Biosensor for respiratory virus detection and method thereof

Abstract

The invention provides a biosensor for respiratory virus detection and a method thereof, and relates to the field of sensors, the biosensor for respiratory virus detection comprises a transistor, a phospholipid membrane is attached to the surface of the transistor, a synthesized DNA nanochannel is embedded into the phospholipid membrane, a bionic sensing interface is constructed, and the biosensor is used for detecting respiratory viruses. According to the biosensor for respiratory virus detection and the method thereof, by combining the carbon nanotube field effect transistor and the DNA nano channel, high-sensitivity detection of respiratory virus nucleic acid is achieved, the detection sensitivity and specificity are improved, and the biosensor has the advantages of being easy to operate, capable of achieving one-step reading, good in use effect and the like.

Description

Technical Field

[0001] The present invention relates to the field of sensors, and in particular to a biosensor for respiratory virus detection and its method. Background Art

[0002] Respiratory Viruses (RV) are a group of viruses that specifically infect human respiratory epithelial cells. From the upper respiratory tract such as the nasal cavity and pharynx to the lower respiratory tract regions such as the bronchi and alveoli, they may all become the targets of virus attacks, thereby triggering a series of respiratory diseases with varying degrees of severity. This group of viruses has diverse members, including influenza viruses with frequent antigenic variations and different subtypes every year; coronaviruses that have caused several global public health crises and have the ability to cross species and strong population adaptability; respiratory syncytial virus (RSV) that is likely to cause respiratory diseases in infants and young children, is highly prevalent in the low-age child population, and often causes severe lower respiratory diseases such as bronchiolitis and pneumonia; adenovirus can not only cause respiratory infections but is also related to diseases in multiple parts such as the eyes and gastrointestinal tract, and its multiple serotypes increase the complexity of infection; human metapneumovirus has also been gradually recognized for its pathogenic role in respiratory diseases in children and the elderly since its discovery, especially during the alternation of winter and spring, it is likely to cause an epidemic.

[0003] Respiratory viruses are highly contagious, which is attributed to their diverse transmission routes. Among them, airborne droplets and contact transmission are the main diffusion routes, and the high variability of the virus also increases its transmission efficiency. Especially in the cold and dry autumn and winter seasons when people's indoor activities increase and ventilation is poor, it becomes a high-incidence period for the epidemic of respiratory viruses. The immune function of the elderly declines, and the immune system of children is not yet perfect. The two become high-risk groups threatened, with significantly higher morbidity and mortality rates than other age groups, making the research and prevention and control of respiratory viruses a difficult problem that the global public health field is determined to overcome.

[0004] Currently, many detection methods for respiratory viruses have been developed, including polymerase chain reaction technology (PCR), enzyme-linked immunosorbent assay technology (ELISA), immunochromatography, or immunofluorescence method, etc. Among them, the PCR technology is a nucleic acid amplification-based technology. Using viral nucleic acid as a template, specific nucleic acid fragments are amplified through a cyclic reaction using primers, DNA polymerase, etc., and then the amplified products are detected to determine whether there is viral nucleic acid. This technology has extremely high specificity, can accurately identify the virus species, and has high sensitivity, can detect very low concentrations of viral nucleic acid. However, when implementing the detection, it requires professional laboratory equipment, skilled technicians, has a relatively high detection cost, a relatively complex operation process, takes a relatively long time, and the sample is easily interfered by inhibitors, and false negative results may occur.

[0005] ELISA technology is based on the principle of specific binding between antigens and antibodies. Through the reaction of enzyme-labeled secondary antibodies with substrates, color changes occur, and the presence of viral antigens or antibodies in the sample is judged by absorbance values. The operation is relatively simple and can be batch-detected, but the sensitivity and specificity are slightly inferior, and cross-reactions may occur, leading to false positives. When detecting antibodies, it cannot be used for early diagnosis.

[0006] Immunochromatography or immunofluorescence assay is a technology based on the specific binding of antigens and antibodies. Immunochromatography technology uses nitrocellulose membrane as a carrier. After the viral antigens or antibodies in the sample bind to the label, they move on the membrane and develop color to form bands, which is a rapid detection technology for judging the presence of viruses. The operation is simple and fast, and no special instruments are required. It can be read with the naked eye, but the sensitivity is low, and only qualitative or semi-quantitative detection can be performed.

[0007] Immunofluorescence assay uses antibodies labeled with fluorescein to specifically bind to viral antigens in the sample. The presence or absence of viruses is determined by observing fluorescence through a fluorescence microscope. This method has strong specificity, can quickly detect antigens and localize them, but requires a fluorescence microscope, has high technical requirements for personnel, limited sensitivity, and strong subjectivity in interpretation.

[0008] Therefore, it is necessary to provide a new biosensor and its method for detecting respiratory viruses to solve the above technical problems. Summary of the Invention

[0009] To solve the technical problems of the above PCR technology, such as relatively high detection cost, complex operation process, relatively long time-consuming, and the sample is easily interfered by inhibitors, which may result in false negative results; for ELISA technology, the sensitivity and specificity are slightly inferior, cross-reactions may occur, leading to false positives, and it cannot be used for early diagnosis when detecting antibodies; and for immunofluorescence assay, it requires a fluorescence microscope, has high technical requirements for personnel, limited sensitivity, and strong subjectivity in interpretation, the present invention provides a biosensor and its method for detecting respiratory viruses.

