CN108828212B - Immunoreaction monopulse detection method based on mass spectrometry technology - Google Patents
Immunoreaction monopulse detection method based on mass spectrometry technology Download PDFInfo
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Abstract
The invention discloses an immunoreaction single-pulse detection method based on a mass spectrometry technology, which realizes single-pulse signal detection by adopting an inductively coupled plasma or microwave plasma mass spectrometry technology, labels immunoreaction by adopting metal nanoparticles, and further detects a pulse signal of metal ions for labeling immunoreaction by utilizing the inductively coupled plasma or microwave plasma mass spectrometry technology, thereby realizing the aim of indirectly detecting an object to be detected.
Description
Technical Field
The invention belongs to the field of medical inspection, but is not limited to the field, and particularly relates to an immunoreaction single-pulse detection method based on a mass spectrometry technology.
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 immunoreaction detection method based on mass spectrometry.
In order to realize the aim of the invention, the invention provides an immunoreaction monopulse detection method based on a mass spectrometry technology, which comprises the following steps:
1) adding a sample containing a substance to be detected, one antibody of the substance to be detected marked with one of a pair of substances with specific affinity and the other antibody of the substance to be detected marked with small nanoparticles into a reaction pool, and incubating for 1-120min (preferably 5-60min) to form an immune complex with one end marked with small nanoparticles and the other end marked with one of the pair of substances with specific affinity;
2) then adding the other marked large nano microsphere in 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 immune complex in the step 1) 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 immune complexes in the step 1) combined on the outer surface of the large nano microsphere;
3) directly adding the mixture reacted in the step 2) into a matched instrument, wherein a strong pulse intensity signal can be generated by characteristic metal elements in a large number of small nanoparticles on the immune complex bound to the surface of the large nanoparticle and characteristic metal elements in small nanoparticles on another antibody not participating in the reaction simultaneously, a detector in the matched instrument captures signals of the characteristic metal elements in the small nanoparticles, and pulse signals of the characteristic metal elements in the small nanoparticles on the immune complex bound to the surface of the large nanoparticle are obtained by filtering pulse intensity signals of the metal elements in the small nanoparticles on the other antibody not participating in the reaction; the quantity of characteristic metal element pulse signals in the small nanoparticles on the immune complex combined with the surface of the large nanoparticle has positive correlation with the content of the object to be detected, and the content of the object to be detected in the sample can be obtained through calculation of a standard curve of the object to be detected.
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 the above-mentioned metal compound nanoparticles, or the silica microspheres, or the 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, etc. 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 a polymer, or nanoparticles of a core/shell structure or a doped structure formed of the above-described metal compounds and silica or a polymer; 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-microsphere and one antibody of the object to be detected is chemical coupling or physical adsorption; the connection mode of the small nano-particles and another antibody strain of the substance to be detected is chemical coupling or physical adsorption.
Further, an antibody of the substance to be detected is 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 the other 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 another antibody of the analyte labeled by the unreacted small nanoparticles and then dispersing the antibody with a citric acid-sodium citrate buffer solution is also included between the step 2) and the step 3); in the step 3), a process of filtering out characteristic metal element pulse signals in small nanoparticles on another antibody which does not participate in reaction is omitted, namely, substances dispersed in a buffer solution are added into a matched instrument, metal elements in the small nanoparticles on a large number of immune complexes combined with the outer surface of the large nanoparticle can simultaneously generate strong pulse intensity signals, a detector in the matched instrument captures the pulse signals of the metal elements, in the method, the number of the metal element pulse signals in the small nanoparticles on the large number of immune complexes combined with the outer surface of the large nanoparticle has positive correlation with the content of the object to be detected, and the content of the object to be detected can be obtained through the standard curve calculation of the object to be detected.
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 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 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 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 a single pulse signal 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 pulse signals of the characteristic metal elements in the small nano particles on another antibody which does not participate in reaction to obtain the pulse signals of the characteristic metal elements in the small nano particles on an immune complex combined with the surface of a large nano microsphere, thereby omitting a washing process, improving the signal-to-noise ratio and improving the detection sensitivity of a system.
4. The method for detecting the single pulse signal after washing removes the small nano particles on the other antibody which does not participate in the reaction by adopting a washing mode, and the single pulse signal analysis means identifies 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, thereby reducing the interference generated by the pulse signal of the characteristic metal element in the small nano particles on the other antibody which does not participate in the reaction and improving the sensitivity.
