CN111465856A - Composition for tuberculosis diagnosis and method for tuberculosis diagnosis based on change in optical properties - Google Patents
Composition for tuberculosis diagnosis and method for tuberculosis diagnosis based on change in optical properties Download PDFInfo
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- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
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- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
- G01N33/54326—Magnetic particles
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
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- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
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- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
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- G01N33/587—Nanoparticles
Abstract
The present invention relates to a composition for tuberculosis diagnosis and a method for tuberculosis diagnosis based on changes in optical properties. The method for diagnosing tuberculosis is characterized by changing optical characteristics based on (a) a fluorescent nano substance-antibody complex and a fluorescent nano substance-antibody complex or (b) a fluorescent nano substance-antibody complex and a metal nanoparticle-antibody complex. Since the optical characteristic change-based tuberculosis diagnosis method according to the present invention uses the change in fluorescence intensity of the metal nanoparticles and the fluorescent nanomaterial, tuberculosis having a low detection limit can be rapidly and accurately measured.
Description
Technical Field
The present invention relates to a composition for tuberculosis diagnosis and a method for tuberculosis diagnosis based on changes in optical properties.
Background
In general, tuberculosis, which is a disease with the leading incidence among korean legal infectious diseases, is a disease caused by infection with Mycobacterium tuberculosis (Mycobacterium tuberculosis). Therefore, it is important to be able to obtain reliable information in a short time from a patient suspected of being infected with tuberculosis for both early diagnosis and treatment.
A conventional method for tuberculosis diagnosis is a Tuberculin Skin Test (TST) method, but has low reliability in tuberculosis diagnosis due to its low specificity. However, it is still widely used due to its advantages of high sensitivity and low cost. Culture methods are also widely used as detection methods with the highest reliability among various tuberculosis diagnosis methods, and as methods for determining the presence or absence of tuberculosis by culturing tubercle bacillus, as the best standard method capable of determining whether tuberculosis is diagnosed or not. However, since the bacteria must be isolated and cultured, the risk of infection of the experimenter cannot be eliminated, and the bacteria culture takes at least 4 to 6 weeks, which results in the disadvantage that early diagnosis cannot be performed.
Recently, BACTEC system using liquid medium or Mycobacterium Growth Indicator Tube (MGIT) method is being performed. This method can confirm the proliferation of tubercle bacillus in about 2 weeks by shortening the culture period using a liquid medium, but has a disadvantage that additional diagnostic tests are required to identify the tubercle bacillus from atypical tubercle bacillus. In addition, there are problems such as waste disposal problems due to the use of radioactive isotopes, risk of infection by tubercle bacillus, and the need for expensive equipment.
The molecular diagnosis method in the tuberculosis diagnosis method is a method of extracting DNA of tubercle bacillus and then confirming the presence or absence of tubercle bacillus, and is used as an auxiliary diagnosis method for karyotic smear examination or culture examination. Korean registered patent No. 1794212 discloses a primer set (primer set) for tubercle bacillus detection and tuberculosis diagnosis, a composition comprising the same, and a kit, and korean registered patent No. 1765677 discloses a primer set for tuberculosis and non-tuberculous acid-fast bacteria detection and a detection method using the same, but the molecular biological method has a problem that immediate diagnosis cannot be made on site.
Tuberculosis can also be diagnosed using immunosensors based on antigen-antibody binding. Due to the specific binding of antibodies to antigens, antibodies are used, in particular, for the detection of biomarkers, immobilized on the surface of an immunosensor, or the like. Korean registered patent No. 1700891 discloses an antigen composition for tuberculosis diagnosis comprising CFP-10 and ESAT-6 fusion protein, and korean laid-open patent No. 2017-0011200 discloses a fusion protein composition of tubercle bacillus specific antigens comprising a combination of tubercle bacillus specific antigens for serological diagnosis of tuberculosis.
On the other hand, quantum dots are also attractive materials in the field of biosensors due to their excellent photostability and the ability to perform continuous monitoring in real time. However, its application in the field of biosensors is limited because its photoluminescence quantum yield is significantly reduced when it is converted from a hydrophobic to a hydrophilic form or is included with other materials. Recently, research on quantum dots has focused on developing sensors for a portion of target analytes that serve as electron donors for Fluorescence Resonance Energy Transfer (FRET) between quantum dots and acceptor molecules.
In addition, up-conversion nanoparticles (UCNPs) have chemical stability, no extinction, and unlike quantum dots commonly used in biochemistry, the maximum emission wavelength is not dependent on size, but multicolor luminescence can be easily performed by changing Host crystals (Host crystals) and Rare earth (Rare earth) dopant materials. Using these characteristics, UCNPs are used for flow cytometry (flow cytometry), photodynamic therapy (photodynamic therapy), diagnosis, etc., and are used as luminescent markers in biological analysis such as immunoassay and genetic analysis, and are also sufficiently used for chemical detection/cell imaging.
However, a technique for diagnosing tuberculosis based on a change in optical characteristics of the fluorescent nanomaterial-antibody complex has not been developed.
In contrast, the present inventors have made an effort to solve the above problems, and as a result, have confirmed that tuberculosis can be diagnosed by a change in optical properties of the fluorescent nanomaterial-antibody complex and the fluorescent nanomaterial-antibody complex or the fluorescent nanomaterial-antibody complex and the metal nanoparticle-antibody complex, thereby completing the present invention.
