A bacterial detection device and method based on single droplet generator

Abstract

The application belongs to the technical field of biological detection, and particularly relates to a kind of bacterial detection device and method driven by class field effect water drop generator, single droplet generator, single droplet generating device, reaction device and signal acquisition device;Single droplet generating device is placed directly above single droplet generator, and signal acquisition device is directly connected with single droplet generator.The bacterial detection method utilizes polystyrene microspheres attached to the electrode of single droplet generator, and forms additional parasitic capacitance between polystyrene microspheres and single droplet generator, which can directly detect bacterial samples.And the change of this parasitic capacitance will directly affect the output voltage of water drop generator, so that bacterial samples can be quickly and conveniently detected and observed through the change of output voltage of single droplet generator.The detection method has the advantages of rapid detection, no external power supply, no waiting energy collection process, simple operation and the like.

Description

Technical Field

[0001] The present invention belongs to the field of biological detection technology, and in particular relates to a bacteria detection device and method driven by a field-effect water droplet generator. Background Art

[0002] Marine corrosion, food contamination, water pollution, and chronic infectious diseases caused by bacterial contamination have brought huge losses to human society. For example, Pseudomonas aeruginosa, a facultative anaerobic, Gram-negative bacterium, is an important bacterium that causes marine corrosion and bacterial infections. Therefore, it is very important to develop a fast and convenient bacterial detection method. At present, the commonly used bacterial detection method is a biochemical detection method based on bacterial culture, but the application of this method is limited by the long time, high cost and labor-intensive procedures. Emerging bacterial detection methods, such as polymerase chain reaction, surface-enhanced Raman spectroscopy and enzyme-linked immunosorbent assay, although these methods have the advantages of fast detection speed and simple operation, still require expensive testing equipment and professionally trained testers to operate.

[0003] Single-droplet generators (SDGs) have attracted significant research attention due to their significantly higher instantaneous power density than solid-liquid triboelectric nanogenerators (TNGNs). Currently, their primary application is energy harvesting. While some SDGs have been used to detect temperature or humidity, these applications rely on powering the sensor after energy harvesting. No research has considered directly exploiting the performance variations of SDGs for self-powered detection. Recent research indicates that when electrodes in a SDG come into contact with other materials, a new capacitance structure, known as parasitic capacitance, forms between them. This parasitic capacitance exhibits a reverse shunting effect, significantly reducing the SDG's output voltage. To address this parasitic capacitance phenomenon, researchers have proposed various solutions to reduce it. However, the significant variations in parasitic capacitance within SDGs, which can be directly reflected in their output voltage, make them promising candidates for bacterial detection. Summary of the Invention

[0004] To address the above issues, this application provides a novel bacteria detection method based on a single droplet generator. This bacteria detection method utilizes the changes in the parasitic capacitance of the single droplet generator itself and displays it through the output voltage. It can directly detect bacterial samples and has the advantages of rapid detection, no need for external power supply, no need to wait for energy collection, and simple operation.

[0005] To achieve the above object, the technical solution adopted by the present invention is:

[0006] A bacteria detection device based on a single droplet generator, a single droplet generator, a single droplet generating device, a reaction device and a signal acquisition device;

[0007] The single droplet generating device is placed directly above the single droplet generator, and the signal acquisition device is directly connected to the single droplet generator.

[0008] The single-droplet generator includes a friction layer, a conductive layer, a supporting substrate and an electrode; the conductive layer is fixed on the supporting substrate, the friction layer is fixed on the conductive layer, and the electrode is placed on the friction layer; the electrode is modified with an aptamer for specifically identifying and capturing target bacteria; a polystyrene microsphere solution is provided in the reaction device, and the surface of the polystyrene microspheres is modified with a specific aptamer for the target bacteria, which is used to bind to the target bacteria and cause changes in the parasitic capacitance of the single-droplet generator itself. The signal acquisition device collects the signal emitted by the single-droplet generator and displays it in the form of an output voltage.

[0009] Preferably, the friction material is a perfluoroethylene propylene copolymer film, the supporting substrate is a PVC board, and the electrode is a gold wire.