Drawings
FIG. 1 is a schematic diagram of an immunoreaction single-pulse detection method based on mass spectrometry, wherein 1-an analyte, 2-an antibody of the analyte labeled with one of a pair of substances with specific affinity, 3-another antibody of the analyte labeled with small nanoparticles, 4-an immune complex labeled with small nanoparticles at one end and one of a pair of substances with affinity at the other end, 5-large nanoparticles labeled with the other of a pair of substances with specific affinity, and 6-a large amount of the immune complex in step 1) is bound to the outer surface of the large nanoparticles;
FIG. 2 is a standard curve of AFP obtained by direct examination of the mixture after the reaction of step 2);
FIG. 3 is a standard curve of AFP obtained by washing the mixture after the reaction of step 2) and then testing it.
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 an immunoreaction single-pulse detection method based on a mass spectrometry technology, which comprises the following steps:
1) adding a sample containing a substance to be detected 1, an antibody 2 of the substance to be detected marked by one of a pair of substances with specific affinity and another antibody 3 of the substance to be detected marked by small nanoparticles into a reaction tank, and incubating for 5-60min to form an immune complex 4 with one end marked with small nanoparticles and the other end marked with one of the pair of substances with specific affinity;
2) then adding the large nanosphere 5 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 immune complex in the step 1) 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 immune complexes 6 in the step 1) on the outer surface of the large nanosphere;
3) directly adding the mixture reacted in the step 2) into a matched instrument, wherein a characteristic metal element in the small nano-particle on the immune complex bound on the surface of the large nano-microsphere and a characteristic metal element in the small nano-particle on the other antibody not participating in the reaction can simultaneously generate a strong pulse intensity signal, a detector in the matched instrument captures the signal of the characteristic metal element in the small nano-particle and obtains the pulse signal of the characteristic metal element in the small nano-particle on the immune complex bound on the surface of the large nano-microsphere by filtering the pulse intensity signal of the characteristic metal element in the small nano-particle on the other antibody not participating in the reaction; the quantity of the pulse signals generated by the characteristic metal elements has positive correlation with the content of the object to be detected, and the content of the object to be detected in the sample can be obtained through calculation of a standard curve of the object to be detected.
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 In the above metal material and silica or polymer, or nanoparticles of a core/shell structure or a doped structure formed In the above metal compound and silica or 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-microsphere and one antibody of the object to be detected is chemical coupling or physical adsorption; the connection mode of the small nano-particles and another antibody strain of the substance to be detected is chemical coupling or physical adsorption.
Wherein an antibody of the object to be detected is 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 the other antibody of the object 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 object to be detected is chemical coupling or physical adsorption.
Wherein, the step between the step 2) and the step 3) further comprises the steps of washing the mixture after the reaction in the step 2) by using a washing solution to remove another antibody of the analyte marked by the unreacted small nano particles, and then dispersing by using a citric acid-sodium citrate buffer solution. And then adding the substances dispersed in the buffer solution into a matched instrument, wherein metal elements in the small nanoparticles on the large immune complexes combined on the outer surface of the large nano microspheres can simultaneously generate strong pulse intensity signals, a detector in the matched instrument captures the pulse signals of the metal elements, the number of the metal element pulse signals in the small nanoparticles on the large immune complexes combined on the outer surface of the large nano microspheres in the method has positive correlation with the content of the object to be detected, and the content of the object to be detected can be obtained through the standard curve calculation of the object to be detected.
Wherein the washing is performed by centrifugal separation or standing precipitationThe manner of (a); 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 sandwich method)
Preparation of Pt nanoparticles:
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 magnetic microspheres:
washing 0.5mL and 2.5mg/mL magnetic microspheres (with particle size of 800nm, available from Technology Ltd of Tianjincel group) with PBS buffer solution (pH 7.4) for 2 times, and then suspending in 10mL PBS buffer solution; 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, continuously adding the blocking solution, blocking for 30min, washing with PBS buffer solution, and then suspending in 10mL of PBS buffer solution for later use.
