KR101721796B1 - Method for absorbing pathogenic microorganism using micro-bead coated with graphene oxide - Google Patents

Abstract

The present invention relates to a method for adsorbing pathogenic microorganisms by using micro-beads coated with graphene oxide. The method comprises the steps of: 1) washing micro-beads; 2) treating the washed micro-beads by a 3-aminopropyltriethoxysilane (APTES) solution; 3) producing micro-beads coated with graphene oxide by reacting the micro-beads treated by the APTES solution with graphene oxide; and 4) making a sample including pathogenic microorganisms come in contact with the micro-beads coated with graphene oxide. According to the present invention, pathogenic microorganisms existing in an environmental sample can be detected by being rapidly adsorbed and concentrated. Pathogenic microorganisms included in a food or a beverage can be easily filtered and refined.

Description

TECHNICAL FIELD [0001] The present invention relates to a method for adsorbing pathogenic microorganisms using microbeads coated with oxidized graphene,

The present invention relates to a method of adsorbing pathogenic microorganisms using micro grains coated with oxidized graphene, and more particularly, to a method of adsorbing pathogenic microorganisms on a micro bead coated with glass grains in order to detect pathogenic microorganisms from environmental samples .

Recently, a variety of biological detection methods have been developed to increase sensitivity and specificity. Biological signal amplification techniques using enzymes or DNA molecules and chemical labeling techniques using liposomes or dendritic linkages have been used to increase the signal to noise ratio. For decades, nucleic acid-based biological sensing technologies have centered around tools that can amplify nucleic acids, such as polymerase chain reaction and nuclear acid sequence based amplification. Techniques for amplifying these signals and samples have already been commercialized and are widely used in the medical field to diagnose various diseases. However, when diagnosing disease early, the amount of target substance present in the sample solution is absolutely insufficient, so the problem of increasing the sensitivity is still rising.

In particular, in the case of environmental samples, at least 1 liter volume of solution is required to detect pathogens. Methods related to concentration such as centrifugation, flocculation, filtration, adsorption, and immunomagnetic separation with antibodies have increased the measurement of pathogenic bacteria in samples. After concentration of the pathogenic bacteria, they were sufficiently dissolved and amplified by polymerase chain reaction or isothermal nucleic acid amplification reaction. However, these methods have a problem in that they can not be directly applied to environmental samples because of the interaction between the pathogen and the surface. Therefore, in order to rapidly adsorb pathogens present in aqueous solution, a substance with a new surface is needed.

Graphene is a carbon allotrope with two-dimensional atomic-scalelattices, and is seen as the most fundamental component in the fabrication of several nanostructures. Particularly, graphene oxide is characterized in that it has a biologically structured surface due to a mixture of a hydrophilic moiety due to a carboxyl group and a residue having a hydrophobic action. Oxidative graphene is a quencher that is used in biological sensing technology based on the phenomenon of energy transfer between two specific color materials (Forster resonance energy transfer), and as a single-stranded DNA-labeled adsorption template Is also being used. The negative charge of the graphene oxide in the wide pH range is applied to the removal of heavy metal ions present in water. However, as far as the details of the interaction between bacteria and oxidative graphene are yet to be elucidated, the technology of adsorbing and concentrating bacteria has not yet been applied.

Korean Patent Publication No. 10-2015-0044280 (published on April 24, 2014)

An object of the present invention is to provide a method for adsorbing pathogenic microorganisms using micro grains coated with oxidized graphene. In order to detect pathogenic microorganisms from environmental samples, graphene oxide is coated on glass micro beads and adsorbed thereon a pathogenic microorganism .

In order to achieve the above object, the present invention provides a method for manufacturing a microbead, comprising: 1) cleaning microbeads; 2) treating the washed microbeads with 3-aminopropyltriethoxysilane (APTES) solution; 3) reacting the microbead treated with the APTES solution with the oxidized graphene to prepare a microbead coated with the oxidized graphene; And 4) contacting the sample containing the pathogenic microorganism with the gravid microparticles coated with the graphene oxide. The present invention also provides a method for adsorbing pathogenic microorganisms using the gravid coated microbeads.

The present invention also provides a pathogenic microorganism adsorption kit comprising micro grains coated with oxidized graphene.

