KR20160114412A - Sensor for Influenza Detection - Google Patents

Abstract

The present invention relates to a sensor for the label-free detection of the existence of influenza in liquid or gas, or for quantifying the amount of the influenza. The sensor of the present invention has remarkable stability and sensitivity which a conventional sensor failed to achieve, and provides a stable and sensitive portable device having high resolution. The sensor comprises: a sensor probe; and a detector connected to the sensor probe. A sialylglycopolymer is attached to a surface of the sensor probe via covalent bonding.

Description

Sensor for Influenza Detection [0002]

The present invention relates to a sensor for detecting or quantifying the presence of influenza in a liquid or gas.

Influenza viruses are RNA viruses surrounded by membranes, composed of segmented segments of negative strand RNA, and are avians and the main source of human infection (Lamb et al., Annu Rev Biochem 52: 467-506 (1983)).

Influenza causes a pandemic flu that can kill thousands to tens of thousands of people every season, and can cause about one million deaths worldwide. In the 20th century, there were three global epidemics of flu due to new influenza, and tens of millions of people died. This strain occurs when an infection occurs from another animal to a human, when the host, the host, receives the gene from a host that hosts another animal.

Influenza is divided into three of five genus, which are contained in the RNA virus, Orthomyxoviridae, and are designated influenza A, B and C.

The overall structure of each of influenza A, B, and C is the same. Virions are 80-120 nm in diameter, and the viral envelopes surrounding the central nucleus are two distinct glycoproteins. The nucleus is composed of viral RNA, (Viral proteins) that are necessary for the production of recombinant proteins.

As a virus, infrequently, the genome of influenza is not a fragment of the nucleic acid. The genome is a negative sense RNA that is segmented into seven or eight, each RNA has one or two genes. This RNA includes hemagglutinin (HA), neuraminidase (NA), neucleoprotein (NP), M1, M2, NS1, NS2 (NEP), PA, PB1, PB1-F2 , And PB2.

Hemagglutinin and neuraminidase are large glycoproteins located outside the virion. The lectin, hemagglutinin, helps the virus invade the cell, and neuraminidase assists in the release of virions from the cells by scavenging sugars that bind to mature virions. Antiviral agents block the activity of these glycoproteins. In addition, these glycoproteins also act as important antigens, and hemagglutinin in particular plays a crucial role in the entry of virus into host cells.

In order for influenza to penetrate the host cell, hemagglutinin of influenza must bind to sialic acid present in the host cell membrane (step 1). When hemagglutinin is cleaved into proteases, the cells absorb the virus into the endosome. The acidic environment of endosomes causes two changes, part of hemagglutinin binding the coat of virion to the vacuole membrane of the cell. The other is to allow the M2 ion channel to move bilaterally through the envelope to acidify the virion nucleus. When the nuclei are acidified and degraded, viral RNA and proteins are released. Eventually, viral RNA, accessory proteins, and RNA-dependent RNA polymerase are released into the cytoplasm (Step 2).

The proteins and RNA of the released nucleus are transferred into the nucleus of the infected cells to form a complex. Inside the nucleus, RNA-dependent RNA polymerase begins to transcribe complementary positive strand RNA (steps 3a and 3b). vRNA is translated into the cytoplasm and translated (step 4), and remains in the nucleus. Newly synthesized viral proteins (neuraminidase and hemagglutinin) are secreted into the cell surface through Golgi bodies (step 5b) or sent to the nucleus to bind to vRNA to create new viral genes. Pre-infected viral proteins perform a number of tasks in the host cell. For example, the original mRNA may be degraded to use the product nucleotide for the synthesis of vRNA, or translation of the original mRNA of the infected cell may be inhibited.

These new virions are composed of negative strand vRNA, RNA-dependent RNA polymerase, and viral proteins. VRNA and viral proteins migrate into the compartment formed between the hemagglutinin and the neuramidase formed on the cell surface (step 6). The new virion is released into the spherical form with the phospholipid layer of the cell and the glycoprotein of the formed envelope (step 7). Birion has the property of attaching to host cells with hemagglutinin, and neuraminidase breaks the bond and helps release. At the end of the virus release, the host cell bursts to death.

