CN108845120B - Metal ion labeled immunoreaction monopulse detection method - Google Patents
Metal ion labeled immunoreaction monopulse detection method Download PDFInfo
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- 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
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/62—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating the ionisation of gases, e.g. aerosols; by investigating electric discharges, e.g. emission of cathode
- G01N27/626—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating the ionisation of gases, e.g. aerosols; by investigating electric discharges, e.g. emission of cathode using heat to ionise a gas
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/62—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating the ionisation of gases, e.g. aerosols; by investigating electric discharges, e.g. emission of cathode
- G01N27/64—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating the ionisation of gases, e.g. aerosols; by investigating electric discharges, e.g. emission of cathode using wave or particle radiation to ionise a gas, e.g. in an ionisation chamber
Abstract
The invention discloses a single pulse detection method for immunoreaction marked by metal ions, which realizes single pulse signal detection by adopting an inductively coupled plasma or microwave plasma mass spectrometry analysis technology, and further detects pulse signals of the metal ions for marking immunoreaction by adopting a metal nanoparticle marked immunoreaction and further utilizing the inductively coupled plasma or microwave plasma mass spectrometry analysis technology, thereby realizing the aim of indirectly detecting an object to be detected.
Description
Technical Field
The invention belongs to the field of medical examination, but is not limited to the field, and particularly relates to a metal ion labeled immunoreaction monopulse detection method.
Background
Currently, in the immunodiagnosis aspect, radioimmunoassay, enzyme-linked immunosorbent assay, chemiluminescence assay and electrochemiluminescence assay are commonly used clinically; meanwhile, in the field of rapid inspection, a colloidal gold method, a fluorescence immunochromatography method, a new microfluidic chip detection mode and the like are commonly used. However, the radioimmunoassay has radioactive pollution and great harm to human bodies; the enzyme-linked immunosorbent assay and the chemiluminescence assay are complex to operate and long in detection period; the electrochemical detection system is matched with electrode equipment and a precise signal analysis system, and the detection equipment is expensive; the colloidal gold method and the fluorescence immunochromatography method have large batch variation and poor stability of the marker; the microfluidic chip method has not yet been commercially applied.
Mass spectrometry is an analysis method for measuring the mass-to-charge ratio (mass-to-charge ratio) of ions, and its basic principle is to ionize each component in a sample in an ion source to generate charged ions with different charge-to-mass ratios, and the charged ions are accelerated by an electric field to form an ion beam, which enters a mass analyzer. In the mass analyzer, the mass is determined by dispersing the generated opposite velocities by an electric field and a magnetic field, and focusing them to obtain mass spectra.
Inductively coupled plasma mass spectrometry (ICP-MS) is an inorganic element and isotope analysis testing technology developed in the 80 th 20 th century, which combines the high temperature ionization characteristics of inductively coupled plasma with the advantages of sensitive fast scanning of mass spectrometers with a unique interface technology to form a high-sensitivity analysis technology. The plasma used in the ICP-MS instrument is essentially the same as that used in emission spectroscopy except for the azimuth and coil grounding modes. The mass analyzer, ion detector and data acquisition system used was again similar to a quadrupole GC-MS instrument. The mass analyzer mostly adopts a quadrupole mass spectrometer, and also adopts a double-focusing fan-shaped magnetic field mass spectrometer with high resolution, a time-of-flight mass spectrometer and the like. The technology is characterized in that: the sensitivity is high; the speed is high, and the quantitative determination of dozens of elements can be completed within a few minutes; the spectral line is simple, and the interference is less compared with the spectral technology; the linear range can reach 7 to 9 orders of magnitude; the preparation and introduction of the sample is simple relative to other mass spectrometry techniques; the method can be used for element analysis and can also be used for quickly measuring the isotope composition; the measurement precision (RSD) can reach 0.1%.
The microwave plasma torch is a new type of plasma generator proposed and developed by kinkindheim et al. The microwave plasma torch mass spectrum (MPT-MS) comprises a microwave power source, an atomization desolventizing device, a microwave plasma torch and an ion mass analysis device. The sample solution is pumped into the atomization desolventizing device by a peristaltic pump for atomization desolventizing, and the generated dry aerosol enters the torch flame from the central channel of the rectangular tube and is ionized under the action of the microwave power source to generate ions for the mass spectrometer to analyze. The device has the characteristics of low power, gas consumption saving and easy popularization and application of medical detection.
The ICP-MS and MPT-MS mass spectrometry technology has the advantages of wide dynamic linear range, low detection limit, high sensitivity and high measurement precision, and is widely applied to the fields of biology, medicine, environment, food and the like.
Disclosure of Invention
In view of the above, the present invention provides a single pulse detection method for metal ion labeled immunoreaction.
In order to achieve the purpose of the invention, the invention provides a metal ion labeled immunoreaction monopulse detection method, which comprises the following steps:
1) adding a sample containing a substance to be detected, a complete antigen of the substance to be detected marked with one of a pair of substances with specific affinity and an antibody of the substance to be detected marked with small nanoparticles into a reaction tank, incubating for 1-120min to form a first immune complex marked with small nanoparticles and a second immune complex marked with one of the pair of substances with specific affinity at one end and at the other end;
2) then adding the large nano-microsphere marked by the other of the pair of substances with specific affinity into the mixture reacted in the step 1), wherein one of the pair of substances with specific affinity marked at one end of the second immune complex is specifically combined with the other of the pair of substances with specific affinity marked on the surface of the large nano-microsphere to form a large amount of second immune complexes combined on the outer surface of the large nano-microsphere;
3) finally, the mixture reacted in the step 2) is added into a matched instrument, the characteristic metal elements in the small nanoparticles on the large quantity of second immune compounds combined on the outer surface of the large nano microspheres, the characteristic metal elements in the small nanoparticles on the first immune compounds and the characteristic metal elements in the small nanoparticles on one antibody of the object to be detected which does not participate in the reaction can simultaneously generate strong pulse intensity signals, a detector in the matched instrument captures signals of the characteristic metal elements, pulse signals of the characteristic metal elements in the small nanoparticles on the large quantity of second immune compounds combined on the outer surface of the large nano microspheres are obtained by filtering the characteristic metal elements in the small nanoparticles on the first immune compounds and the pulse signals of the characteristic metal elements in the small nanoparticles on one antibody of the object to be detected which does not participate in the reaction, and pulse signals of the characteristic metal elements in the small nanoparticles on the large quantity of second immune compounds combined on the outer surface of the large nano microspheres are obtained The number of the numbers has negative correlation with the content of the substance to be detected, and the content of the substance to be detected can be obtained through standard curve calculation.
