CN108828212A - A kind of immune response pulse detection method based on analytical technique of mass spectrum - Google Patents
A kind of immune response pulse detection method based on analytical technique of mass spectrum Download PDFInfo
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- CN108828212A CN108828212A CN201810394668.7A CN201810394668A CN108828212A CN 108828212 A CN108828212 A CN 108828212A CN 201810394668 A CN201810394668 A CN 201810394668A CN 108828212 A CN108828212 A CN 108828212A
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- G—PHYSICS
- G01—MEASURING; TESTING
- 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
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
- G01N33/54326—Magnetic particles
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- 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
Abstract
The immune response pulse detection method based on analytical technique of mass spectrum that the invention discloses a kind of, single pulse signal detection is realized by using inductively coupled plasma body or microwave plasma analytical technique of mass spectrum, it is reacted by using metal nanoparticle labelled immune, further using inductively coupled plasma body or the pulse signal of the metal ion of microwave plasma analytical technique of mass spectrum detection labelled immune reaction, to realize the purpose of detection determinand indirectly.
Description
Technical field
The invention belongs to field of medical examination, but are not limited to this field, and in particular to one kind is based on analytical technique of mass spectrum
Immune response pulse detection method.
Background technique
Currently, clinically there are commonly radioimmunology, enzyme-linked immunization, chemoluminescence method, electricity in terms of immunodiagnosis
Chemoluminescence method；Field is quickly being examined simultaneously, there are commonly colloidal gold methods, fluorescence immune chromatography method and emerging micro-fluidic
Chip detection mode etc..However, there are radiocontaminations for radioimmunology, human body is damaged big；Enzyme-linked immunization and chemiluminescence
The cumbersome of method, detection cycle are long；The signal resolution of the matching used electrode equipment of electrochemical process detection system and precision
System, detection device are expensive；Colloidal gold method, fluorescence immune chromatography method batch variation are big, the stability of marker is poor；Micro-fluidic core
Piece method not yet realizes commercial applications at present.
Mass spectral analysis is a kind of analysis method for measuring ion mass-to-charge ratio (mass-charge ratio), the basic principle is that making to try
Each component ionizes in an ion source in sample, generates the electrically charged ion of different charge-mass ratios, the effect of accelerated electric field, shape
At ion beam, into mass analyzer.In the mass analyser, electric and magnetic fields are recycled to make that opposite velocity dispersion occurs,
They are focused respectively and obtains mass spectrogram, so that it is determined that its quality.
Inductivity coupled plasma mass spectrometry (ICP-MS) is the inorganic elements and isotope to grow up the 1980s
Analysis and testing technology, it is with unique interfacing by the sensitive of the thermal ionization characteristic of inductively coupled plasma body and mass spectrograph
Quickly the advantages of scanning, combines and is formed a kind of analytical technology of high sensitivity.Plasma used in ICP-MS instrument removes
It is and used in emission spectrum essentially identical outside orientation and coil earthing mode.Used mass analyzer, ion detection
Device and data collection system are again similar with quadrupole rod GC-MS instrument.Mass analyzer mostly uses quadrupole mass filter, also adopts
With with high-resolution double focusing magnetic sector mass spectrometer, Time-of-flight mass spectrometer etc..The characteristics of technology：High sensitivity；Speed
Degree is fast, can complete the quantitative determination of tens elements in a few minutes；Spectral line is simple, and interference will be lacked relative to spectral technique；Line
Property range is up to 7~9 orders of magnitude；The preparation and introducing of sample are simple relative to other mass-spectrometric techniques；It can be not only used for element point
Analysis, can also carry out the quick measurement of isotopics；Measuring precision (RSD) can be to 0.1%.
Microwave plasma torch is a kind of novel plasma generating device that Jin Qinhan etc. is proposed and developed.Microwave etc. from
Daughter torch mass spectrum (MPT-MS) includes microwave power source, is atomized and removes dissolving device, microwave plasma torch and mass of ion analytical equipment.
Sample solution by peristaltic pump be pumped into atomization go dissolving device carry out atomization go it is molten, the dry aerosol of generation by quarter bend central passage
It into torch flame, is ionized under the action of microwave power source, generates the ion for spectrometer analysis.Its, gas consumption low with power
It saves, be easy to the characteristics of medicine detection promotes and applies.