Initial registration of influenza virus is one of the most effective ways to protect against influenza. It is important to provide sensitive virus detection in this public place. Sensors are portable, automatic, and require no-label detection with long life, high sensitivity and reproducibility. The standard sensor consists of a transducer and a receptor layer.

Today, virus sensors use various types of transducers, such as electrical, mechanical, and optical. Various biosensors targeting specific receptor antibodies, sugar chains, aptamers, glycans and proteins have been used. Receptors target RNA and DNA from viruses or surface proteins.

On the other hand, microcantilevers have started to receive the spotlight as biosensors by enabling label-free detection of proteins, DNA, and cells.

Recently, a microcantilever has been used to detect a marker protein such as PSA, CRP, and myoglobin. In addition, the microcantilever can detect a chemical substance as a non-labeled substance at a sub-nanogram level level, (US 2015/0065364 A1). Also, quantum mechanical phenomena using micro cantilever has been actively studied.

Numerous references are referenced throughout the specification and are cited therein. The disclosure of the cited document is incorporated herein by reference in its entirety to more clearly describe the state of the art to which the present invention pertains and the content of the present invention.

U.S. Patent Publication No. 2015/0065364

The present inventors have sought to provide an anti-virus detection sensor for influenza viruses for use in a field-ready, high-resolution, stable, sensitive portable device. As a result, by fabricating a sensor for detecting influenza with excellent stability and sensitivity that can not be achieved by existing sensors, which can accurately detect influenza at levels from nanograms to sub-nanograms present in a liquid or gas, It was completed.

Therefore, an object of the present invention is to provide a high-resolution, stable and sensitive anti-label detection sensor for influenza virus.

It is another object of the present invention to provide a method for detecting and quantifying influenza in a liquid or a gas by using the sensor for detecting influenza.

Other objects and advantages of the present invention will become more apparent from the following detailed description of the invention, claims and drawings.

According to an aspect of the present invention, there is provided a sensor probe comprising: a sensor probe having only one end fixed; And a detector connected to the sensor probe, wherein a sialylglycopolymer is attached to the surface of the sensor probe by a covalent bond.

The sensors of the present invention can be used to detect or quantify the presence of influenza in a liquid or gas.

Sialyl glycol polymer

In the sensor for detecting influenza of the present invention, the use of a sialyl glycol is required to ensure the high stability and sensitivity of the sensor probe.

The sensitivity of the sensor probe depends on the value of the surface stress occurring in the sensor layer due to the specific binding of the target molecule. Surface stresses are caused by the interaction of complex formation, and receptor molecules can be included in the matrix layer to increase the response of the sensor.

In this case, surface stress is influenced not only by the binding of the receptor molecule to the target molecule, but also by other factors such as the interaction between the receptor molecule and the matrix molecule, and the stability of the complex.

Although it is well known that influenza virus specifically binds to the sialic acid moiety, the present inventors have found that by using the method of directly bonding the sialic acid monomer or sialyl oligosaccharide itself to the probe surface, It can not be done.

Further, in order to introduce a large amount of sialyl moiety into the sensor surface, the oligosaccharide containing sialyl group, the hydrogel material and the surface of the sensor probe are simultaneously reacted in one solution so that the sialyl oligosaccharide is potentially randomly incorporated into the hydrogel Attempts have been made to attach the sialyl moiety to the probe surface at high density through the porous matrix in the form of a crosslinked hydrated gel.

However, in this case, it is impossible to control the density of the sialyl moiety at the exact amount and ratio required for virus detection, and in particular, it is disadvantageous from the standpoint of the stability of the sensor probe, and it is impossible to secure enough stability to be applicable to a dynamic cantilever.

Thus, the present inventors have found that a method for developing a virus detector with high sensitivity, accuracy and stability is a method for producing a sialylglycopolymer comprising a sialyl oligosaccharide as an influenza receptor (binding moiety) . ≪ / RTI >

In the present invention, the influenza virus specifically recognizes and binds to the sialyl group of the sialyliolysaccharide in the sialyl glycopolymer.