In order to achieve the purpose of the invention, the invention also provides another metal ion labeled immunoreaction single pulse detection method, which comprises the following steps:
1) simultaneously adding a sample containing a substance to be detected, an antibody of a pair of substances with specific affinity for marking the substance to be detected, and a small nano-particle marked complete antigen of the substance to be detected into a reaction tank, incubating for 1-120min, wherein the substance to be detected competes with the small nano-particle marked complete antigen of the substance to be detected to react with the antibody of the pair of substances with specific affinity for marking the substance to be detected, so as to form a first immune complex with one end marked with one of the pair of substances with specific affinity and a second immune complex with one end marked with the small nano-particle and the other end marked with one of the pair of substances with specific affinity;
2) adding the large nanosphere labeled by the other of the pair of substances with specific affinity into the reaction pool, wherein one of the pair of substances with specific affinity labeled at one end of the first immune complex is combined with the other of the pair of substances with specific affinity labeled on the surface of the large nanosphere, and one of the pair of substances with specific affinity labeled at one end of the second immune complex is combined with the other of the pair of substances with specific affinity labeled on the surface of the large nanosphere to form a large amount of first immune complexes and a second immune complex labeled with small nanoparticles on the outer surface of the large nanosphere;
3) finally, the mixture reacted in the step 2) is added into a matched instrument, the characteristic metal elements in a large number of small nano-particles on the second immune complex combined with the outer surface of the large nano-microsphere and the characteristic metal elements on the complete antigen of the object to be detected which does not participate in the reaction can generate strong pulse intensity signals at the same time, a detector in the matched instrument captures the pulse signals of the characteristic metal elements, pulse signals of characteristic metal elements in a large number of small nanoparticles on the second immune complex combined with the outer surface of the large nano microsphere are obtained by filtering pulse signals of the characteristic metal elements on the complete antigen of the object to be detected which does not participate in the reaction, the quantity of the pulse signals of the characteristic metal elements in the small nanoparticles on the large number of second immune complex combined with the outer surface of the large nano microsphere has negative correlation with the content of the object to be detected, and the content of the object to be detected can be obtained by calculating through a standard curve.
Further, the matched instrument is an instrument capable of quantitatively detecting metal elements, such as a microwave plasma torch mass spectrometer (MPT-MS) or an inductively coupled plasma mass spectrometer (single quadrupole or triple quadrupole ICP-MS, high-resolution ICP-MS and ICP-TOF-MS); the immune reaction is homogeneous or heterogeneous.
Further, the material of the macro-nano microsphere is alkali metal (Li, Na, K, Rb, Cs Se, francium Fr), alkaline earth metal (Be, Mg, Ca, Sr, Ba, Ra), lanthanide metal (La, Ce, Pr, Nd, Pm, Sm, Eu, Gd, Tb, Dy, Ho, Er, Tm, Yb, Lu), actinide metal (Ac, Th, protactinium Pa, uranium U, Np, plutonium Pu, americium Am, curium Cm, Bk, Cf, Es, Fm, Md, Cd No, ClR), transition metal (Sc, TiTi, V, Cr, Mn, Fe, Co, Ni, Cu, Zn, Y, Nb, Zr, Nb, Ag, Pd, Ag, Pd, Pt, Pd, Pt, Pd, Ta, Tb, Dy, Re, Te, Pt, and Co, Pt, Main group metals (aluminum Al, gallium Ga, indium In, tin Sn, thallium Tl, lead Pb, bismuth Bi, Uut, Uuq, Uup, Uuh), metalloids (boron B, silicon Si, germanium Ge, arsenic As, antimony Sb, tellurium Te, polonium Po), and the like include, but are not limited to, the above-mentioned metal microspheres, or nanoparticles of the above-mentioned metal compounds, or silica, or polyethylene, polystyrene, polyvinyl fluoride, silicone, melamine, polyvinyl chloride, polylactic acid, epoxy resin, phenol resin, polyester, polyacrylonitrile, polyacrylic acid, polyamide, chitosan, cellulose, polyaniline, polyacetylene, poly (L-glutamic acid), polyimide, polypyrrole, β -cyclodextrin polymer microspheres, and the like, including but not limited to the above-mentioned polymer microspheres, or microspheres of core/shell structure or doped structure formed by any two or more of the above substances; and the particle size of the large nano-microsphere is 100 nm-100 mu m.
