CN113588602A - Rapid parathyroid hormone detection method - Google Patents
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- CN113588602A CN113588602A CN202110772347.8A CN202110772347A CN113588602A CN 113588602 A CN113588602 A CN 113588602A CN 202110772347 A CN202110772347 A CN 202110772347A CN 113588602 A CN113588602 A CN 113588602A
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- 108090000445 Parathyroid hormone Proteins 0.000 title claims abstract description 55
- 239000000199 parathyroid hormone Substances 0.000 title claims abstract description 47
- 229960001319 parathyroid hormone Drugs 0.000 title claims abstract description 47
- 238000001514 detection method Methods 0.000 title claims abstract description 33
- 102000003982 Parathyroid hormone Human genes 0.000 title claims description 46
- 239000010931 gold Substances 0.000 claims abstract description 40
- 229910052737 gold Inorganic materials 0.000 claims abstract description 40
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 claims abstract description 38
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 claims abstract description 21
- 239000002105 nanoparticle Substances 0.000 claims abstract description 6
- 239000013307 optical fiber Substances 0.000 claims abstract description 6
- 238000005259 measurement Methods 0.000 claims abstract description 4
- 239000000523 sample Substances 0.000 claims abstract description 4
- 102100036893 Parathyroid hormone Human genes 0.000 claims abstract 9
- 239000000427 antigen Substances 0.000 claims description 11
- 102000036639 antigens Human genes 0.000 claims description 11
- 108091007433 antigens Proteins 0.000 claims description 11
- 238000003756 stirring Methods 0.000 claims description 9
- 150000001875 compounds Chemical class 0.000 claims description 7
- 239000011521 glass Substances 0.000 claims description 6
- 239000000758 substrate Substances 0.000 claims description 6
- NOGFHTGYPKWWRX-UHFFFAOYSA-N 2,2,6,6-tetramethyloxan-4-one Chemical compound CC1(C)CC(=O)CC(C)(C)O1 NOGFHTGYPKWWRX-UHFFFAOYSA-N 0.000 claims description 4
- 239000002245 particle Substances 0.000 claims description 4
- 239000002253 acid Substances 0.000 claims description 3
- 238000009835 boiling Methods 0.000 claims description 3
- 239000008367 deionised water Substances 0.000 claims description 3
- 229910021641 deionized water Inorganic materials 0.000 claims description 3
- 238000010438 heat treatment Methods 0.000 claims description 3
- NLJMYIDDQXHKNR-UHFFFAOYSA-K sodium citrate Chemical compound O.O.[Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O NLJMYIDDQXHKNR-UHFFFAOYSA-K 0.000 claims description 3
- 239000001509 sodium citrate Substances 0.000 claims description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 3
- 102000004169 proteins and genes Human genes 0.000 abstract description 10
- 108090000623 proteins and genes Proteins 0.000 abstract description 10
- 238000000034 method Methods 0.000 abstract description 9
- 239000012535 impurity Substances 0.000 abstract description 2
- 238000006303 photolysis reaction Methods 0.000 abstract description 2
- 230000015843 photosynthesis, light reaction Effects 0.000 abstract description 2
- 238000004519 manufacturing process Methods 0.000 abstract 1
- 239000000243 solution Substances 0.000 description 31
- 239000002953 phosphate buffered saline Substances 0.000 description 5
- 239000008280 blood Substances 0.000 description 4
- 210000004369 blood Anatomy 0.000 description 4
- 239000012528 membrane Substances 0.000 description 4
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 description 2
- 239000011575 calcium Substances 0.000 description 2
- 229910052791 calcium Inorganic materials 0.000 description 2
- 239000012634 fragment Substances 0.000 description 2
- 238000003018 immunoassay Methods 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 239000002184 metal Substances 0.000 description 2
- 229910052751 metal Inorganic materials 0.000 description 2
- 238000001000 micrograph Methods 0.000 description 2
- NMHMNPHRMNGLLB-UHFFFAOYSA-N phloretic acid Chemical compound OC(=O)CCC1=CC=C(O)C=C1 NMHMNPHRMNGLLB-UHFFFAOYSA-N 0.000 description 2
- 230000035945 sensitivity Effects 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- OAICVXFJPJFONN-UHFFFAOYSA-N Phosphorus Chemical compound [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 description 1
- 208000035965 Postoperative Complications Diseases 0.000 description 1
- 208000024770 Thyroid neoplasm Diseases 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000005540 biological transmission Effects 0.000 description 1
- 210000000988 bone and bone Anatomy 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 description 1
- 210000003372 endocrine gland Anatomy 0.000 description 1
- 229940088597 hormone Drugs 0.000 description 1
- 239000005556 hormone Substances 0.000 description 1
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 230000001483 mobilizing effect Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 239000003960 organic solvent Substances 0.000 description 1
- 210000002990 parathyroid gland Anatomy 0.000 description 1
- 230000002093 peripheral effect Effects 0.000 description 1
- 239000011574 phosphorus Substances 0.000 description 1
- 229910052698 phosphorus Inorganic materials 0.000 description 1
- 238000002331 protein detection Methods 0.000 description 1
- 230000003248 secreting effect Effects 0.000 description 1
- 238000004904 shortening Methods 0.000 description 1