[0010] The biosensor and its method for detecting respiratory viruses provided by the present invention include: a transistor, on the surface of which a phospholipid membrane is attached, and the synthesized DNA nanochannel is embedded in the phospholipid membrane to construct a biomimetic sensing interface.

[0011] Preferably, the transistor is a carbon nanotube field effect transistor, denoted as CNT FET, where CNT is a carbon nanotube.

[0012] The preparation method of the biosensor for detecting respiratory viruses includes the following operating steps:

[0013] Step 1. Preparation of CNT FET: First, select the Si / SiO2 structure deposited with CNT thin film as the basis. After selection, spin-coat the surface of the Si / SiO2 structure deposited with CNT thin film to enhance the adhesion of photoresist. Use photolithography to expose the shape and size pattern of the CNT channel layer, and remove the unprotected part by etching to form the CNT channel layer. Spin-coat again on the surface of the etched Si / SiO2 structure, and deposit metal using electron beam technology to form the source electrode S, drain electrode D, and metal leads. Subsequently, perform photolithography and exposure on the patterns of the source electrode S, drain electrode D, and metal leads. After exposure, deposit the hafnium oxide dielectric layer on the surface of the structure with electrodes and leads deposited using atomic layer deposition technology. After deposition, photolithograph and etch the hafnium oxide dielectric layer;

[0014] Step 2. Synthesis of DNA nanochannel: After the CNT FET is prepared, select a variety of oligonucleotides, dissolve them in phosphate buffer solution (PBS) respectively, and mix them in equimolar ratio to obtain the mixed oligonucleotide solution. At this time, heat it to 80°C - 100°C and keep it for 8 - 12 minutes. Subsequently, slowly cool it to 4°C at a rate of 1°C per minute to allow the oligonucleotide chains to self-assemble into a DNA nanochannel structure;

[0015] Step 3. Synthesis of phospholipid membrane: After obtaining the DNA nanochannel structure, first select the appropriate phospholipid powder (DOPC) and dissolve it in chloroform to transform the morphology of phospholipid molecules. After transformation, blow dry the phospholipid solution with nitrogen to remove the residual chloroform, and redissolve the dried phospholipid in deionized water and perform ultrasonic treatment at an appropriate temperature to obtain the phospholipid solution with lipids dispersed. At this time, use a manual extruder to repeatedly extrude the phospholipid solution with lipids dispersed through a polycarbonate membrane to form a uniform single-layer fused liposome to obtain the phospholipid membrane;

[0016] Step 4. Embedding DNA nanochannel into phospholipid membrane: After the phospholipid membrane is synthesized, take out an appropriate amount of phospholipid solution and DNA nanochannel solution, use an ozone instrument to treat the surface of the HfO2 dielectric layer of the CNT FET to increase hydrophilicity, drop the phospholipid solution onto the surface of the CNT FET, and incubate at room temperature for 1 - 3 hours. After incubation, rinse to remove the excess phospholipid solution. At this time, drop the DNA nanochannel solution onto the surface of the phospholipid bilayer and incubate at room temperature for 1.5 hours. Rinse to remove the excess DNA nanochannel solution.

[0017] It should be noted that through the above steps, a biosensor for respiratory virus detection has been successfully constructed. This sensor combines the high sensitivity of carbon nanotube field-effect transistors and the high specificity of DNA nanochannels, achieving efficient detection of respiratory virus nucleic acids. At the same time, the phospholipid membrane acts as an isolation layer, effectively shielding non-specific signal interference and improving the accuracy and reliability of detection.

[0018] Preferably, the specific operation for preparing the CNT FET is as follows:

[0019] S1. Preparation of the CNT channel layer: First, select the Si / SiO2 structure deposited with the CNT thin film. Use a spin coater to perform spin coating on the surface of the Si / SiO2 structure deposited with the CNT thin film. Use photolithography to expose the pattern of the shape and size of the CNT channel layer, and etch the CNT thin film to obtain the CNT channel layer.

[0020] S2. Preparation of the source electrode, drain electrode, and metal leads: After the CNT channel layer is prepared, perform spin coating on the surface of the etched Si / SiO2 structure again. Use an electron beam process to deposit metal to form the source electrode S, drain electrode D, and metal leads, and expose the patterns of the source electrode S, drain electrode D, and metal leads.

[0021] S3. Preparation and etching of the dielectric layer: Use atomic layer deposition to deposit a hafnium oxide dielectric layer on the surface of the structure after depositing the source and drain electrodes S and the metal leads. Here, hafnium oxide is denoted as HfO2. Use a spin coater to perform spin coating on the surface of the hafnium oxide dielectric layer, and use photolithography to expose the pattern of the shape and size of the hafnium oxide dielectric layer. At this time, etch away the dielectric layer except for the CNT channel layer.

[0022] Preferably, the synthesis of the DNA nanochannel includes the following operation steps:

[0023] Step 1. Preparation stage: First, select a variety of oligonucleotides and dissolve each of the oligonucleotides in a phosphate buffer solution (0.01X PBS) solvent.

[0024] Step 2. Mix the oligonucleotide solutions: After the preparation stage is completed, mix the various oligonucleotide solutions in an equimolar ratio to obtain the mixed oligonucleotide solution.