Further, the material of the small nanoparticles is alkali metal (Li, Na, K, Rb, Cs Se, francium Fr), alkaline earth metal (Be, Mg, Ca, Sr, Ba, Ra), lanthanide metal (La, Ce, Pr, Nd, Pm, Sm, Eu, Gd, Tb, Dy, Ho, Er, Tm, Yb, Lu), actinide metal (Ac, Th, Pm Pa, U, Np, Pu, Am, Cm, Bk, F, Es, Fm, Md, Cd, Lr), transition metal (Sc, Ti, V, Cr, Mn, Fe, Co, Ni, Cu, Zn, Y, Nb, Zr, Nb, Ag, Pd, Ag, Pd, Pt, Pd, Pt, Pd, Ta, Tb, Dy, Rh, Pt, and Pt, and Pt, and Co, Pt, and Co, Fe, Pt, Co, Fe, Cu, Fe, Pt, Fe, Cu, Pt, Cu, Pt, Ti, Cu, Ti, Pt, Ti, Cu, Ti, Cu, Ti, main group metals (aluminum Al, gallium Ga, indium In, tin Sn, thallium Tl, lead Pb, bismuth Bi, Uut, Uuq, Uup, Uuh), metalloids (boron B, silicon Si, germanium Ge, arsenic As, antimony Sb, tellurium Te, polonium Po), and the like include, but are not limited to, the above-described metal microspheres, or nanoparticles of the above-described metal compounds, or nanoparticles of a core/shell structure or a doped structure formed of the above-described metal materials and silica or polymers, or nanoparticles of a core/shell structure or a doped structure formed of the above-described metal compounds and silica or polymers; and the particle size of the small nano-particles is 0.1 nm-800 nm.
Further, the metal elements in the large nano-microspheres and the small nano-particles are not the same metal at the same time; and the particle size of the large nano-microsphere is larger than that of the small nano-particle.
Further, wherein the pair of substances having specific affinity is biotin and streptavidin, biotin and avidin, fluorescein and anti-fluorescein, an antibody, and a secondary antibody specifically binding to the antibody;
the connection mode between the pair of substances with specific affinity, the large nano-microspheres and the complete antigen of the object to be detected is chemical coupling or physical adsorption; the connection mode of the small nano-particles and an antibody of the object to be detected is chemical coupling or physical adsorption.
Further, the complete antigen of the analyte is directly connected to the surface of the large nanoparticle in a chemical coupling or physical adsorption manner, and is not connected by a pair of substances with specific affinity;
the small nano-particles are connected with an antibody of the substance to be detected through the bridge action of a pair of substances with specific affinity, the connection mode between one of the pair of substances with specific affinity and the small nano-particles is chemical coupling or physical adsorption, and the connection mode between the other of the pair of substances with specific affinity and the antibody of the substance to be detected is chemical coupling or physical adsorption.
Further, a step of washing the mixture after the reaction in the step 2) with a washing solution to remove the small nanoparticle-labeled first immune complex and one antibody of the unreacted small nanoparticle-labeled analyte, and then dispersing the mixture with a citric acid-sodium citrate buffer solution is further included between the step 2) and the step 3); in the step 3), the process of filtering out the pulse signals of the characteristic metal elements in the small nanoparticles on the first immune complex and the characteristic metal elements in the small nanoparticles on one antibody of the unreacted analyte is omitted.
Further, the washing adopts a centrifugal separation mode or a standing precipitation mode; or when the material of the large nano-microspheres is magnetic Fe3O4、γ-Fe2O3Pt, Ni or Co microspheres, or Fe which is magnetic3O4、γ-Fe2O3And Pt, Ni or Co and inorganic matter or organic matter to form core/shell structure or doped structure microsphere, and magnetic separation mode is adopted for washing
The invention is based on homogeneous/heterogeneous reaction, collects the pulse signal of the metal nano-particles by combining mass spectrum, converts the pulse signal into the content of the object to be detected, and has the advantages of high detection speed and stable, accurate and reliable detection result.
The invention has the following beneficial effects:
1. the invention realizes the single pulse signal detection method by adopting the inductively coupled plasma or microwave plasma mass spectrometry, and further detects the pulse signal of the metal ions for marking the immunoreaction by adopting the metal nanoparticle marking immunoreaction and the inductively coupled plasma or microwave plasma mass spectrometry, thereby realizing the aim of indirectly detecting the object to be detected;
2. the inductively coupled plasma or microwave plasma mass spectrometry adopted by the invention has the advantages of wide linear detection range, low detection line, high detection speed, high sensitivity, high measurement precision and the like, and expands the application of the inductively coupled plasma or microwave plasma mass spectrometry in the field of medical inspection;
3. the invention relates to a method for directly detecting single pulse signals without washing, which adopts a single pulse signal analysis means to identify pulse signals of characteristic metal elements in small nano particles and further filters the characteristic signals of the metal elements on free substances in a system to obtain signals of the characteristic metal elements in the small nano particles on immune complexes combined on the surfaces of large nano microspheres, thereby saving the washing process, improving the signal-to-noise ratio and improving the detection sensitivity of the system.
4. According to the method for detecting the washed single pulse signal, the free substances in the system are removed in a washing mode, the signal of the characteristic metal element in the small nano-particles on the immune complex combined on the surface of the large nano-microsphere is identified by a single pulse signal analysis means, the interference generated by the pulse signal of the characteristic metal element in the free substances is reduced, and the sensitivity is improved.
Drawings
FIG. 1 is a schematic diagram of a single-pulse metal ion labeled immunoreaction detection method of the present invention, wherein 1-an analyte, 2-an antibody of the analyte labeled with small nanoparticles, 3-a complete antigen of the analyte labeled with one of a pair of substances having specific affinity, 4-a first immune complex, and 5-a second immune complex; 6-the other marked large nano microsphere in the pair of substances with specific affinity, and 7-the outer surface of the large nano microsphere is combined with a large amount of second immune complexes;
FIG. 2 shows FT obtained by directly detecting the mixture after the reaction in step 2)3A standard curve of (a);
FIG. 3 shows FT detected after washing the mixture after the reaction of step 2)3The standard curve of (2).
Detailed Description
The present invention is explained below with reference to examples, which are merely illustrative of the present invention. Unless otherwise specified, the technical means used in the present invention are well known to those skilled in the art. In addition, the embodiments should be considered illustrative, and not restrictive, of the scope of the invention, which is defined solely by the claims. It will be apparent to those skilled in the art that various changes or modifications in the components and amounts of the materials used in these embodiments can be made without departing from the spirit and scope of the invention.