- 239000002356 single layer Substances 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 238000001356 surgical procedure Methods 0.000 description 1
- 201000002510 thyroid cancer Diseases 0.000 description 1
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
- G01N21/55—Specular reflectivity
- G01N21/552—Attenuated total reflection
- G01N21/553—Attenuated total reflection and using surface plasmons
- G01N21/554—Attenuated total reflection and using surface plasmons detecting the surface plasmon resonance of nanostructured metals, e.g. localised surface plasmon resonance
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- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Nanotechnology (AREA)
- Physics & Mathematics (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Investigating Or Analysing Materials By Optical Means (AREA)
- Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
Abstract
The invention discloses a rapid parathyroid hormone detection method, which comprises the following steps of (1) preparing a nano gold solution; (2) configuring the DSP to a concentration of 1nmol by using DMSO (dimethyl sulfoxide), and adsorbing DSP molecules to the surface of the gold nanoparticles to obtain a DSP-AUNPS solution; (3) forming PTH antibody-DSP-AuNPs complexes; (4) adopting a surface plasma instrument to carry out wavelength shift measurement and display, wherein the surface plasma instrument is connected with a gold film nanopore SPR chip through an optical fiber probe to manufacture a standard curve; the detection time is shortened, the surface plasma detection is realized, the fluorescent mark is not needed, the influences of impurity protein, background fluorescence, photolysis of protein to be detected and photobleaching are avoided, the detection process is simpler and more convenient, and the detection result is more accurate.
Description
Technical Field
The invention belongs to the technical field of parathyroid hormone detection, and particularly relates to a rapid parathyroid hormone detection method.
Background
Parathyroid gland is an endocrine gland of the human body and has the main functions of secreting parathyroid hormone (PTH), mobilizing bone calcium to enter blood, increasing blood calcium level and decreasing blood phosphorus level. The content of PTH is rapidly determined in the operation and compared with the hormone level before the operation, and the treatment is timely adopted, so that the postoperative complications of thyroid cancer can be effectively reduced.
The traditional method for detecting PTH in clinic is mainly a chemiluminescence immunoassay method, the detection needs 4-6 hours, the half-life period of PTH is only 2-4 minutes, the chemiluminescence method mainly detects the fragment of PTH after metabolism, the instant concentration of PTH after the operation cannot be truly reflected,
surface plasmon is an electron wave oscillating back and forth across the surface of a metal film. Such surface waves can be excited by a beam of ordinary white light, which transmits light that loses one color. Even if only a slight amount of foreign matter adheres to the surface of the metal film, the lost color shifts with an increase in the amount of foreign matter. The micro color change is detected to sensitively sense the micro content of foreign matters in the environment, and the micro color change is a chemical molecule detection platform with high sensitivity and low detection limit. If an antibody for detecting a certain protein is previously modified on the surface of the gold film, the protein will bind to the antibody when the gold film is placed in a solution containing the protein, and any other substance in the solution will not bind to the antibody. The protein content can be determined by measuring the color shift of the incident light relative to the color of the protein before binding and comparing the color shift with a calibration value
With the gradual maturity of the optical fiber surface plasma technology, the optical fiber surface plasma technology shows very good detection sensitivity and specificity in protein detection, utilizes a nanogold particle to amplify a detection signal, combines free PTH in plasma by a double-antibody sandwich method, and combines a surface plasma rapid detection technology to develop a method for rapidly detecting PTH in blood.
Disclosure of Invention
The invention aims to provide a parathyroid hormone rapid detection method, which well solves the problems of long PTH detection time and complicated process, can rapidly detect PTH in an operation site, shortens the detection time and realizes the instant detection.
In order to solve the technical problems, the invention is realized by the following technical scheme:
a method for quickly detecting parathyroid hormone includes such steps as,
(1) preparing a nano gold solution: heating 1mL of 1% chloroauric acid and 99mL of deionized water in a container to boil, then adding 4mL of 1% sodium citrate solution, and continuing stirring for 15min under the condition of boiling to obtain a nanogold solution;
(2) preparing DSP into 1nmol concentration by DMSO, adding 10ul into 10ml of nano-gold solution, stirring for 20min to enable DSP molecules to be adsorbed on the surfaces of the gold nanoparticles, and standing overnight at 4 ℃ to obtain a DSP-AUNPS solution;
(3) and adding a proper amount of PTH antibody into the DSP-AuNPs solution, and stirring at room temperature for 20min to form the PTH antibody-DSP-AuNPs compound.