Disclosure of Invention
Problems to be solved
The purpose of the present invention is to provide a composition for diagnosing tuberculosis and a diagnostic method, which can simply and accurately judge whether tuberculosis occurs or not.
Means for solving the problems
In order to achieve the above object, the present invention provides a composition for tuberculosis diagnosis comprising (a) a fluorescent nano substance-antibody complex and a fluorescent nano substance-antibody complex or (b) a fluorescent nano substance-antibody complex and a metal nanoparticle-antibody complex.
The present invention also provides a method for diagnosing tuberculosis based on changes in optical characteristics of (a) the fluorescent nanomaterial-antibody complex and the fluorescent nanomaterial-antibody complex or (b) the fluorescent nanomaterial-antibody complex and the metal nanoparticle-antibody complex.
In addition, the present invention also provides a tuberculosis diagnosis method, which includes the steps of: (a) measuring a fluorescence value of a composition for tuberculosis diagnosis including a fluorescent nanomaterial-antibody complex to which a monoclonal first antibody that specifically binds to a mycobacterium-derived tuberculosis antigen is bound and a metal nanoparticle-antibody complex to which a second antibody is bound; (b) reacting the biological sample with the tuberculosis diagnosis composition whose fluorescence value has been measured; and (c) confirming the change of the fluorescence value of the formed [ fluorescent nano-substance-antibody complex ] - [ tuberculosis antigen ] - [ metal nano-particle-antibody complex ].
In addition, the present invention also provides a tuberculosis diagnosis method, which includes the steps of: (a) measuring a fluorescence value of a composition for tuberculosis diagnosis including a fluorescent nanomaterial-antibody complex to which a monoclonal first antibody specifically binding to a mycobacterium-derived tuberculosis antigen is bound and a fluorescent nanomaterial-antibody complex to which a second antibody is bound; (b) reacting the biological sample with the tuberculosis diagnosis composition whose fluorescence value has been measured; and (c) confirming the change of the fluorescence value of the formed [ fluorescent nano-substance-antibody complex ] - [ tuberculosis antigen ] - [ fluorescent nano-particle-antibody complex ].
In addition, the present invention also provides a tuberculosis diagnosis method, which includes the steps of: (a) reacting a biological sample with a magnetic nanomaterial-antibody complex to which a monoclonal first antibody specifically binding to a mycobacterium-derived tuberculosis antigen is bound; (b) reacting the formed [ magnetic nanomaterial-antibody complex ] - [ tuberculosis antigen ] with a fluorescent nanomaterial-antibody complex to which a second antibody is bound; and (c) separating the formed [ magnetic nano material-antibody complex ] - [ tuberculosis antigen ] - [ fluorescent nano material-antibody complex ] by a magnetic separation method, and then measuring the fluorescence value of the separated [ magnetic nano material-antibody complex ] - [ tuberculosis antigen ] - [ fluorescent nano material-antibody complex ].
In the present invention, the antibody specifically binds to a tubercle bacillus antigen selected from the group consisting of CFP10, Ag85B and L AM.
In the present invention, the metal nanoparticles are selected from the group consisting of magnetic nanoparticles, silver nanoparticles, and gold nanoparticles.
In the present invention, the fluorescent nanomaterial is selected from the group consisting of an upconversion nanoparticle, a quantum dot, a quantum nanorod, a quantum nanowire, and a graphene oxide quantum dot.
ADVANTAGEOUS EFFECTS OF INVENTION
Since the optical characteristic change-based tuberculosis diagnosis method according to the present invention utilizes the change in fluorescence intensity of the metal nanoparticles and the fluorescent nanomaterials, tuberculosis having a low detection limit can be rapidly and accurately measured.
Drawings
Fig. 1 is an explanatory diagram of [ upconversion nanoparticle-antibody complex ] - [ tuberculosis antigen ] - [ quantum dot-antibody complex ] according to an embodiment of the present invention.
Fig. 2 is an explanatory diagram of [ upconversion nanoparticle-antibody complex ] - [ tuberculosis antigen ] - [ graphene oxide quantum dot-antibody complex ] according to an embodiment of the present invention.
Fig. 3 is an illustrative diagram of [ upconversion nanoparticle-antibody complex ] - [ tuberculosis antigen ] - [ gold nanoparticle-antibody complex ] according to an embodiment of the present invention.
Fig. 4 is an explanatory diagram of [ upconversion nanoparticle-antibody complex ] - [ tuberculosis antigen ] - [ magnetic nanoparticle-antibody complex ] according to an embodiment of the present invention.
FIG. 5 shows the results of SDS-PAGE and Western blotting (Western blot) analysis for confirming the expression of tubercle bacillus-specific antigen CFP10 and antibodies G2 and G3 against it according to an embodiment of the present invention.
Fig. 6 is a TEM photograph (a) of gold nanoparticles synthesized according to an embodiment of the present invention, the result of measuring the average particle diameter (B) and the charge (C).
FIG. 7 is an absorption spectrum (A) and FT-IR (B) measurement results of gold nanoparticles prepared according to an embodiment of the present invention and a gold nanoparticle-G2 antibody complex.
