KR101523077B1 - methods of sensing for biomaterials using commercial pH meter - Google Patents

Abstract

The present invention relates to a method and apparatus for detecting a bio-material, and more particularly, to a method for rapidly detecting a bio-material using a pH meter or a commercial pH measuring apparatus such as a pH indicator.
The present invention provides a method for changing the pH of a standard reagent containing an ester compound using a biomaterial-magnetic nanoparticle-acetylcholinesterase complex.

Description

[0001] The present invention relates to a method for detecting a biomaterial using a pH meter,

The present invention relates to a biomaterial detection method, and more particularly, to a biomaterial detection method using a pH meter such as a pH meter or a pH indicator.

Biosensors for effectively detecting various bio-substances directly or indirectly related to life phenomena such as proteins, enzymes, nucleic acids, cells, pathogens, etc. have been developed. Immunoassay using an antigen antibody using a radioisotope or a fluorescent substance as a label, a surface-plasmon resonance biosensor, a total internal reflection ellipsometry biosensor as a label-free biosensor ), And optical waveguide biosensors (optical waveguide biosensors). Korean Patent Laid-Open Publication No. 2009-0061270 discloses a method for detecting a biomolecule by using a nanocomposite and a resonant reflection light filter.

However, conventional methods for detecting biomaterials have a disadvantage that expensive equipment is required, and there is a continuing need for a method for measuring biomaterials using a more economical measuring device such as a commercial pH meter.

A problem to be solved by the present invention is to provide a method for detecting a biomaterial using pH.

In order to solve the above problems, a detection method according to the present invention is characterized in that a magnetic nanoparticle and an acetylcholinesterase are bound to a biomaterial to form a complex, and the pH of the standard sample containing the ester compound is changed And analyzing it.

The present invention relates to a method for producing a biosensor, comprising: injecting magnetic nanoparticles into a test sample containing a biomaterial to bind magnetic nanoparticles to the biomaterial and separating the magnetic nanoparticles using a magnet;

Forming a biomaterial-magnetic nanoparticle-acetylcholinesterase complex by binding acetylcholinesterase to the separated biomaterial together with magnetic nanoparticles;

Introducing the biomaterial-magnetic nanoparticle-acetylcholinesterase complex into a standard reagent containing an ester compound to change the pH; And

and quantifying using a pH change.

Although it is not theoretically limited, when the content of the biomaterial contained in the test sample increases, the content of the combined substance of the biomaterial and the magnetic nanoparticle separated from the sample increases, and the biomaterial is further reacted with acetylcholinesterase The content of biomaterial-magnetic nanoparticle-acetylcholinesterase complex also increases. The increase in the content of the complex increases the amount of change in the pH due to the hydrolysis of the ester compound in the acetylcholine-containing standard sample-the degree of change and / or rate of change-thereby increasing the qualities of the biomaterials contained in the sample Quantitative analysis becomes possible.

In the present invention, the biomaterial is a substance directly or indirectly related to a life phenomenon, such as a protein, a nucleic acid, a cell, a bacterium, or a pathogen.

In the practice of the present invention, the protein may be selected from the group consisting of Troponin I, heart disease markers Troponin C, Troponin T, cancer markers Alpha fero-Protein (AFP), prostate specific protein (PSA), carcinoembryonic antigen And Interleukin protein, which is furthermore a marker of immune response to infection.

In another embodiment of the present invention, the nucleic acid includes DNA and RNA, and includes food poisoning bacteria, Mycobacterium tuberculosis, meningitis bacterium, and pneumococcal DNA.

In another embodiment of the present invention, the cell is one of the body's defense mechanisms, such as a white blood cell or cancerous tumor cell that causes cancer.

In another embodiment of the present invention, the pathogen is selected from the group consisting of pathogenic Escherichia coli, Listeria monocytogenes, Vibrio bacteria, Norovirus, Salmonella, Staphylococcus aureus, Enteritis Vibrio, enteric haemorrhagic Escherichia coli O157, Bacteria, Welsh onion, Botryuris bacteria, etc.

In one embodiment of the present invention, the biomaterial is a protein such as troponin I. Troponin I is known as a protein bound to the myocardium. When the myocardium is injured, troponin I is removed from the myocardium, causing the blood vessels to float and the concentration of troponin I in the blood is several tens pg / mL. / mL < / RTI > Thus, the increase in troponin I concentration in the blood vessels can be detected to know the prognosis of heart disease.

In another embodiment of the present invention, Salmonella is capable of causing the food poisoning.

In the present invention, the magnetic nanoparticles are understood as magnetic fine particles which react with magnetic force. It is understood that the magnetic nanoparticles have a size of 1 to 10000 nanometers, preferably 50 to 1000 nanometers, and more preferably 100 to 500 nanometers. In the practice of the present invention, the magnetic nanoparticles can be manufactured and used by a known method, and commercially available. For example, γ-Fe ₂ O ₃ can be used as the magnetic nanoparticles have.

In the present invention, the magnetic nanoparticles are magnetic nanoparticles having a functional group capable of binding to a bio material. The functional groups may vary depending on the kind of the biomaterial. For example, when the target biomolecule is a nucleic acid such as DNA or RNA, a nucleic acid having a base sequence capable of complementary binding thereto may be used. In addition, for example, when the biomaterial is an antigen or protein or cell in which an immune response is produced, it may be an antibody corresponding thereto.

In the practice of the present invention, the magnetic nanoparticles may be formed with functional groups capable of binding to troponin I on the surface or inside thereof so as to be able to bind to troponin I. Functional groups capable of binding to troponin I are antibodies capable of binding to troponin I, and various antibodies capable of binding to troponin I are known. A method of forming a troponin I binding antibodies on the surface of the nanoparticles, Jun is incorporated by reference in full document in the present invention ha etc., Biomaterials 32 (2011) 1177 ~ 1184 of "A multicomponent recognition and separation system established via fluorescent, magnetic, dualencoded multifunctional bioprobes ".

In one embodiment of the present invention, the antibodies can use known antibodies capable of binding to troponin I. In this case, the binding between troponin I and the magnetic nanoparticles can be made by antigen-antibody binding, and one troponin I can have a plurality of magnetic particles, or a plurality of tropinins can be bound to one magnetic particle.

In the present invention, the acetylcholinesterase may be an antibody or a complementary base sequence capable of binding to a functional group capable of binding with a biomaterial, for example, a biomaterial.

The functional groups capable of binding to the biomaterial can be bound through a linking material known in acetylcholinesterase. The linking material may be a substance that can be biochemically bound to the biomaterial by, for example, an antigen-antibody reaction, and examples thereof include ethoxysilane-based hydrocarbons having 3 to 9 carbon atoms or methanes having 2 to 9 carbon atoms Ethoxy silane based hydrocarbons.

In the practice of the invention, the acetylcholine esterase is immobilized on a functional group capable of binding to troponin I, for example, an antibody capable of binding to troponin I to acetylcholinesterase to form troponin I- Lt; / RTI > Methods for forming an antibody-immobilized acetylcholinesterase are described in Nature Chemistry, 2011, 3, 697-703, which is incorporated herein by reference in its entirety. The binding of troponin I to the invertase may be via antigen-antibody binding, and a plurality of acetylcholinesterases may be conjugated to a single troponin I. In the practice of the present invention, a known linking substance such as 1,4-phenylene diisothiocyanate (PDITC) can be used for binding between the acetylcholinesterase and the tropinin I antibody.

In the present invention, it is understood that the complex is a complex in which magnetic nanoparticles and acetylcholinesterase are bound to a biomaterial, and which maintains the activity of acetylcholinesterase.

The term "maintaining the activity of the acetylcholinesterase" means that the activity is at least 50%, preferably at least 70%, and most preferably at least 80%, as compared to the activity of the unbound acetylcholinesterase. do.

In the present invention, the ester compound contained in the standard sample means a compound that can be hydrolyzed by acetylcholinesterase. Reactants capable of causing pH change by hydrolysis include acetylcholine, triacetin, ethyl acetate and the like. Preferably, acetylcholine is most reactive with acetylcholinesterase, so triacetin is particularly preferred in that three acetic acid materials are produced upon one-time degradation.

In the present invention, the decomposition of the ester compound may be carried out at an elevated temperature, preferably within a range of from room temperature to 65 ° C, within a range in which denaturation of the acetylcholinesterase does not occur. The activity difference of acetylcholinesterase according to the temperature is not high and it is preferable to be performed at room temperature.

In the present invention, the pH change due to the hydrolysis of acetylcholine can be measured using a pH meter or pH paper. In the case of a pH meter, the pH change of the solution can be measured in real time. In the case of using pH paper, pH can be confirmed.

The present invention provides a novel substance that binds a biomaterial, magnetic nanoparticles, and acetylcholinesterase, and changes the pH of a standard sample containing an ester compound.

According to the present invention, there is provided a method for quantitatively measuring biomaterials using a commercial pH measuring device. The measuring method according to the present invention can easily measure at room temperature since the range of variation according to the temperature is not large.

Figure 1 is a flow chart of a commercial pH meter based detection method. (b) binding of acetylcholinesterase; (c) addition of acetylcholine to a standard sample and hydrolysis; and (d) measurement of pH change.
FIG. 2 is a graph showing the results of a decomposition reaction test (black: acetylcholine, red: triacetin, blue: ethyl acetate
FIG. 3 shows the result of decomposition reaction test by temperature
FIG. 4 shows the results of the decomposition reaction test with and without the enzyme under basic conditions
FIG. 5 shows the results of (a) decomposition reaction test by pH (b) decomposition reaction test by presence or absence of enzyme at low pH
FIG. 6 shows the results of Troponin I detection using acetylcholinesterase. (A) pH change (black: 0 ng / mL, purple: 0.01 ng / mL, orange: 0.1 ng / 1 ng / mL, blue: 10 ng / mL, red: 100 ng / mL) (b)
FIG. 7 shows the results of the detection of food poisoning bacteria using acetylcholinesterase
(a) pH change in concentration per reaction time (black: 0 cfu / mL, Orange: 10 ² cfu / mL, Green: 10 ⁴ cfu / mL, blue: 10 ⁶ cfu / mL, red: 10 ⁸ cfu / mL ) (b) pH value by concentration at 10 minutes after the reaction

Hereinafter, the present invention will be described in detail with reference to examples. The following examples illustrate the present invention in detail, but are for the purpose of illustrating the present invention and are intended to limit the scope of the present invention, and it is to be noted that the scope of the present invention is defined by the claims.

Reagents and equipment

Iron (III) chloride, FeCl ₃ , Urea, sodium citrate, polyacrylamide, [3-Aminopropyl] triethoxysilane , APTES, glutaraldehyde, bovine serum albumin (BSA), acetylcholinesterase, 1,4-phenylene diisothiocyanate, PDITC). 1-Butanol, dimethylformamide (DMF), acetylcholine, human serum were purchased from Sigma-Aldrich. Antibodies to Troponin I were purchased from HyTest.

Antibodies against Salmonella were purchased from ABCam.

The Amicon-100K and Amicon-300K filters used to obtain the purified antibody-enzyme conjugate were purchased from Amicon.

Commercial pH meter, portable pH meter and pH indicator for measuring pH change were purchased from inoLab, HANNA instruments and Merck respectively.

Deionized distilled water was obtained by using a commercial ultrapure water production system (Human Science, Korea) and was used to synthesize magnetic nanoparticle clusters and to make phosphate buffer solutions.

The neodymium magnet used for the separation of non-bound Troponin I and non-bound acetylcholinesterase was purchased from Seoul Magnetics Co. The intensity of the magnet measured by Gauss meter was 30 milli tesla.

Example 1. Detection of Troponin I using a pH meter

Stage 1. Synthesis of Fe ₃ O ₄ magnetic nanoparticle clusters (hereinafter referred to as magnetic nanoparticles) and immobilization of antibodies

Fe ₃ O ₄ magnetic nanoparticles were synthesized using a one-pot solvothermal method. First, 4 millimoles of iron chloride, 12 millimoles of element and 8 millimoles of sodium citrate are dissolved in 80 milliliters of water. Thereafter, 0.6 g of polyacrylamide is added while stirring continuously using a magnet rod. The reaction solution thus prepared is transferred to a 100 ml volume Teflon autoclave vessel and maintained at a temperature of 200 ° C for 12 hours to cause a synthesis reaction. After cooling the solution to room temperature, the synthesized particles are collected using a magnet, and the remaining reactants are discarded and washed several times with ethanol and water to finally obtain Fe ₃ O ₄ magnetic nanoparticles. The size of the obtained nanoparticles is about 200 nanometers.

The synthesized magnetic nanoparticles react with APTES (3- (aminopropyl) triethoxysilane) and glutaraldehyde, respectively, so that amine groups on the surface are activated and can bind to the antibody. Thereafter, the antibody is immobilized on the particle surface by mixing the Troponin-I antibody and the magnetic nanoparticles, and then treated with BSA (Bovine Serum Albumin) and Tween-20 to prevent non-specific binding .

Step 2. Acetylcholinesterase and antibody fixation

The fixation of the acetylcholinesterase and the antibody is performed using a linking substance called 1,4-phenylene diisothiocyanate (PDITC). 0.2 micromolar antibody is dispersed in 100 microliters of buffer solution and then added to 1 milliliter of dimethylformamide (DMF) containing 20 micrograms of PDITC dissolved therein. The mixed solution thus obtained is stirred at room temperature for 2 hours while being shielded from light, and then 5 milliliters of water and 5 milliliters of 1-butanol are added to the mixed solution. When centrifugation is performed, the mixed solution is divided into the upper organic solution and the lower aqueous solution, and then the upper organic solution containing unreacted PDITC is removed. Subsequently, 3 milliliters of 1-butanol is added and centrifuged, and furthermore, the organic solution layer of the upper layer is removed several times. The PDITC-antibody conjugate can be purified from an aqueous solution that has undergone the above procedure using an Amicon-100K filter. The purified conjugate was again dispersed in a buffer solution, added with 10 milligrams of acetylcholinesterase, and reacted at room temperature for 48 hours. Subsequently, the PDITC-antibody not bound to the enzyme is removed using an Amicon-300K filter to obtain a purified acetylcholinesterase-antibody conjugate.

Step 3. Troponin I - Magnetic nanoparticle complex formation

Add 20 micrograms of magnetic nanoparticles to human serum containing Troponin I. After stirring at room temperature for 1 hour, the Troponin I-magnetic nanoparticle complex is separated using a permanent magnet, and the remaining material is removed. The separated composite is redispersed in the buffer solution, and if the unremoved material remains, the separation and redispersion of the composite using the permanent magnet is repeated.

Step 4. Combination of Troponin I-Magnetic Nanoparticle Complex and Antibody-Fixed Acetylcholinesterase

10 μl of antibody-immobilized acetylcholinesterase was added to the Troponin I-Magnetic Nanoparticle Complex solution obtained in Step 3, and the mixture was stirred at room temperature for about 1 hour. Then, the complex of Troponin I-magnetic nanoparticle-acetylcholinesterase is separated using a permanent magnet and then redispersed in a buffer solution. The detection of troponin I using the complex is the same as in step 5.

Step 5. Select the best reactants

Acetylcholinesterase can hydrolyze various ester substances. Based on these properties, a test was conducted to find the reactants that could cause the most pH changes. Acetylcholine, triacetin, and ethyl acetate were selected as candidates. Acetylcholine is the most reactive substance with acetylcholinesterase, and triacetin has the advantage of producing three acetic acid substances once decomposed. As a result of the test, the pH change by acetylcholine was the largest among the three substances (FIG. 2).

Step 6. Test of reaction temperature pH change in detection of troponin

Acetylcholinesterase increased the enzyme reactivity with temperature and measured the pH change by acetylcholine degradation by reaction temperature to find the optimum reaction temperature. As shown in Fig. 3, it was confirmed that the difference in reactivity between room temperature (25 ° C) and 55 ° C before the denaturation occurred was only about 10%. This is because acetylcholinesterase already has excellent enzyme reactivity capable of decomposing about 100,000 molecules per minute and decomposes sufficiently many molecules even at room temperature. Therefore, the optimum reaction temperature was set at room temperature, which can be reacted without any additional equipment.

Step 7. Optimum pH condition setting

Acetylcholinesterase has the highest reactivity at pH above neutrality, but in this case, there is a problem that acetylcholine is also hydrolyzed by hydroxyl ion. The hydrolysis reaction by the hydroxyl ion acts as a noise for the hydrolysis reaction by the enzyme, making accurate measurement difficult. Therefore, an experiment was conducted to find a pH condition that can reduce nonspecific hydrolysis reaction by hydroxyl ion while maintaining enzyme activity. As shown in FIG. 4, it was confirmed that (a) the non-specific hydrolysis reaction by the hydroxyl ion was inhibited at a pH of 6 and the enzyme activity was maintained to some extent, and the detection was proceeded by setting the pH to 6 . (Fig. 4) (b)

Step 8. Hydrolysis and pH measurement method using isolated Troponin I-magnetic nanoparticle-acetylcholinesterase complex

The complex of Troponin I-magnetic nanoparticle-acetylcholinesterase obtained in step 4 is added to a 1 milliliter solution of 25 millimoles of acetylcholine. The acetylcholinesterase in the complex promotes the production of acetic acid as a catalyst for the hydrolysis reaction of acetylcholine. The activity of acetylcholinesterase increases with increasing temperature until denaturation at 65 ° C and the reaction temperature can be elevated to promote the hydrolysis reaction. The pH change due to the hydrolysis of acetylcholine can be measured using a pH meter or pH paper. In the case of a pH meter, the pH change of the solution can be measured in real time. In the case of using pH paper, . (Fig. 6)

Example 2. Detection of Salmonella using pH meter

Stage 1. Magnetic nanoparticles and antibody fixation

Salmonella antibodies were used to synthesize Fe ₃ O ₄ magnetic nanoparticle clusters (hereinafter referred to as magnetic nanoparticles) as in step 1 of Example 1, and the antibodies were immobilized.

Step 2. Immobilization of acetylcholine esterase and Salmonella antibodies

Was performed in the same manner as in step 2 of Example 1, except that Salmonella antibody was used.

Step 3. Salmonella-Magnetic Nanoparticle Complex Formation and Isolation

Add 20 micrograms of magnetic nanoparticles to 10 mL of milk containing Salmonella. After stirring at room temperature for 1 hour, the salmonella-magnetic nanoparticle complex is separated using a permanent magnet, and the remaining material is removed. The separated composite is redispersed in the buffer solution, and if the unremoved material remains, the separation and redispersion of the composite using the permanent magnet is repeated.

Step 4. Salmonella - Reaction of Magnetic Nanoparticle Complex with Antibody-Fixed Acetylcholinesterase

To the salmonella-magnetic nanoparticle complex solution obtained in Step 3, 10 microliters of the antibody-immobilized acetylcholinesterase were added and stirred at room temperature for about 1 hour. Then, the salmonella-magnetic nanoparticle-acetylcholinesterase complex is separated using a permanent magnet and redispersed in a buffer solution. The process of detecting Salmonella using the complex is the same as step 5.

Step 5. Quantification by hydrolysis and pH measurement using salmonella-magnetic nanoparticle-acetylcholinesterase complex

The complex of salmonella-magnetic nanoparticle-acetylcholinesterase obtained in step 4 is added to a solution of 1 milliliter of 25 millimoles of acetylcholine. The acetylcholinesterase in the complex promotes the production of acetic acid as a catalyst for the hydrolysis reaction of acetylcholine. The activity of acetylcholinesterase increases with increasing temperature until denaturation at 65 ° C and the reaction temperature can be elevated to promote the hydrolysis reaction. The pH change due to the hydrolysis of acetylcholine can be measured using a pH meter or pH paper. In the case of a pH meter, the pH change of the solution can be measured in real time. In the case of using pH paper, . (Fig. 7)

Claims (17)

The method comprising: forming a complex by binding a bio-material, magnetic nanoparticle, and acetylcholinesterase; and changing the pH of a standard sample containing the ester compound as the complex,
The bio-
A protein selected from the group consisting of Troponin I, Troponin C, Troponin T, Alpha fero-Protein (AFP), Prostate Specific Protein (PSA), and Carcinoembryonic antigen (CEA)
One or more selected from the group consisting of pathogenic Escherichia coli, Listeria, Vibrio, Norovirus, Salmonella, Staphylococcus aureus, Vibrio parahaemolyticus, Enteric haemorrhagic Escherichia coli O157, Campylobacter bacteria, Bacillus cerauss, Lt; RTI ID = 0.0 >
Method of detecting biomaterial. The method of claim 1, wherein the complex hydrolyzes the ester compound. The method according to claim 1, wherein the ester compound comprises at least one of acetylcholine, triacetin, and ethyl acetate. The method according to claim 1, wherein the complex is formed by binding the magnetic nanoparticles and the acetylcholinesterase to the biomaterial. 5. The method according to claim 4, wherein the magnetic nanoparticles and the acetylcholinesterase are formed with functional groups capable of binding to the biomaterial. [Claim 5] The method according to claim 5, wherein the functional group is an antibody when the biomaterial is an antigen, and a complementary base chain when the biomaterial is DNA or RNA. 6. The method of claim 5, wherein the functional groups are bonded through a linking material. delete delete delete A step of injecting magnetic nanoparticles into a test sample containing a bio material to bind the magnetic nanoparticles to the biomaterial and separating the magnetic nanoparticles using a magnet;
Forming a biomaterial-magnetic nanoparticle-acetylcholinesterase complex by binding acetylcholinesterase to the separated biomaterial together with magnetic nanoparticles;
Introducing the biomaterial-magnetic nanoparticle-acetylcholinesterase complex into a standard reagent containing an ester compound to change the pH; And
The step of quantifying using the pH change
Lt; / RTI >
The bio-
A protein selected from the group consisting of Troponin I, Troponin C, Troponin T, Alpha fero-Protein (AFP), Prostate Specific Protein (PSA), and Carcinoembryonic antigen (CEA)
One or more selected from the group consisting of pathogenic Escherichia coli, Listeria, Vibrio, Norovirus, Salmonella, Staphylococcus aureus, Vibrio parahaemolyticus, Enteric haemorrhagic Escherichia coli O157, Campylobacter bacteria, Bacillus cerauss, Wherein the microorganism is selected from the group consisting of bacteria,
Method of detecting biomaterial. delete delete delete delete delete delete

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