As shown in FIG. 1, the invention provides a single pulse detection method of metal ion labeled immunoreaction, comprising the following steps:
1) adding a sample containing a substance to be detected 1, a substance complete antigen 3 marked by one of a pair of substances with specific affinity and an antibody 2 of the substance to be detected marked by small nanoparticles into a reaction tank, incubating for 5-60min to form a first immune complex 4 marked by the small nanoparticles and a second immune complex 5 marked by one of a pair of substances with specific affinity at one end;
2) then adding the large nanosphere 6 marked by the other of the pair of substances with specific affinity into the mixture reacted in the step 1), wherein one of the pair of substances with specific affinity marked at one end of the second immune complex is specifically combined with the other of the pair of substances with specific affinity marked on the surface of the large nanosphere to form a large amount of second immune complexes 7 on the outer surface of the large nanosphere;
3) finally, the mixture reacted in the step 2) is added into a matched instrument, the characteristic metal elements in the small nanoparticles on the large quantity of second immune compounds combined on the outer surface of the large nano microspheres, the characteristic metal elements in the small nanoparticles on the first immune compounds and the characteristic metal elements in the small nanoparticles on one antibody of the object to be detected which does not participate in the reaction can simultaneously generate strong pulse intensity signals, a detector in the matched instrument captures signals of the characteristic metal elements, pulse signals of the characteristic metal elements in the small nanoparticles on the large quantity of second immune compounds combined on the outer surface of the large nano microspheres are obtained by filtering the characteristic metal elements in the small nanoparticles on the first immune compounds and the pulse signals of the characteristic metal elements in the small nanoparticles on one antibody of the object to be detected which does not participate in the reaction, and pulse signals of the characteristic metal elements in the small nanoparticles on the large quantity of second immune compounds combined on the outer surface of the large nano microspheres are obtained The number of the numbers has negative correlation with the content of the substance to be detected, and the content of the substance to be detected can be obtained through standard curve calculation.
The invention also provides another immunoreaction monopulse detection method marked by metal ions, which comprises the following steps:
1) simultaneously adding a sample containing a substance to be detected, an antibody of a pair of substances with specific affinity for marking the substance to be detected, and a small nanoparticle marked complete antigen of the substance to be detected into a reaction tank, incubating for 5-60min, wherein the substance to be detected competes with the small nanoparticle marked complete antigen of the substance to be detected to react with the antibody of the pair of substances with specific affinity for marking the substance to be detected, so as to form a first immune complex with one end marked with one of the pair of substances with specific affinity and a second immune complex with one end marked with the small nanoparticle and the other end marked with one of the pair of substances with specific affinity;
2) adding the large nanosphere labeled by the other of the pair of substances with specific affinity into the reaction pool, wherein one of the pair of substances with specific affinity labeled at one end of the first immune complex is combined with the other of the pair of substances with specific affinity labeled on the surface of the large nanosphere, and one of the pair of substances with specific affinity labeled at one end of the second immune complex is combined with the other of the pair of substances with specific affinity labeled on the surface of the large nanosphere to form a large amount of first immune complexes and a second immune complex labeled with small nanoparticles on the outer surface of the large nanosphere;
3) finally, the mixture reacted in the step 2) is added into a matched instrument, the characteristic metal elements in a large number of small nano-particles on the second immune complex combined with the outer surface of the large nano-microsphere and the characteristic metal elements on the complete antigen of the object to be detected which does not participate in the reaction can generate strong pulse intensity signals at the same time, a detector in the matched instrument captures the pulse signals of the characteristic metal elements, pulse signals of characteristic metal elements in a large number of small nanoparticles on the second immune complex combined with the outer surface of the large nano microsphere are obtained by filtering pulse signals of the characteristic metal elements on the complete antigen of the object to be detected which does not participate in the reaction, the quantity of the pulse signals of the characteristic metal elements in the small nanoparticles on the large number of second immune complex combined with the outer surface of the large nano microsphere has negative correlation with the content of the object to be detected, and the content of the object to be detected can be obtained by calculating through a standard curve.
The matched instrument can be a microwave plasma torch mass spectrometer (MPT-MS), an inductively coupled plasma mass spectrometer (a single quadrupole rod or triple quadrupole rod ICP-MS, a high-resolution ICP-MS, an ICP-TOF-MS) and other instruments capable of quantitatively detecting metal elements; the immune reaction is homogeneous or heterogeneous.
Wherein the macro-nano-microsphere is made of alkali metals (Li, Na, K, Rb, Cs Se, francium Fr), alkaline earth metals (Be, Mg, Ca, Sr, Ba, Ra), lanthanide metals (La, Ce, Pr, Nd, Pm, Sm, Eu, Gd, Tb, Dy, Ho, Er, Tm, Yb, Lu), actinide metals (Ac, Th, Pa, U, Np, Pu, Amur, Cm, Bk, Cf, Li Es, Fm, Md, No, Lr), transition metals (Sc, Ti, V, Cr, Mn, Fe, Co, Ni, Cu, Zn, Y, Mo, Nb, W, Ag, Re, Au, Ag, Pd, Pt, Pd, Pt, Pd, Pt, Pd, Pt, Gallium Ga, indium In, tin Sn, thallium Tl, lead Pb, bismuth Bi, Uut, Uuq, Uup, Uuh), metalloids (boron B, silicon Si, germanium Ge, arsenic As, antimony Sb, tellurium Te, polonium Po), and the like include, but are not limited to, the above-mentioned metal microspheres, or nanoparticles of the above-mentioned metal compounds, or silica, or polyethylene, polystyrene, polyvinyl fluoride, silicone, melamine, polyvinyl chloride, polylactic acid, epoxy resin, phenol resin, polyester, polyacrylonitrile, polyacrylic acid, polyamide, chitosan, cellulose, polyaniline, polyacetylene, poly (L-glutamic acid), polyimide, polypyrrole, β -cyclodextrin polymer microspheres, and the like, including but not limited to the above-mentioned polymer microspheres, or microspheres of core/shell structure or doped structure formed by any two or more of the above substances; and the particle size of the large nano-microsphere is 100 nm-100 mu m.