Fig. 8 is a result of sem (a) and FT-ir (b) analyzing a series of upconversion nanoparticles and particle complexes immobilized with tuberculosis antibody G3 according to an embodiment of the present invention.
Fig. 9 is a result of confirming upconversion fluorescence of the carboxyl group-treated upconversion nanoparticle (PAA-UCNP) and the upconversion nanoparticle-G3 antibody complex (UCNP @ CFP10G3) prepared according to an embodiment of the present invention.
Fig. 10 is a result of analyzing the detection limit of CFP10 antigen of the upconverting nanoparticle-G3 antibody complex and the gold nanoparticle-G2 antibody complex prepared according to an embodiment of the present invention.
Best mode for carrying out the invention
In the present invention, when a fluorescent nanomaterial to which an antibody against a tubercle bacillus secretory antigen is bound and a metal nanoparticle are used, it is intended to confirm that tuberculosis can be measured quickly and accurately based on a change in optical characteristics.
In the present invention, after preparing a metal nanoparticle-antibody complex using magnetic nanoparticles and gold nanoparticles and a fluorescent nanomaterial-antibody complex using upconversion nanoparticles, whether tuberculosis is present or not can be confirmed based on the change in optical characteristics after reaction with a biological sample containing a tuberculosis antigen.
Accordingly, the present invention relates to a composition for tuberculosis diagnosis, which includes (a) a fluorescent nano-substance-antibody complex and a fluorescent nano-substance-antibody complex or (b) a fluorescent nano-substance-antibody complex and a metal nanoparticle-antibody complex.
In the present invention, the antibody may be used without particular limitation as long as it is capable of specifically binding to a tubercle bacillus antigen, but CFP10, Ag85B, L AM, and the like are preferably used.
The metal nanoparticles may be exemplified by magnetic nanoparticles, silver nanoparticles, gold nanoparticles, etc., but are not limited thereto, and preferably have a diameter of about 5nm to about 200 nm.
The fluorescent nano-substance may be exemplified by Upconversion nanoparticles (UCNP), Quantum dots (Quantum dots), Quantum nanorods (Quantum nano), Quantum nanowires (Quantum nanowires), Graphene Oxide Quantum dots (Graphene Oxide Quantum dots), etc., but is not limited thereto.
In another aspect, the present invention relates to a tuberculosis diagnosis method based on changes in optical characteristics of (a) the fluorescent nanomaterial-antibody complex and the fluorescent nanomaterial-antibody complex or (b) the fluorescent nanomaterial-antibody complex and the metal nanoparticle-antibody complex.
In the tuberculosis diagnosis method, the tuberculosis diagnosis may be determined with reference to an optical characteristic change that occurs in proportion or inverse proportion to the amount of binding between the fluorescent nanomaterial-antibody complex to which the first antibody that changes by the amount of the tubercle bacillus-specific antigen is bound and the fluorescent nanomaterial-antibody complex to which the second antibody is bound, or between the fluorescent nanomaterial-antibody complex to which the first antibody is bound and the metal nanoparticle-antibody complex to which the second antibody is bound.
That is, (1) when the magnetic nanoparticles and the upconversion nanoparticles are used, whether or not tuberculosis can be confirmed by the fluorescence intensity increased according to the antigen concentration, (2) when the quantum dots, quantum nanorods, quantum nanowires, or graphene oxide quantum dots and upconversion nanoparticles are used, whether or not tuberculosis is confirmed by the increased fluorescence intensity of the quantum dot, quantum nanorod, quantum nanowire or graphene oxide quantum dot by absorbing the fluorescence of the upconversion nanoparticle due to resonance of the wavelength emitting the fluorescence of the upconversion nanoparticle and the wavelength absorbing the fluorescence of the quantum dot, quantum nanorod, quantum nanowire or graphene oxide quantum dot, (3) when the gold nanoparticle or silver nanoparticle and upconversion nanoparticle are used, whether or not tuberculosis is present can be confirmed by the optical signal that the gold nanoparticles or silver nanoparticles absorb fluorescence of the upconversion nanoparticles and the fluorescence intensity increases or decreases.
For example, gold nanoparticles, although absorbing at different wavelengths depending on the size and shape of the particle, generally have an absorption wavelength of 550nm, and upconverting nanoparticles are excited at 980nm (exitatiti on) and exhibit fluorescence at 550nm and 660 nm. Therefore, when a biological sample containing a tuberculosis antigen is reacted to the upconverting nanoparticle-antibody complex to which the first antibody is bound and the gold nanoparticle-antibody complex to which the second antibody is bound, the distance between the upconverting nanoparticle and the gold nanoparticle becomes short by the mediation of the tuberculosis antigen, and as a result, the fluorescence intensity at 550nm decreases.
Thus, the present invention relates in another aspect to a method of tuberculosis diagnosis comprising the steps of: (a) measuring a fluorescence value of a composition for tuberculosis diagnosis including a fluorescent nano-substance-antibody complex to which a monoclonal first antibody specifically binding to a mycobacterium-derived tuberculosis antigen is bound and a metal nanoparticle-antibody complex to which a second antibody is bound; (b) reacting the biological sample with the composition for tuberculosis diagnosis having the measured fluorescence value; and (c) confirming the change of the fluorescence value of the formed [ fluorescent nano-substance-antibody complex ] - [ tuberculosis antigen ] - [ metal nano-particle-antibody complex ].