3. Biotin labeling a mouse anti-human alpha-fetoprotein (AFP) monoclonal antibody:
firstly, diluting a strain of mouse anti-human AFP monoclonal antibody into 1mg/mL by using a sodium carbonate buffer solution, stirring for 4 hours at room temperature (25 +/-5 ℃) in the dark by using the sodium carbonate buffer solution, and dialyzing; 6-Aminohexanoic acid-N-hydroxysuccinimide-Biotin (BCNHS) was then formulated with N, N-Dimethylamide (DMF) to 1 mg/mL; adding 125-66.7 microliter DMF solution into 1mL of a strain of mouse anti-human AFP 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 nano-particle labeled another mouse anti-human AFP monoclonal antibody
Firstly, diluting another mouse anti-human AFP monoclonal antibody into 1mg/mL by using a sodium carbonate buffer solution, and stirring for 4 hours in the dark by using the sodium carbonate buffer solution at room temperature (25 +/-5 ℃), and dialyzing; adding 1mL of another mouse anti-human AFP monoclonal antibody solution into 5mL of prepared Pt nanoparticle colloidal solution, magnetically stirring at 25 +/-5 ℃ for 30min in the dark, 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. Detection process
(1) Adding 10 mu L of a sample to be detected, 10 mu L of a mouse anti-human AFP monoclonal antibody marked by biotin and 10 mu L of another mouse anti-human AFP monoclonal antibody marked by Pt nanoparticles into a reaction tank, and carrying out incubation reaction for 30min at 37 ℃ to form an AFP immune complex with one end marked with Pt nanoparticles and the other end marked with biotin;
(2) continuously adding streptavidin-labeled magnetic microspheres into the mixture reacted in the step 1), and specifically binding biotin labeled at one end of the immune complex and streptavidin labeled on the surfaces of the magnetic microspheres to form a large amount of AFP immune complexes labeled with Pt nanoparticles on the surfaces of the magnetic microspheres;
(3) detecting the mixture reacted in the step 2) by using an inductively coupled plasma mass spectrometer (ICP-MS), wherein Pt nanoparticles on a large amount of immune complexes combined on the surface of the magnetic microsphere and Pt nanoparticles on another strain of mouse anti-human AFP monoclonal antibody which does not participate in the reaction can simultaneously generate a strong pulse intensity signal, a detector in the ICP-MS captures the pulse signal of the Pt nanoparticles, and pulse signals of Pt nanoparticles on a large number of immune complexes combined on the surface of the magnetic microsphere are obtained by filtering pulse signals of Pt nanoparticles on another strain of mouse anti-human AFP monoclonal antibody which does not participate in the reaction, the number of the pulse signals of the Pt nanoparticles on the large number of immune complexes combined on the surface of the magnetic microsphere has positive correlation with AFP, and the content of AFP in the sample is obtained by calculating through a standard curve of the AFP.
6. Establishment of a Standard Curve
The AFP calibration material with the concentration of 0, 5, 10, 50, 150 and 600ng/mL is prepared to be used for establishing an AFP standard curve, the detection sensitivity is 5ng/mL, the detection range is 5-600 ng/mL, the detection result is shown in table 1, and the standard curve is shown in fig. 2.
TABLE 1
| AFP calibrator (ng/mL) | 0 | 5 | 10 | 50 | 150 | 600 |
| Pt pulse Signal number (cps) | 513 | 9486 | 21964 | 89762 | 301456 | 1323598 |
Example 2 (washing Single pulse sandwich method)
Process for preparing Pt nanoparticles
The same procedure as in "preparation of Pt nanoparticles" of example 1;
2. streptavidin-labeled magnetic microspheres:
the same procedure as in 2 of example 1 for "streptavidin-labeled magnetic microspheres";
3. a strain of mouse anti-human AFP monoclonal antibody marked by biotin comprises:
the same procedure as that of the "biotin-labeled mouse anti-human AFP monoclonal antibody" in example 1 was used;
pt nanoparticles labeling another strain of mouse anti-human AFP monoclonal antibody:
the same procedure as that of the "Pt nanoparticle labeled another strain of mouse anti-human AFP monoclonal antibody" in example 1 was performed;
5. detection process
(1) The same procedure as in 5(1) of example 1;
(2) the same procedure as in 5(2) of example 1;
(3) washing the mixture reacted in the step (2) in a magnetic separation mode to remove another strain of mouse anti-human AFP monoclonal antibody marked by Pt nanoparticles which do not participate in the reaction, and then dispersing the Pt nanoparticles in a citric acid-sodium citrate (LM) buffer solution;
(4) and (3) detecting the substance dispersed in the LM buffer solution in the step (3) by using ICP-MS, wherein a large number of Pt nano-particles on the AFP immune complex combined on the surface of the magnetic microsphere can generate a strong pulse signal at the same time, a detector in the instrument captures the pulse signal of Pt on the AFP immune complex combined on the surface of the magnetic microsphere, the number of the pulse signals of the Pt nano-particles on the AFP immune complex combined on the surface of the magnetic microsphere has positive correlation with the content of APF in the sample, and the content of AFP in the sample can be obtained by calculating an AFP standard curve.
