a triboelectric nanogenerator according to an embodiment of the invention;

FIG. 3 is an equivalent circuit diagram of a DNA biosensor driven by a friction nanogenerator according to an embodiment of the invention;

FIG. 4(a) is the output voltage of the triboelectric nanogenerator;

FIG. 4(b) is the stability of the triboelectric nanogenerator;

FIG. 5 is a representation of the ITO conductive glass after modification; wherein a is an XPS diagram of ITO conductive glass modified silane, b is an XPS diagram of ITO conductive glass modified silane and then connected with a capture probe, c is an XPS diagram of a carbon nano tube, d is an XPS diagram of a signal probe modified by the carbon nano tube, and e is an XPS diagram of ITO conductive glass modified silane and the capture probe;

FIG. 6 is a feasibility analysis of the triboelectric nanogenerator-driven DNA biosensor;

FIG. 7a shows the variation of output voltage with DNA concentration;

FIG. 7b is a linear fit after error bars from triplicate experiments with different concentrations of DNA;

FIG. 7c is an SEM image of ITO conductive glass surfaces at different concentrations of DNA and blank sets.

FIG. 8 is a 3D diagram of a portable device of the DNA biosensor driven by the tribo nanogenerator.

Detailed Description

In order to facilitate an understanding of the invention, the invention is described in more detail below with reference to the accompanying drawings and specific examples. Preferred embodiments of the present invention are shown in the drawings. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.

Example 1 this example provides a triboelectric nanogenerator driven DNA biosensor, the structure of which is shown in fig. 1. As shown in fig. 1, the sensor includes a triboelectric nanogenerator 100, a resistive biosensor 200, a variable resistor 300, and an LED lamp 400. The friction nano-generator 100 serves to convert external mechanical energy into electrical energy to output an electrical signal to an external circuit. The resistive biosensor 200 is electrically connected to the frictional nanogenerator 100 such that the frictional nanogenerator 100 supplies power to the resistive biosensor 200 for detecting a sensing signal. The variable resistor 300 is connected in series with the resistive biosensor 200 to divide a portion of the voltage. The LED lamp 400 is connected in parallel across the variable resistor 300 for emitting an optical alarm signal when the sensing signal is greater than a certain signal threshold.

Fig. 2 shows a structural representation of the triboelectric nanogenerator 100. As shown in fig. 2, the tribo nanogenerator 100 comprises two support substrates, an elastic element 160, a first conductive layer 103, a second conductive layer 105, and a first friction layer 104. The two support substrates are a first support substrate 101 and a second support substrate 102, respectively, and the support substrates are made of PVC plates. The elastic elements 106 are connected between two support substrates, in the embodiment shown in fig. 2, the elastic elements 106 are springs, the number of which is four. The first conductive layer 103 is fixed to the lower surface of the first support substrate 101, and the second conductive layer 105 is fixed to the upper surface of the second support substrate 102. The material of the first conductive layer 103 and the second conductive layer 105 is an aluminum thin film with a thickness of 50 μm. The lower surface of the first conductive layer 103 is provided with a first friction layer 104, the first friction layer 104 is a perfluoroethylene propylene copolymer film, the thickness of the perfluoroethylene propylene copolymer film is 50 micrometers, and the size and shape of the first friction layer are the same as those of the two conductive layers. The second conductive layer 105 can be used as an electrode material and a second friction layer to generate electricity by friction with the first friction layer 104. The first conductive layer 103 and the second conductive layer 105 are adhered to the two support substrates by Kapton adhesive.

The resistive biosensor 200 is made of etched ITO conductive glass, as shown in fig. 1, a gap with a width of 50 μm is etched in the middle of the ITO conductive glass, and the etching depth is equal to the thickness of the ITO film layer. The etched ITO conductive glass has infinite resistance due to the fault in the middle of the ITO film layer.

The surface of the etched ITO conductive glass needs to be modified by silane and a DNA capture probe in sequence so as to specifically identify and combine target DNA.

The sensing system for bacteria detection driven by the friction nano generator provided by the embodiment has the working principle that: the first supporting substrate 101 is pressed to make the first friction layer 104 and the second conductive layer 105 (second friction layer) perform periodic contact friction, so as to convert mechanical energy into electrical energy, and output current to the system circuit through the first conductive layer 103 and the second conductive layer 105, and an equivalent circuit diagram is shown in fig. 3. The output voltage was measured by a programmable multifunction electrometer (Keithley, model 6514), and whether it was the target DNA could be identified by whether the LED light was on or off.

In the initial state, the resistance biosensor 200 is an etched ITO conductive glass, and the etched ITO conductive glass is not conductive and is connected in a circuit, and the resistance is infinite.