As another example, the quantum dot generally has an absorption wavelength of 550nm, has a characteristic that the quantum dot exhibits fluorescence at wavelengths other than a predetermined absorption wavelength, and does not emit fluorescence at wavelengths other than the predetermined absorption wavelength, although the absorption wavelength differs depending on the size and shape of the particle, and the upconversion nanoparticle is excited at 980nm (excitation) and exhibits fluorescence at 550nm and 660 nm. Therefore, when a biological sample containing a tuberculosis antigen is reacted with the upconversion nanoparticle-antibody complex bound with the first antibody and the quantum dot-antibody complex bound with the second antibody, the distance between the upconversion nanoparticle and the quantum dot becomes shorter mediated by the tuberculosis antigen, and when an absorption wavelength of the upconversion nanoparticle other than the absorption wavelength of the quantum dot is applied, the fluorescence intensity increases.
Thus, another aspect of the present invention relates to a method for diagnosing tuberculosis, comprising the steps of: (a) measuring a fluorescence value of a composition for tuberculosis diagnosis including a fluorescent nanomaterial-antibody complex to which a monoclonal first antibody specifically binding to a mycobacterium-derived tuberculosis antigen is bound and a fluorescent nanomaterial-antibody complex to which a second antibody is bound; (b) reacting the biological sample with the composition for tuberculosis diagnosis having the measured fluorescence value; and (c) confirming the change of the fluorescence value of the formed [ fluorescent nano-substance-antibody complex ] - [ tuberculosis antigen ] - [ fluorescent nano-particle-antibody complex ].
As another example, magnetic nanoparticles have the property of being attracted by magnets, and without specific absorption wavelengths in the UV/Vis (visible light) range, upconversion nanoparticles are excited at 980nm (excitation) and show fluorescence at 550nm as well as at 660 nm. Therefore, when a biological sample containing a tuberculosis antigen is reacted with the upconverting nanoparticle-antibody complex to which the first antibody is bound and the magnetic nanoparticle-antibody complex to which the second antibody is bound, the upconverting nanoparticle is bound to the magnetic nanoparticle using the tuberculosis antigen as a medium, and when the washing process is repeated after separation using a magnet, only the upconverting nanoparticle captured by the magnetic nanoparticle remains. Therefore, tuberculosis can be diagnosed by a negative detection method of measuring a decreased fluorescence intensity by measuring a result of fluorescence before reacting tuberculosis antigens and measuring fluorescence after separating and washing with a magnet after reacting tuberculosis antigens, or by a positive detection method of measuring fluorescence of up-converted nanoparticles after separating and washing with a magnet after reacting tuberculosis antigens.
Accordingly, another aspect of the present invention relates to a tuberculosis diagnostic method comprising the steps of: (a) reacting a biological sample with a magnetic nanomaterial-antibody complex to which a monoclonal first antibody specifically binding to a mycobacterium-derived tuberculosis antigen is bound; (b) reacting the formed [ magnetic nanomaterial-antibody complex ] - [ tuberculosis antigen ] with a fluorescent nanomaterial-antibody complex to which a second antibody is bound; and (c) separating the formed [ magnetic nano material-antibody complex ] - [ tuberculosis antigen ] - [ fluorescent nano material-antibody complex ] by a magnetic separation method, and then measuring the fluorescence value of the separated [ magnetic nano material-antibody complex ] - [ tuberculosis antigen ] - [ fluorescent nano material-antibody complex ].
Detailed Description
The present invention will be described in more detail below with reference to examples. These examples are only for illustrating the present invention, and it should be apparent to those skilled in the art that the scope of the present invention should not be construed as being limited by these examples.
Example 1 preparation of tubercle bacillus specific antigen CFP10 and recombinant antibodies against it G2 and G3
Referring to korean registered patent No. 1631054, tuberculosis antigen C FP10 to be used in the examples and two Group2(G2, Group 2) and Group3(G3, Group 3) antibody genes capable of binding thereto were expressed in escherichia coli and isolated and purified for use.
For this, the control vector pET22, the CFP10 antigen expression vector pET22-CFP10, the G2 antibody expression vector pET22-GBP-CFP10G2, the G3 antibody expression vector pET22-CFP10G3 were transformed into E.coli B L21 (DE3) strain, each strain was cultured to OD 0.4 at 37 ℃ in 100ml of L B (1% (w/v) tryptone, 1% NaCl, 0.5% Yeast extract (Yeast extra t)) + ampicillin (ampicillin) medium, protein expression was carried out by adding 0.1mM IPTG, and after 6 hours of culture, the strains were collected by centrifugation (3,500rpm, 4 ℃, 10 minutes).
The cultured strain was sonicated in a lysis (lysis) (50mM sodium phosphate (pH7.5), 5% (w/v), 50mM Na Cl) buffer, and then centrifuged (15,000g, 4 ℃, 10 minutes) to obtain a protein, which was analyzed by SDS-PAGE and Western blot analysis (Western blot analysis). As shown in FIG. 5, the expression of recombinant CFP10 antigen and G2(G BP-CFP10G2) and G3(CFP10G3) antibodies could be confirmed.