Wherein the small nanoparticles are made of alkali metals (Li, Na, K, Rb, Se, francium Fr), alkaline earth metals (Be, Mg, Ca, Sr, Ba, Ra), lanthanide metals (La, Ce, Pr, Nd, Pm, Sm, Eu, Gd, Tb, Dy, Ho, Er, Tm, Yb, Lu), actinides (Ac, Th, Pa, U, Np, Pu, Amur, Cm, Bk, Cf, Li Es, Fm, Md, No, Lr), transition metals (Sc, Ti, V, Cr, Mn, Fe, Co, Ni, Cu, Zn, Y, Mo, Nb, W, Ag, Re, Ag, Pd, Al, Pd, Pt, Pd, Pt, Pd, Pt, gallium Ga, indium In, tin Sn, thallium Tl, lead Pb, bismuth Bi, Uut, Uuq, Uup, Uuh), metalloids (boron B, silicon Si, germanium Ge, arsenic As, antimony Sb, tellurium Te, polonium Po), and the like including but not limited to the above metal microspheres, or nanoparticles of the above metal compound, or nanoparticles of a core/shell structure or a doped structure formed by the above metal material and silica or a polymer, or nanoparticles of a core/shell structure or a doped structure formed by the above metal compound and silica or a polymer; and the particle size of the small nano-particles is 0.1 nm-800 nm.
Wherein the metal elements in the large nano-microspheres and the small nano-particles are not the same metal at the same time; and the particle size of the large nano-microsphere is larger than that of the small nano-particle.
Wherein the pair of substances with specific affinity is biotin and streptavidin, biotin and avidin, fluorescein and anti-fluorescein, an antibody and a second antibody specifically binding to the antibody;
the connection mode between the pair of substances with specific affinity, the large nano-microspheres and the complete antigen of the object to be detected is chemical coupling or physical adsorption; the connection mode of the small nano-particles and an antibody of the object to be detected is chemical coupling or physical adsorption.
The complete antigen of the object to be detected can be directly connected to the surface of the large nano microsphere in a chemical coupling or physical adsorption mode without being connected by a pair of substances with specific affinity;
the small nano-particles are connected with an antibody of the substance to be detected through the bridge action of a pair of substances with specific affinity, the connection mode between one of the pair of substances with specific affinity and the small nano-particles is chemical coupling or physical adsorption, and the connection mode between the other one of the pair of substances with specific affinity and the antibody of the substance to be detected is chemical coupling or physical adsorption.
The method also comprises the steps of washing the mixture reacted in the step 2) by using a washing solution to remove the first immune complex marked by the small nanoparticles and an antibody of the analyte marked by the unreacted small nanoparticles, and then dispersing the mixture by using a citric acid-sodium citrate buffer solution, wherein the step of filtering pulse signals of characteristic metal elements in the small nanoparticles on the first immune complex and the characteristic metal elements in the small nanoparticles on the antibody of the analyte not participating in the reaction is omitted in the step 3).
Wherein the washing adopts a centrifugal separation or standing precipitation mode; or when the material of the large nano-microspheres is magnetic Fe3O4、γ-Fe2O3Pt, Ni or Co microspheres, or Fe which is magnetic3O4、γ-Fe2O3And when Pt, Ni or Co and inorganic matter or organic matter form the microsphere with core/shell structure or doped structure, then the washing adopts the magnetic separation mode.
The present invention will be further described with reference to specific examples.
Example 1 (No-Wash Single pulse Competition method)
1, preparing Pt nano particles;
0.15g of polyvinylpyrrolidone (PVP) and 33.9mg of chloroplatinic acid (H) were sequentially added2PtCl6.6H2O), 20mL of ultrapure water and 20mL of ethylene glycol are added into a reaction vessel, and the mixture is refluxed and reacted for 3 hours at the temperature of 60 ℃ at the rotating speed of 240r/min to obtain a brownish black Pt nano particle colloidal solution.
2. Streptavidin labeled SiO2Microspheres
2mL of 2.5% w/v SiO2Microspheres (particle size 1 μm, sienna millennium biotechnology limited) were washed 2 times with PBS buffer (pH 7.4) and resuspended in 10mL of PBS buffer; continuously adding 5mg of EDC and 5mg of NHS into the washed magnetic microsphere solution, and activating for 1h under stirring; and washing with PBS buffer solution, then suspending in 10mL of PBS buffer solution, adding the blocking solution continuously, blocking for 30min, washing with PBS buffer solution, and then suspending in 10mL of PBS buffer solution for later use.
3. Biotin-labeled Free Triiodothyronine (FT)3) Complete antigen
Firstly, a strain FT is treated by sodium carbonate buffer solution3Diluting the complete antigen to 1mg/mL, stirring with sodium carbonate buffer solution at room temperature (25 +/-5 ℃) for 4 hours in the dark, and dialyzing; 6-Aminohexanoic acid-N-hydroxysuccinimide-Biotin (BCNHS) was then formulated with N, N-Dimethylamide (DMF) to 1 mg/mL; one FT in 1mL3Adding 125-66.7 microliter DMF solution into complete antigen solution, mixing in glass bottle, stirring at room temperature (25 +/-5 deg.C) in dark2 hours; adding 9.6 mu L of 1mol/L ammonium chloride solution, and stirring for 10 minutes at room temperature (25 +/-5 ℃) in the dark; the mixed solution was transferred to a dialysis bag and dialyzed overnight at 4 ℃ against phosphate buffer. And finally taking out and adding the same amount of glycerol to the mixture, and preserving the mixture at the temperature of 20 ℃.
