A method for detecting hepatitis b virus antigen by nanopore

Abstract

The present application relates to a kind of nano-pore detection hepatitis B virus antigen method, belong to electrochemical detection technical field.The present application discloses a kind of nano-pore detection hepatitis B virus antigen method, by setting nano-pore in electrochemical detection analysis device, the electric signal generated by the action of two hepatitis B virus antigen proteins, hepatitis B virus antigen plasmid transfection cell lysate and hepatitis B virus infection cell lysate with nano-pore is collected, the amplitude of current signal and current block time characteristics and signal amount and other characteristics are analyzed, to detect the difference of two hepatitis B virus antigen proteins, distinguish normal cell and virus antigen transfection cell, virus infection cell, repeatability is good, high reliability.The present application is a kind of single molecule level detection, does not need chemical marking and direct detection method of immunoreaction, more convenient, and selectivity and sensitivity are higher.Nano-pore single molecule detection technology is applied on hepatitis B virus infection cell for the first time.

Description

Technical Field

[0001] This invention belongs to the field of electrochemical detection technology and relates to a method for detecting hepatitis D virus antigen using nanopores. Background Technology

[0002] In 1970, researchers in Turin, Italy, discovered a novel immune product in patients with severe chronic hepatitis B. In 1977, this product was named delta factor and first formally reported by Rizzetto et al. In 1978, the National Institutes of Health in the United States began related research and discovered that delta factor was associated with a novel viral particle. In 1980, this viral particle was formally named hepatitis D virus (HDV). HDV was found to be a defective single-stranded negative-sense RNA virus that requires hepatotropic DNA viruses such as hepatitis B virus (HBV) to provide its outer shell for replication. HDV is present in the hepatocyte nuclei and serum of HDV-infected individuals who are positive for hepatitis B surface antigen (HBsAg), and primarily replicates within hepatocytes. HDV is mainly transmitted through blood transfusions and blood products, similar to the transmission route of HBV. HDV infection is mostly seen in HBV-infected individuals, but sporadic HDV infections can also occur. Over-infection with HDV and HBV can exacerbate liver damage and easily lead to chronic active hepatitis, cirrhosis, and fulminant hepatitis.

[0003] Currently, there are various clinical methods for detecting hepatitis D virus (HDV), the most common being liver function tests, HDV RNA testing, HDV antibody (-Ab) testing, and ELISA testing for HDV antigen (-Ag). However, liver function tests can only rule out the presence of viral infection in the liver and cannot detect a specific type of hepatitis D virus in a single test. HDV RNA testing presents significant challenges for primer design due to the high variability of the HDV genome, and there is currently no unified testing standard. HDV antibody testing primarily targets HDV-IgG, but HDV-IgG is closely related to hepatocyte damage and inflammatory activity, often resulting in inconsistent results between acute and chronic infections. HDV antigen testing is currently the gold standard for clinical detection of hepatitis D virus, but ELISA kits require large sample volumes and suffer from short window periods and low sensitivity and specificity.

[0004] In view of the above, there is an urgent need for a highly selective, sensitive, and specific detection method to detect antigens in hepatitis D virus. Summary of the Invention

[0005] In view of this, the purpose of the present invention is to provide a method for detecting hepatitis D virus antigen using nanopores.

[0006] To achieve the above objectives, the present invention provides the following technical solution: A method for detecting hepatitis D virus antigen using nanopores, the method being as follows: A nanopore electrochemical detection device is formed by placing nanopores within an electrochemical detection apparatus. An electrolyte is added to the device, and the sample solution is placed on the cis side (cis end) of the nanopore. An external electric field is applied, and the current signal of the sample is acquired. The amplitude, retardation time, and frequency characteristics of the current signal are analyzed to detect hepatitis D antigen at the single-molecule or cellular level. The sample includes hepatitis D virus antigen protein, hepatitis D virus antigen plasmid transfected cell lysate, and hepatitis D virus-infected cell lysate. The hepatitis D virus antigen includes hepatitis D virus large antigen and hepatitis D virus small antigen. The sequence of the hepatitis D virus large antigen is as follows: MSRSSESRKNRGGREEILEQWVAGRKKLEELERDLRKTKKKLKKIEDENPWLGNIKGILGKKDKDGEGAPPAKRARTDQMEVDSGPGKRPLRGGFTDKERQDHRRRKALENKK KQLSAGGKNLSKEEEEELRRLTEEDERRERRVAGPPVGGVNPLEGGSRGAPGGGFVPNLQGVPESPFSRTGEGLDIRGNQGFPWDILFPADPPFSPQSCRPQHHHHHH, as in SEQ Shown as ID NO:1.