The surface of the sensor probe of the present invention may be adhered with a single or a plurality of independent sialylglycolic polymer molecules, wherein the sialylglycolic polymer molecules are not crosslinked.

Here, since each of the sialicylicol polymer molecules is an independent polymer previously prepared by covalently linking sialyloligosaccharide to a polymeric polymer prior to attaching to the surface of the sensor probe, the influenza virus particle And an oligosaccharide sequence in a specific ratio and amount.

In addition, each of the sialylglycol polymer molecules of the present invention is previously prepared with molecules independent of the attachment of the sensor probe surface, and thus has predetermined characteristics in relation to molecular weight, solubility, matrix flexibility, stability, So that it is possible to secure not only the high sensitivity and accuracy of the sensor itself but also excellent stability.

In one embodiment, the sialylglycol polymer molecule of the invention is characterized in that it comprises a sialyl (? 2-3) galactosyl moiety or a sialyl oligosaccharide having a sialyl (? 2-6) galactosyl moiety do.

Sialyl (sialyl) saccharides having sialyl (a2-3) galactose or sialyl (a2-6) galactose ends have different molecular shapes and are thereby distinguished from each other. Human and avian influenza viruses distinguish these differences.

It is generally recognized that human viruses preferentially bind to receptors with a Sia (? 2-6) Gal-terminus preferentially and bind to receptors with the avian Sia (? 2-3) Gal-terminus (Yoshihiro, Caister Academic Press, United Kingdom, 2006, 372p). The third saccharide may also affect the affinity of other subtype receptors of influenza virus (Gambaryan AS et al., Virology, 2005, 334, 2, pages 276-283). However, all species of viruses in other hosts can bind different types of sialylglycan receptors to varying degrees, so that the sequence of the sialylolysaccharide as the influenza receptor molecule contained in the sensor probe of the present invention is only Quot; is intended to unduly limit the scope of the legitimate rights of the present invention.

Thus, the sialyloligosaccharide as an influenza receptor molecule contained in the sensor probe of the present invention can be used without limitation as long as it contains a sialyl group, and examples thereof include Neu5Acα2-3Galβ1-4Glcβ (3'SL), Neu5Acα2-6Galβ1 Neu5Acα2-3Galβ1-4GlcNAcβ (6'SLN), Neu5Gcα2-6Galβ1-4GlcNAcβ (6 '(Neu5Gc) LN), Neu5Acα2-3Galβ1-4GlcNAc (3'SLN), Neu5Gcα2-3Galβ1-4GlcNAc Neu5Acα2-3Galβ1-4- (6-Su) GlcNAc (Su-3'SLN), Neu5Acα2-3Galβ1-3GlcNAc (SiaLe ^c ), Neu5Acα2-3Galβ1-3- (6-Su) GlcNAc ^{(Su-SiaLe c), (} Neu5Acα2-3Galβ1-4) (Fucα1-3) GlcNAc (SiaLe x), (Neu5Acα2-3Galβ1-4) (Fucα1-3) (6-Su) GlcNAc (Su-SiaLe x), Neu5Acα2-6Galβ1-4GlcNAc and Neu5Acα2-6Galβ1-4- (6-Su) GlcNAc (Su-6'SLN), but the present invention is not limited thereto.

Also, as the surface glycoprotein structure is not clearly described in the prior art (Gambaryan AS et al. Virology. 1997, 9, 232 (2), pages 345-350) Different preferences can be found. It also offers the opportunity to develop universal receptors for several types of viruses by combining different receptors on one polymer matrix. The use of polymeric matrices as vehicles for biologically active carbohydrate ligands is useful due to their non-reactivity and excellent stability. It is believed that oligosaccharide stability does not affect the overall stability of the receptor (Rune Slimestada et al., Journal of Chromatography A, 1118, Issue 2, 2006, Pages 281-284, RS Shallenberger et al, Food Chemistry, 1983, Pages 159-165).