Pt nanoparticle-labeled mouse anti-human FT3Monoclonal antibodies
First, another mouse was treated with anti-human FT using sodium carbonate buffer3Diluting the monoclonal antibody to 1mg/mL, stirring with sodium carbonate buffer solution at room temperature (25 +/-5 ℃) for 4 hours in the dark, and dialyzing; 1mL of another mouse anti-human FT was added to 5mL of the prepared Pt nanoparticle colloidal solution3The monoclonal antibody solution is magnetically stirred for 30min at 25 +/-5 ℃ in the dark, and then the mixed solution is transferred into a dialysis bag and dialyzed overnight at 4 ℃ by using a phosphate buffer. And finally taking out and adding the same amount of glycerol to the mixture, and preserving the mixture at the temperature of 20 ℃.
5. No-wash single-pulse competition method detection process
(1) Taking 10 mu L of sample of the substance to be detected and 10 mu L of biotin-labeled strain FT3Complete antigen and 10 mu L Pt nano particle labeled mouse anti-human FT3Monoclonal antibody, performing competitive reaction for 30min at 37 deg.C, and detecting FT in sample3Mouse anti-human FT labeled with Pt nanoparticles3Immunoreactive binding of monoclonal antibodies to form primary FT3Immune complex, biotin-labeled FT3Complete antigen and Pt nanoparticle labeled mouse anti-human FT3Immunoreaction of the monoclonal antibody to form a second FT3An immune complex;
(2) continuously adding streptavidin marked SiO into the reaction tank2Microsphere, biotin labeled at one end of the second immunocomplex and SiO2Specific binding of streptavidin on the surface of the microsphere occurs to form SiO2Second FT with a large number of Pt nano particles bonded on the outer surface of the microsphere3Immune complex, first FT3Immune complexes do not participate in the response;
(3) detecting the reacted mixture in the step (2) by using ICP-MS, and detecting a large number of second FTs bound on the outer surface of the large nanospheres3On immune complexesPt nanoparticles, first FT3Pt nanoparticles on immune complexes and unreacted one strain of FT3The Pt nanoparticles on the complete antigen will simultaneously generate a strong pulse signal, the detector in the ICP-MS instrument captures the Pt metal element signal, and the first FT is filtered3Pt nanoparticles on immune complexes and unreacted one strain of FT3Pulse signal of Pt nano-particles on complete antigen obtains a large number of second FTs combined on the outer surface of the large nano-microsphere3Pulse signal of Pt nanoparticles on immune complexes, bulk second FT bound to the outer surface of the large nanospheres3Quantity of Pt nanoparticle pulse signal on immune complex and FT in sample3Has a negative correlation, and FT can be obtained by calculating a standard curve3The content of (a).
6. Establishment of a Standard Curve
FT was prepared at concentrations of 0, 1.8, 4.5, 7.5, 12, 40pg/mL3Calibrant for creating FT3The detection sensitivity of the standard curve is 1.8pg/mL, the detection range is 1.8-40 pg/mL, the detection result is shown in Table 1, and the standard curve is shown in FIG. 2.
TABLE 1
| FT3Calibrator (pg/mL) | 0 | 1.8 | 4.5 | 7.5 | 12 | 40 |
| Number of Pt pulse signals (cps) | 101564 | 55248 | 21380 | 13422 | 7125 | 2342 |
Example 2 (Wash Single pulse Competition method)
1. The same procedure as in "preparation of Pt nanoparticles" of example 1;
2. streptavidin-labeled SiO as in 2 of example 12The process of microsphere is the same;
3. biotin-labeled mouse anti-human FT3Monoclonal antibodies:
firstly, a rat anti-human FT is treated by sodium carbonate buffer solution3Diluting the monoclonal antibody to 1mg/mL, stirring with sodium carbonate buffer solution at room temperature (25 +/-5 ℃) for 4 hours in the dark, and dialyzing; 6-Aminohexanoic acid-N-hydroxysuccinimide-Biotin (BCNHS) was then formulated with N, N-Dimethylamide (DMF) to 1 mg/mL; one mouse in 1mL is used for resisting human FT3Adding 125-66.7 microliter of the DMF solution into the monoclonal antibody solution, mixing in a glass bottle, and stirring at room temperature (25 +/-5 ℃) for 2 hours in the dark; adding 9.6 mu L of 1mol/L ammonium chloride solution, and stirring for 10 minutes at room temperature (25 +/-5 ℃) in the dark; the mixed solution was transferred to a dialysis bag and dialyzed overnight at 4 ℃ against phosphate buffer. And finally taking out and adding the same amount of glycerol to the mixture, and preserving the mixture at the temperature of 20 ℃.
Pt nanoparticle labeled one strain of FT3Complete antigen:
firstly, using sodium carbonate buffer solution to make another strain FT3Diluting the complete antigen to 1mg/mL, stirring with sodium carbonate buffer solution at room temperature (25 +/-5 ℃) for 4 hours in the dark, and dialyzing; to 5mL of the prepared Pt nanoparticle solution was added 1mL of another FT3Complete antigen solution, magnetic force shielding at 25 +/-5 deg.CStirring for 30min, transferring the mixed solution into a dialysis bag, and dialyzing with phosphate buffer at 4 ℃ overnight. And finally taking out and adding the same amount of glycerol to the mixture, and preserving the mixture at the temperature of 20 ℃.