[0010] Preferably, the single droplet generating device includes a control switch and a dripping device for generating a single droplet or continuous droplets.

[0011] Preferably, the diameter of the gold wire is 10-50 microns; the diameter of the polystyrene microspheres is 300-800 nanometers.

[0012] A bacterial detection method based on a single droplet generator involves immersing a gold wire modified with an aptamer in a test solution containing target bacteria. The gold wire then specifically captures the target bacteria through the specific recognition and capture capabilities of the aptamer.

[0013] After capturing the target bacteria, the gold wire is immersed in a solution of polystyrene microspheres modified with aptamers. The target bacteria attached to the gold wire then specifically recognize the aptamers and adsorb the polystyrene microspheres.

[0014] Due to the attachment of polystyrene microspheres, the parasitic capacitance of the single-droplet generator will change and can be displayed in the form of output voltage, making the signal acquisition and detection process convenient; the single-droplet generator sends the signal to the information acquisition device, which can reflect the target bacterial contamination situation based on the change of the parasitic capacitance of the single-droplet generator.

[0015] (1) Immerse the gold wire in the sample to be tested and incubate at 37°C for 120 min;

[0016] (2) Take out the gold wire and soak it in a solution of polystyrene microspheres modified with aptamers at 37°C for 120 min;

[0017] (3) Remove the gold wire, rinse with deionized water to remove unbound polystyrene microspheres, and air dry naturally;

[0018] (4) The gold wire is placed on the friction layer of a single droplet generator to control the drop of a single droplet. The output signal of the single droplet generator is tested and compared with the output signal when the original gold wire is used as an electrode to analyze the bacterial contamination situation.

[0019] Preferably, after polystyrene microspheres are bound to the electrodes of the single-droplet generator, they will enhance the parasitic capacitance of the single-droplet generator and store the transferred charge in the single-droplet generator through the reverse shunting effect, thereby reducing the output voltage of the single-droplet generator. Therefore, the output voltage of the single-droplet generator can reflect the parasitic capacitance and thus the bacterial concentration. The fitting relationship between the output voltage of the single-droplet generator and the bacterial concentration is as follows:

[0020]

[0021] Among them, U0 represents the initial output voltage of the water drop generator; U C Represents the actual output voltage of the water drop generator; where S Au 、S ps , ε water , ε ps , K F 、C B represent the coverage area between water and gold wire-F23, the total area of ​​attached polystyrene microspheres, the dielectric constant of water, the dielectric constant of polystyrene microspheres, the Freundlich isotherm constant, and the bacterial concentration, respectively; when the bacterial type is determined, b and a are two constants.

[0022] Preferably, the signal acquisition device is connected to an alarm system, and when the voltage signal exceeds a threshold, the alarm system sounds an alarm.

[0023] Beneficial effects

[0024] 1. In the present invention, the single droplet generator can effectively convert mechanical energy into electrical energy, eliminating the need for external power supply, improving convenience and reducing operational difficulty;

[0025] 2. In this invention, the parasitic capacitance phenomenon in a single droplet generator is used for the first time to detect bacteria, breaking through the limitation of the traditional single droplet generator that requires energy collection before use.

[0026] 3. The bacteria-specific detection method of the present invention uses aptamer probe technology to specifically detect target bacteria;

[0027] 4. Aptamers can be selectively replaced for different bacteria, so this method can achieve specific detection of different bacteria by replacing aptamers or molecular probes;

[0028] 5. This method is quick and simple and does not require external power supply. It only takes a few hours to complete the operation. Compared with traditional methods, this method has better safety and efficiency.

[0029] 6. The bacterial detection method of the present invention has a low production cost and is applicable to various environments. At the same time, it can convert the voltage signal into a visual signal in conjunction with the alarm system for easy observation. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 This is a schematic structural diagram of a bacteria detection method based on a single droplet generator according to an embodiment of the present invention;

[0031] Figure 2 Schematic diagram of a single droplet generator according to an embodiment of the present invention;

[0032] Figure 3 Schematic diagram of the working principle of the bacteria detection method based on a single droplet generator according to an embodiment of the present invention;

[0033] Figure 4 The output voltage of the single droplet generator with different droplet falling heights;

[0034] Figure 5 The output voltage of the gold wire modified with the aptamer (name) as the electrode of the single droplet generator when detecting the absence of the target bacteria (name);

[0035] Figure 6 The output voltage of the single droplet generator changes with the bacterial concentration and the fitting diagram.