Meanwhile, the sialylglycolic polymer used in the sensor probe of the present invention may be a polymer prepared by covalently linking sialyloligosaccharide to an acrylic polymer.

The acrylic polymer is a polymer prepared by polymerizing various acrylic monomers such as acrylic acid, acrylamide, acrylate, methacrylate, alkyl acrylate, alkyl methacrylate, cyanoacrylate, acrylonitrile, and derivatives of CH ₂ = CHCOOR Or a copolymer, and may be a copolymer of one or more acrylic units with other units.

In a preferred embodiment, the acrylic polymer may be a polyacrylamide, a polyacrylate or a copolymer of polyacrylamide and polyacrylate, but is not necessarily limited thereto.

The sialylglycolic polymer of the present invention is stably attached to the surface of the sensor probe by covalent bonding. For example, disulfide bonds, diamine bonds, sulfide-amine bonds, carboxy-amine But are not limited to, carboxyl-amine bonds, ester bonds, and the like.

sensor Probe

The sensor probe should be interpreted as having a submicroimeter or micrometer-sized flexible structure connected with the detection means so that the deformation of the structure can be measured. These deformations include all kinds of deformations that cover any movement of the sensor probe. The deformation of the sensor probe structure is a concept that includes all kinds of static and dynamic deformation such as bending, twisting, and vibration.

The membrane transducer attached to the sensor probe can be designed and configured to be attached to the sensor probe in a configuration that allows it to be attached to a measurement chamber in a liquid processing system, a gas processing system, or any other system where actual processing occurs.

In an embodiment of the present invention, the sensor probe may be a single-layer or multi-layer structure.

For example, the sensor probe of the present invention includes a supporting layer; A thiol, silane or silanetran linker having functional groups; And a sialyl glycol polymer layer covalently linked to the support layer through the functional group of the linker.

Here, the shape and size of the support layer may vary depending on the condition and the measuring operation, and the support layer may be fixed at one end by one or several points or sides. The material of the support layer may be composed of various materials such as silicon, polymer, aluminum oxide, and glass.

The linker is provided for stably connecting the support layer and the sialyl glycol polymer layer through the functional group.

Functional groups of linkers include reactive hydrocarbon groups such as alkenyl, alkynyl; Haloalkyl groups such as haloalkyl, fluoroalkyl; Oxygen-containing reactive groups such as hydroxyl, carbonyl, aldehyde, haloformyl, ester, carboxylate, carboxyl, alkoxy, peroxy, ether, acetal, ketal; Amines, amides, imines, imides, azides, azo, cyanates, nitrates. Nitrogen-containing reactive groups such as nitrile, nitrite, nitro, and nitroso; Sulfur containing reactive groups such as thiol, sulfide, disulfide, sulfoxide, sulfone, sulfonate, thiocyanate, thion, and thall; Phosphorus-containing reactive groups such as phosphine, phosphate, and phosphono; Boron-containing functional groups such as boron, boronate, boronate, borino, borinate, but are not necessarily limited thereto.

In one embodiment, the linker can be a silane / silane linker comprising an amine group as a functional group, wherein the sialyl glycol polymer can be stably and easily immobilized to the amine group present in the linker. The carboxy group of sialic acid can also react with amine groups present on the surface through amide bond formation.

When the support is composed of quartz, silicon or a glass material, a silane / silane such as (3-aminopropyl) triethoxysilane, 3-aminopropyltrimethoxysilane or 3- (1-aminopropyl) Linker may be used, and the sialyl glycol polymer layer may be bonded to the surface of the sensor probe through the amino group of the linker.

When the support layer is made of gold or silver, a thiol linker such as 4-aminothiophenol may be used as the linker.

In addition, the sialyl glycol polymer layer is not randomly introduced through cross-linking by reacting sialyl oligosaccharide in a single solution containing the polymer molecules and the surface of the sensor probe at the same time, And a sialylglycolic polymer molecule previously prepared so as to contain a sialyl moiety in an amount of not less than 10% by weight, and is not in the form of a crosslinked gel between polymer molecules.