[0007] The sequence of the hepatitis D virus small antigen is: MSRSESRKNRGGREEILEQWVAGRKKLEELERDLRKTKKKLKKIEDENPWLGNIKGILGKKDKDGEGAPPAKRARTDQMEVDSGPGKRPLRGGFTDKERQDHRRRKALENKKKQLSAGGKNLSKEEEEELRRLTEEDERRERRVAGPPVGGVNPLEGGSRGAPGGGFVPNLQGVPESPFSRTGEGLDIRGNQGFPHHHHHH, such as SEQ ID NO:2 shown.

[0008] Preferably, the nanopore electrochemical detection device is constructed as follows: A support film embedded with nanopores is placed between two solution chambers containing electrolytes, and a current loop is constructed between the two solution chambers. Specifically, two electrodes are placed in the two solution chambers respectively, and a power supply, an ammeter, and other devices are connected in sequence between the two electrodes to form a current loop, thus forming the nanopore electrochemical detection device.

[0009] The supporting film is a Teflon film.

[0010] The chamber material is resin.

[0011] The nanopores are MspA bio-nanopores.

[0012] The electrode is a silver electrode with silver chloride coated on its surface.

[0013] More preferably, the electrolyte in the solution chamber is a mixed solution of 1-butyl-3-methylimidazolium chloride (BMIMCl) and tris(hydroxymethyl)aminomethane (Tris) dissolved in water; The electrolyte contains 1-butyl-3-methylimidazolium chloride at a concentration of 1-1.5 M and tris(hydroxymethyl)aminomethane at a concentration of 10 mM. The concentration of 1-butyl-3-methylimidazolium chloride differs between the two solution chambers.

[0014] The electrolyte has a pH of 7.4.

[0015] Further preferably, the specific construction of the cell lysis buffer for hepatitis D virus large and small antigen plasmid transfection is as follows: Hepatitis D virus large antigen overexpression plasmid modified with a fluorescent tag or hepatitis D virus small antigen overexpression plasmid is transfected into human liver cancer cells (Huh7), cultured, and the cells are collected and stored at -80℃. Then, PBS + 1× protease inhibitor + 1× phosphatase inhibitor is added to the transfected cells, and the cells are frozen at -80℃. After repeated freeze-thaw shaking to completely lyse the cells, the supernatant is collected by centrifugation and stored at -80℃.

[0016] A further preferred embodiment of the hepatitis D virus-infected cell lysate is constructed as follows: Huh7 cells stably expressing NTCP are infected with HDV, and cells are collected on the second day after HDV infection. Then, PBS + 1× protease inhibitor + 1× phosphatase inhibitor are added to the infected cells, and the cells are frozen at -80°C. After repeated freeze-thaw cycles and shaking to completely lyse the cells, the supernatant is collected by centrifugation and stored at -80°C.

[0017] The beneficial effects of this invention are as follows: This invention discloses a method for detecting hepatitis D virus antigens at the single-molecule and cellular levels using a nanopore sensing system. By setting up nanopores in an electrochemical detection device, the method collects signals from the interaction between the hepatitis D virus antigen protein, hepatitis D virus antigen plasmid-transfected cell lysates, or hepatitis D virus-infected cell lysates and the nanopores. The method analyzes characteristics such as the current amplitude, signal retardation time, and signal generation quantity of the electrical signals to detect information such as the content of large and small hepatitis D virus antigens and their charged properties. This method can detect pure samples of two hepatitis D virus antigens (L-HDAg and S-HDAg) and distinguish between the two proteins based on the electrical signals generated in the electrochemical detection device. This method can also detect hepatitis D virus antigen overexpression plasmid-transfected cells and hepatitis D virus-infected cell lysates, distinguishing between normal cells and virus-infected cells, thus realizing the cross-application of nanopores with biomedicine. This method fully utilizes the advantages of nanopore detection systems—label-free, rapid, low-cost, and convenient detection—accurately recording minute current changes caused by different proteins, with high reliability, high sensitivity, and good repeatability.