(4) Adopting a surface plasma instrument to carry out wavelength shift measurement and display, wherein the surface plasma instrument is connected with a gold film nanopore SPR chip through an optical fiber probe;
firstly, introducing a PBS solution into a sensing area of a gold membrane nanopore SPR chip, introducing a PTH antibody-DSP-AuNPs compound solution for a period of time after a base line is stable, introducing PTH antigen solutions with the PTH antigen concentrations of 0, 1pmol, 0.01nmol, 0.1nmol, 1nmol, 0.01 μmol, 0.1 μmol and 1 μmol from low to high after a signal base line is stable, and recording a wavelength offset value; making a standard curve;
(5) and (3) detecting the concentration of the PTH antigen to be detected: and (3) introducing the solution to be detected into a sensing area of the gold film nanopore SPR chip, detecting a wavelength shift value, and contrasting the wavelength shift value with a standard curve to obtain the accurate PTH antigen concentration in the solution to be detected.
Further, the diameter of the nano gold particles in the step (1) is about 17 nm.
Further, the gold film nanopore SPR chip comprises a glass substrate, and a gold film is arranged on the detection surface of the glass substrate.
Further, the self-assembled benzoic anhydride is arranged on the plasma sensing surface of the gold film.
The invention has the following beneficial effects:
1, shortening the detection time: the most common PTH detection method at present is a chemiluminescence method, the detection time is 4-6 hours, PTH is easy to degrade, a PTH fragment is mainly determined by a chemiluminescence immunoassay method, and the timely concentration of PTH after surgery cannot be truly reflected.
2, surface plasma detection without fluorescent marking avoids the influence of impurity protein, background fluorescence, photolysis of protein to be detected and photobleaching, so that the detection process is simpler and more convenient, and the detection result is more accurate.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the description of the embodiments will be briefly introduced below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art that other drawings can be obtained according to the drawings without creative efforts.
FIG. 1: the invention relates to a nano gold particle projection electron microscope image.
FIG. 2: schematic structure diagram of gold film nanopore SPR chip
FIG. 3: the invention relates to a wavelength shift graph caused by PTH with different concentrations.
FIG. 4: the PTH of the present invention is plotted over the linear range of 1pM to 1. mu.M.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
In the description of the present invention, it is to be understood that the terms "opening," "upper," "lower," "thickness," "top," "middle," "length," "inner," "peripheral," and the like are used in an orientation or positional relationship that is merely for convenience in describing and simplifying the description, and do not indicate or imply that the referenced component or element must have a particular orientation, be constructed and operated in a particular orientation, and thus should not be considered as limiting the present invention.
A rapid detection method of parathyroid hormone is characterized in that: comprises the following steps of (a) carrying out,
(1) preparing a nano gold solution: heating 1mL of 1% chloroauric acid and 99mL of deionized water in a container to boil, then adding 4mL of 1% sodium citrate solution, and continuing stirring for 15min under the condition of boiling to obtain a nanogold solution; the lower panel is a transmission electron microscope image of synthetic gold nanoparticles, which can be seen to be about 17nm in diameter, as shown in FIG. 2.
(2) Preparing DSP into 1nmol concentration by DMSO, adding 10ul into 10ml of nano-gold solution, stirring for 20min to enable DSP molecules to be adsorbed on the surfaces of the gold nanoparticles, and standing overnight at 4 ℃ to obtain a DSP-AUNPS solution;
wherein DMSO is dimethyl sulfoxide, and DSP is N-hydroxysuccinimide ester. Overnight at 4 ℃ was in a 4 ℃ freezer overnight for adequate binding of DSP and gold nanoparticles.
DMSO is used as an organic solvent, also called a universal solvent, DSP is directly added into the colloidal gold solution and is not easy to dissolve, so the DSP is dissolved by the DMSO firstly and then added into the nanogold solution.
(4) Adding a proper amount of PTH antibody into the DSP-AuNPs solution, and stirring at room temperature for 20min to form a PTH antibody-DSP-AuNPs compound; as shown in fig. 1.
PTH is parathyroid hormone, and DSP-AuNPs solution is nano gold solution adsorbed with N-hydroxysuccinimide ester.
(4) Adopting a surface plasma instrument to carry out wavelength shift measurement and display, wherein the surface plasma instrument is connected with a gold film nanopore SPR chip through an optical fiber probe;
firstly, introducing a PBS solution into a sensing area of a gold membrane nanopore SPR chip, introducing a PTH antibody-DSP-AuNPs compound solution for a period of time (20 minutes) after a base line is stable, introducing PTH antigen solutions with the PTH antigen concentrations of 0, 1pmol, 0.01nmol, 0.1nmol, 1nmol, 0.01 mu mol, 0.1 mu mol and 1 mu mol from low to high after a signal base line is stable, and recording a wavelength offset value; making a standard curve; as shown in fig. 3-4. The PBS solution was phosphate buffered saline.