5. And (3) detection process:
(1) taking 10 mu L of sample of the substance to be detected and 10 mu L of biotin-labeled anti-mouse anti-human FT3Monoclonal antibody and 10 mu L Pt nanoparticle labeled FT3Complete antigen, competitive reaction at 37 deg.C for 30min, and FT in the sample to be tested3Mouse anti-human FT labeled with biotin3Immunoreactive binding of monoclonal antibodies to form primary FT3Immune complex, Pt nanoparticle labeled one strain of FT3Complete antigen and biotin-labeled mouse anti-human FT3Immunoreactive binding of the monoclonal antibody to form a second FT3An immune complex;
(2) continuously adding streptavidin marked SiO into the reaction tank2Microspheres, labelled at first FT3Biotin labeled at one end of immune complex and labeled in second FT3Biotin at one end of immune complex is respectively marked on SiO2Specific binding of streptavidin on the surface of the microsphere occurs to form SiO2The microspheres have a plurality of first FTs bonded to the outer surface thereof3Immune complex and second FT labeled with Pt nanoparticle3An immune complex;
(3) washing the mixture reacted in the step 2) by a centrifugal separation mode to remove a strain of FT marked by unreacted Pt nano particles3Complete antigen, then dispersed in LM buffer;
(4) the material dispersed in LM buffer was detected using ICP-MS, SiO2The exterior surface of the microspheres having incorporated therein a plurality of second FTs3The Pt nano particles on the immune complex can simultaneously generate a strong pulse signal, and a detector in the ICP-MS captures the pulse signal of the Pt nano particles, namely SiO2The exterior surface of the microspheres having incorporated therein a plurality of second FTs3Quantity of Pt nanoparticle pulse signal on immune complex and FT in sample3Has a negative correlation, and FT can be obtained by calculating a standard curve3The content of (a).
6. Establishment of a Standard Curve
FT was prepared at concentrations of 0, 1.8, 4.5, 7.5, 12, 40pg/mL3Calibrant for creating FT3The detection sensitivity of the standard curve is 1.8pg/mL, the detection range is 1.8-40 pg/mL, the detection result is shown in Table 2, and the standard curve is shown in FIG. 3.
TABLE 2
| FT3Calibrator (pg/mL) | 0 | 1.8 | 4.5 | 7.5 | 12 | 40 |
| Number of Pt pulse signals (cps) | 201564 | 130358 | 44915 | 31983 | 17153 | 4598 |
(1) PBS buffer solution
(2) Citric acid-sodium citrate buffer (LM)
(3) Sealing liquid
(4) Sodium Carbonate Buffer (CB)
Sodium carbonate 4.33g
Sodium bicarbonate 2.96g
The purified water is fixed to the volume of 1000 mL;
(5) phosphoric acid buffer solution (PB)
Sodium dihydrogen phosphate 0.99g
Disodium hydrogen phosphate 5.16g
The purified water is fixed to the volume of 1000 mL;
(6) cleaning liquid
Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.
Claims (10)
1. A metal ion labeled immunoreaction monopulse detection method is characterized by comprising the following steps:
1) adding a sample containing a substance to be detected, a complete antigen of the substance to be detected marked with one of a pair of substances with specific affinity and an antibody of the substance to be detected marked with small nanoparticles into a reaction tank, and incubating for 1-120min to form a first immune complex marked with small nanoparticles and a second immune complex marked with one of the pair of substances with specific affinity at one end and at the other end;
2) then adding the large nanospheres marked by the other one of the pair of substances with specific affinity into the mixture reacted in the step 1), wherein one of the pair of substances with specific affinity marked at one end of the second immune complex is specifically combined with the other one of the pair of substances with specific affinity marked on the surface of the large nanospheres to form a large amount of second immune complexes in the step 1) on the outer surface of the large nanospheres;
3) finally, the mixture reacted in the step 2) is directly added into a matched instrument, the characteristic metal elements in the small nanoparticles on the large quantity of second immune complexes combined with the outer surface of the large nanoparticles, the characteristic metal elements in the small nanoparticles on the first immune complexes and the characteristic metal elements in the small nanoparticles on one antibody of the object to be detected which does not participate in the reaction can simultaneously generate strong pulse intensity signals, a detector in the matched instrument captures pulse signals of the characteristic metal elements, pulse signals of the characteristic metal elements in the small nanoparticles on the large quantity of second immune complexes combined with the outer surface of the large nanoparticles are obtained by filtering the characteristic metal elements in the small nanoparticles on the first immune complexes and the pulse signals of the characteristic metal elements in the small nanoparticles on one antibody of the object to be detected which does not participate in the reaction, and the characteristic metal elements in the small nanoparticles on the large quantity of second immune complexes combined with the outer surface of the large nanoparticles The number of the element pulse signals has negative correlation with the content of the object to be detected, and the content of the object to be detected can be obtained through standard curve calculation;
the material of the large nano-microsphere comprises alkali metal, alkaline earth metal, lanthanide series metal, actinide series metal, transition metal, main group metal or metalloid but is not limited to the metal microsphere, or is a nano-microsphere of the metal compound, or is silicon dioxide, or comprises polyethylene, polystyrene, polyvinyl fluoride, organic silicon, melamine, polyvinyl chloride, polylactic acid, epoxy resin, phenolic resin, polyesters, polyacrylonitrile, polyacrylic acid, polyamides, chitosan, celluloses, polyaniline, polyacetylenes, poly (L-glutamic acid), polyimide, polypyrrole and beta-cyclodextrin polymer microspheres but is not limited to the polymer microsphere, or is a microsphere with a core/shell structure or a doped structure formed by any two or more of the substances; the particle size of the large nano-microspheres is 100 nm-100 mu m;
the small nano particles are made of Pt; the particle size of the small nano-particles is 0.1 nm-800 nm;
the metal elements in the large nano-microspheres and the small nano-particles are not the same metal at the same time; and the particle size of the large nano-microsphere is larger than that of the small nano-particle.
2. The method for detecting the metal ion-labeled immunoreaction monopulse according to claim 1, wherein the matched instrument is a microwave plasma torch mass spectrometer or an inductive coupling plasma mass spectrometer; the immune reaction is homogeneous or heterogeneous.