ICP-MS and MPT-MS analytical technique of mass spectrum are wide with dynamic linear response range, detection limit is low, high sensitivity, measurement essence
High advantage is spent, the fields such as biology, medicine, environment, food are widely used in.
Summary of the invention
In view of this, the object of the present invention is to provide a kind of immune response pulse detection side based on analytical technique of mass spectrum
Method.
In order to achieve the object of the present invention, the present invention provides a kind of immune response pulse inspection based on analytical technique of mass spectrum
Survey method, includes the following steps：
1) by the one of the determinand of a label in the sample containing determinand, a pair of of substance with pathoklisis
Another strain antibody of strain antibody and the determinand of small nanoparticle label is added in reaction tank, is incubated for 1-120min (preferably
5-60min), formed one end be marked with small nano particle, the other end be marked in a pair of of substance with pathoklisis one
A immune complex；
2) another in a pair of of substance with pathoklisis is then added into the mixture after step 1) reaction
The big nanosphere of label marks one in step 1) in a pair of of substance with pathoklisis of immune complex one end
It is a to be specifically bound with another in special sexual compatibility a pair of substance on big nanosphere surface with label, shape
The immune complex in a large amount of steps 1) is combined at big nanosphere outer surface；
3) mixture after reaction in step 2) is added directly into necessary instrument, big nanosphere surface combines big
In small nano particle on amount immune complex characteristic metal element and the small nanometer on another strain antibody of reaction is not participated in
Characteristic metal element in grain can generate flash strength signal simultaneously, and the detector in necessary instrument captures in small nano particle
The signal of characteristic metal element and by filtering out metallic element in small nano particle on another strain antibody for not participating in reaction
Pulse strength signal obtain characteristic metal member in the small nano particle on the immune complex that big nanosphere surface combines
The pulse signal of element；Characteristic metal element pulse letter in the small nano particle on immune complex that big nanosphere surface combines
Number quantity and determinand content have positive correlation, by the calculating of the standard curve of determinand can obtain in sample to
Survey the content of object.
Further, wherein the necessary instrument be microwave plasma torch mass spectrograph (MPT-MS) or inductive coupling etc. from
Daughter mass spectrograph (single quadrupole rod or triple level four bars ICP-MS, high-resolution ICP-MS, ICP-TOF-MS) etc. can be examined quantitatively
Survey the instrument of metallic element；The immune response is homogeneous or heterogeneous.
Further, wherein the material of the big nanosphere is alkali metal (lithium Li, sodium Na, potassium K, rubidium Rb, caesium Se, francium
Fr), alkaline-earth metal (beryllium Be, magnesium Mg, calcium Ca, strontium Sr, barium Ba, radium Ra), lanthanide series metal (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, lutetium Lu), actinide metals (actinium Ac, thorium Th, protactinium
Pa, uranium U, neptunium Np, plutonium Pu, americium Am, curium Cm, berkelium, californium Cf, einsteinium Es, fermium Fm, mendelevium Md, nobelium No, lawrencium Lr), transition metal (scandium Sc,
Titanium Ti, vanadium V, chromium Cr, manganese Mn, iron Fe, cobalt Co, nickel, 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, mercury Hg), main group metal (aluminium Al,
Gallium Ga, indium In, tin Sn, thallium Tl, lead Pb, bismuth Bi, Uut, Uuq, Uup, Uuh), metalloid (boron, silicon Si, germanium Ge, arsenic As, antimony
Sb, tellurium Te, polonium Po) etc. including but not limited to above-mentioned metallic microspheres, be perhaps above-mentioned metal compound nanoparticles or be two
Silicon oxide microsphere, or for polyethylene, polystyrene type, polyvinyl fluoride, organosilicon, melamine, polyvinyl chloride, polylactic acid,
Epoxy resin, phenolic resin, polyesters, polyacrylonitrile, polyacrylic acid, polyamide-based, chitosan class, cellulose family, polyaniline
Class, carbene class, poly(L-glutamic acid), polyimides, polypyrrole, beta cyclo dextrin polymer microballoon etc. are including but not limited to above-mentioned poly-
Object microballoon is closed, the microballoon of the core/shell structure or doped structure that are either formed for two or more above-mentioned any substance；And
And the partial size of big nanosphere is 100nm~100 μm.