[0025] Step 3. Thermal annealing treatment: Add the mixed oligonucleotide solution to a thermal cycler for thermal annealing treatment. The thermal annealing program generally includes the following operation steps:

[0026] Step1. First, heat the mixed oligonucleotide solution to 80°C - 100°C and hold for 8 - 12 minutes to completely denature it, that is, unwind it into a single-stranded state.

[0027] Step 2. After the strand separation is completed, slowly cool down to 4 °C at a rate of 1 °C per minute, so that the oligonucleotide strands are gradually annealed and self-assembled to form a DNA nanochannel structure during the cooling process.

[0028] Preferably, the synthesis of the phospholipid membrane includes the following operating steps:

[0029] a1. Dissolution of phospholipid powder: Weigh 8 - 12 mg of dioleoylphosphatidylcholine powder and dissolve it in 1 mL of chloroform (CHCl3) to form a liquid mixture with a concentration of 8 mg / ml - 12 mg / ml.

[0030] a2. Removal of solvent: After the transformation of phospholipid molecules is completed, blow dry the phospholipid solution with nitrogen to remove the residual chloroform.

[0031] a3. Re - dissolution and dispersion of phospholipid: After the removal, redissolve the dried phospholipid in step a2 in phosphate buffer at 2 mg / mL and perform ultrasonic treatment at 25 °C for 4 - 7 minutes to obtain a phospholipid solution after lipid dispersion.

[0032] a4. Formation of phospholipid membrane: Use a manual extruder to repeatedly extrude the phospholipid solution after lipid dispersion through a polycarbonate membrane to form a uniform single - layer fused liposome, and obtain the phospholipid membrane.

[0033] Preferably, the embedding of the synthesized DNA nanochannel into the phospholipid membrane includes the following stages:

[0034] b1. Surface pretreatment stage: First, pretreat the hafnium oxide surface with an ultraviolet ozone cleaner.

[0035] b2. Preparation stage: Select the phospholipid solution after extrusion treatment and the DNA nanochannel solution obtained by self - assembly, and evenly drop them onto the surface of the carbon nanotube field - effect transistor.

[0036] b3. Spreading stage of phospholipid bilayer: After the preparation stage is completed, incubate the CNT FET device dropped with the mixed solution of phospholipid and DNA nanochannel at room temperature for 1.5 - 2 hours. After incubation, rinse the CNT FET device with phosphate buffer and blow it with nitrogen to remove the unbound excess phospholipid solution, so that only the tightly bound phospholipid bilayer remains on its surface.

[0037] b4. Stage of embedding DNA nanochannels into the phospholipid bilayer: Take another 10 μL of the self-assembled DNA nanochannel solution, and evenly drop the prepared DNA nanochannel solution onto the surface of the CNT FET with a spread phospholipid bilayer. At this time, the hydrophobic cholesterol groups on the surface of the DNA nanochannels have specific interactions with the lipid molecules in the phospholipid bilayer, prompting the DNA nanochannels to be smoothly inserted into the phospholipid bilayer to form a stable embedded structure;

[0038] b5. Post-treatment stage: After the dropping is completed, let the CNT FET device prepared in b4 stand and incubate at room temperature for 1.5 - 2 hours, and rinse the CNT FET device with phosphate buffer (0.01×PBS) to remove the excess unbound DNA nanochannel solution, obtaining a biomimetic sensing interface.

[0039] Compared with related technologies, the biosensor and its method for respiratory virus detection provided by the present invention have the following beneficial effects:

[0040] 1. By combining carbon nanotube field effect transistors with DNA nanochannels, the present invention realizes highly sensitive detection of respiratory virus nucleic acids. The phospholipid membrane, as an isolation layer, can effectively shield non-specific signal interference, while the DNA nanochannels precisely match the target virus nucleic acids through their specific blocking sequences, ensuring high specificity of the detection. This combination significantly improves the sensitivity and specificity of the detection, overcoming the false negative or false positive results that occur in traditional detection methods such as PCR and ELISA;

[0041] 2. The detection scheme of the present invention adopts a label-free and one-step readout mode, greatly shortening the detection time. Compared with the complex operation process and long detection time of PCR technology, the present invention can complete the detection in a shorter time, providing the possibility for the timely treatment of patients. At the same time, due to the simple and fast detection process, it is also suitable for on-site point-of-care testing needs, solving the problems of immunochromatography or immunofluorescence methods, such as the need for a fluorescence microscope, high requirements for personnel technology, limited sensitivity, and strong subjectivity in interpretation.