[0036] In the figure, 100 is a single droplet generator, 200 is a single droplet generating device, 300 is a signal acquisition device, 101 is a friction layer, 102 is a conductive layer, 103 is a supporting substrate, and 104 is an electrode. DETAILED DESCRIPTION

[0037] Example 1 provides a bacteria detection device based on a single droplet generator. Figure 1 The structure of the detection device is shown. Figure 1 As shown, the detection device includes a single droplet generator 100, a single droplet generating device 200, and a signal acquisition device 300. The single droplet generator 100 affects the efficiency of converting the energy generated by a single droplet into electrical energy through changes in its own parasitic capacitance, and generates a detection signal. The single droplet generating device is placed in a suitable position directly above the single droplet generator. The single droplet generation device can be controlled by a control switch to generate a single droplet, which is used to provide a single droplet. The signal acquisition device 300 is directly connected to the single droplet generator and includes a voltage detection device and an alarm system for monitoring signal changes of the single droplet generator, analyzing the data, and issuing an alarm signal.

[0038] like Figure 2 As shown, the single droplet generator 100 includes a friction layer 101, a conductive layer 102, a support substrate 103 and electrodes 104. The friction layer 101 is made of a perfluoroethylene propylene copolymer film, the conductive layer is made of a metal material (preferably copper foil, iron, silver, etc.), the support substrate is a PVC board, and the electrodes are gold wires (preferably with a diameter of 20 microns). Figure 2 In the example shown, the conductive layer is fixed to a support substrate, while the friction layer is fixed to the conductive layer. The electrodes are positioned appropriately on the friction layer. The support substrate, conductive layer, and friction layer are all of the same size. The conductive layer is adhered to the support substrate using Kapton adhesive, while the friction layer is directly adhered to the adhesive side of the conductive layer.

[0039] The conductive layer in the single-droplet generator is considered the source, the electrode is considered the drain, and the single droplet is considered the gate. Its operating mechanism is very similar to that of a field-effect transistor (FET). The contact between the droplet (gate) and the gold electrode (drain) dynamically controls the on / off state of the single-droplet generator. The electrode is modified with an aptamer to specifically identify and capture the target bacteria. The reaction device contains a solution of polystyrene microspheres, whose surfaces are modified with aptamers specific for the target bacteria. These microspheres bind to the target bacteria, causing changes in the parasitic capacitance of the single-droplet generator.

[0040] The diameter of the polystyrene microspheres is 500 nanometers.

[0041] The selected electrode material needs to be modified with aptamers to facilitate the specific recognition and capture of bacteria.

[0042] The single droplet generating device 200 includes a droplet device with a switch and a control switch. By controlling the switch, a single droplet or continuous droplets can be generated.

[0043] This embodiment provides a bacterial detection method based on a single droplet generator. The operating principle is as follows: When a single droplet generated by a single droplet generator 200 falls onto the friction layer, the droplet completely contacts the friction layer and the electrode, generating charge transfer, which can convert mechanical energy into electrical energy. The electrical signal is then transmitted to the signal acquisition device and alarm system 300 via the conductive layer 102 and the electrode 104. When parasitic capacitance forms between the electrode and the polystyrene microspheres in the single droplet generator, the reverse shunting effect of the parasitic capacitance causes the amount of charge transferred by the droplet generator to decrease, which can be displayed as an output voltage. The output voltage can be measured using a programmable multifunctional electrometer (Keithley, 6514), and the voltage signal is converted into a visual signal using an alarm program written in LabVIEW. This visual signal can be an indicator light, a buzzer, a voice or text message alert, etc.

[0044] Common tests also include the specific detection of Gram-positive bacteria (environmental pollution) through vancomycin, the detection of Gram-negative bacteria (environmental pollution) through concanavalin, the detection of Staphylococcus aureus (medical pollution) through Staphylococcus aureus-related antibodies, etc.