Further, wherein the material of the small nano particle is alkali metal (lithium Li, sodium Na, potassium K, rubidium Rb, caesium Se, francium
Fr), alkaline-earth metal (beryllium Be, magnesium Mg, calcium Ca, strontium Sr, barium Ba, radium Ra), lanthanide series metal (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, lutetium Lu), actinide metals (actinium Ac, thorium Th, protactinium
Pa, uranium U, neptunium Np, plutonium Pu, americium Am, curium Cm, berkelium, californium Cf, einsteinium Es, fermium Fm, mendelevium Md, nobelium No, lawrencium Lr), transition metal (scandium Sc,
Titanium Ti, vanadium V, chromium Cr, manganese Mn, iron Fe, cobalt Co, nickel, 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, mercury Hg), main group metal (aluminium Al,
Gallium Ga, indium In, tin Sn, thallium Tl, lead Pb, bismuth Bi, Uut, Uuq, Uup, Uuh), metalloid (boron, silicon Si, germanium Ge, arsenic As, antimony
Sb, tellurium Te, polonium Po) etc. including but not limited to above-mentioned metallic microspheres, be perhaps above-mentioned metal compound nanoparticles or to be upper
State core/shell structure that metal material and silica or polymer the are formed perhaps nano particle of doped structure or be above-mentioned
The nano particle of core/shell structure or doped structure that metallic compound and silica or polymer are formed；And small nanometer
The partial size of particle is 0.1nm~800nm.
Further, wherein the metallic element in the big nanosphere and small nano particle is not same gold simultaneously
Belong to；And the big nanosphere partial size is greater than small nano particle.
Further, wherein a pair of of substance with pathoklisis is biotin and streptavidin, biology
Element and Avidin, fluorescein and anti-fluorescein, antibody and the secondary antibody for specifically binding this antibody；
And a pair of of substance with pathoklisis and big nanosphere, determinand a strain antibody between company
The mode of connecing is chemical coupling or is physical absorption；Another strain antibody connection type of small nano particle and determinand is that chemistry is even
Connection is physical absorption.
Further, wherein a strain antibody of the determinand is directly connected by way of chemical coupling or physical absorption
It is connected on the surface of big nanosphere, is not attached by a pair of of substance with pathoklisis；
Pass through a pair of of substance with pathoklisis between the small nano particle and another strain antibody of determinand
Function served as bridge is attached, and the connection type between small nano particle of one in a pair of of substance with pathoklisis is to change
Coupling or physical absorption are learned, another connection between the antibody of determinand in a pair of of substance with pathoklisis
Mode is chemical coupling or physical absorption.
Further, wherein further including by the mixture cleaning solution after step 2) reaction between step 2) and step 3)
Another strain antibody of the determinand of the small nanoparticle label of reaction is not participated in washing removal, then uses citric acid-sodium citrate
The step of buffer disperses；The small nano particle filtered out on another strain antibody for not participating in reaction is eliminated in the step 3)
The process of middle characteristic metal element pulse signal, that is, the substance that will be dispersed in buffer are added in matched instrument, receive greatly
It is strong that the metallic element in the small nano particle on a large amount of immune complexs that the outer surface meter Wei Qiu combines can generate simultaneously flash
Signal is spent, the detector in necessary instrument captures the pulse signal of golden prime element, and big nanosphere outer surface combines in this method
A large amount of immune complexs on small nano particle in metallic element pulse signal quantity and determinand content have positive
Guan Xing calculates the content that can obtain determinand by the standard curve of determinand.
Further, wherein the washing is by the way of centrifuge separation or staticly settling；Or when big nanosphere
Material is magnetic Fe3O4、γ-Fe2O3, Pt, Ni or Co microballoon, or be magnetic Fe3O4、γ-Fe2O3, Pt, Ni or Co with
When the microballoon for the core/shell structure or doped structure that inorganic matter or organic matter are formed, then the washing is by the way of Magneto separate.