Since the recombinant CFP10 antigen and antibody expressed above have a 6x-His tag, purification was performed using HisTrap FF column and fast protein liquid chromatography (FP L C) (GE Healthcare, general electric medical group) for the next experiment.
Example 2 preparation of "Metal nanoparticle-antibody Complex" and "fluorescent Nanopaterial-antibody Complex" against Mycobacterium tuberculosis specific antigen CFP10
2-1: preparation of Metal nanoparticle-antibody Complex
2-1-1: preparation of magnetic nanoparticle-antibody complexes
2-1-1-1: amine functional substitution of magnetic nanoparticles
After 9m L of methanol was added to 1m L of APTES solution and mixed well, 10mg of magnetic nanoparticles were added and dispersed by an ultrasonic mill, and stirred for 17 hours to form amine functional groups on the surface, after which, the magnetic nanoparticles were collected by a magnet, the supernatant was discarded, washed 3 times with methanol (10 m L for each washing) and a magnet to prepare magnetic nanoparticles substituted with amine groups, and then stored at 4 ℃.
2-1-1-2: preparation of magnetic nanoparticle-G2 antibody complex
Magnetic nanoparticles substituted with amine groups were dispersed in 8m L glutaraldehyde (ph7.4) solution and stirred for 1 hour, the magnetic nanoparticles were collected with a magnet and then the supernatant was discarded, and after washing 3 times with 10m L per washing with 10mM PBS (ph7.4) solution and a magnet, the magnetic nanoparticles were dispersed in 2m L10 mM PBS (ph7.4) solution, a G2 antibody capable of binding to the tuberculosis antigen CFP10 prepared in example 1 was added to perform a reaction with gentle shaking at room temperature for 14 hours so that the concentration of the magnetic nanoparticle solution activated with glutaraldehyde (glutaraldehyde) became 5.0 μ G/m L per 1m L, after that, the supernatant was discarded after collecting the magnetic nanoparticles with a magnet, a magnetic nanoparticle solution (ph7.4) solution (10 m L per washing with 10m and a magnet was washed 3 times, 0.5% PBS (PBS) dissolved in 0.5% PBS was added to prepare a capping magnetic nanoparticle complex, and after preserving the capping antibody at 364 ℃ for 30 minutes.
2-1-2: preparation of gold nanoparticle-antibody complex
2-1-2-1 preparation of gold nanoparticles
By HAuCl4Gold nanoparticles (AuNP) used in the examples were synthesized by citrate reduction (citrate reduction) method. 100ml of 1mM HAuCl4The solution was stirred vigorously under reflux (ref lux), when the temperature of the solution reached 90 ℃, 10ml of 38.8mM trisodium citrate dihydrate (trisodium citrate) was added rapidly and reacted at the same temperature for 15 minutes. When dispersed gold nanoparticles were synthesized in the case where the solution immediately changed from deep purple to deep red, the reaction solution was rapidly cooled in ice and stored at 4 ℃ until use.
As shown in fig. 6, the shape of the synthesized gold nanoparticles was observed by TEM, and it was confirmed that gold nanoparticles having an average diameter of about 15nm were synthesized and negatively charged.
2-1-2-2: preparation of gold nanoparticle-G2 antibody complex
1m L gold nanoparticles prepared in 2-1-2-1 were washed 2 times with DI and 0.5mM K2CO3The solution was titrated to pH 9.0. to increase the stability of gold nanoparticles, 10% (w/v) PEG8000 of 25. mu. L was added, then, 6ml of 300ug/ml G2(GBP-CFP10G2) antibody prepared in example 1 was added, and reacted at room temperature for 2 hours with gentle stirring, after centrifugation (13,000rpm, 4 ℃, 30 minutes) and 2-time repeated washing of the prepared gold nanoparticle-G2 antibody complex, suspended in TBST buffer (0.5% Tween (Tween)20) and stored at 4 ℃ until use.
Absorption spectra and FT-IR (Fourier transform spectrum) of gold nanoparticles (AuNPs) and the prepared gold nanoparticle-G2 antibody complex (Au NPs @ GBP-CFP10G2) were measured, and the results thereof are shown in FIG. 7.
As shown in FIG. 7, it was found that gold nanoparticles (AuNP) and the prepared gold nanoparticle-G2 antibody complex (AuNP @ GBP-CFP10G2) had the same absorption wavelength of 550nm band in 2-1-1, and it was found that the G2 antibody was successfully combined with each other by FT-IR spectroscopy, and thus surface treatment was successfully performed.
2-2: preparation of fluorescent Nanometric substance-antibody Complex
2-2-1: preparation of upconversion nanoparticles
At 0.2M L N (NO)3)3×H2O(Ln：Y3+，Yb3+，Er3+) Then, 3ml of ultrapure water (DI), 22.5ml of ethanol and 150mg of CTAB were added to the solution while stirring vigorously in this order, 1.0M NaF was added dropwise, the resulting solution was crystallized while stirring vigorously at room temperature for 2 hours, then, 1.5M L nitric acid was added, and the mixed solution was transferred to a 23M L Teflon-lined autoclave (Teflon-lined autoclave) and stirred at 180 ℃The reaction was carried out for 8 hours. The generated upconversion nanoparticles (UCNPs) were collected by centrifugation for 20 minutes, and dried at 60 ℃ in a drying oven after being washed with ethanol and ultrapure water (DI) (1:1, v/v).
50mg of polyacrylic acid (Poly (acrylic acid)) (PAA, MW 1800) having carboxyl groups was added to 9M L ultrapure water (DI), titrated to pH8 using 0.2M NaOH, and after dropping 1M L dispersion of upconversion nanoparticles (UCNP), the resultant solution was stirred additionally for 5 hours, the dispersion was dissolved in 10M L DEG and stirred at 105 ℃ for 1 hour to remove water, and finally, the mixture was transferred to 23M L Teflon-lined autoclave (Teflon-lined autocl ave) and incubated at 160 ℃ for 2 hours, particles were collected by centrifugal separation and washed with ultrapure water (DI) and ethanol (1:1, v/v%), and dried at 60 ℃ in a dry air oven, thereby preparing upconversion nanoparticles (UCPAA-UCNP) treated with carboxyl groups.
The shape of the prepared carboxyl group-treated upconversion nanoparticles (PAA-UCNP) was confirmed by TEM, and the average particle size was confirmed, and the result is shown in fig. 8A.
As shown in fig. 8A, it was confirmed that the average diameter of the upconversion nanoparticles treated with carboxyl groups (PAA-UCNP) was about 57 nm.
2-2-2: preparation of Up-converting nanoparticle-antibody complexes
200mg of EDC and 200mg of sulfo-NHS were dissolved in 5m L of MES buffer, 1m L of carboxyl-treated upconversion nanoparticles (PAA-UCNP) were added thereto, and the mixture was stirred at room temperature for 1 hour, then washed with 13,000 MES buffer for five minutes by a centrifuge for 3 times, and then dispersed in 5m L of HEPES buffer by an ultrasonic mill, and then, G3 antibody capable of binding to the tuberculosis antigen CFP10 prepared in example 1 was added thereto, and the mixture was stirred at room temperature for 17 hours to react so that the concentration of the final upconversion nanoparticle solution per 1m L became 5.0. mu.g/m L, thereby preparing an upconversion nanoparticle-G3 antibody complex (UCNP @ CF P10G 3). after the reaction, the mixture was washed with 10mM PBS buffer (7.4) for 3 times at 13,000rpm for five minutes by a centrifuge, and then stored at 4 ℃.
FT-IR of the prepared upconverting nanoparticles (UCNP), the upconverting nanoparticles treated with carboxyl groups (PAA-UCNP), and the upconverting nanoparticle-G3 antibody complex (UCNP @ CFP10G3) were measured, and the results thereof are shown in fig. 8B.
As shown in fig. 8B, it was found that the carboxyl group and the G3 antibody were successfully bound to the upconversion nanoparticle (UCNP) and surface-treated.
In order to confirm the optical properties of the upconverting nanoparticles, the carboxyl group-treated upconverting nanoparticles (PAA-UCNP) and the upconverting nanoparticle-G3 antibody complex (UCNP @ CFP10G3) were excited (excitation) at 980nm, and then the region up to 400 to 800nm was scanned (emission scanning), and the results are shown in fig. 9.
As shown in fig. 9, both the carboxyl-treated upconverting nanoparticles (PAA-UCNP) and the upconverting nanoparticle-G3 antibody complex (UCNP @ CFP10G3) can observe fluorescence wavelengths in the wavelength range of about 550nm and 660 nm. Therefore, the optical properties of the upconverting nanoparticle-G3 antibody complex (UCNP @ CFP10G3) were completely retained, and thus it was confirmed that the upconverting nanoparticle-G3 antibody complex was sufficiently used for diagnosing tubercle bacillus by utilizing the properties.
Example 3 detection of Mycobacterium tuberculosis Using "Metal nanoparticle-antibody Complex" and "fluorescent Nanobody-antibody Complex
3-1: tuberculosis (TB) detection using magnetic nanoparticle-antibody complex and upconversion nanoparticle-antibody complex
After 0.5m L upconverting nanoparticle-G3 antibody complex prepared in 2-2-2 and 0.3m L magnetic nanoparticle-G2 antibody complex prepared in 2-1-1-2 were put in a 1.5m L microtube and mixed well, 0.05m L solution containing tuberculosis antigen (CFP-10) was added, the reaction was performed by shaking gently at room temperature for 30 minutes, the upconverting nanoparticle-magnetic nanoparticle bound by the tuberculosis antigen and the unbound magnetic nanoparticle were collected by a magnet, the supernatant was discarded, and the fluorescence intensity by irradiating a 980nm light source was measured after washing 3 times with 10mM PBS (pH7.4) solution (3 m L for each washing) and a magnet.
As a result, when usedWhen nanoparticle-G3 antibody complex and magnetic nanoparticle-G2 antibody complex were converted, R20.9914, which is very reliable, and it was confirmed that the target tuberculosis antigen CFP-10 could be detected with a detection limit of 2.68pg/m L.