[0045] The detection method is as follows:

[0046] (1) Immerse the gold wire in the test solution at 37°C for 120 min.

[0047] (2) Take out the gold wire and soak it in a solution of polystyrene microspheres modified with aptamers at 37°C for 120 min;

[0048] (3) Remove the gold wire, rinse gently with deionized water to remove unbound polystyrene microspheres, and dry;

[0049] (4) The gold wire is placed on the friction layer of a single droplet generator to control the drop of a single droplet. The output signal of the single droplet generator is tested and compared with the output signal when the original gold wire is used as an electrode to analyze the bacterial contamination.

[0050] Take the detection of Pseudomonas aeruginosa as an example:

[0051] A gold wire modified with aptamer F23 is immersed in a test solution containing Pseudomonas aeruginosa. The gold wire, through the specific recognition and capture capabilities of aptamer F23, specifically captures Pseudomonas aeruginosa. After capturing Pseudomonas aeruginosa, the gold wire is then immersed in a solution of polystyrene microspheres modified with aptamer F23. The target Pseudomonas aeruginosa attached to the gold wire then specifically recognizes and adsorbs the polystyrene microspheres with the aptamer F23. The attachment of the polystyrene microspheres increases the parasitic capacitance of the single-droplet generator. This parasitic capacitance has a reverse shunt effect, storing the transferred charge generated by the single-droplet generator and reducing the output voltage signal monitored by the external circuit. When the concentration of Pseudomonas aeruginosa is high, the number of polystyrene microspheres attached to the gold wire surface also increases, and the parasitic capacitance of the single-droplet generator also increases. At this time, the single-droplet generator can store more transferred charge, reducing the output voltage generated by the single-droplet generator. Therefore, the voltage signal generated by the single-droplet generator 100 can reflect the parasitic capacitance state of the single-droplet generator and thus reflect the contamination status of Pseudomonas aeruginosa. By replacing aptamers or other molecular recognition probes, specific detection of different target bacteria can be performed, such as aptamers specific for Staphylococcus aureus, aptamers specific for Escherichia coli, and galactosamine specific for Pseudomonas aeruginosa.

[0052] like Figure 3As shown, when polystyrene microspheres are attached to the electrode surface of the water droplet generator through Pseudomonas aeruginosa, the water droplet generator will generate parasitic capacitance. The parasitic capacitance has a reverse shunt effect, which can store the transferred charge generated by the single droplet generator, causing the output voltage of the single droplet generator to drop.

[0053] Figure 4 The output performance of the single-droplet generator and the effect of different single-droplet falling heights on the output performance of the single-droplet generator. The aptamer modification on the electrode described in this experiment and the specific aptamer on the surface of the polystyrene microspheres belong to the same aptamer F23, which can specifically identify Pseudomonas aeruginosa. It can be seen that when the droplet falling height is 30cm, the output voltage of the single-droplet generator is relatively stable and the output is high. When the droplet falling height is too high, the droplet will break and produce many small droplets at the moment of contact with the friction layer, which will make the output voltage baseline unstable. Therefore, it is most appropriate to use 30cm as the droplet falling height. It can be seen that the output voltage of the single-droplet generator is about -60V.

[0054] Figure 5 It was demonstrated that the gold wire modified with aptamer F23 had no effect on the output performance of the single-droplet generator, and that the output performance of the single-droplet generator did not change when the test solution did not contain Pseudomonas aeruginosa.

[0055] The voltage changes of Pseudomonas aeruginosa detection method based on single droplet generator at different concentrations of Pseudomonas aeruginosa are shown in the figure. Figure 6 As shown in a in Figure 1. As the concentration of Pseudomonas aeruginosa increases, the absolute value of the output voltage of the single-droplet generator continues to decrease. This is because as the concentration of Pseudomonas aeruginosa increases, the number of polystyrene microspheres adhering to the gold wire also increases, causing the parasitic capacitance of the single-droplet generator to increase continuously and the voltage to decrease. Figure 6 As shown in (b), there is a good direct correlation between the output voltage of the single-droplet generator and the concentration of Pseudomonas aeruginosa. A mathematical model can be established to infer the bacterial concentration.