The present invention is based on homogeneous/heterogeneous reaction, the pulse signal of combination mass spectrum acquisition metal nanoparticle, by pulse
Signal is converted into the content of determinand, and detection speed is fast, and testing result is stable, accurate, reliable.
The invention has the advantages that：
1, the present invention realizes simple venation by using inductively coupled plasma body or microwave plasma analytical technique of mass spectrum
Signal detection is rushed, is reacted by using metal nanoparticle labelled immune, inductively coupled plasma body or microwave are further utilized
Plasma Mass Spectrometry analysis technology detect labelled immune reaction metal ion pulse signal, thus realize detect indirectly it is to be measured
The purpose of object；
2, the present invention uses inductively coupled plasma body or microwave plasma analytical technique of mass spectrum have the line of detection
Property the advantages that range is wide, the low detection speed of detection line is fast, high sensitivity, high measurement accuracy, expanded inductively coupled plasma body
Or application of the microwave plasma analytical technique of mass spectrum in field of medical examination；
3, the present invention does not wash the detection method for directly carrying out single pulse signal, is identified using single pulse signal analysis means
The pulse signal of characteristic metal element in small nano particle, and further filter out small on another strain antibody for not participating in reaction
The pulse signal of characteristic metal element small is received in nano particle come obtain on the immune complex that big nanosphere surface combines
The pulse signal of characteristic metal element in rice grain eliminates washing process, promotes signal-to-noise ratio, so as to improving the detection spirit of system
Sensitivity.
4, the detection method of single pulse signal, is removed by the way of washing and is not participated in the another of reaction after the present invention washs
Small nano particle on strain antibody, single pulse signal analysis means identify on the immune complex that big nanosphere surface combines
The signal of characteristic metal element in small nano particle reduces special in the small nano particle on another strain antibody for not participating in reaction
The interference that the pulse signal of metallic element generates is levied, sensitivity is improved.
Detailed description of the invention
Fig. 1 is the immune response pulse detection method schematic diagram of the present invention based on analytical technique of mass spectrum, wherein
1- determinand, 2- have a strain antibody of the determinand of a label in a pair of of substance of pathoklisis, the small nanometer of 3-
Another strain antibody of the determinand of grain label, the one end 4- is marked with small nano particle, the other end is marked with affinity one
To one immune complex in substance, 5- has the big nanometer of another label in a pair of of substance of pathoklisis micro-
Ball, 6- is big, and nanosphere outer surface combines the immune complex in a large amount of step 1)；
Fig. 2 is the standard curve that the mixture after step 2) reaction is directly carried out to detected AFP；
Fig. 3 is the standard curve that detected AFP is carried out after washing the mixture after step 2) reaction.
Specific embodiment
The present invention is explained below with reference to embodiment, and case study on implementation is merely to illustrate the present invention.Unless stated otherwise, of the invention
Used in technological means be method known in those skilled in the art.In addition, embodiment be interpreted as it is illustrative,
The range being not intended to limit the present invention, the spirit and scope of the invention are limited only by the claims that follow.For those skilled in the art
Member for, under the premise of without departing substantially from spirit and scope of the present invention, in these embodiments material component and dosage carry out
Various changes or change also belong to protection scope of the present invention.
As shown in Figure 1, the present invention provides a kind of immune response pulse detection method based on analytical technique of mass spectrum, packet
Include following steps：
1) by the determinand of a label in the sample containing determinand 1, a pair of of substance with pathoklisis
Another strain antibody 3 of one strain antibody 2 and the determinand of small nanoparticle label is added in reaction tank, is incubated for 5-60min, is formed
One end is marked with small nano particle, the other end is marked with one immune complex in a pair of of substance with pathoklisis
4；
2) another in a pair of of substance with pathoklisis is then added into the mixture after step 1) reaction
The big nanosphere 5 of label, label is in step 1) in a pair of of substance with pathoklisis of immune complex one end
One is specifically bound on big nanosphere surface with another in special sexual compatibility a pair of substance with label,
It forms big nanosphere outer surface and combines immune complex 6 in a large amount of steps 1)；
3) mixture after reaction in step 2) is added directly into necessary instrument, what big nanosphere surface combined exempts from
It characteristic metal element and is not participated in small nano particle on another strain antibody of reaction in small nano particle on epidemic disease compound
Characteristic metal element can generate flash strength signal simultaneously, and the detector in necessary instrument captures feature gold in small nano particle
Belong to the signal of element and by filtering out characteristic metal element in the small nano particle on another strain antibody for not participating in reaction
Characteristic metal element in small nano particle on immune complex of the pulse strength signal to obtain big nanosphere surface combination
Pulse signal；Characteristic metal element generate pulse signal quantity and determinand content have positive correlation, by
The calculating for surveying the standard curve of object can obtain the content of determinand in sample.