[0018] This invention is a direct detection method at the single-molecule level, requiring no chemical labeling or immune reaction, making it more convenient and offering higher selectivity and sensitivity. It represents the first application of nanopore single-molecule detection technology to hepatitis D virus-infected cells.

[0019] Other advantages, objectives, and features of the invention will be set forth in part in the description which follows, and in part will be apparent to those skilled in the art from the following examination, or may be learned from practice of the invention. The objectives and other advantages of the invention can be realized and obtained through the following description. Attached Figure Description

[0020] To make the objectives, technical solutions, and advantages of the present invention clearer, the preferred embodiments of the present invention will be described in detail below with reference to the accompanying drawings, wherein: Figure 1 The nanopore electrochemical detection and analysis device used in the examples; Figure 2 The simulated structural diagrams of the large and small antigens (L-HDAg and S-HDAg) of hepatitis D virus in the embodiments are shown. Figure 3 The detection signals of hepatitis D virus large antigen protein (L-HDAg) and hepatitis D virus small antigen protein (S-HDAg) in a nanopore electrochemical detection and analysis device using basic buffer A and basic buffer B as electrolytes are shown in Figure a. The current graph of the L-HDAg detection signal is shown in Figure b. The current graph of the S-HDAg detection signal is shown in Figure c. and Figure d are bar charts showing the signal amplitude and signal quantity of the two different proteins. Figure 4 A bar chart comparing the signals detected by the large antigen protein of hepatitis D virus (L-HDAg) and the small antigen protein of hepatitis D virus (S-HDAg) in a nanopore electrochemical detection device in terms of current amplitude (a), retardation time (b), and signal generation frequency (c). Figure 5 The data represent the detection signals of normal cells and cells transfected with hepatitis D virus antigen overexpression plasmid in a nanopore electrochemical detection and analysis device using basal buffer A and basal buffer B as electrolytes. Here, a is the current graph of the detection signal of the lysate of normal cells (not transfected with viral antigen plasmid) as a control group; b is the current graph of the detection signal of the lysate of cells transfected with hepatitis D virus large antigen (L-HDAg) overexpression plasmid; c is the current graph of the detection signal of the lysate of cells transfected with hepatitis D virus small antigen (S-HDAg) overexpression plasmid; and df is a bar chart showing the signal amplitude and signal quantity for the three different cell lysates. Figure 6 The data represent the detection signals of normal cells and hepatitis D virus-infected cells in a nanoporous electrochemical detection and analysis device using basal buffer A and basal buffer B as electrolytes. a is the current graph of the detection signal of the lysate of uninfected normal cells as a control group; b is the current graph of the detection signal of the lysate of hepatitis D virus-infected cells; c and d are bar charts showing the signal amplitude and signal quantity corresponding to the two different cell lysates. Detailed Implementation

[0021] The following specific examples illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and various details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention.

[0022] The hepatitis D virus large antigen involved in the following examples has the following sequence: MSRSSESRKNRGGREEILEQWVAGRKKLEELERDLRKTKKKLKKIEDENPWLGNIKGILGKKDKDGEGAPPAKRARTDQMEVDSGPGKRPLRGGFTDKERQDHRRRKALEN KKKQLSAGGKNLSKEEEEELRRLTEEDERRERRVAGPPVGGVNPLEGGSRGAPGGGFVPNLQGVPESPFSRTGEGLDIRGNQGFPWDILFPADDPPFSPQSCRPQHHHHHH.

[0023] The sequence of the hepatitis D virus small antigen is as follows: MSRSESRKNRGGREEILEQWVAGRKKLEELERDLRKTKKKLKKIEDENPWLGNIKGILGKKDKDGEGAPPAKRARTDQMEVDSGPGKRPLRGGFTDKERQDHRRRKALENKKKQLSAGGKNLSKEEEEELRRLTEEDERRERRVAGPPVGGVNPLEGGSRGAPGGGFVPNLQGVPESPFSRTGEGLDIRGNQGFPHHHHHH.

[0024] Example 1 The nanopore electrochemical method was used to detect and differentiate between large and small antigen proteins of hepatitis D virus, as detailed below: 1. Prepare the solution: (1) Prepare the basic buffer solution: (a) Dissolve 1-butyl-3-methylimidazolium chloride (BMIMCL) and tris(hydroxymethyl)aminomethane (Tris) in deionized water and adjust the pH to 7.4 to form a basic buffer A containing 1-butyl-3-methylimidazolium chloride (BMIMCL) and tris(hydroxymethyl)aminomethane (Tris) (where the concentration of 1-butyl-3-methylimidazolium chloride is 1 M and the concentration of tris(hydroxymethyl)aminomethane is 10 mM). (b) Dissolve 1-butyl-3-methylimidazolium chloride (BMIMCL) and tris(hydroxymethyl)aminomethane (Tris) in deionized water and adjust the pH to 7.4 to form a basic buffer B containing 1-butyl-3-methylimidazolium chloride (BMIMCL) and tris(hydroxymethyl)aminomethane (Tris) (where the concentration of 1-butyl-3-methylimidazolium chloride is 1.5 M and the concentration of tris(hydroxymethyl)aminomethane is 10 mM).

[0025] All solutions were filtered using a 0.22 μM filter before use.

[0026] (2) Prepare protein samples: The synthesized large and small antigen proteins of hepatitis D virus were dissolved in enzyme-free deionized water to obtain a 20 μM solution, which was then stored in a refrigerator at -20℃ for later use. 2. Construction of a nanoporous electrochemical detection and analysis device: A supporting film containing MspA bio-nanopores (where the supporting film is a Teflon membrane) is placed in a solution chamber containing electrolytes (the chamber material is resin). The entire chamber is divided into two smaller chambers. Two electrodes (both of which are silver electrode materials with silver chloride coating) are then placed in the two smaller chambers respectively. A power supply, an ammeter, etc., are connected sequentially between the two electrodes to form a nanopore electrochemical detection and analysis device (e.g., Figure 1 (As shown).

[0027] 3. Detection of large and small antigen proteins of hepatitis D virus: (1) The prepared large and small antigen protein sample solutions are added to one chamber (cis side (cis end) of the nanopore electrochemical device. The two chambers are filled with the above-mentioned basic buffer A and basic buffer B respectively. An external electric field is applied, and under the action of the electric field force, the large and small antigen proteins in the protein sample solution are driven to interact with the nanopore to generate a signal.

[0028] (2) The current signal collected in step (1) is amplified by a low-noise current amplifier (Axon Axopatch 200B). The collected electrical signal file is selected by the Clamfit software to select the characteristic signal. Then, statistical analysis is performed in the Origin software to analyze the current amplitude and current lag time of the electrical signal and obtain the corresponding protein sample feature map.

[0029] Protein analysis and comparison are performed, enabling the analysis and detection of proteins using a nanopore electrochemical detection device, such as... Figure 3 As shown, the detection signals of hepatitis D virus large antigen protein (L-HDAg) and hepatitis D virus small antigen protein (S-HDAg) in a nanopore electrochemical detection and analysis device using basic buffer A and basic buffer B as electrolytes are presented. Among them, a is the current graph of the L-HDAg detection signal, b is the current graph of the S-HDAg detection signal, and c and d are bar charts showing the signal amplitude and signal quantity of the two different proteins.

[0030] The L-HDAg and S-HDAg protein samples were analyzed and compared, such as... Figure 4 As shown, a is a bar chart comparing the current amplitude of L-HDAg and S-HDAg, b is a bar chart comparing the current lag time of L-HDAg and S-HDAg, and c is a bar chart comparing the current signal generation frequency of L-HDAg and S-HDAg. Figure 4As can be seen, the two differ significantly in current amplitude, signal delay time, and signal generation frequency. In terms of current amplitude: L-HDAg Ⅱ>S-HDAg Ⅰ>L-HDAg Ⅰ; in terms of signal delay time: L-HDAg Ⅰ>L-HDAg Ⅱ>S-HDAg Ⅰ; and in terms of signal generation frequency: L-HDAg Ⅱ>L-HDAg Ⅰ>S-HDAg Ⅰ. L-HDAg Ⅰ and L-HDAg Ⅱ represent the two specific signals generated when detecting L-HDAg, named L-HDAg Ⅰ and L-HDAg Ⅱ respectively.

[0031] The above results demonstrate that the nanopore electrochemical detection device has high selectivity and high sensitivity, and can successfully detect and distinguish between hepatitis D virus large antigen protein (L-HDAg) and hepatitis D virus small antigen protein (S-HDAg).

[0032] Example 2 Nanopore electrochemical method was used to detect and distinguish normal cells from cells transfected with HDAg overexpression plasmid, as detailed below: 1. Prepare the solution: (1) Prepare the basic buffer solution: (a) Dissolve 1-butyl-3-methylimidazolium chloride (BMIMCL) and tris(hydroxymethyl)aminomethane (Tris) in deionized water and adjust the pH to 7.4 to form a basic buffer A containing 1-butyl-3-methylimidazolium chloride (BMIMCL) and tris(hydroxymethyl)aminomethane (Tris) (where the concentration of 1-butyl-3-methylimidazolium chloride is 1 M and the concentration of tris(hydroxymethyl)aminomethane is 10 mM). (b) Dissolve 1-butyl-3-methylimidazolium chloride (BMIMCL) and tris(hydroxymethyl)aminomethane (Tris) in deionized water and adjust the pH to 7.4 to form a basic buffer B containing 1-butyl-3-methylimidazolium chloride (BMIMCL) and tris(hydroxymethyl)aminomethane (Tris) (where the concentration of 1-butyl-3-methylimidazolium chloride is 1.5 M and the concentration of tris(hydroxymethyl)aminomethane is 10 mM).

[0033] All solutions were filtered using a 0.22 μM filter before use.

[0034] (2) Prepare cell lysis buffer samples: ①Construction of cells transfected with HDAg overexpression plasmid: HDAg-Flag overexpression plasmids: Flag-L-HDAg plasmid (modified with fluorescent tag flag and expressing large form of HDV Delta Antigen, synthesized by Universal Biotech (Anhui)) and Flag-S-HDAg (modified with fluorescent tag flag and expressing small form of HDV Delta Antigen, synthesized by Universal Biotech (Anhui)).

[0035] The empty vector control p3xFlag-CMV-10 was purchased from General Biotechnology (Anhui).

[0036] Human liver cancer cells (Huh7) were induced at a concentration of 1.5 × 10⁻⁶. 5 Cells were seeded per well in 12-well cell culture plates and cultured in a 37°C cell culture incubator containing 5% CO2. The day after cell seeding, the HDAg-Flag overexpression plasmid or the empty vector control p3xFlag-CMV-10 was transfected into Huh7 cells. After 6 hours, the culture medium in the wells was aspirated, and 1 mL of DMEM complete medium was added for further culture. 48 hours after transfection, the culture medium in the wells was aspirated, and the cells were washed twice with PBS. 400 μL of PBS was added to each well to collect the cells into 1.5 mL enzyme-free EP tubes. The tubes were then subjected to three freeze-thaw cycles at -80°C, centrifuged at 12000 rpm for 5 minutes at 4°C, and the supernatant was transferred to new 1.5 mL enzyme-free EP tubes and stored at -80°C.

[0037] ②Preparation of cell lysis buffer samples: After transfecting, wash the cells twice with PBS. Add 400 μL of PBS + 1× protease inhibitor (TargetMol, C0001) + 1× phosphatase inhibitor (TargetMol, C0002) to each well and freeze at -80°C. Repeat the freeze-thaw cycle three times, vortexing for 60 seconds each time to completely lyse the cells. Centrifuge at 4°C for 5 minutes at 12,000 rpm. Transfer the supernatant to enzyme-free EP tubes and store at -80°C.

[0038] 2. Construction of a nanoporous electrochemical detection and analysis device: A supporting film containing MspA bio-nanopores (where the supporting film is a Teflon membrane) is placed in a solution chamber containing electrolytes (the chamber material is resin). The entire chamber is divided into two smaller chambers. Two electrodes (both of which are silver electrode materials with silver chloride surface coating) are then placed in the two smaller chambers respectively. A power supply and an ammeter are connected sequentially between the two electrodes to form a nanopore electrochemical detection and analysis device (e.g., Figure 1 (As shown).

[0039] 3. Detection of normal cells not transfected with viral antigen plasmids and cell lysates transfected with HDAg overexpression plasmids: (1) Normal cells that have not been transfected with viral antigen plasmids and the sample solution of the HDAg overexpression plasmid transfected cell lysate prepared above are added to one chamber of the above nanopore electrochemical detection device. The above-mentioned basic buffer A and basic buffer B are used in the two chambers respectively. An external electric field is applied, and under the action of the electric field force, the large and small antigen proteins in the protein sample solution are driven to interact with the nanopore to generate a signal.

[0040] (2) The current signal collected in step (1) was amplified by a low-noise current amplifier (Axon Axopatch 200B). The collected electrical signal file was selected by the Clamfit software to select the characteristic signal. Then, statistical analysis was performed in the Origin software to analyze the current amplitude of the electrical signal and obtain the corresponding cell lysate sample characteristic map.

[0041] The samples were analyzed and compared, and a nanoporous electrochemical detection device was used to analyze and detect three types of cell lysates, such as... Figure 5 The figures show the detection signals of normal cells not transfected with viral antigen plasmids and cells transfected with hepatitis D virus antigen (HDAg) overexpression plasmids in a nanopore electrochemical detection and analysis device using basal buffer A and basal buffer B as electrolytes. Figure a shows the current graph of the detection signal of the lysate from normal cells not transfected with viral antigen plasmids as a control group; figure b shows the current graph of the detection signal from the lysate of cells transfected with the hepatitis D virus large antigen (L-HDAg) overexpression plasmid; figure c shows the current graph of the detection signal from the lysate of cells transfected with the hepatitis D virus small antigen (S-HDAg) overexpression plasmid; and figure df shows the bar charts of signal amplitude and signal quantity for the three different cell lysate samples. Figure 5It can be seen that when normal cell lysate samples without viral antigen plasmid transfection were added to the nanopore electrochemical detection and analysis device, three characteristic signals were generated: two of them were pore-blocking signals with long retardation times, requiring an external electric field to reverse the voltage before the current level returned to normal (current amplitude percentages were 92.7% and 94.6%), and one signal had a large current amplitude but a short retardation time (current amplitude percentage was 64.8%). Cell lysate samples transfected with hepatitis D virus large antigen (L-HDAg) overexpression plasmid were analyzed using the nanopore electrochemical detection and analysis device. Two characteristic signals were generated after the device was applied: one was a pore-blocking signal with a long lag time, requiring an external electric field to reverse the voltage before the current level returned to normal (current amplitude accounted for 85.1%), and the other was a signal with a moderate current amplitude but a short lag time (current amplitude accounted for 54.4%). When cell lysate samples transfected with hepatitis D virus small antigen (S-HDAg) overexpression plasmid were added to the nanopore electrochemical detection and analysis device, a characteristic signal was generated: a signal with a moderate current amplitude and a short lag time (current amplitude accounted for 55.7%). By comparing the signals from the three cell lysate samples, the signals from normal cells and cells transfected with HDAg overexpression plasmids could be distinguished based on the current amplitude percentage and signal type. This further demonstrates the advantages of the nanopore electrochemical detection method in multiple dimensions, exhibiting excellent selectivity and high resolution, and successfully distinguishing between normal cells and cells transfected with HDAg overexpression plasmids.

[0042] Example 3 Nanopore electrochemical methods are commonly used to detect and differentiate normal cells from hepatitis D virus (HDV)-infected cells in solutions, as detailed below: 1. Prepare the solution: (1) Prepare the basic buffer solution: (a) Dissolve 1-butyl-3-methylimidazolium chloride (BMIMCL) and tris(hydroxymethyl)aminomethane (Tris) in deionized water and adjust the pH to 7.4 to form a basic buffer A containing 1-butyl-3-methylimidazolium chloride (BMIMCL) and tris(hydroxymethyl)aminomethane (Tris) (where the concentration of 1-butyl-3-methylimidazolium chloride is 1 M and the concentration of tris(hydroxymethyl)aminomethane is 10 mM). (b) Dissolve 1-butyl-3-methylimidazolium chloride (BMIMCL) and tris(hydroxymethyl)aminomethane (Tris) in deionized water and adjust the pH to 7.4 to form a basic buffer B containing 1-butyl-3-methylimidazolium chloride (BMIMCL) and tris(hydroxymethyl)aminomethane (Tris) (where the concentration of 1-butyl-3-methylimidazolium chloride is 1.5 M and the concentration of tris(hydroxymethyl)aminomethane is 10 mM).

[0043] All solutions were filtered using a 0.22 μM filter before use.

[0044] (2) Prepare cell lysis buffer samples: ① Construction of cells infected with hepatitis D virus (HDV): Two days prior to HDV infection, Huh7 cells stably expressing NTCP (Huh7-NTCP cells, a gift from Researcher Chen Xinwen of the Wuhan Institute of Virology, Chinese Academy of Sciences) were injected with 1.2 × 10⁻⁶ cells. 5 Cells were seeded per well in 24-well cell culture plates treated with type I collagen and cultured in a cell culture incubator at 37°C with 5% CO2. One day before HDV infection, cells were incubated for 24 h with 350 μL William's E complete medium (Procell, PM151221) + 4% PEG8000 + HDV virus suspension (MOI: 4). Twenty-four h after HDV infection, cells were washed three times with PBS and cultured again with 500 μL William's E complete medium. Cells were harvested on day 2 after HDV infection.

[0045] ② Preparation of hepatitis D virus-infected cell lysate samples: After transfecting, wash the cells twice with PBS. Add 400 μL of PBS + 1× protease inhibitor (TargetMol, C0001) + 1× phosphatase inhibitor (TargetMol, C0002) to each well and freeze at -80°C. Repeat the freeze-thaw cycle three times, vortexing for 60 seconds each time to completely lyse the cells. Centrifuge at 4°C for 5 minutes at 12,000 rpm. Transfer the supernatant to enzyme-free EP tubes and store at -80°C.

[0046] 2. Construction of a nanoporous electrochemical detection and analysis device: A supporting film containing MspA bio-nanopores (where the supporting film is a Teflon membrane) is placed in a solution chamber containing electrolytes (the chamber material is resin). The entire chamber is divided into two smaller chambers. Two electrodes (both of which are silver electrode materials with silver chloride surface coating) are then placed in the two smaller chambers respectively. A power supply and an ammeter are connected sequentially between the two electrodes to form a nanopore electrochemical detection and analysis device (e.g., Figure 1 (As shown).

[0047] 3. Detection of uninfected normal cells and hepatitis D virus (HDV) infected cell lysates: (1) Uninfected normal cells and the prepared hepatitis D virus (HDV) infected cell lysate sample solution are added to one chamber of the above-mentioned nanopore electrochemical detection device. The above-mentioned basic buffer A and basic buffer B are used in the two chambers respectively. An external electric field is applied, and under the action of the electric field force, the large and small antigen proteins in the protein sample solution are driven to interact with the nanopore to generate a signal.

[0048] (2) The current signal collected in step (1) was amplified by a low-noise current amplifier (Axon Axopatch 200B). The collected electrical signal file was selected by the Clamfit software to select the characteristic signal. Then, statistical analysis was performed in the Origin software to analyze the current amplitude of the electrical signal and obtain the corresponding cell lysate sample characteristic map.

[0049] The samples were analyzed and compared, enabling the analysis and detection of two cell lysates using nanopores, such as... Figure 6 As shown, the detection signals of uninfected normal cells and hepatitis D virus-infected cells in a nanopore electrochemical detection and analysis device using basal buffer A and basal buffer B as electrolytes are presented. a is the current graph of the detection signal of the lysate from uninfected normal cells (as a control group), b is the current graph of the detection signal from the lysate of hepatitis D virus-infected cells, and c and d are bar charts showing the signal amplitude and signal quantity corresponding to the two different cell lysates. Figure 6 It can be seen that after adding the nanopore electrochemical detection and analysis device to the normal cell lysate sample without virus infection, only one characteristic signal with a moderate current amplitude and short retardation time was generated (current amplitude accounted for 56.0%); after adding the HDV-infected cell lysate sample to the nanopore electrochemical detection and analysis device, two other signals were generated, which were similar to those detected by the pure hepatitis D virus large and small antigens (L-HDAg, S-HDAg). Figure 3 The signals were identical (current amplitude percentages were 75.7% and 86.2%). By comparing the signals of the two cell lysate samples, the signals produced by the lysate samples of normal cells and HDV-infected cells could be distinguished based on the current amplitude percentage and signal type. Furthermore, the presence of both large and small hepatitis D virus antigens (L-HDAg and S-HDAg) in HDV infection could be confirmed based on the current amplitude percentage and signal type. The surface nanopore electrochemical detection method further demonstrates high selectivity, high sensitivity, high repeatability, and high reliability, successfully distinguishing between normal cells and HDV-infected cells.

[0050] In summary, this invention discloses a method for detecting hepatitis D virus antigens using nanopores. By incorporating nanopores in an electrochemical detection and analysis device, the electrical signals generated by the interaction between the sample and the nanopores are collected. The characteristics of these electrical signals, such as current amplitude, retardation time, and signal generation quantity, are analyzed to detect information such as sample content and electrical properties. This method can detect large and small hepatitis D virus antigens at both the single-molecule and cellular levels. The electrochemical analysis device collects electrochemical signals, distinguishing between large and small hepatitis D virus antigens (L-HDAg and S-HDAg) at the single-molecule level and between normal cells and cells transfected with hepatitis D virus overexpression plasmids (L-HDAg and S-HDAg), or between normal cells and hepatitis D virus (HDV)-infected cells, at the cellular level. This achieves the cross-application of nanopore sensing technology with biomedicine. It fully leverages the advantages of low cost, label-free operation, and speed of nanopore electrochemical devices, accurately recording minute current changes caused by different protein or cellular samples, with good repeatability and high reliability. This invention is a direct detection method at the single-molecule level, requiring no chemical labeling or immune reaction, making it more convenient and offering higher selectivity and sensitivity. It represents the first application of nanopore single-molecule detection technology to hepatitis D virus-infected cells.

[0051] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. A method for detecting hepatitis D virus antigen using nanopores, characterized in that, The method is as follows: A nanopore electrochemical detection device is formed by placing nanopores in an electrochemical detection apparatus. An electrolyte is added to the nanopore electrochemical detection apparatus, and the sample solution to be tested is placed on the cis side of the nanopore. An external electric field is applied, and the current signal of the sample is collected. The amplitude, retardation time, and frequency characteristics of the current signal are analyzed to detect hepatitis D virus antigen at the single-molecule or cellular level. The sample includes hepatitis D virus antigen protein, hepatitis D virus antigen plasmid transfected cell lysate, and hepatitis D virus infected cell lysate. The hepatitis D virus antigen includes hepatitis D virus large antigen and hepatitis D virus small antigen. The sequence of the hepatitis D virus large antigen is shown in SEQ ID NO:1, and the sequence of the hepatitis D virus small antigen is shown in SEQ ID NO:

2. The nanopore electrochemical detection device is constructed as follows: a support film embedded with nanopores is placed between two solution chambers containing electrolytes, and a current loop is constructed between the two solution chambers to form the nanopore electrochemical detection device. The electrolyte in the solution chamber is a mixed solution of 1-butyl-3-methylimidazolium chloride and tris(hydroxymethyl)aminomethane dissolved in water; The electrolyte contains 1-butyl-3-methylimidazolium chloride at a concentration of 1-1.5 M and tris(hydroxymethyl)aminomethane at a concentration of 10 mM; the concentrations of 1-butyl-3-methylimidazolium chloride in the electrolyte differ between the two solution chambers. The electrolyte has a pH of 7.4; The nanopores are MspA bio-nanopores.

2. The method according to claim 1, characterized in that, The construction of the current loop is specifically achieved by placing two electrodes in two solution chambers respectively, and connecting a device including a power supply and an ammeter between the two electrodes in sequence to form a current loop.

3. The method according to claim 2, characterized in that, The electrode is a silver electrode with silver chloride coated on its surface.

4. The method according to claim 1, characterized in that, The chamber material is resin.

5. The method according to claim 1, characterized in that, The cell lysis buffer for hepatitis D virus antigen plasmid transfection was constructed as follows: Hepatitis D virus large antigen overexpression plasmid or hepatitis D virus small antigen overexpression plasmid was transfected into human liver cancer cells, cultured, and the cells were collected; then PBS, protease inhibitor and phosphatase inhibitor were added to the transfected cells, and the cells were frozen at -80℃. After repeated freeze-thaw shaking, the cells were completely lysed, and the supernatant was collected by centrifugation.

6. The method according to claim 1, characterized in that, The following steps were taken to construct a cell lysate for hepatitis D virus infection: Huh7 cells stably expressing NTCP were infected with HDV and the cells were collected on the second day after HDV infection. Then, PBS, protease inhibitors, and phosphatase inhibitors were added to the infected cells, and the cells were frozen at -80°C. After repeated freeze-thaw cycles and shaking to completely lyse the cells, the supernatant was collected by centrifugation.

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