6. Establishment of a Standard Curve
The AFP calibration material with the concentration of 0, 5, 10, 50, 150 and 600ng/mL is prepared to be used for establishing an AFP standard curve, the detection sensitivity is 5ng/mL, the detection range is 5-600 ng/mL, the detection result after washing is shown in Table 2, and the detection result is shown in figure 3.
TABLE 2
| AFP calibrator (ng/mL) | 0 | 5 | 10 | 50 | 150 | 600 |
| Pt pulse Signal number (cps) | 1124 | 12032 | 25064 | 103861 | 321265 | 1518227 |
(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. An immunoreaction monopulse detection method based on a mass spectrometry technology is characterized by comprising the following steps of:
1) adding a sample containing a substance to be detected, an antibody of the substance to be detected marked by one of a pair of substances with specific affinity and another antibody of the substance to be detected marked by small nanoparticles into a reaction tank, and incubating for 1-120min to form an immune complex of which one end is marked with small nanoparticles and the other end is marked with one of the pair of substances with specific affinity;
2) then adding the other marked large nano microsphere in 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 immune complex in the step 1) 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 immune complexes in the step 1) combined on the outer surface of the large nano microsphere;
3) finally, directly adding the mixture reacted in the step 2) into a matched instrument, wherein the characteristic metal elements in the small nanoparticles on the immune complex bound to the surface of the large nanoparticle and the characteristic metal elements in the small nanoparticles on the other antibody not participating in the reaction can simultaneously generate a strong pulse intensity signal, a detector in the matched instrument captures the pulse signals of the characteristic metal elements in the small nanoparticles, and the pulse intensity signals of the characteristic metal elements in the small nanoparticles on the immune complex bound to the surface of the large nanoparticle are obtained by filtering the pulse intensity signals of the characteristic metal elements in the small nanoparticles on the other antibody not participating in the reaction; the quantity of pulse signals generated by characteristic metal elements in small nano particles on the immune complex combined with the surface of the large nano microsphere has positive correlation with the content of the object to be detected, and the content of the object to be detected in the sample can be obtained through calculation of a standard curve of the object to be detected;
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 a silica microsphere, or comprises polyethylene, polystyrene, polyvinyl fluoride, organic silicon, melamine, polyvinyl chloride, polylactic acid, epoxy resin, phenolic resin, polyester, polyacrylonitrile, polyacrylic acid, polyamide, chitosan, cellulose, polyaniline, polyacetylene, poly (L-glutamic acid), polyimide, polypyrrole and beta-cyclodextrin polymer microsphere 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-microsphere 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 single pulse detection method of claim 1, wherein the associated instrument is a microwave plasma torch mass spectrometer or an inductively coupled 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 having 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-microsphere and one antibody of the object to be detected is chemical coupling or physical adsorption; the connection mode of the small nano-particles and the other antibody strain of the substance to be detected is chemical coupling or physical adsorption.
6. The single pulse immunoreaction detecting method of claim 1, wherein one antibody of the substance to be detected is directly connected to the surface of the large nanometer microsphere through a chemical coupling or physical adsorption mode without being connected through a pair of substances with specific affinity;
the small nano particles are connected with the other antibody strain 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 other antibody strain of the substance to be detected is chemical coupling or physical adsorption.
7. The single pulse detection method of immune response 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 single pulse detection method of claim 1, wherein the particle size of the small nanoparticle is 0.1 to 800nm, and the particle size of the large nanoparticle is 100 μm.
9. The single pulse detection method of claim 1 to 8, further comprising a step of washing the mixture after the reaction in step 2) with a washing solution to remove another antibody of the analyte labeled with unreacted small nanoparticles, and then dispersing the antibody with a citric acid-sodium citrate buffer solution, between step 2) and step 3); in the step 3), the process of filtering out the characteristic metal element pulse signals in the small nanoparticles on another antibody strain which does not participate in the reaction is omitted.
10. The method for detecting an immune response monopulse according to claim 9, wherein said washing is performed by centrifugation or static precipitation; 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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