TECHNICAL FIELD

The present disclosure relates to a biosensor and a method for detecting an influenza. A virus in a sample. According to the biosensor of the present disclosure, it is possible to provide a cost-effective and rapid enzyme immunoassay for influenza A virus. The present disclosure also provides highly sensitive analytical method capable of accurately and selectively quantifying influenza A viruses.

BACKGROUND ART

The flu, which is caused by various influenza viruses, is a contagious respiratory illness. Most people with an influenza virus infection have mild symptoms and can recover within two weeks. However, in serious cases, which are likely to occur in children under the age of 5, adults over the age of 65, pregnant women, and people who have medical conditions, patients are likely to either be hospitalized or even die.

The major types of influenza viruses that routinely spread among people are influenza A and B viruses. Influenza A viruses are classified according to 15 different hemagglutinin (HA) subtypes and 9 different neuraminidase (NA) subtypes while influenza B virus is not divided into subtypes. Various types of influenza A viruses created by many different combinations of HA and NA proteins have been reported. It is well-known that influenza A viruses such as H1N1, H1N2, and H3N2 commonly infect people. In fact, the Centers for Disease and Control and Prevention (CDC) reported that the first global influenza pandemic was caused by influenza A (H1N1) virus (https://www.cdc.gov/flu/about/season/flu-season-2017-2018.htm).

It is difficult to diagnose a specific type of influenza A virus based on only the symptoms. Thus, various methods for the diagnosis of specific influenza A viruses have been developed. The sandwich enzyme immunoassays (EIA) with two antibodies are capable of selectively capturing a specific type of influenza A virus. Currently, electrochemical and optical sensors such as chemiluminescence, colorimetric, and fluorescence are widely applied as a detection method of sandwich EIA operated with a capture antibody and a detection antibody conjugated with horseradish peroxidase (HRP) or alkaline phosphatase (AP). In order to rapidly analyze influenza A viruses with high sensitivity and selectivity, EIAs using nanoparticles, such as gold, platinum, silver, and magnetic beads have been developed. However, Most of EIAs reported so far are complicated, expensive, and time-consuming.

As shown inFIG.1, neuraminidase (NA), a subtype of influenza A virus, can be applied as an enzyme to devise biosensors with fluorescence detection. 2′-(4-Methylumbelliferyl)-α-D-N-acetylneuraminic acid (MUNANA), a fluorogenic substrate, is converted to 4-methyllumbelliferone (4MU) in the presence of NA at acidic condition (pH 4.5-6). The concentration of 4MU formed in this reaction is dependent on the concentration of NA in a sample containing influenza A virus. In other words, the fluorescence intensity of 4MU formed from the reaction of NA and MUNANA (seeFIG.1) is proportionally enhanced with the increase of NA in a sample. 4MU excited at 365 nm emits blue light at 450 nm. Biosensors devised using the scheme inFIG.1were applied for the quantitative and qualitative analyses of influenza A viruses. However, it was not possible to detect a specific type of influenza A virus in a sample because the biosensors were operated without any antibodies capable of selectively capturing a specific type of influenza A virus.

Therefore, a biosensor and a method which can effectively and rapidly detect a specific type of influenza A virus in a sample have yet to be developed.

SUMMARY

According to one aspect of the present invention, a biosensor for detecting an influenza. A virus in a sample is provided, which comprises: an influenza A virus antibody immobilized on a surface of Au—Fe3O4composite; wherein the antibody binds with the influenza A virus in the sample, which converts 4-methylumbelliferyl-N-acetyl-α-D-neuraminic acid (MUNANA) to 4-methylumbelliferone (4-MU), wherein the 4-MU emits green light at pH of 5.5-6.5; and wherein the 4-MU emits blue light at pH of 9.3-11.3. In the biosensor, 1,1′-oxalyldiimidazole chemiluminescence (ODI-CL) reagent may be utilized to emit the blue and green lights. The 1,1′-oxalyldiimidazole chemiluminescence (ODI-CL) reagent may comprise an ODI and H2O2. The sample may be plasma or whole blood.

In accordance with another aspect of the present invention, a kit for detecting an influenza A virus in a sample is provided, the kit comprises: the above biosensor; and a container. The kit may further comprise: a sodium phosphate buffer for adjusting the pH between 5.5 to 6.5; a sodium hydroxide buffer for adjusting the pH between 9.3-11.3; and 1,1′-oxalyldiimidazole chemiluminescence (ODI-CL) reagent. The 1,1′-oxalyldiimidazole chemiluminescence (ODI-CL) reagent may comprise an ODI and H2O2.

In yet another aspect of the present invention, a method of detecting an influenza A virus in a sample is provided, the method comprises: immobilizing an influenza A virus antibody on a surface of Au—Fe3O4composite; mixing the immobilized antibody with 4-methylumbelliferyl-N-acetyl-α-D-neuraminic acid (MUNANA), and incubating the mixture to form 4-methylumbelliferone (4-MU); adjusting pH of the incubated mixture between 5.5 to 6.5 and measuring light intensity derived from the 4-MU by using a first enzyme assay with 1,1′-oxalyldiimidazole chemiluminescence (ODI-CL) detection; and adjusting pH of the incubated mixture between 9.3-11.3 and measuring light intensity of derived from the 4-MU by using a second enzyme assay with 1,1′-oxalyldiimidazole chemiluminescence (ODI-CL) detection. In this method, a sodium phosphate buffer may be used for adjusting the pH between 5.5 to 6.5; and a sodium hydroxide buffer may be used for adjusting the pH between 9.3-11.3. The first and second enzyme assays with 1,1′-oxalyldiimidazole chemiluminescence (ODI-CL) detection may be performed by using ODI and H2O2. The influenza A virus antibody may be a hemagglutinin (HA) subtype antibody. The influenza A virus may be H1N1 type, 1-T3N2 type or H5N1 type influenza A virus. The above step of incubating the mixture may be performed for 6-12 minutes at room temperature.

These and other aspects will be appreciated by one of ordinary skill in the art upon reading and understanding the following specification.