[0042] 3. By flexibly replacing the blocking sequences in the DNA nanochannels, specific detection of multiple viruses can be achieved. Moreover, the phospholipid membrane self-assembled on the surface of the transistor uses its excellent shielding effect to significantly enhance the anti-pollution ability and non-specific adsorption resistance of the device, enabling the present invention to maintain stable detection performance in complex sample environments and improving the reliability and accuracy of the detection. Description of the Drawings

[0043] Figure 1 It is a schematic diagram of the structure of the CNT FET device;

[0044] Figure 2Schematic illustration of the preparation of CNT FET Figure 1 ;

[0045] Figure 3 Schematic illustration of the preparation of CNT FET Figure 2 ;

[0046] Figure 4 Schematic illustration of the preparation of CNT FET Figure 3 ;

[0047] Figure 5 Schematic structure illustration of the two-dimensional map of DNA nanopore Figure 1 ;

[0048] Figure 6 Schematic structure illustration of the two-dimensional map of DNA nanopore Figure 2 ;

[0049] Figure 7 Schematic structure diagram of the target virus sequence;

[0050] Figure 8 Transmission characteristic curve of the transistor incubated only with phospholipid;

[0051] Figure 9 Transmission characteristic curve of the transistor incubated with phospholipid embedded with NP-O;

[0052] Figure 10 Response comparison chart of two incubation tests;

[0053] Figure 11 Transmission characteristic curve of the closed DNA nanopore;

[0054] Figure 12 Transmission characteristic curve of the open DNA nanopore;

[0055] Figure 13 Schematic diagram of the response comparison of the closed and open DNA channel tests;

[0056] Figure 14 Transmission characteristic curve of different concentrations of non-target virus nucleic acid solution;

[0057] Figure 15 Transmission characteristic curve of different concentrations of target virus nucleic acid solution;

[0058] Figure 16 Response comparison chart of non-target virus nucleic acid and target virus nucleic acid. Detailed implementation method

[0059] Next, the technical solutions in the embodiments of the present application will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present application. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present application without creative efforts belong to the protection scope of the present application.

[0060] In some embodiments, as Figure 1 shown, it includes a carbon nanotube field-effect transistor, the carbon nanotube field-effect transistor is of P-type, a phospholipid membrane is attached to the surface of the P-type carbon nanotube field-effect transistor, and the synthesized DNA nanochannel is embedded in the membrane through hydrophobic interaction to construct a biomimetic sensing interface.

[0061] Among them, the P-type carbon nanotube field-effect transistor is denoted as CNT FET, where CNT is carbon nanotube.

[0062] It should be noted that CNT FET is a three-terminal device with a carbon nanotube as the channel, and its working principle mainly depends on the regulation of the carrier concentration in the channel by the gate voltage. In the liquid gate structure, when a gate bias is applied, ions in the electrolyte solution form a double-layer electric field on the surface of the dielectric layer, generating an electric field perpendicular to the channel, thereby effectively regulating the carrier distribution in the carbon nanotube channel, and further regulating the current between the source and the drain.

[0063] In some embodiments, as Figures 2 to 4 shown, the specific operation for preparing the CNT FET is as follows:

[0064] S1. Preparation of the CNT channel layer: First, select the Si / SiO2 structure deposited with the CNT thin film, use a spin coater to perform spin coating on the surface of the Si / SiO2 structure deposited with the CNT thin film, use photolithography to expose the pattern of the shape and size of the CNT channel layer, and etch the CNT thin film to obtain the CNT channel layer;

[0065] Among them, for the photolithography process, first operate the laser direct writing lithography machine, select the required layout, carry out the exposure operation on a carbon sheet with a size of 1.1×1.1 cm, and set the exposure energy in the range of 30 - 300 mJ / cm 2 interval.

[0066] After the exposure is completed, put the CNT FET device into the developer and develop for 1 minute. After the development is completed, immediately transfer it to a phosphate buffer solution (0.01×PBS) for dissolution and fixation, and thus a CNT FET device with a channel size of 25×10 -6 ×65×10 -6 m can be obtained.

[0067] Finally, with the aid of a microscope, observe the specific situation of the pattern transfer of the CNT FET device for subsequent use.

[0068] Specifically, when etching the CNT film to obtain the CNT channel layer, use a reactive ion etcher (RIE) to etch with oxygen for 60 s under the condition of a power of 80 W to remove the excess carbon nanotubes.

[0069] S2. Preparation of source, drain electrodes and metal leads: After the CNT channel layer is prepared, spin-coat the surface of the etched Si / SiO2 structure again through a spin coater, deposit metal using an electron beam process to form a source electrode S, a drain electrode D and metal leads, and expose the patterns of the source electrode S, the drain electrode D and the metal leads;

[0070] S3. Preparation and etching of the dielectric layer: Use atomic layer deposition technology to deposit a hafnium oxide (HfO2) dielectric layer on the surface of the structure after depositing the source and drain electrodes S and the metal leads, spin-coat the surface of the hafnium oxide (HfO2) dielectric layer through a spin coater, expose the pattern of the shape and size of the hafnium oxide (HfO2) dielectric layer through a laser direct writing lithography machine, and then use a reactive ion etcher to etch away the dielectric layer except for the CNT channel layer.

[0071] In some embodiments, such as Figure 1 、 Figure 5 、 Figure 6 and Figure 7 shown, the synthesis of the DNA nanochannel includes the following operating steps:

[0072] Step 1. Preparation stage: First, select 6 kinds of oligonucleotides, and dissolve various oligonucleotides in a phosphate buffer solution (0.01X PBS) solvent respectively.

[0073] Specifically, as Figure 5 shown, among them, the 6 kinds of oligonucleotides are respectively:

[0074]

[0075] Step 2. Mix the oligonucleotide solutions: After the preparation stage is completed, mix the 6 kinds of oligonucleotide solutions in an equimolar ratio to obtain the mixed oligonucleotide solution;

[0076] Step 3. Thermal annealing treatment: Add the mixed oligonucleotide solution to a thermal cycler for thermal annealing treatment, and the thermal annealing program usually includes the following operating steps:

[0077] Step1. First, heat the mixed oligonucleotide solution to 95 °C and keep it for 10 minutes to make it completely denatured, that is, unwind into a single-stranded state.