The present invention relates to a method for adsorbing pathogenic microorganisms using micro grains coated with oxidized graphene. In order to detect pathogenic microorganisms from environmental samples, graphene oxide is coated on glass micro beads, and then pathogenic microorganisms are adsorbed thereon, And a method for extracting nucleic acid by dissolving it by bead pulverization method. According to the present invention, pathogenic microorganisms present in environmental samples can be quickly adsorbed and concentrated and detected, and pathogenic microorganisms contained in foods and beverages can be easily filtered and purified.

FIG. 1 is a graph showing the presence or absence of graphene oxide coated on a microbead using Raman spectroscopy.
FIG. 2 shows the result of observing the surface of a gravitationally coated microbead using an atomic force microscope.
FIG. 3 shows the results of a comparison of the gram-negative bacterial adsorption rate with the flow rate.
FIG. 4 shows the adsorption rate of gram negative bacteria according to the amount of oxidized graphene-coated microbeads.
FIG. 5 shows the results of confirming the gram-negative bacterial adsorption rate at various concentrations.
FIG. 6 shows the results of comparing the adsorption rate of Gram-positive bacteria with pH.
Figure 7 shows the results of Gram-positive bacteria adsorption rate versus flow rate.
Fig. 8 shows the results of comparison of virus adsorption rate according to pH.
9 shows the results of comparison of the virus adsorption rate according to the flow rate.

The present inventors have evaluated the ability of bacteria to adsorb on the surface where oxidized graphene is present. More specifically, graphene oxide was coated on a glass bead of microbeads and confirmed by atomic force microscopy (Raman spectroscopy) and Raman spectroscopy. The ability of bacteria to adsorb to oxidized graphene was determined by flowing various concentrations of bacterial samples under various fluid flow conditions. Bacteria adsorbed to the graphene oxide were visually observed by bacterial fluorescent staining. The extracted nucleic acid was quantitatively determined by polymerase chain reaction and the present invention was completed.

The present invention provides: 1) cleaning microbeads; 2) treating the washed microbeads with 3-aminopropyltriethoxysilane (APTES) solution; 3) reacting the microbead treated with the APTES solution with the oxidized graphene to prepare a microbead coated with the oxidized graphene; And 4) contacting the sample containing the pathogenic microorganism with the gravid microparticles coated with the graphene oxide. The present invention also provides a method for adsorbing pathogenic microorganisms using the gravid coated microbeads.

Preferably, the APTES solution of step 2) may be treated with 10 to 100 mM of APTES solution dissolved in ethanol for 1 to 2 hours at room temperature, but is not limited thereto.

Preferably, the reaction with the graphene oxide in the step 3) may be performed by reacting the graphene oxide dispersed in ethanol at a concentration of 0.1 to 1 mg / ml at room temperature for 0.5 to 5 hours, but is not limited thereto.

Preferably, in step 4), the sample containing the pathogenic microorganism may be contacted with the graphene-coated microbead at a flow rate of 1 to 20 ml / min. However, the present invention is not limited thereto.

Preferably, the sample containing the pathogenic microorganism may include, but is not limited to, 10 ³ to 10 ⁷ cfu / ml pathogenic microorganisms.

Preferably, the pathogenic microorganism may be a bacterium or a virus, more preferably the bacterium may be Salmonella typhimurium or Staphylococcus aureus, the virus may be Adeno But are not limited to, viruses (Adenoviruses).

The present invention also provides a pathogenic microorganism adsorption kit comprising micro grains coated with oxidized graphene.

Preferably, the pathogenic microorganism may be a bacterium or a virus, more preferably the bacterium may be Salmonella typhimurium or Staphylococcus aureus, the virus may be Adeno But are not limited to, viruses (Adenoviruses).

Also, the size of the microbeads used in the present invention may be 30 to 300 탆, preferably 100 탆, but the numerical ranges are shown for illustrative purposes only, and the present invention is not limited thereto .

Hereinafter, the present invention will be described in detail with reference to embodiments which do not limit the present invention. It should be understood that the following embodiments of the present invention are only for embodying the present invention and do not limit or limit the scope of the present invention. It is therefore to be understood that within the scope of the appended claims, the invention may be practiced otherwise than as specifically described herein.

< Example  1> Micro glass material On the bead  Oxidation Graffin  How to coat

Prior to the coating of the graphene oxide, beads (hereinafter referred to as microbeads) having a diameter of 100 μm of glass were immersed in a piranha solution mixed with sulfuric acid and hydrogen peroxide in a volume ratio of 3: 1 for 30 minutes, Followed by rinsing and drying at 70 ° C.

The grained microbeads were coated with oxidized graphene using the following two methods. The first method for coating the microgrid with the oxidized graphene was that the microbead was treated at room temperature for 1 hour in a solution of 10 mM 3-aminopropyltriethoxysilane (APTES) prepared on an acetone basis, The solution excluding the microbeads was removed. The grafted oxide grains dispersed in distilled water at a concentration of 1 mg / ml were treated at 80 ° C for 12 hours in the microbeads treated with APTES.

In the second method, 10 mM APTES solution prepared on ethanol - based microbeads was treated at room temperature for 1 hour, and then the solution excluding microbeads was removed. The APTES-treated microbid was again reacted with graphene oxide, which was dispersed in ethanol at a concentration of 1 mg / ml, at room temperature for 2 hours. The microwave beads coated with the oxidized graphene produced by each method were ultrasonicated for 10 minutes and then dried at 80 ° C.

The presence of graphene oxide coated on the microbeads was confirmed using Raman spectroscopy (Fig. 1). In the ^first position and 1600 cm ^{-1 -} As a result, the microbeads are not processed anything in or've found only typical peak representing the characteristics of the glass microbeads treated graphene oxide appears below 1200 cm ^-1 1330 cm It was confirmed that peaks common to all the graphite materials appear. Therefore, it was confirmed that oxide graphene was successfully coated on the microbeads. As a first method, graphene oxide graphene microbeads showed weak peaks at 1330 cm ^-1 and 1600 cm ^-1 , which are characteristic of graphene, compared with graphene graphene microbeads coated by the second method, A method of manufacturing graphene graphene microbeads to be used for adsorbing the grains is to use the second method.

Also, as shown in FIG. 2, when observing the surface of the graphene-coated microbead using an atomic force microscope, the surface of the graphene graphene microbead prepared by the second method was measured to be about 5.5 nm , And the surface of the graphene microbeads prepared by the first method was measured to be about 1.7 nm. As a result, it was confirmed that the second method increases the surface roughness of the graphene microbeads.

< Example  2> Oxidation Grapina  Coated micro Bead  Bacteria and virus adsorption method used

The bacteria were cultured in a 3% Tryptic soy broth (TSB) medium at 37 ° C in the presence of Gram-negative bacteria Salmonella typhimurium and Gram-positive bacteria Staphylococcus aureus. To prepare environmental samples containing gram negative bacteria, distilled water was prepared at a concentration of 10 ⁴ cfu / ml. First, in order to confirm the amount of bacteria adsorbed by the flow rate, a pneumatic pump was used to flow a gas containing graphene-coated microbeads at various rates (1, 5, 10, 20 ml / min) . The quantitative determination method of bacteria adsorbed on oxidized graphene-coated microbeads is as follows. Bacteria adsorbed on oxidized graphene - coated microbeads were extracted by bead collision method, and the extracted nucleic acid was quantitatively determined by polymerase chain reaction. As a result, as shown in FIG. 3, about 60% of the bacteria were adsorbed to the glass microbeads at a rate of 1 ml / min, and the bacteria adsorbed on the glass microbeads decreased by about 20% as the flow rate was increased. In the case of graphene oxide microbeads coated by the first method, the adsorption rate of bacteria was about 40% at a rate of 1 ml / min than that of free microbeads. The graphene oxide microbeads coated by the second method showed a bacterial adsorption rate of about 90% regardless of flow rate.

To determine the amount of adsorbed by bacterial concentration, various amounts of oxidized graphene microbeads (0.01, 0.05, 0.1 g) and various bacterial concentrations (10 ⁷ , 10 ⁶ , 10 ⁵ , 10 ⁴ , 10 ³ cfu / ml) was pumped at 20 ml / min using a pneumatic pump. First, as shown in FIG. 4, the optimal amount of graphene oxide-coated microbeads was found to be 80% or more in the amount of graphene-coated microbeads coated with 0.05 g or more. Based on these results, the amount of graphene oxide coated microbeads was determined to be 0.1 g in order to maintain the optimum adsorption rate. In addition, LIVE / DEAD® ^TM in order to determine the bacteria adsorbed to the oxidized graphene-coated microbeads visually As a result of checking with the kit, it was visually confirmed that bacteria stained with green fluorescence showing survival were successfully adsorbed on oxidized graphene-coated microbeads, as shown in Fig.

As shown in FIG. 5, the adsorption rate at various bacterial concentrations was about 80 to 100% even in the concentration range of 10 ⁷ to 10 ³ cfu / ml, considering the presence of a low concentration of bacteria. Of the bacteria.

In the case of Gram-positive bacteria, the amount of adsorbed on oxidized graphene-coated microbeads at various pHs (3, 5, 7, 9) was first ascertained. Gram-positive bacteria were incubated at a concentration of 10 ⁴ cfu / ml in each pH solution (pH 3; 10 mM Glycine, pH 5; 10 mM Acetate, pH 7; 10 mM Tris-HCl, pH 9; 10 mM Tris- Ready. The prepared bacteria were passed through a gravid coated microbead at a flow rate of 1 ml / min. The nucleic acid was extracted using a bead collision method and the extracted nucleic acid was quantitatively determined using a polymerase chain reaction. As a result, as shown in Fig. 6, the adsorption rate was about 95% or more at all pH values. To determine the amount of bacteria adsorbed by the flow rate, the cells were flowed at various rates (1, 5, 10, and 20 ml / min) using a pneumatic pump to tubes containing oxidized graphene coated microbeads. As a result, the bacteria adsorption rate was about 90% or more regardless of the flow rate as shown in FIG.

In the case of adenovirus, the amount of adsorbed on graphene-coated microbeads for various pHs (3, 5, 7, 9) was first determined. As a result, as shown in FIG. 8, the adsorption rate was about 75% or more at the acidic condition. Likewise, in order to confirm the adsorption amount of virus according to the flow rate, it was flowed at various rates (1, 5, 10, 20 ml / min) under acidic conditions. As a result, as shown in FIG. 9, about 80% of the virus was adsorbed to the glass microbeads at a rate of 1 ml / min, and the virus adsorbed on the glass microbeads decreased by about 50% as the flow rate was increased.

Claims (12)

1) cleaning microbeads;
2) treating the washed microbeads with 3-aminopropyltriethoxysilane (APTES) solution;
3) reacting the microbead treated with the APTES solution with the oxidized graphene to prepare a microbead coated with the oxidized graphene; And
4) contacting the sample containing the pathogenic microorganism with the microbead coated with the oxidized graphene, wherein the microbead is coated with the oxidized graphene. [2] The method according to claim 1, wherein the APTES solution of step 2) is treated with 10 to 100 mM of APTES solution dissolved in ethanol at room temperature for 1 to 2 hours. Method of adsorbing microorganisms. The method according to claim 1, wherein the reaction with the graphene oxide in the step 3) is carried out at room temperature for 0.5 to 5 hours by reacting the graphene oxide dispersed in ethanol at a concentration of 0.1 to 1 mg / (Method of Adsorbing Pathogenic Microorganisms Using the Coated Microbeads). [3] The method of claim 1, wherein the step 4) comprises contacting the sample containing the pathogenic microorganism to the micro grabbing coated with the oxidized graphene at a flow rate of 1 to 20 ml / min. Method of adsorbing pathogenic microorganisms using beads. The method of claim 1, wherein the sample containing the pathogenic microorganism comprises 10 ³ to 10 ⁷ cfu / ml of a pathogenic microorganism. The method for adsorbing a pathogenic microorganism according to any one of claims 1 to 5, wherein the pathogenic microorganism is bacteria or virus. [Claim 7] The method according to claim 6, wherein the bacteria is Salmonella typhimurium or Staphylococcus aureus. [Claim 7] The method according to claim 6, wherein the virus is adenovirus. A pathogenic microorganism adsorption kit comprising microparticles coated with oxidized graphene. The pathogenic microorganism adsorption kit according to claim 9, wherein the pathogenic microorganism is bacteria or virus. 11. The pathogenic microorganism adsorption kit according to claim 10, wherein the bacterium is Salmonella typhimurium or Staphylococcus aureus. The pathogenic microorganism adsorption kit according to claim 10, wherein the virus is adenovirus.

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