[0056]

[0057] Among them, U0 represents the initial output voltage of the water drop generator; U C Represents the actual output voltage of the water drop generator; where S Au 、S ps , ε water , ε ps , K F 、C BThey represent the coverage area between water and gold wire-F23, the total area of ​​attached polystyrene microspheres, the dielectric constant of water, the dielectric constant of polystyrene microspheres, the Freundlich isotherm constant, and the bacterial concentration, respectively. b and a are two constants (aK F =218.143, b=0.2347). The bacteria detection method based on the single droplet generator of this example can detect any biological material, not limited to bacteria, fungi, viruses, animal and plant cells or biological macromolecules.

[0058] The mathematical model in this example is applicable to the detection of other bacteria. For the mathematical models of other bacteria, the constant K F , b and a will change.

[0059] The above description is merely a preferred embodiment of the present application and is not intended to limit the present application. Various modifications and variations are possible for those skilled in the art. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present application shall be included within the scope of protection of the present application.

Claims

1. A bacteria detection device based on a single droplet generator, characterized in that: The bacteria detection device includes a single droplet generator, a single droplet generating device, a reaction device and a signal acquisition device; The single droplet generation device is placed directly above the single droplet generator, and the signal acquisition device is directly connected to the single droplet generator; The single-droplet generator includes a friction layer, a conductive layer, a support substrate, and electrodes. The conductive layer is fixed to the support substrate, the friction layer is fixed to the conductive layer, and the electrodes are placed on the friction layer. The electrodes are gold wires modified with aptamers for specific identification and capture of target bacteria. A polystyrene microsphere solution is placed within the reaction device. The polystyrene microspheres are modified with aptamers specific for the target bacteria, which bind to the target bacteria and cause changes in the parasitic capacitance of the single-droplet generator. A signal acquisition device collects the signal emitted by the single-droplet generator and displays it in the form of an output voltage. When polystyrene microspheres are bound to the electrodes of the single-droplet generator, they enhance the parasitic capacitance of the single-droplet generator and reduce the output voltage of the single-droplet generator through the reverse shunt effect. Therefore, the output voltage of the single-droplet generator can reflect the parasitic capacitance and thus the bacterial concentration. The fitting relationship between the output voltage of the single droplet generator and the bacterial concentration is as follows: ； in, Represents the initial output voltage of the water drop generator; Represents the actual output voltage of the water drop generator; Respectively represent the coverage area between water and gold wire-aptamer, the total area of ​​attached polystyrene microspheres, the dielectric constant of water, the dielectric constant of polystyrene microspheres, the Freundlich isotherm constant, and the bacterial concentration; when the bacterial type is determined, b and a are two constants.

2. The bacteria detection device based on a single droplet generator according to claim 1, characterized in that: The friction layer material is a perfluoroethylene propylene copolymer film, and the supporting substrate is a PVC board.

3. The bacteria detection device based on a single droplet generator according to claim 1, characterized in that: The single droplet generating device includes a control switch and a dripping device, which is used to generate a single droplet or continuous droplets.

4. The bacteria detection device based on a single droplet generator according to claim 2, characterized in that: The diameter of the gold wire is 10-50 microns; the diameter of the polystyrene microspheres is 300-800 nanometers.

5. The bacteria detection device based on a single droplet generator according to claim 1, wherein the signal acquisition device is connected to an alarm system, and when the voltage signal exceeds a threshold, the alarm system issues an alarm.

6. A detection method using the device according to any one of claims 1 to 5, characterized in that: (1) Immerse the gold wire in the sample to be tested and incubate at 37°C for 120 min; (2) Take out the gold wire and immerse it in a solution of polystyrene microspheres modified with aptamers at 37°C for 120 min. (3) Remove the gold wire, rinse with deionized water to remove unbound polystyrene microspheres, and air dry naturally; (4) Place the above-mentioned gold wire on the friction layer of the single droplet generator, control the falling of the single droplet, test the output signal of the single droplet generator, and compare it with the output signal when the original gold wire is used as an electrode to analyze the bacterial contamination situation.

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