Wherein the necessary instrument can be microwave plasma torch mass spectrograph (MPT-MS), inductively coupled plasma constitution
Spectrometer (single quadrupole rod or triple level four bars ICP-MS, high-resolution ICP-MS, ICP-TOF-MS) etc. being capable of quantitative detection metal
The instrument of element；The immune response is homogeneous or heterogeneous.
Wherein the material of the big nanosphere be alkali metal (lithium Li, sodium Na, potassium K, rubidium Rb, caesium Se, francium Fr), alkaline earth gold
Belong to (beryllium Be, magnesium Mg, calcium Ca, strontium Sr, barium Ba, radium Ra), lanthanide series metal (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, lutetium Lu), actinide metals (actinium Ac, thorium Th, protactinium Pa, uranium U, neptunium Np,
Plutonium Pu, americium Am, curium Cm, berkelium, californium Cf, einsteinium Es, fermium Fm, mendelevium Md, nobelium No, lawrencium Lr), transition metal (scandium Sc, titanium Ti, vanadium V, chromium
Cr, manganese Mn, iron Fe, cobalt Co, nickel, 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, mercury Hg), main group metal (aluminium Al, gallium Ga, indium In, tin
Sn, thallium Tl, lead Pb, bismuth Bi, Uut, Uuq, Uup, Uuh), metalloid (boron, silicon Si, germanium Ge, arsenic As, antimony Sb, tellurium Te, polonium Po)
Deng including but not limited to above-mentioned metallic microspheres, perhaps for above-mentioned metal compound nanoparticles or be silica, Huo Zhewei
Polyethylene, polystyrene type, polyvinyl fluoride, organosilicon, melamine, polyvinyl chloride, polylactic acid, epoxy resin, phenolic resin,
Polyesters, polyacrylonitrile, polyacrylic acid, polyamide-based, chitosan class, cellulose family, polyaniline compound, carbene class, poly- (L- paddy ammonia
Acid), polyimides, polypyrrole, beta cyclo dextrin polymer microballoon etc. is including but not limited to above-mentioned polymer microballoon, or be above-mentioned
The microballoon of core/shell structure or doped structure that two or more any substance is formed；And the partial size of big nanosphere
For 100nm~100 μm.
Wherein the material of the small nano particle be alkali metal (lithium Li, sodium Na, potassium K, rubidium Rb, caesium Se, francium Fr), alkaline earth gold
Belong to (beryllium Be, magnesium Mg, calcium Ca, strontium Sr, barium Ba, radium Ra), lanthanide series metal (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, lutetium Lu), actinide metals (actinium Ac, thorium Th, protactinium Pa, uranium U, neptunium Np,
Plutonium Pu, americium Am, curium Cm, berkelium, californium Cf, einsteinium Es, fermium Fm, mendelevium Md, nobelium No, lawrencium Lr), transition metal (scandium Sc, titanium Ti, vanadium V, chromium
Cr, manganese Mn, iron Fe, cobalt Co, nickel, 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, mercury Hg), main group metal (aluminium Al, gallium Ga, indium In, tin
Sn, thallium Tl, lead Pb, bismuth Bi, Uut, Uuq, Uup, Uuh), metalloid (boron, silicon Si, germanium Ge, arsenic As, antimony Sb, tellurium Te, polonium Po)
Deng including but not limited to above-mentioned metallic microspheres, be perhaps above-mentioned metal compound nanoparticles or be above-mentioned metal material with
The core/shell structure formed in silica or polymer perhaps the nano particle of doped structure or be above-mentioned metal compound
The nano particle of the core/shell structure or doped structure that are formed in object and silica or polymer；And small nano particle
Partial size be 0.1nm~800nm.