[0078] Step 2. After the strand separation is completed, slowly cool down to 4 °C at a rate of 1 °C per minute, so that the oligonucleotide strands are gradually annealed and self-assembled to form a DNA nanochannel structure during the cooling process;

[0079] Specifically, the two-dimensional diagram of the DNA nanopore shows that, as Figure 5 (NP-O), Figure 6 (NP-C) and Figure 7 The double-stranded structure between the closed sequence and the target virus nucleic acid sequence. The components of the DNA strand are represented by straight lines, and the 5' end and 3' end are marked with a square and a triangle respectively. The hairpin loop containing T4 is presented as a dotted line, and the position of cholesterol modification is marked with an asterisk. The target virus nucleic acid sequence can hybridize complementarily with the closed sequence, thereby changing the channel from the closed state to the open state.

[0080] The synthesis of the phospholipid membrane includes the following operating steps:

[0081] a1. Dissolution of phospholipid powder: Weigh 10 mg of dioleoylphosphatidylcholine powder and dissolve it in 1 mL of chloroform to form a liquid mixture of 10 mg / ml;

[0082] a2. Removal of solvent: After the transformation of phospholipid molecules is completed, blow dry the phospholipid solution with nitrogen to remove the residual chloroform;

[0083] a3. Re-dissolution and dispersion of phospholipid: After the removal, re-dissolve the dried phospholipid in a2 in phosphate buffer at 2 mg / mL and perform ultrasonic treatment at 25 °C for 5 minutes to obtain a phospholipid solution after lipid dispersion;

[0084] a4. Formation of phospholipid membrane: Use a manual extruder to repeatedly extrude the phospholipid solution after lipid dispersion through a polycarbonate membrane with a pore size of 100 nm for 21 times to form a uniform monolayer of fused liposomes to obtain the phospholipid membrane.

[0085] Among them, the manual extruder is: a filter of Avant, equipped with a filter membrane with a pore size of 0.05 μm, and the phospholipid solution in a3 is extruded and filtered 21 times.

[0086] In some embodiments, as Figure 1 shown, the embedding of the synthesized DNA nanochannel into the phospholipid membrane includes the following stages:

[0087] b1. Surface pretreatment stage: First, use an ultraviolet ozone cleaning machine to pretreat the hafnium oxide surface.

[0088] Among them, during the pretreatment, the temperature is set at 25 °C, and the CNT FET device is cleaned for 5 min to prepare for subsequent experimental applications.

[0089] b2. Preparation stage: Select the extruded phospholipid solution and the self-assembled DNA nanochannel solution, and evenly drop them onto the surface of the carbon nanotube field-effect transistor;

[0090] b3. Spreading stage of the phospholipid bilayer: After the preparation stage is completed, incubate the CNT FET device dropped with 10 μL of phospholipid and 10 μL of DNA nanochannel mixed solution at room temperature for 1.5 hours. After incubation, rinse the incubated device 2 - 3 times with phosphate buffer at room temperature of 25 °C, and gently blow the surface with nitrogen to remove the phospholipid molecules not bound to the surface of the CNT FET device, so that only the tightly bound phospholipid bilayer remains on its surface;

[0091] b4. Embedding stage of DNA nanochannels into the phospholipid bilayer: Take another 10 μL of the self-assembled DNA nanochannel solution, and evenly drop the prepared DNA nanochannel solution onto the surface of the carbon nanotube field-effect transistor with the phospholipid bilayer already spread. At this time, the hydrophobic cholesterol groups on the DNA nanochannels embed into the lipid hydrophobic tail region of the phospholipid bilayer by hydrophobic interaction. Meanwhile, hydrogen bonds are formed between the DNA nanochannels and the relevant groups on the phospholipid bilayer. Coupled with the synergistic influence of van der Waals forces between atoms and molecules, specific interactions occur between the hydrophobic cholesterol groups on the DNA nanochannels and the lipid molecules in the phospholipid bilayer, prompting the DNA nanochannels to be successfully inserted into the phospholipid bilayer to form a stable embedding structure;

[0092] Specifically, after the specific interaction occurs, the process of the DNA nanochannels approaching the phospholipid bilayer is accelerated under the synergistic action of thermodynamics and concentration gradient, and then it is successfully inserted into the phospholipid bilayer.

[0093] b5. Post-treatment stage: After dropping, let the carbon nanotube field-effect transistor device prepared in b4 stand and incubate at room temperature for 1.5 hours, and rinse the carbon nanotube field-effect transistor device with phosphate buffer to remove the excess unbound DNA nanochannel solution, obtaining a biomimetic sensing interface.

[0094] Specifically, finally, the structure as shown in Figure 1 is formed: The DNA nanochannels and the phospholipid bilayer are tightly bound through non-covalent interactions, successfully constructing a stable and highly biomimetic biosensing interface. This interface is evenly spread on the surface of the carbon nanotube field-effect transistor, endowing the carbon nanotube field-effect transistor with specific biorecognition and sensing functions, and realizing highly sensitive and highly specific virus detection.