3-2: tuberculosis (TB) detection using gold nanoparticle-antibody complex and upconversion nanoparticle-antibody complex
After 0.5m L upconverting nanoparticle-G3 antibody complex prepared in 2-2-2 and 0.3m L gold nanoparticle-G2 antibody complex prepared in 2-1-2-2 were put in a 1.5m L microtube and sufficiently mixed, in this state, the fluorescence intensity generated by irradiating a 980nm light source was measured and recorded, then, 0.05m L solution containing a tuberculosis antigen (CFP-10) was added, reaction was performed with gentle shaking at normal temperature for 30 minutes, and a 980nm light source was again irradiated to measure the fluorescence intensity of the upconverting nanoparticle according to the decrease in the concentration of the tuberculosis antigen, and the result is shown in FIG. 10.
From FIG. 10, it was confirmed that when the upconverting nanoparticle-G3 antibody complex and the gold nanoparticle-G2 antibody complex were used, R was20.9922, it was confirmed that CFP-10, the target tuberculosis antigen, could be detected with a detection limit of 1.80pg/m L with very high reliability.
Since specific portions of the present invention have been described in detail, it is obvious to those skilled in the art that these specific techniques are merely preferred embodiments and the scope of the present invention is not limited thereto. Accordingly, the substantial scope of the present invention will be defined by the appended claims and equivalents thereof.
[ possibility of Industrial utilization ]
Since the optical characteristic change-based tuberculosis diagnosis method according to the present invention utilizes the change in fluorescence intensity of the metal nanoparticles and the fluorescent nanomaterials, tuberculosis having a low detection limit can be rapidly and accurately measured, thereby being commercially useful.
Claims (12)
1. A composition for the diagnosis of tuberculosis, wherein,
the composition includes (a) a fluorescent nanomaterial-antibody complex and a fluorescent nanomaterial-antibody complex or (b) a fluorescent nanomaterial-antibody complex and a metal nanoparticle-antibody complex.
2. The composition for tuberculosis diagnosis according to claim 1,
the antibody specifically binds to a tubercle bacillus antigen selected from the group consisting of CFP10, Ag85B, and L AM.
3. The composition for tuberculosis diagnosis according to claim 1,
the metal nanoparticles are selected from the group consisting of magnetic nanoparticles, silver nanoparticles, and gold nanoparticles.
4. The composition for tuberculosis diagnosis according to claim 1,
the fluorescent nanomaterial is selected from the group consisting of up-conversion nanoparticles, quantum dots, quantum nanorods, quantum nanowires, and graphene oxide quantum dots.
5. A method for diagnosing tuberculosis, characterized in that,
the tuberculosis diagnosis method is based on the change of optical characteristics of (a) a fluorescent nano-substance-antibody complex and a fluorescent nano-substance-antibody complex or (b) a fluorescent nano-substance-antibody complex and a metal nanoparticle-antibody complex.
6. The tuberculosis diagnostic method according to claim 5,
the antibody specifically binds to a tubercle bacillus antigen selected from the group consisting of CFP10, Ag85B, and L AM.
7. The tuberculosis diagnostic method according to claim 5,
the metal nanoparticles are selected from the group consisting of magnetic nanoparticles, silver nanoparticles, and gold nanoparticles.
8. The tuberculosis diagnostic method according to claim 5,
the fluorescent nanomaterial is selected from the group consisting of up-conversion nanoparticles, quantum dots, quantum nanorods, quantum nanowires, and graphene oxide quantum dots.
9. The tuberculosis diagnostic method according to claim 5,
the method for diagnosing tuberculosis is determined based on the amount of binding between a fluorescent nanomaterial-antibody complex to which a first antibody that changes according to the amount of a tubercle bacillus-specific antigen is bound and a fluorescent nanomaterial-antibody complex to which a second antibody is bound, or the amount of binding between a fluorescent nanomaterial-antibody complex to which a first antibody is bound and a metal nanoparticle-antibody complex to which a second antibody is bound, and the change in optical characteristics due to the amount of binding.
10. A method for diagnosing tuberculosis, comprising the steps of:
(a) measuring a fluorescence value of a composition for tuberculosis diagnosis including a fluorescent nano-substance-antibody complex to which a monoclonal first antibody specifically binding to a mycobacterium-derived tuberculosis antigen is bound and a metal nanoparticle-antibody complex to which a second antibody is bound;
(b) reacting the biological sample with the tuberculosis diagnosis composition whose fluorescence value has been measured; and
(c) the change in the fluorescence value of the formed [ fluorescent nanomaterial-antibody complex ] - [ tuberculosis antigen ] - [ metal nanoparticle-antibody complex ] was confirmed.
11. A method for diagnosing tuberculosis, comprising the steps of:
(a) measuring a fluorescence value of a composition for binding diagnosis including a fluorescent nanomaterial-antibody complex to which a monoclonal first antibody that specifically binds to a mycobacterium-derived tuberculosis antigen is bound and a fluorescent nanomaterial-antibody complex to which a second antibody is bound;
(b) reacting the biological sample with the tuberculosis diagnosis composition whose fluorescence value has been measured; and
(c) the change in the fluorescence value of the formed [ fluorescent nanomaterial-antibody complex ] - [ tuberculosis antigen ] - [ fluorescent nanomaterial-antibody complex ] was confirmed.