Wherein the metallic element in the big nanosphere and small nano particle is not same metal simultaneously；And it is described big
Nanosphere partial size is greater than small nano particle.
Wherein a pair of of substance with pathoklisis be biotin and streptavidin, biotin and affine
Element, fluorescein and anti-fluorescein, antibody and the secondary antibody for specifically binding this antibody；
And a pair of of substance with pathoklisis and big nanosphere, determinand a strain antibody between company
The mode of connecing is chemical coupling or is physical absorption；Another strain antibody connection type of small nano particle and determinand is that chemistry is even
Connection is physical absorption.
Wherein a strain antibody of the determinand is directly directly connected to by way of chemical coupling or physical absorption
On the surface of big nanosphere, it is not attached by a pair of of substance with pathoklisis；
Pass through a pair of of substance with pathoklisis between the small nano particle and another strain antibody of determinand
Function served as bridge is attached, and the connection type between small nano particle of one in a pair of of substance with pathoklisis is to change
It learns coupling or is physical absorption, another company between the antibody of determinand in a pair of of substance with pathoklisis
The mode of connecing is chemical coupling or is physical absorption.
It wherein further include that the mixture after step 2) reaction is washed into removal with cleaning solution between step 2) and step 3)
Another strain antibody of the determinand of the small nanoparticle label of reaction is not participated in, then with citric acid-sodium citrate buffer solution point
Scattered step.Then the substance that will be dispersed in buffer is added in matched instrument, what big nanosphere outer surface combined
The metallic element in small nano particle on a large amount of immune complexs can generate flash strength signal simultaneously, in necessary instrument
Detector captures the pulse signal of golden prime element, in this method on a large amount of immune complexs of big nanosphere outer surface combination
The quantity of metallic element pulse signal and the content of determinand have positive correlation in small nano particle, pass through the standard of determinand
Curve calculates the content that can obtain determinand.
Wherein the washing is by the way of centrifuge separation or staticly settling；Or when the material of big nanosphere is magnetism
Fe3O4、γ-Fe2O3, Pt, Ni or Co microballoon, or be magnetic Fe3O4、γ-Fe2O3, Pt, Ni or Co and inorganic matter or have
When the microballoon for the core/shell structure or doped structure that machine object is formed, then the washing is by the way of Magneto separate.
The present invention is further detailed below by way of specific embodiment.
Embodiment 1 (does not wash pulse sandwich method)
The preparation of 1.Pt nano particle：
Successively by 0.15g polyvinylpyrrolidone (PVP), 33.9mg chloroplatinic acid (H2PtCl6.6H2O), 20mL ultrapure water,
20mL ethylene glycol is added in reaction vessel, and under the revolving speed of 240r/min, back flow reaction 3h, obtains brownish black at 60 DEG C
Pt nanoparticle colloid solution.
2. streptavidin marks magnetic microsphere：
0.5mL is taken, 2.5mg/mL magnetic microsphere (partial size 800nm is purchased from Tianjin Sai Er groups of Science and Technology Ltd.s) uses PBS
(pH=7.4) is resuspended in the PBS buffer solution of 10mL after washing 2 times in buffer solution；To the magnetic microsphere solution after washing
In continuously add 5mg EDC and 5mg NHS, stir and lower activate 1h；10mL PBS buffering is resuspended in after being washed with PBS buffer solution
In solution, continuously add confining liquid closing 30min, be resuspended in after being washed with PBS buffer solution in 10mL PBS buffer solution to
With.