[0095] Experiment 1. Test the transfer characteristic curve of the CNT FET device;

[0096] 1. Drop a buffer solution (0.01×PBS) on the dielectric layer region of the CNT FET device and measure an initial curve.

[0097] 2. Rinse off the buffer solution, then drop the test solution (NaCl solutions with different concentration gradients) on the dielectric layer region of the CNT FET device and start measuring after a few seconds.

[0098] 3. Compare the measured curves. If the curve current decreases, it means that the ion permeability of the channel or the channel changes from closed to open, attracting positive charge ions (Na+) close to the dielectric layer, triggering the above electrical characteristics.

[0099] In some embodiments, as Figures 8 to 10 shown, specifically, Figure 8 shows the test results of 100 μM to 1 M NaCl solutions after incubating a phospholipid membrane on the transistor surface. The figure shows that the response change of the transistor current is extremely small, indicating that the phospholipid membrane can effectively shield the ions in the solution. The mechanism is that the high shielding effect of the phospholipid membrane prevents charged ions from penetrating the bilayer, making it impossible to form an electric double layer on the surface of the dielectric layer or change the carrier distribution in the channel, so that the current of the device remains almost unchanged. This result shows that the device with only a spread phospholipid membrane is insensitive to ion concentration changes, verifying the effectiveness of the phospholipid membrane in shielding environmental interference and reducing non-specific adsorption.

[0100] Specifically, Figure 9 shows the test results of incubating a phospholipid membrane embedded with open nanochannels (NP-O) on the transistor surface. Compared with the device incubated with only a phospholipid membrane, the response of the transistor current increases significantly. This phenomenon indicates that the open nanochannels allow positive charge ions in the solution to pass through and accumulate in the hydration layer between the membrane and the dielectric layer, thus changing the carrier distribution in the carbon nanotube channel and resulting in a decrease in current. This result verifies the key role of DNA nanochannels in regulating ion transport and enhancing the device response;

[0101] Specifically, Figure 10 Through the response comparison bar chart under two incubation conditions, it intuitively shows that the response of the device embedded with DNA nanochannels (NP-O) is significantly higher than that of the device without DNA nanochannels. This comparison further confirms that the addition of DNA nanochannels enables the ions in the solution to pass through the channels, thus enhancing the sensitivity of the transistor to ions.

[0102] In some embodiments, as Figures 11 to 13 shown, specifically, Figure 11It is a test diagram of incubating phospholipids embedded with closed DNA nanochannels (NP-C) on a transistor. It can be seen from the diagram that when the DNA nanochannels are successfully closed by the closing sequence, the current response of the transistor is very small, indicating that ions are effectively shielded outside the phospholipid membrane and cannot pass through the nanochannels. This phenomenon shows that the closing sequence has successfully achieved the closing of the channels and prevented the passage of ions;

[0103] Specifically, Figure 12 It shows a phospholipid membrane embedded with closed DNA nanochannels (NP-C) incubated on a transistor. By adding a test liquid containing the nucleic acid of the virus to be detected, the target virus nucleic acid undergoes complementary hybridization with the closing sequence, causing the nanochannels to change from the closed state to the open state. Subsequently, electrical tests of NaCl solutions with different concentration gradients are performed on the open channels. The results show that Na + ions can pass through the channels and approach the transistor dielectric layer, causing significant changes in the electrical signal. This change in the electrical signal reflects the restoration of ion transport and the transition of the channel state, further verifying the effective regulation of the opening of the channels by the hybridization of the target virus nucleic acid and the closing sequence. Among them, V GS (v) is the gate voltage of the CNT FET device, and I (uA) is the source-drain current of the CNT FET device;

[0104] Specifically, 20 μL of heat-inactivated virus preservation reagent is added dropwise, and the target virus nucleic acid undergoes complementary hybridization with the closing sequence.

[0105] Furthermore, virus inactivation usually makes the virus lose its infectivity through physical methods such as heating and radiation or chemical methods. However, this process does not necessarily destroy the nucleic acid structure of the virus. In most cases, heat-inactivating the virus denatures the virus protein through high temperature, thereby destroying its structure and preventing its replication and infection. However, the nucleic acid of the virus still remains relatively intact and will not affect the above experiments.

[0106] Specifically, Figure 13 It is a bar chart comparing the responses of DNA nanochannel closing and DNA nanochannel opening, from which it can be intuitively seen the difference in current responses of DNA nanochannels in the closed state and the open state. This difference in current response further confirms the specific complementary hybridization between the target virus nucleic acid and the closing sequence, successfully inducing the opening of the nanochannels, indicating the key role of the target virus nucleic acid in channel regulation.

[0107] Experiment 2: Test the transfer characteristic curve of the CNT FET device.

[0108] 1. Drop a buffer solution (0.01×PBS) in the dielectric layer area of the device and measure an initial curve.

[0109] 2. Rinse off the buffer solution, and then start the measurement after dropping the test solutions (target virus nucleic acid solutions and non-target virus nucleic acid solutions with different concentration gradients) onto the dielectric layer region of the device for dozens of seconds. It should be noted that DNA hybridization generally takes more than ten seconds.

[0110] 3. Compare the measured curves. If the curve current decreases, it means the channel is open, attracting the positive charge ions (Na+) in the solution to approach the dielectric layer, triggering the above electrical characteristics.