12. A method for diagnosing tuberculosis, comprising the steps of:
(a) reacting a biological sample with a magnetic nanomaterial-antibody complex to which a monoclonal first antibody specifically binding to a mycobacterium-derived tuberculosis antigen is bound;
(b) reacting the formed [ magnetic nanomaterial-antibody complex ] - [ tuberculosis antigen ] with a fluorescent nanomaterial-antibody complex to which a second antibody is bound; and
(c) after separating the formed [ magnetic nanomaterial-antibody complex ] - [ tuberculosis antigen ] - [ fluorescent nanomaterial-antibody complex ] by a magnetic separation method, the fluorescence value of the separated [ magnetic nanomaterial-antibody complex ] - [ tuberculosis antigen ] - [ fluorescent nanomaterial-antibody complex ] is measured.
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| KR1020180158404A KR102098030B1 (en) | 2017-12-11 | 2018-12-10 | Composition for Tuberculosis Diagnosis and Diagnostic Method for Tuberculosis based on Optical Properties |
| PCT/KR2018/015675 WO2019117585A2 (en) | 2017-12-11 | 2018-12-11 | Composition for diagnosing tuberculosis and method for diagnosing tuberculosis on basis of change in optical characteristics |
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Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20050064604A1 (en) * | 2001-11-05 | 2005-03-24 | Bayer Technology Services Gmbh | Assay based on doped nanoparticles |
| CN1606691A (en) * | 2001-09-06 | 2005-04-13 | 基因描绘系统有限公司 | Rapid and sensitive detection of cells and viruses |
| US20080124738A1 (en) * | 2005-03-01 | 2008-05-29 | Pritest, Inc | Compositions and methods of testing for tuberculosis and mycobacterium infection |
| KR20110124489A (en) * | 2010-05-11 | 2011-11-17 | 부산대학교 산학협력단 | Magnetophoresis nanobiosensor for immunoassays |
| CN102636649A (en) * | 2012-03-04 | 2012-08-15 | 无锡市第四人民医院 | Kit for detecting carcinoembryonic antigen based on antibody functionalized magnetic nanometer material and up-conversion fluorescence nanometer material |
| KR20130059579A (en) * | 2011-11-29 | 2013-06-07 | (주)유 바이오메드 | Composition for diagnosis of multi-cancer |
| CN104374764A (en) * | 2013-08-15 | 2015-02-25 | 中国科学技术大学 | Analysis probe, preparation method and uses thereof |
| WO2015128830A1 (en) * | 2014-02-26 | 2015-09-03 | Stellenbosch University | Method for diagnosing tuberculosis |
Family Cites Families (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US9295003B2 (en) * | 2007-03-19 | 2016-03-22 | Apple Inc. | Resource allocation in a communication system |
| WO2011030160A1 (en) | 2009-09-11 | 2011-03-17 | Isis Innovation Limited | Detection of mycobacteria |
| EP2629095A1 (en) * | 2012-02-17 | 2013-08-21 | Biosure R & T Cell Co. | Detection of mycobacterial protein targets using quantum dots (QDs) and immunomagnetic seperation |
| WO2015122391A1 (en) | 2014-02-13 | 2015-08-20 | 国立大学法人静岡大学 | Quantum dot fluorescence enhanced immunoassay |
| KR101994515B1 (en) * | 2016-04-06 | 2019-06-28 | 중앙대학교 산학협력단 | Bovine tuberculosis diagnosing strip and diagnosing method using the same |
-
2018
- 2018-12-10 KR KR1020180158404A patent/KR102098030B1/en active IP Right Grant
- 2018-12-11 CN CN201880079556.0A patent/CN111465856A/en active Pending
Patent Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1606691A (en) * | 2001-09-06 | 2005-04-13 | 基因描绘系统有限公司 | Rapid and sensitive detection of cells and viruses |
| US20050064604A1 (en) * | 2001-11-05 | 2005-03-24 | Bayer Technology Services Gmbh | Assay based on doped nanoparticles |
| US20080124738A1 (en) * | 2005-03-01 | 2008-05-29 | Pritest, Inc | Compositions and methods of testing for tuberculosis and mycobacterium infection |
| KR20110124489A (en) * | 2010-05-11 | 2011-11-17 | 부산대학교 산학협력단 | Magnetophoresis nanobiosensor for immunoassays |
| KR20130059579A (en) * | 2011-11-29 | 2013-06-07 | (주)유 바이오메드 | Composition for diagnosis of multi-cancer |
| CN102636649A (en) * | 2012-03-04 | 2012-08-15 | 无锡市第四人民医院 | Kit for detecting carcinoembryonic antigen based on antibody functionalized magnetic nanometer material and up-conversion fluorescence nanometer material |
| CN104374764A (en) * | 2013-08-15 | 2015-02-25 | 中国科学技术大学 | Analysis probe, preparation method and uses thereof |
| WO2015128830A1 (en) * | 2014-02-26 | 2015-09-03 | Stellenbosch University | Method for diagnosing tuberculosis |
Non-Patent Citations (2)
| Title |
|---|
| EUN JU KIM等: "An easy and sensitive sandwich assay for detection of Mycobacterium tuberculosis Ag85B antigen using quantum dots and gold nanorods", vol. 87, pages 150 - 156, XP055629770 * |
| SANG-HYUN HWANG ET AL.: "Upconversion nanoparticle-based Förster resonance energy transfer for detecting the IS6110 sequence of Mycobacterium tuberculosis complex in sputum", BIOSENSORS AND BIOELECTRONICS, vol. 53, pages 112 * |
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