On the other hand, the sensor probe of the present invention may further include an additional layer. The additional layer may be a metal layer such as silver, gold, platinum, palladium, aluminum, chromium, titanium, copper, ruthenium, rhodium or combinations or alloys with such metals or other metals.

The additional layer may be provided by deposition, precipitation, laser ablation, electron beam evaporation, electroplating, shadow masking, or any other suitable technique for providing a metal layer. The additional layer may have a thickness of between a few hundred nanometers and a few nanometers.

The additional layer may be provided on the outer surface of the sensor probe. The outer surface may be present on the sensor probe membrane, or may be a part or the bottom surface of the upper probe.

The additional layer may be provided as a buried layer in the probe. By providing the additional layer as a buried layer, the possible bimorph effect can be avoided or minimized. In principle, an additional layer of a probe made of a material that reflects at least a part of the incident light or has piezoelectric resistance characteristics may be provided and configured as a buried layer.

standard Probe

In addition to the sensor probe described above, the sensor for influenza detection of the present invention may further include a reference probe used for acquiring a reference signal. If the sensor comprises at least two membrane probes depending on the situation of use, at least one of the probes may be a reference probe used to obtain a reference signal. Since the reference signal can be used to filter, for example, noise such as fluctuations in flow rate, variations in concentration, changes in the optical properties of the liquid, heat drift due to nonspecific coupling, or artificial sensor signals resulting from transient phenomena, And obtains a reference signal at the same time.

The sensor for detecting influenza of the present invention may include several thousand or more membrane probes such as 2-3, 2-5, 5-10, 10-20, 20-50 or even more. The aligned probes may be similar in terms of their mechanical and chemical properties, similar membrane groups may be provided, or all of the aligned probes may have different mechanical and / or chemical properties.

Providing similar probes has the advantage of enabling more accurate measurement of probe deformation by comparing and analyzing signals derived from different probes with similar characteristics.

Providing membrane probes with different characteristics from each other has the advantage of providing a multifunctional sensor divider.

Detector

A common method for measuring probe deformation is to measure the change in deflection in the sensor probe that occurs as the detection target couples to the sensor probe using detection means.

Here, the detector shines a light beam on a probe and monitors light reflected from the light source and the probe through a sensor by a photosensitive detecting means such as a photodiode, and optically records the light. The detector may thus include a light emitter and a light detector.

Another method of measuring the probe deformation is to measure the change in the resonance frequency of the sensor probe, which occurs as the detection target is coupled to the sensor probe, using detection means.

Wherein the deformation of the probe may be detected by reading a piezoelectric or piezoresistive resistor. In this case, the material of the probe may include additional layers with added piezoelectric or piezoresistive properties.

The constituent features of the influenza detection sensor of the present invention have been described so far.

The sensor of the present invention may be a micro cantilever. Cantilever and other membrane surface stress sensors establish a sensitive system with high specificity. They do not indicate the problem of nonspecific gravimetric signals, they are small in size and easy to produce. Previously, cantilevers and other types of surface stressed membrane transducers have been introduced (Shengbo Sang et al. Biosensors and Bioelectronics 51 (2014) 124-135).

In the cantilever sensor, when the influenza virus binds to the oligosaccharide sequence, a change in the side stress in the sialyl glycol polymer layer occurs, which leads to a change in the membrane structure. The detection of the signal can be performed by reading all of the above (light, electricity, etc.).

The cantilever of the present invention is a static cantilever for measuring the change of the deflection of the cantilever caused by the stress due to the molecular interaction when the molecule to be detected is adsorbed, Or may be a dynamic cantilever that performs a cantilever operation.

It has been proposed that the use of a dynamic microcantilever rather than a static microcantilever allows for more sensitive non-label detection in the detection of the target material.

The dynamic microcantilever, especially as the length of the cantilever is smaller, results in further deviations from the theoretical predicted value of the resonance frequency measured in the experiment. In this case, the difference between the resonance frequency measured in the experiment and the theoretical value is due to the fact that the geometry can not be accurately processed in a cantilever having a very small size. Specifically, the etching process causes a change in the thickness of the cantilever, which results in a change in the resonance frequency. Particularly, as the cantilever having a very small size, a small thickness change greatly affects the resonance frequency change.