PBS is a common buffer used to dissolve and dilute standard proteins and samples to be tested, while setting a zero value as a blank.
(5) And (3) detecting the concentration of the PTH antigen to be detected: and (3) introducing the solution to be detected into a sensing area of the gold film nanopore SPR chip, detecting a wavelength shift value, and contrasting the wavelength shift value with a standard curve to obtain the accurate PTH antigen concentration in the solution to be detected.
The gold film nanopore SPR chip comprises a glass substrate, and a gold film is arranged on the detection surface of the glass substrate.
And the self-assembled benzoic anhydride is arranged on the plasma sensing surface of the gold film. For single-layer membrane capture of PTH antibodies.
Hydroxyl on benzoic anhydride can be esterified with carboxyl at the end of PTH antibody protein, thus helping gold membrane capture antibody.
In the description herein, references to the description of "one embodiment," "an example," "a specific example" or the like are intended to mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the invention. In this specification, the schematic representations of the terms used above do not necessarily refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples.
The preferred embodiments of the invention disclosed above are intended to be illustrative only. The preferred embodiments are not intended to be exhaustive or to limit the invention to the precise embodiments disclosed. Obviously, many modifications and variations are possible in light of the above teaching. The embodiments were chosen and described in order to best explain the principles of the invention and the practical application, to thereby enable others skilled in the art to best utilize the invention. The invention is limited only by the claims and their full scope and equivalents.
Claims (4)
1. A rapid detection method of parathyroid hormone is characterized in that: comprises the following steps of (a) carrying out,
(1) preparing a nano gold solution: heating 1mL of 1% chloroauric acid and 99mL of deionized water in a container to boil, then adding 4mL of 1% sodium citrate solution, and continuing stirring for 15min under the condition of boiling to obtain a nanogold solution;
(2) preparing DSP into 1nmol concentration by DMSO, adding 10ul into 10ml of nano-gold solution, stirring for 20min to enable DSP molecules to be adsorbed on the surfaces of the gold nanoparticles, and standing overnight at 4 ℃ to obtain a DSP-AUNPS solution;
(3) adding a proper amount of PTH antibody into the DSP-AuNPs solution, and stirring at room temperature for 20min to form a PTH antibody-DSP-AuNPs compound;
(4) adopting a surface plasma instrument to carry out wavelength shift measurement and display, wherein the surface plasma instrument is connected with a nano gold film SPR chip through an optical fiber probe;
firstly, introducing a PBS solution into a nano gold film SPR chip sensing area, introducing a PTH antibody-DSP-AuNPs compound solution for a period of time after a base line is stable, introducing a PTH antibody-DSP-AuNPs compound solution with PTH antigen concentrations of 0, 1pmol, 0.01nmol, 0.1nmol, 1nmol, 0.01 μmol, 0.1 μmol and 1 μmol from low to high after a signal base line is stable, and recording a wavelength offset value; making a standard curve;
(5) and (3) detecting the concentration of the PTH antigen to be detected: and (3) introducing the solution to be detected into a sensing area of the gold film nanopore SPR chip, detecting a wavelength shift value, and contrasting the wavelength shift value with a standard curve to obtain the accurate PTH antigen concentration in the solution to be detected.
2. The rapid parathyroid hormone detection method according to claim 1, wherein: the diameter of the nano gold particles in the step (1) is about 17 nm.
3. The rapid parathyroid hormone detection method according to claim 1, wherein: the gold film nanopore SPR chip comprises a glass substrate, and a gold film is arranged on the detection surface of the glass substrate.
4. The rapid detection method of parathyroid hormone according to claim 3, wherein: and the self-assembled benzoic anhydride is arranged on the plasma sensing surface of the gold film.
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Citations (7)
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| US20030082179A1 (en) * | 2001-07-03 | 2003-05-01 | Hutchison James Scott | Parathyroid hormone antibodies and related methods |
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| US20140012224A1 (en) * | 2006-04-07 | 2014-01-09 | The Regents Of The University Of California | Targeted hollow gold nanostructures and methods of use |
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- 2021-07-08 CN CN202110772347.8A patent/CN113588602B/en active Active
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| US20030082179A1 (en) * | 2001-07-03 | 2003-05-01 | Hutchison James Scott | Parathyroid hormone antibodies and related methods |
| US20140012224A1 (en) * | 2006-04-07 | 2014-01-09 | The Regents Of The University Of California | Targeted hollow gold nanostructures and methods of use |
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| EP2221603A1 (en) * | 2009-02-18 | 2010-08-25 | Koninklijke Philips Electronics N.V. | Sensing device for detecting a target substance |
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