After the resistive biosensor 200 is soaked in a solution containing target DNA, the DNA capture probe modified on the surface of the resistive biosensor 200 can specifically recognize and capture the target DNA by clipping and complementation, and after the target DNA is captured, the target DNA is soaked in a carbon nanotube solution modified by a DNA signal probe, and at this time, the target DNA captured by the resistive biosensor 200 is complementarily combined with the DNA signal probe to adsorb the carbon nanotube, and the resistance of the resistive biosensor 200 is reduced because the carbon nanotube is conductive. When the target DNA concentration is high, the number of carbon nanotubes combined with the resistive biosensor 200 is also large, the resistance of the resistive biosensor 200 is smaller, and the voltage divided between the two ends of the resistive biosensor 200 is also smaller, so that the concentration of the target DNA can be reflected according to the voltage value between the two ends of the resistive biosensor 200. The sensitivity can also be lower when the base sequence of the target DNA is longer, and now the target DNA of 22bp is detected, and the lower limit of the detection is 1 pM.

Fig. 4a and 4b show the output voltage and power stability of the friction nano-generator, respectively. As can be seen from fig. 4, the operating voltage of the friction nanogenerator according to the invention is about 160V. The characterization of the ITO conductive glass and the characterization of the carbon nanotubes are shown in fig. 5, wherein a and b are ITO conductive glass modified silane and capture probes, respectively. c. d is the carbon nano tube and the carbon nano tube after the signal probe is modified. Figure 5e demonstrates that the modified silane as well as the capture probe had no effect on the output. To further determine the feasibility of the invention, the feasibility analysis of the invention was performed as shown in FIG. 6. It is clear that the absence of either probe is not detectable and that the voltage drops only when the DNA of interest is present.

The voltage variation of self-driven DNA sensor based on friction nano-generator to detect different concentrations of DNA of interest is shown in FIG. 7 a. Based on the dependence of the output voltage on the continuous change of the DNA concentration, a concentration detection mode is provided, the voltage is continuously reduced along with the increase of the target DNA concentration, the lower detection limit is 1pM, because the quantity of the adhered carbon nanotubes is increased along with the increase of the target DNA concentration, the conductivity of the ITO conductive glass is increased, and the voltage is reduced. Error bars obtained from three replicates are shown in figure 7 b. And a correlation coefficient. Has good linear relation and high sensitivity. FIG. 7 is a drawing showing the adhesion of carbon nanotubes by SEM, and as shown in FIGS. 7c, I and II are SEM images of the case where the signal probe is not modified on the carbon nanotube and the case where the target DNA is not ligated, respectively, it can be seen from the sequential decrease in the concentrations of the target DNA III to IX in FIG. 7c that the adhered carbon nanotubes are indeed decreased as the concentration of the target DNA is decreased. In conclusion, the output voltage is analyzed, the concentration of the target DNA can be judged, and the method is based on the working principle of the DNA sensor of the friction nano generator.

The DNA biosensor of the embodiment can detect any DNA material, is not limited to bacteria, fungi, viruses, animals, plants and the like, and can realize rapid qualitative and quantitative detection of DNA.

Embodiment 2 this example provides a bacterial DNA detection method driven by a triboelectric nanogenerator, comprising the steps of:

FIG. 8 is a 3D diagram of a portable device of the DNA sensor of the friction nano-generator of the present invention, the sensor is placed in the middle of the portable device, the LED lamp is installed in the round hole on the upper surface of the portable device, whether the target DNA is detected can be simply distinguished, and ITO conductive glass and a variable resistor are respectively placed in the inner groove.

Claims

1. A triboelectric nanogenerator-driven DNA biosensor comprising:

the surface of the resistance biosensor is modified with a DNA capture probe;

and the surface of the carbon nano tube is modified with a DNA signal probe.

2. The DNA biosensor driven by friction nanogenerator according to claim 1, wherein the friction nanogenerator comprises two conductive layers, one of which has a friction layer on its surface; the friction layer is used as a negative electrode of the power generation layer, the other conducting layer is used as a positive electrode of the power generation layer, and the positive electrode of the power generation layer and the negative electrode of the power generation layer generate power through friction under the action of external force.

3. The DNA biosensor driven by tribo-nanogenerator according to claim 2, wherein the tribo-layer is a perfluoroethylene propylene copolymer film with a thickness of 50 μm.

4. The DNA biosensor driven by a tribo nanogenerator according to claim 2, wherein the conductive layer is an aluminum thin film with a thickness of 50 μm.

5. The DNA biosensor driven by a tribo nanogenerator according to claim 2, wherein a support substrate is provided on a surface of the conductive layer opposite to the frictional surface thereof; an elastic element is connected between the two supporting substrates.

6. The DNA biosensor driven by a tribo nanogenerator according to claim 1, wherein the resistive sensor is made of etched ITO conductive glass, a gap with a width of 50 μm is etched in the middle of the ITO conductive glass, and the etching depth is equal to the thickness of the ITO film layer; and silane is modified on the surface of the etched ITO conductive glass.

7. The tribo nanogenerator driven DNA biosensor as in claim 1, further comprising a variable resistance in series between the tribo nanogenerator and the resistive biosensor.

8. The DNA biosensor driven by friction nanogenerator according to claim 7, wherein the two ends of the variable resistor are connected in parallel with an alarm device.

9. Use of a tribo nanogenerator driven DNA biosensor according to any of claims 1 to 8 for the detection of bacterial DNA.

10. A DNA detection method driven by a friction nano generator is characterized by comprising the following steps: