CN102507395A - Method for monitoring virus particles in solution in real time - Google Patents

Abstract

The invention relates to a method for real-time monitoring of virus particles in a solution, which can realize rapid detection of virus particles. The method for real-time monitoring of the virus particles in the solution is to make the sample solution containing the virus particles pass through the nanopore under the action of a bias voltage, detect the electrical signal, and compare the electric signal obtained when the solution containing the standard virus passes through the nanopore. Ratio, to determine the information on the virus particles contained in the sample solution. Preferably, determining that the sample solution contains virus particles is determined by comparing at least one of the amplitude, duration, time distribution, frequency, waveform characteristics or phase characteristics of the electrical signal obtained when the solution containing the standard virus passes through the nanopore Information. The invention has the characteristics of dynamic real-time, high sensitivity and high throughput, and meets the requirements of virus counting, classification and real-time analysis in the environment.

Description

Method for real-time monitoring of viral particles in solution

technical field

The invention relates to a method for real-time monitoring of virus particles in a solution.

Background technique

Viruses are the smallest of many microorganisms, have special structures, and are highly pathogenic. Widely present in air, water, soil, and human blood, body fluids, and feces, it poses a great threat to public health. Taking the water environment as an example, among the more than 700 water-borne viruses that have been discovered so far, rotavirus, hepatitis virus and enterovirus are dominant, which can cause various diseases such as diarrhea and hepatitis. Among them, rotavirus causes 11.4 billion cases of diarrhea and 870,000 deaths per year in developing countries [Albert, M.J., et al., Bangladesh. Journal of clinical microbiology, 1999.37(11): p.3458]. Infection of hepatitis A and E viruses is usually related to substandard water supply, and hepatitis E is particularly serious in pregnant women, with a mortality rate of up to 25% [Aggarwal, R. and K. Krawczynski, Journal of gastroenterology and hepatology, 2000.15 (1): p.9-20]. In 2004, infectious diseases broke out in Sichuan Province of my country due to drinking water pollution, and 123 students suffered from hepatitis A [Ren Jinfa. Chinese Journal of Health Inspection, 2009(004): p.942-944]. It can be seen that the virus is extremely harmful to the human body, and polluting water sources will pose a great threat to public health. The hepatitis virus, HIV virus, etc. that exist in the blood, and the influenza virus, rubella virus, etc. that exist in the air will also affect people's health. In order to ensure the health and safety of the environment and the human body, methods for real-time detection of viruses in various environments and small portable devices have become an urgent need in clinical and daily life.

At present, the commonly used clinical virus detection methods include: electron microscope observation, electrophoretic separation and indirect enzyme-linked immunoassay (ELISA), respectively, from the size and shape of virus particles, charge properties and surface specific recognition to detect and identify the types and characteristics of viruses . However, these methods must be carried out under laboratory conditions, and it takes a long time to achieve real-time detection. The technical process is relatively complicated, and it is difficult to promote clinically. Therefore, a method and portable device for detecting viruses in the environment with high sensitivity in real time are urgently needed to be developed. Nanopore technology, as a new generation of single-molecule detection hotspot, can also be used in high-sensitivity virus detection.

In 1996, Kasianowicz and colleagues first reported [Kasianowicz, J.J., et al., Proceedings of the National Academy of Sciences of the United States of America, 1996.93(24): p.13770-13773] the single-stranded DNA or RNA in the electric field It passes through the α-hemolysin nanopore under the action, and the phenomenon of blockade current (blockade current) is generated when the molecule passes through the hole, and the magnitude of the blockade current when each base passes through the nanopore can be obtained in turn. Since the blocking current generated by different bases has a corresponding decrease, four bases can be distinguished according to this, and the sequence components of DNA or RNA molecules can be obtained. In addition, the length of blocking the entire molecule can also be calculated from the duration of blocking current. After decades of continuous efforts, the research on nanopore as a method for single molecule detection is increasing day by day. The main reason is that this method has the advantages of convenience, rapidity and low cost.

Contents of the invention

The invention provides a method for real-time monitoring of virus particles in a solution, which can realize rapid detection of virus particles.

The applicant has found through research that in virus detection, the particle size and shape of virus particles can also be obtained by analyzing the size of the blocking current and the duration of the signal; in addition, the specific antibody modification of the pore wall can also realize the detection of specific virus species. Targeted capture to distinguish it from other particles. Compared with traditional electron microscope observation, electrophoretic separation and indirect enzyme-linked immunoassay (ELISA), it has the characteristics of fast detection speed, convenient operation and high sensitivity, and is an attractive virus detection method. Through this method, in principle, rapid detection of virus particles can be realized, and the type, quantity and state can be distinguished through the amplitude and duration of blocking current formed when the virus particles pass through the nanopore.

The method for real-time monitoring of the virus particles in the solution is to make the sample solution containing the virus particles pass through the nanopore under the action of a bias voltage, detect the electrical signal, and compare the electric signal obtained when the solution containing the standard virus passes through the nanopore. Ratio, to determine the information on the virus particles contained in the sample solution. The virus particles are preferably rotavirus, hepatitis B virus or human herpes virus.

Preferably, determining that the sample solution contains virus particles is determined by comparing at least one of the amplitude, duration, time distribution, frequency, waveform characteristics or phase characteristics of the electrical signal obtained when the solution containing the standard virus passes through the nanopore Information.

Preferably, the size of the nanopores is 100-250 nm. More preferably, the surface of the nanopore is modified by coupling agent molecules, DNA single- or double-stranded molecules, RNA molecules, PNA molecules or protein molecules.

As an improvement of the present invention, before the sample solution containing virus particles passes through the nanopore, it is pretreated through a submicron filter to filter out bacteria and other large particle impurities.

The detection device used in the present invention is the prior art, including: separating the two solution pools with a chip with nanopores, so that the two solution pools are only connected through the nanopores. An electrode is placed in each of the two solution pools, and a detection system is formed in series with shielded wires and data acquisition and analysis.

The real-time virus detection method includes all or some steps in the following process:

Sample preparation: Prepare the samples to be tested (air, soil, aqueous solution of collected materials in water samples, blood and biological fluids, secretions) into potassium chloride solution, and filter it with a submicron membrane filter.

Signal detection: install the nanopore in the above detection system. A bias voltage is applied across the electrodes to record electrical signals.

Result comparison: take the known common virus solution as the standard virus solution, pass through the nanopore, detect its electrical signal, and obtain the characteristic value of the blocking current of the standard virus under different conditions (including the amplitude, duration, and frequency of the blocking current) , waveform and phase). Statistical analysis is performed on the obtained results to determine the presence and quantity of specific viruses in unknown samples.

The invention has the characteristics of dynamic real-time, high sensitivity and high throughput, and meets the requirements of virus counting, classification and real-time analysis in the environment.

Description of drawings

Fig. 1 shows solid nanopore perspective view (1a) and central sectional view (1b);

Figure 2 shows a schematic diagram of a detection device connected to a filter device;

Figure 3 shows a schematic diagram of a system in which solid-state nanopores are used for biosensing detection;

Fig. 4 shows the experimental result that nanopore virus detection method detects rotavirus in water;

Figure 5 shows the experimental results of the nanopore virus detection method detecting hepatitis B virus in water;

Fig. 6 shows the current signal graph when the human herpes virus passes through the nanopore modified by EBV-Ab.

Among them, 1: nanopore; 2: silicon nitride film; 3: chip with nanopore; 4: electrode; 5: electrolyte solution; 6: submicron filter; 7: signal acquisition and analysis system.

Detailed ways

Example 1

Detection methods of main viruses in water:

Sample preparation: Add 0.0745g of potassium chloride to 10ml of ultrapure water, 10ml of target virus standard (such as rotavirus, hepatitis B virus) and 10ml of water sample to be tested to form 100mM potassium chloride solution A, Standard virus solution B and test sample solution C. Filter the two liquids with a 220nm filter membrane to remove bacteria and other large particle impurities.

Device: a chip 3 with a nanopore 1 with a diameter of 100 nm is used to separate the two solution pools so that the two solution pools are only connected through the nanopore. An electrode 4 is placed in each of the two solution pools, and the electrodes are connected to the analysis system 7 through shielded wire data acquisition.

Standard product test: at room temperature, add 50 μl potassium chloride solution and rotavirus standard virus solution into the chambers on both sides of the chip respectively, insert electrodes, connect with the data acquisition system, and close the shielding box. Adjust the bias voltage applied by the electrode to 200mV, and record the amplitude, duration, frequency, and waveform of the standard virus via hole blocking current as the standard for judging this virus. Taking rotavirus and hepatitis B virus as examples, the current signals when the two viruses pass through the hole are shown in Figure 4 and Figure 5. The blocking current amplitude, duration and waveform shape all have obvious differences.

Detection process: at room temperature, add 50 μl of potassium chloride solution and sample solution into the chambers on both sides of the chip, insert electrodes, connect with the data acquisition system, close the shielding box, collect and accurately record the standard virus via hole blocking current Amplitude, duration, frequency, waveform and phase characteristics, and statistical analysis, and finally get the characteristics of the blocking current of different viruses.

Therefore, various target viruses in water samples can be detected accurately and quickly.

Example 2

Modified nanopores specifically recognize human herpesviruses

Nanopore modification: put the chip with a diameter of 200nm in a dry and clean 10ml test tube, take 4ml of piranha solution (75mL of concentrated sulfuric acid (98%) and 25mL of hydrogen peroxide solution (30%)) into the test tube, and stir carefully with a gun tip , but do not touch the chip, place it in a water bath at 100°C for 10-15min. After cleaning, the chip was immersed in 2% APTS (3-aminopropyltriethoxysilane) solution, and combined at room temperature for 30 minutes. Take out the chip and wash it several times with methanol and ultrapure water. Subsequently, put the chip in 0.5ml activation buffer (0.1M MES, 0.5M NaCl, pH 6.0), add 0.2mg EDC, 0.3mg NHS, react for 30min, add 0.5ml pH7.2 phosphate buffer Wherein, 0.1 μM human herpesvirus antibody EBV-Ab was added after 5 minutes. Through the reaction of the amino group and the carboxyl group of the amino acid residue, it is combined in the nanopore of the chip.

Sample preparation: Take saliva sample A (containing EBV) to be tested, add 1ml of rotavirus-containing saliva sample B (does not contain EBV, contains rotavirus) and control saliva sample C (does not contain EBV and other viruses), and 9ml 100mM potassium chloride solution and mix well. Filter the two liquids with a 220nm filter membrane to remove bacteria and other large particle impurities.

Detection process: at room temperature, add 50 μl of potassium chloride solution and sample solution into the chambers on both sides of the chip, insert electrodes, connect with the data acquisition system, close the shielding box, collect and accurately record the standard virus via hole blocking current Amplitude, duration, frequency, waveform and phase characteristics, and statistical analysis, and finally get the characteristics of the blocking current of different viruses. The current signal of the human herpes virus through the EBV-Ab modified pore process is shown in Figure 6. The results showed that only when there was EBV, the antigenic virus specifically recognized by EBV-Ab, the blocking current continued to decrease. Through this method, accurate and rapid detection of the virus can be achieved.

Claims (7)

1. real-time method of virion in the monitoring solution; It is characterized in that; Under the bias voltage effect, make the sample solution that comprises virion through nano-pore, detect electric signal; Electric signal through obtaining during through nano-pore with the solution that comprises standard virus is compared, and confirms to comprise in the sample solution information of virion.

2. the method for virion is characterized in that in the real-time monitoring solution as claimed in claim 1, and the information that comprises virion in the said sample solution is at least a information in kind, quantity or the state that comprises virion.

3. the method for virion in the real-time monitoring solution as claimed in claim 1; It is characterized in that; Through at least a the comparing in amplitude, duration, time distribution, frequency, electric signal waveform characteristic or the phase propetry of the electric signal that obtains during through nano-pore with the solution that comprises standard virus, confirm to comprise in the sample solution information of virion.

4. the method for virion in according to claim 1 or claim 2 the real-time monitoring solution is characterized in that said nano-pore is of a size of 100-250nm.

5. the method for virion is characterized in that in the real-time monitoring solution as claimed in claim 4, and said nano-pore is through coupling agent molecule, dna single chain or duplex molecule, RNA molecule, pna molecule or protein molecule finishing.

6. the method for virion is characterized in that said nano-pore is through the antiviral antibody finishing in the real-time monitoring solution as claimed in claim 5.

7. the method for virion in according to claim 1 or claim 2 the real-time monitoring solution is characterized in that, the said sample solution that comprises virion carries out pre-service through before the nano-pore through the submicron order filter.

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