3. The method of claim 2, wherein the alkali metal is lithium Li, sodium Na, potassium K, rubidium Rb, cesium Se, or francium Fr; the alkaline earth metal is beryllium Be, magnesium Mg, calcium Ca, strontium Sr, barium Ba or radium Ra; the lanthanide metal is lanthanum La, cerium Ce, praseodymium Pr, neodymium Nd, promethium Pm, samarium Sm, europium Eu, gadolinium Gd, terbium Tb, dysprosium Dy, holmium Ho, erbium Er, thulium Tm, ytterbium Yb or lutetium Lu; the actinide metal is actinium Ac, thorium Th, protactinium Pa, uranium U, neptunium Np, plutonium Pu, americium Am, curium Cm, berkelium Bk, californium Cf, einsteine Es, fermium Fm, mendelevium Md, nobelium No, or lawrencium Lr; the transition metal is scandium Sc, titanium Ti, vanadium V, chromium Cr, manganese Mn, iron Fe, cobalt Co, nickel Ni, copper Cu, zinc Zn, yttrium Y, zirconium Zr, niobium Nb, molybdenum Mo, technetium Tc, ruthenium Ru, rhodium Rh, palladium Pd, silver Ag, cadmium Cd, hafnium Hf, tantalum Ta, tungsten W, rhenium Re, osmium Os, iridium Ir, platinum Pt, gold Au or mercury Hg; the main group metal is aluminum Al, gallium Ga, indium In, tin Sn, thallium Tl, lead Pb, bismuth Bi, Uut, Uuq, Uup or Uuh; the metalloid is boron B, silicon Si, germanium Ge, arsenic As, antimony Sb, tellurium Te or polonium Po.
4. The method of claim 1, wherein the alkali metal is lithium Li, sodium Na, potassium K, rubidium Rb, cesium Se, or francium Fr; the alkaline earth metal is beryllium Be, magnesium Mg, calcium Ca, strontium Sr, barium Ba or radium Ra; the lanthanide metal is lanthanum La, cerium Ce, praseodymium Pr, neodymium Nd, promethium Pm, samarium Sm, europium Eu, gadolinium Gd, terbium Tb, dysprosium Dy, holmium Ho, erbium Er, thulium Tm, ytterbium Yb or lutetium Lu; the actinide metal is actinium Ac, thorium Th, protactinium Pa, uranium U, neptunium Np, plutonium Pu, americium Am, curium Cm, berkelium Bk, californium Cf, einsteine Es, fermium Fm, mendelevium Md, nobelium No, or lawrencium Lr; the transition metal is scandium Sc, titanium Ti, vanadium V, chromium Cr, manganese Mn, iron Fe, cobalt Co, nickel Ni, copper Cu, zinc Zn, yttrium Y, zirconium Zr, niobium Nb, molybdenum Mo, technetium Tc, ruthenium Ru, rhodium Rh, palladium Pd, silver Ag, cadmium Cd, hafnium Hf, tantalum Ta, tungsten W, rhenium Re, osmium Os, iridium Ir, platinum Pt, gold Au or mercury Hg; the main group metal is aluminum Al, gallium Ga, indium In, tin Sn, thallium Tl, lead Pb, bismuth Bi, Uut, Uuq, Uup or Uuh; the metalloid is boron B, silicon Si, germanium Ge, arsenic As, antimony Sb, tellurium Te or polonium Po.
5. The method of claim 1, wherein the pair of substances with specific affinity is biotin and streptavidin, biotin and avidin, fluorescein and anti-fluorescein, an antibody, and a secondary antibody that specifically binds to the antibody;
the connection mode between the pair of substances with specific affinity, the large nano-microspheres and the complete antigen of the object to be detected is chemical coupling or physical adsorption; the connection mode of the small nano-particles and an antibody of the object to be detected is chemical coupling or physical adsorption.
6. The method for detecting the metal ion labeled immunoreaction monopulse according to claim 1, wherein the complete antigen of the analyte is directly connected to the surface of the large nanosphere by means of chemical coupling or physical adsorption without being connected by a pair of substances with specific affinity;
the small nano-particles are connected with an antibody of the substance to be detected through the bridge action of a pair of substances with specific affinity, the connection mode between one of the pair of substances with specific affinity and the small nano-particles is chemical coupling or physical adsorption, and the connection mode between the other one of the pair of substances with specific affinity and the antibody of the substance to be detected is chemical coupling or physical adsorption.
7. The method of claim 1, wherein the particle size of the large nanoparticle is 100nm to 100 μm and the particle size of the small nanoparticle is 0.1 nm.
8. The method of claim 1, wherein the small nanoparticles have a particle size of 0.1-800 nm and the large nanoparticles have a particle size of 100 μm.
9. The method for detecting metal ion labeled immunoreaction monopulse according to any one of claims 1 to 8, further comprising the step of washing the mixture after the reaction of step 2) with a washing solution to remove the small nanoparticle labeled first immune complex and an antibody of the unreacted small nanoparticle labeled test substance, and then dispersing the mixture with a citric acid-sodium citrate buffer solution between step 2) and step 3); in the step 3), a process of filtering pulse signals of the characteristic metal elements in the small nanoparticles on the first immune complex and the characteristic metal elements in the small nanoparticles on one antibody of the unreacted analyte is omitted.
10. The method of claim 9, wherein the washing is performed by centrifugationA separation or standing precipitation mode; or when the material of the large nano-microspheres is magnetic Fe3O4、γ-Fe2O3Pt, Ni or Co microspheres, or Fe which is magnetic3O4、γ-Fe2O3And when Pt, Ni or Co and inorganic matter or organic matter form the microsphere with core/shell structure or doped structure, then the washing adopts the magnetic separation mode.
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| Detection of HIV-1 p24 antigen using streptavidin –biotin and gold nanoparticles based immunoassay by inductively coupled plasma mass spectrometry;Qian He等;《J. Anal. At. Spectrom》;20140522;第29卷;第1477–1482页 * |
| Gold nanoparticle-based inductively coupled plasma mass spectrometry amplification and magnetic separation for the sensitive detection of a virus-specific RNA sequence;I-Hsiang Hsu等;《Journal of Chromatography A》;20110302;第1795–1801页 * |
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