[0111] In some embodiments, such as Figures 14 to 16 in, specifically, Figure 14 shows the test results of the response to different concentrations of non-target virus nucleic acid after incubating a phospholipid membrane embedded with a closed DNA nanochannel (NP-C) on a transistor. In the experiment, the non-target virus nucleic acid failed to undergo effective complementary hybridization with the closed sequence of the nanochannel, so the channel remained closed. As can be seen from the figure, the current of the transistor showed almost no response, indicating that the ions were effectively shielded outside the phospholipid membrane and could not enter the interior of the transistor through the nanochannel. This result shows that the non-target virus nucleic acid cannot open the channel, further verifying the specific recognition ability of the nanochannel for the target nucleic acid.

[0112] Specifically, Figure 15 shows the test results of the response to different concentrations of target virus nucleic acid after incubating a phospholipid membrane embedded with a closed DNA nanochannel (NP-C) on a transistor. In the experiment, the target virus nucleic acid underwent specific complementary hybridization with the closed sequence of the nanochannel, resulting in the transformation of the nanochannel from the closed state to the open state. Subsequently, the positive charge ions (Na + ) in the solution could smoothly pass through the channel and approach the HfO2 dielectric layer of the CNT FET, thus causing a significant change in the electrical signal of the CNT FET. This result shows that the target virus nucleic acid can effectively regulate the opening of the nanochannel, further verifying the specificity and functionality of the system; among them, it can be seen from Figure 15 that the CNT FET device responds to DNA at the aM concentration level (10-18M), demonstrating the high sensitivity of the CNT FET device.

[0113] Specifically, Figure 15 is a comparative bar chart of the responses of non-target virus nucleic acid and target virus nucleic acid. The experimental results show that only the target virus nucleic acid can open the nanochannel and cause a significant change in electrical characteristics. As the concentration of the target virus nucleic acid increases, the number of open channels increases, resulting in a more significant change in the current response, that is, the more the current drops. This comparative result further confirms the specific recognition ability of the nanochannel for the target virus nucleic acid.

[0114] The above are only embodiments of the present invention, and do not thus limit the patent scope of the present invention. Any equivalent structure or equivalent process transformation made by using the content of the specification and drawings of the present invention, or directly or indirectly applied in other related technical fields, shall similarly be included within the patent protection scope of the present invention.

Claims

1. A biosensor for respiratory virus detection, characterized in that, It includes a transistor, on the surface of which a phospholipid membrane is attached, and the synthesized DNA nanochannel is embedded in the phospholipid membrane to construct a biomimetic sensing interface.

2. The biosensor for respiratory virus detection according to claim 1, wherein The transistor is a carbon nanotube field effect transistor.

3. A method for preparing a biosensor for respiratory virus detection according to any one of claims 1-2, characterized in that, It includes the following operation steps: Step 1: Preparation of the carbon nanotube field effect transistor: First, select the Si / SiO2 structure deposited with a carbon nanotube thin film. After selection, perform spin coating, photolithography, and etching on the surface of the Si / SiO2 structure deposited with the carbon nanotube thin film to form a carbon nanotube channel layer. At this time, spin coat the etched Si / SiO2 structure surface, deposit metal using an electron beam process to form electrodes and leads, and photolithograph the electrode and lead patterns. After exposure, deposit a hafnium oxide dielectric layer on the structure surface coated with electrodes and leads using atomic layer deposition. After deposition, photolithograph and etch the hafnium oxide dielectric layer; Step 2: Synthesis of the DNA nanochannel: After the carbon nanotube field effect transistor is prepared, dissolve various oligonucleotides and mix them in an equimolar ratio to obtain a mixed oligonucleotide solution. At this time, heat it to 80 °C - 100 °C and maintain it for 8 - 12 minutes to completely denature it, and slowly cool it to 4 °C to allow the oligonucleotide chains to self-assemble to obtain a DNA nanochannel structure; Step 3: Synthesis of the phospholipid membrane: After obtaining the DNA nanochannel structure, first select an appropriate phospholipid powder and dissolve it in chloroform to transform the phospholipid molecules. After the transformation is completed, blow dry the phospholipid solution with nitrogen to remove the residual chloroform, redissolve the dried phospholipid in deionized water, and perform ultrasonic treatment at an appropriate temperature to obtain a phospholipid solution with dispersed lipids. At this time, use a manual extruder to repeatedly extrude the phospholipid solution with dispersed lipids through a polycarbonate membrane to form a uniform monolayer fused liposome to obtain the phospholipid membrane; Step 4: Embedding the DNA nanochannel in the phospholipid membrane: After the phospholipid membrane is synthesized, take out an appropriate amount of phospholipid solution and DNA nanochannel solution, treat the surface of the HfO2 dielectric layer of the carbon nanotube field effect transistor with an ozone instrument to increase hydrophilicity, drop the phospholipid solution onto the surface of the carbon nanotube field effect transistor, and incubate at room temperature for 1 - 3 hours. After incubation, rinse to remove the excess phospholipid solution. At this time, drop the DNA nanochannel solution onto the phospholipid bilayer surface and incubate at room temperature for 1.5 hours, and rinse to remove the excess DNA nanochannel solution.