Therefore, unless the stability of the probe receptor molecules and the matrix is ensured, the value of the resonance frequency measured due to the error caused in this process must be different from the theoretical predicted value.

The sensor for detecting influenza of the present invention can secure a high stability and sensitivity because a sialyl glycol polymer precisely prepared in advance is used instead of a crosslinked hydrated gel containing a sialyloligosaccharide or a sialyl moiety randomly, As a result, it can be used as a more sensitive dynamic cantilever.

Also, the surface stresses generated in the stable sensor probes of the present invention depend on the amount of specific binding of the matrix established by the concentration of virus in the matrix. This function provides an opportunity for an invented sensor to provide quantitative measurement of the virus in different substrates.

The surface stresses generated in the matrix can be regenerated by washing solutions, such as carbamide aqueous solutions, of oligosaccharide sequence complexes with viral particles. The use of such receptor molecules in the invented sensor provides an opportunity for reuse.

The present invention provides a non-labeling detection sensor for influenza viruses for high resolution, stable, sensitive portable devices. The influenza detection sensor of the present invention has excellent stability and sensitivity that can not be achieved by conventional sensors that accurately detect influenza at levels from nanograms to sub-nanograms present in a liquid or gas.

Figure 1 shows a schematic of a sensor for influenza detection.
Fig. 2 shows the structure of polyacrylamide modified with Neu5Acα2-3Galβ1-4Glcβ (3'SL-PAA) as an example of a sialylglycolic polymer.
Figure 3 shows a schematic of a setup for providing an in-flow experiment.
Figure 4 shows the results of detection of duck virus using PAA-3'SL receptor molecules. Time dependence of surface stresses was observed at different concentrations. (Black line: 10 ⁸ virus / ml, red line: 10 ⁷ virus / ml, blue line: 10 ⁶ virus / ml, green line: 0 virus / ml.
Figure 5 shows the regeneration of the PAA-3'SL receptor for virus detection. (Blue line: injection of 10 ⁷ virus / ml solution before regeneration, green line: second solution of 10 ⁷ virus / ml solution after regeneration with 10% urea solution).
6 shows the frequency spectrum of the piezoelectric ceramic cantilever. (1: resonance peak corresponding to the bending mode, and 2: resonance peak corresponding to the length mode.)
7 shows the time dependence of the relative resonance frequency shift of the piezoelectric ceramics modified with the PAA-3'SL receptor in the experiment using the virus solution and the umbilical membrane fluid.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are only for describing the present invention more specifically and that the scope of the present invention is not limited by these embodiments.

Example

Cantilever  Manufacture of sensors

The influenza detection sensor used in the experiment was based on the modified membrane shown in Fig. The spacered oligosaccharides were synthesized by the method described in Poly (N-oxysuccinimidyl acrylate) (Tuzikov et al., 2000; Pazynina et al., 2002; Mochalova et al., 2003; Shilova et al., 2005) To prepare a high molecular weight polyacrylamide conjugate having Neu5Acα2-3Gal β1-4Glcβ (3SL) (FIG. 2).

Two rectangular cantilevers (500 x 100 x 1 um) were used as the membrane. Arrow ™ TL1Au (NanoWorld, Switzerland). They were made from monolithic silicon coated with 5 nm titanium / 30 nm gold on one side.

BioScan setup (Biosensor Academy, LLC.) Was used to measure cantilever deflection with optical reading.

A piezoceramic cantilever (1500 X 1000 X 110 um) was fabricated from a piezoelectric diaphragm AW4E6, 5T-70E.

The FemtoScan setup (Advanced Technologies Center) was used to measure resonance frequency changes.

 3-Aminopropyltrimethoxysilane was used for surface modification of the cantilevers. In order to prevent nonspecific adsorption on the sensor cantilever (unmodified gold surface), cantilevers were reacted in BSA aqueous solution for 2 hours before the virus experiment. A denatured cantilever with BSA (chemically attached to the surface via APTMS) was used as a reference in all viral experiments.