4. The preparation method of the biosensor for respiratory virus detection according to claim 3, characterized in that, The specific operation for preparing the carbon nanotube field effect transistor is as follows: S1: Preparation of the carbon nanotube channel layer: First, select the Si / SiO2 structure deposited with a carbon nanotube thin film, perform spin coating on the surface of the Si / SiO2 structure deposited with the carbon nanotube thin film using a spin coater, expose the pattern of the shape and size of the carbon nanotube channel layer using photolithography, and etch the carbon nanotube thin film to obtain the carbon nanotube channel layer; S2. Preparation of source, drain electrodes and metal leads: After the carbon nanotube channel layer is prepared, the surface of the etched Si / SiO2 structure is spin-coated again, and metal is deposited using an electron beam process to form a source electrode S, a drain electrode D, and metal leads, and the patterns of the source electrode S, the drain electrode D, and the metal leads are exposed; S3. Preparation and etching of dielectric layer: After the source and drain electrodes S and metal leads are deposited, a hafnium oxide dielectric layer is deposited on the surface of the structure using atomic layer deposition. The surface of the hafnium oxide dielectric layer is spin-coated using a spin coater, and the pattern of the shape and size of the hafnium oxide dielectric layer is exposed using a photolithography process. At this time, the dielectric layer except for the carbon nanotube channel layer is etched away.

5. The preparation method of the biosensor for respiratory virus detection according to claim 4, characterized in that, The synthesis of the DNA nanochannel includes the following operating steps: Step 1. Preparation stage: First, select a variety of oligonucleotides and dissolve each of the oligonucleotides in a phosphate buffer solvent. Step 2. Mixing oligonucleotide solutions: After the preparation stage is completed, the various oligonucleotide solutions are mixed in an equimolar ratio to obtain a mixed oligonucleotide solution; Step 3. Thermal annealing treatment: The mixed oligonucleotide solution is added to a thermal cycler for thermal annealing treatment. The thermal annealing program generally includes the following operating steps: Step 1. First, heat the mixed oligonucleotide solution to 80°C - 100°C and hold for 8 - 12 minutes to completely denature it, that is, to unwind it into a single-stranded state. Step 2. After the strand separation is completed, slowly cool it to 4°C at a rate of 1°C per minute, so that the oligonucleotide strands gradually anneal and self-assemble to form a DNA nanochannel structure during the cooling process.

6. The preparation method of the biosensor for respiratory virus detection according to claim 5, characterized in that, The synthesis of the phospholipid membrane includes the following operating steps: a1. Dissolution of phospholipid powder: Weigh 8 - 12 mg of dioleoylphosphatidylcholine powder and dissolve it in 1 mL of chloroform to form a liquid mixture of 8 mg / ml - 12 mg / ml; a2. Removal of solvent: After the phospholipid molecules are transformed, the phospholipid solution is dried with nitrogen to remove the residual chloroform; a3. Redissolution and dispersion of phospholipid: After the removal is completed, the dried phospholipid in a2 is redissolved in a phosphate buffer at 2 mg / mL and sonicated at 25°C for 4 - 7 minutes to obtain a phospholipid solution after lipid dispersion; a4. Formation of phospholipid membrane: Using a manual extruder, the phospholipid solution after lipid dispersion is repeatedly extruded through a polycarbonate membrane to form a uniform monolayer of fused liposomes to obtain the phospholipid membrane.

7. The preparation method of the biosensor for respiratory virus detection according to claim 6, characterized in that, Embedding the synthesized DNA nanochannel into the phospholipid membrane includes the following stages: b1. Surface pretreatment stage: First, use an ultraviolet ozone cleaner to pretreat the surface of hafnium oxide. b2. Preparation stage: Select the extruded phospholipid solution and the self-assembled DNA nanochannel solution, and uniformly drip them onto the surface of the carbon nanotube field effect transistor; b3. Spreading stage of the phospholipid bilayer: After the preparation stage is completed, the carbon nanotube field effect transistor device with the mixed solution of phospholipids and DNA nanochannels added dropwise is incubated at room temperature for 1.5 - 2 hours. After incubation, the carbon nanotube field effect transistor device is rinsed with phosphate buffer solution and blown with nitrogen to remove the unbound excess phospholipid solution, leaving only the tightly bound phospholipid bilayer on its surface. b4. Embedding stage of DNA nanochannels into the phospholipid bilayer: Then, take 10 μL of the self-assembled DNA nanochannel solution and evenly drop the prepared DNA nanochannel solution onto the surface of the carbon nanotube field effect transistor with the phospholipid bilayer already spread. At this time, the hydrophobic cholesterol groups on the DNA nanochannels embed into the lipid hydrophobic tail region of the phospholipid bilayer by virtue of hydrophobic interactions. Meanwhile, hydrogen bonds are formed between the DNA nanochannels and the relevant groups on the phospholipid bilayer. Together with the synergistic influence of van der Waals forces between atoms and molecules, the hydrophobic cholesterol groups on the DNA nanochannels have specific interactions with the lipid molecules in the phospholipid bilayer, promoting the smooth insertion of DNA nanochannels into the phospholipid bilayer to form a stable embedded structure. b5. Post-treatment stage: After the dropping is completed, the carbon nanotube field effect transistor device prepared in b4 is left to stand and incubate at room temperature for 1.5 - 2 hours, and then rinsed with phosphate buffer solution to remove the unbound excess DNA nanochannel solution, obtaining a biomimetic sensing interface.

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