4 - aminothiol phenol was used for surface modification of piezoelectric ceramics cantilevers. To prevent nonspecific adsorption on the sensor cantilever, cantilevers were reacted in BSA aqueous solution for 2 hours before the virus experiment.

The sensor and the reference cantilever were fabricated before the experiment according to the protocol described below.

The cantilever Arrow ™ TL1Au (NanoWorld, Switzerland) was prepared for denaturation. The cantilever was washed with a piranha solution (H ₂ SO ₄ .H ₂ O ₂ ). The piranha solution should be prepared prior to each rinsing procedure by adding concentrated H ₂ SO ₄ to a 30% solution of H ₂ O ₂ . The cantilever was washed with distilled water and ethanol, and a 10% ethanol solution of APTMS was prepared. The reaction was carried out in a 10% ethanol solution of APTMS for 1 hour, followed by washing several times with ethanol and Milli-Q water. A solution of 10 ^-3 M PAA ^{3 '} SL was prepared, and the cantilever sensor was reacted with the solution of PAA-3' SL at room temperature for 16 hours. Washed several times with Milli-Q water, and reacted with two cantilevers in the BSA solution for 1 hour. The two cantilevers were then washed several times with water.

Piezoelectric ceramic cantilevers were cut from the piezoelectric diaphragm AW4E6, 5T-70E for denaturation and washed with distilled water and ethanol. A 0.001 M aqueous solution of 4-aminothiophenol was prepared, the cantilever was reacted in this aqueous solution for 12 hours, and then washed several times with Milli-Q water. Aqueous solution of 10 ^-3 M PAA- ^{3 '} SL was prepared, and the piezoelectric ceramic cantilever was allowed to react in a PAA-3' SL aqueous solution at room temperature for 16 hours. It was then washed several times with Milli-Q water, and the piezoelectric ceramic cantilever was allowed to react in the BSA solution for one hour. The piezoelectric ceramic cantilever was washed with water several times.

The experiment was performed by the BioScan cantilever system in a continuous flow. The settings for the measurement were planned with the flow shown in FIG.

A 10 mL syringe syringe pump (New Era Pump Systems, Inc, USA) was connected to the second port of a 6-way valve (Upchurch, USA), the liquid cell of the liquid cell BioScan cantilever system was connected to the third port, A loop of 부 volume was connected to the first and fourth ports, the sample was injected through the fifth port, and the sixth port was used for disposal. In all experiments, the speed of the syringe pump was set at 1 ml / h. The syringe was completely filled with PBS. When a probe is injected, the probe begins to move through the Teflon tube to the cell. It reached the cells in only 7 minutes.

Example  1. Sensor response to different concentrations of virus

Figure 4 shows the evolution of surface stress due to the injection of duck virus present at different concentrations in PBS solution. The dynamic properties of surface stress growth were the same in all experiments at different concentrations. Significant differences were obtained in the different concentration signals of 0, 10 ⁶ , 10 ⁷ and 10 ⁸ virus / ml (v / ml). Allantoic liquid was used as the zero probe. The supernatant fluid contained other proteins but caused slightly less receptor layer stress change than the viral solution.

Example  2. Reproducibility

Initially, the experiment was performed in a flow of injecting the viral solution at the same concentration of 10 ⁷ viral / ml (FIG. 5, blue line). The sensor and the reference cantilever were immersed in a 10% urea aqueous stream at a rate of 1 ml / h for 18 minutes. The urea solution was then injected to replace the probe in the loop. The first experiment was then repeated. The change in surface stress for the second experiment is shown in Fig. 5 (green line). Experimental results show that sensor can be regenerated and reused by washing.

Example  3. Resonance frequency  Measurement of movement

The piezoelectric ceramic cantilevers exhibited a high frequency non-bending resonance mode such as a longitudinal extension mode lacking a non-piezoelectric microcantilever such as a silicon-based micro cantilever as a result of the high piezoelectric effect of the piezoelectric layer (FIG. 6). The mechanical change of the receptor layer fixed on the piezoelectric ceramic cantilever dramatically shifted the resonance peak of the longitudinal extension mode according to the binding of the target molecule.

Virus detection experiments were performed as described in Example 1. The movement of the resonance frequency according to the virus adsorption is shown in Fig. 7 (green line).

Experimental results show that the resonance frequency shift due to influenza adsorption is clearly observed.

Claims (19)

A sensor probe having one end fixed; And a detector connected to the sensor probe, the sensor for influenza detection comprising:
Wherein a sialylglycopolymer is attached to the surface of the sensor probe by a covalent bond.
2. The sensor of claim 1, wherein the influenza virus specifically binds to a sialyl group of the sialylglycol polymer.
The sensor of claim 1, wherein said sensor is used to detect or quantify the presence of influenza in a liquid or gas.
The sensor according to claim 1, wherein a plurality of sialylglycolic polymer molecules are attached to the surface of the sensor probe, and the sialylglycolic polymer molecules are not crosslinked.
The method of claim 1, wherein the sialyl glycol polymer comprises a sialyl (? 2-3) galactosyl moiety or a sialyl oligosaccharide having a sialyl (? 2-6) galactosyl moiety sensor.
6. The method of claim 5, wherein the sialyloligosaccharide is selected from the group consisting of Neu5Acα2-3Galβ1-4Glcβ (3'SL), Neu5Acα2-6Galβ1-4Glcβ (6'SL), Neu5Acα2-6Galβ1-4GlcNAcβ (6'SLN), Neu5Gcα2-6Galβ1- (Neu5Gc) LN), Neu5Acα2-3Galβ1-4- (6-Su) GlcNAc (Su-Glu), Neu5Acα2-3Galβ1-4GlcNAc (3'SLN), Neu5Gcα2-3Galβ1-4GlcNAc (3 '^{3'SLN), Neu5Acα2-3Galβ1-3GlcNAc (SiaLe c)} , Neu5Acα2-3Galβ1-3- (6-Su) GlcNAc (Su-SiaLe c), (Neu5Acα2-3Galβ1-4) (Fucα1-3) GlcNAc (SiaLe x ), (Neu5Acα2-3Galβ1-4) (Fucα1-3) (6-Su) GlcNAc (Su-SiaLe ^x ), Neu5Acα2-6Galβ1-4GlcNAc and Neu5Acα2-6Galβ1-4- SLN). ≪ / RTI >
5. The method of claim 4, wherein each of the sialylglycolic polymer molecules is an independent polymer previously prepared by covalently linking a sialyloligosaccharide to a polymeric polymer before being attached to the surface of the sensor probe .
The sensor according to claim 7, wherein the polymer is an acrylic polymer.
2. The sensor of claim 1, wherein the sensor further comprises a reference probe used to obtain a reference signal.
The sensor according to claim 1, wherein the sensor probe comprises a single layer or a multi-layer structure.
11. The sensor probe of claim 10, wherein the sensor probe comprises: a support layer; A thiol, silane or silanetran linker having functional groups; And a layer of a sialyl glycol polymer covalently linked to the support layer through a functional group of the linker.
12. The sensor according to claim 11, wherein the support layer is a silicon support layer, and the linker is an aminosilane compound.
The sensor according to claim 1, wherein a change in deflection occurs in the sensor probe as the influenza virus binds to the sialyl group.
14. The apparatus of claim 13, wherein the detector comprises a light source for irradiating light to the sensor probe and a sensor for collecting light reflected from the probe, and comparing the positional change of the irradiated light beam with the reflected light beam, Of the sensor.
The sensor according to claim 1, wherein a change occurs in the resonance frequency of the sensor probe as the influenza virus binds to the sialyl group.
16. The sensor of claim 15, wherein the detector measures a piezoresistive or piezoelectric effect of the sensor probe.
The sensor according to claim 1, wherein the sensor probe is a micro cantilever.
18. The sensor of claim 17, wherein the sensor probe is a static cantilever or a dynamic cantilever.
A method for detecting influenza in a liquid or a gas using the sensor according to any one of claims 1 to 18 to provide information on the presence or absence of influenza.

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