3. the one plant of anti-human alpha-fetoprotein of mouse (AFP) monoclonal antibody of biotin labeling:
The anti-human AFP monoclonal antibody of one plant of mouse is first diluted to 1mg/mL with sodium carbonate buffer, and is buffered with sodium carbonate
Liquid chamber temperature (25 DEG C ± 5 DEG C) is dialysed after being protected from light stirring 4 hours；N is then used, N- dimethylformamide (DMF) is by 6-aminocaproic acid-N
HOSu NHS-biotin (BCNHS) is configured to 1mg/mL；Add in the anti-human AFP monoclonal antibody solution of mono- plant of mouse of 1mL
Enter the above-mentioned 125 μ L of μ L~66.7 of DMF solution, mixed in vial, room temperature (25 DEG C ± 5 DEG C) is protected from light stirring 2 hours；It is added
9.6 μ L of 1mol/L ammonium chloride solution, room temperature (25 DEG C ± 5 DEG C) are protected from light stirring 10 minutes；Then mixed solution is transferred to bag filter, uses
4 DEG C of dialysed overnights of phosphate buffer.It finally takes out plus -20 DEG C of equivalent glycerol saves.
The anti-human AFP monoclonal antibody of another plant of mouse of 4.Pt nanoparticle label
The anti-human AFP monoclonal antibody of another plant of mouse is first diluted to 1mg/mL with sodium carbonate buffer, and slow with sodium carbonate
Fliud flushing room temperature (25 DEG C ± 5 DEG C) is protected from light stirring 4 hours and dialyses；It is another that 1mL is added in the Pt nanoparticle colloid solution of 5mL preparation
The anti-human AFP monoclonal antibody solution of one plant of mouse is protected from light magnetic agitation 30min at 25 DEG C ± 5 DEG C, and then mixed solution is transferred to
Bag is analysed, with 4 DEG C of dialysed overnights of phosphate buffer.It finally takes out plus -20 DEG C of equivalent glycerol saves.
5. detection process
(1) by 10 μ L samples to be tested, the anti-human AFP monoclonal antibody of one plant of mouse of 10 μ L biotin labelings, Pt nanometers of 10 μ L
The anti-human AFP monoclonal antibody of another plant of mouse of particle marker is added in reaction tank, the incubation reaction 30min at 37 DEG C, is formed
One end is marked with Pt nano particle, the other end is marked with the AFP immune complex of biotin；
(2) continue that the magnetic microsphere that streptavidin marks is added in the mixture after reacting to step 1), mark upper
It states streptavidin of the biotin of immune complex one end with label on magnetic microsphere surface to specifically bind, forms magnetic
Property microsphere surface combine the AFP immune complex that a large amount of label has nano particle；
(3) mixture after reaction in step 2) is detected using icp ms (ICP-MS), magnetic
Property a large amount of immune complex that combines of microsphere surface on Pt nano particle and do not participate in another plant of anti-human AFP of mouse of reaction
Pt nano particle in monoclonal antibody can generate flash strength signal simultaneously, and the detector in ICP-MS captures Pt nanometers
The pulse signal of grain, and by filtering out the Pt nano particle in the anti-human AFP monoclonal antibody of another plant of mouse for not participating in reaction
Pulse signal come obtain magnetic microsphere surface combination a large amount of immune complex on Pt nano particle pulse signal, magnetic
Property microsphere surface combine a large amount of immune complex on Pt nano particle pulse signal quantity and AFP have be positively correlated
Property, the content for obtaining AFP in sample is calculated by the standard curve of AFP.
6. the foundation of standard curve
Configuration concentration is 0,5,10,50,150, the AFP calibration object of 600ng/mL is for establishing AFP standard curve, detection spirit
Sensitivity is 5ng/mL, and detection range is 5~600ng/mL, and testing result is as shown in table 1, and standard curve is as shown in Figure 2.
Table 1
| AFP calibration object (ng/mL) | 0 | 5 | 10 | 50 | 150 | 600 |
| Pt pulse signal quantity (cps) | 513 | 9486 | 21964 | 89762 | 301456 | 1323598 |
Embodiment 2 (washing pulse sandwich method)
The preparation process of 1.Pt nano particle
It is identical in embodiment 11 process of " preparation of Pt nano particle "；
2. streptavidin marks magnetic microsphere：
It is identical in embodiment 12 process of " streptavidin label magnetic microsphere "；
3. the anti-human AFP monoclonal antibody of one plant of mouse of biotin labeling:
It is identical in embodiment 13 process of " the anti-human AFP monoclonal antibody of one plant of mouse of biotin labeling "；
The anti-human AFP monoclonal antibody of another plant of mouse of 4.Pt nanoparticle label：
It is identical in embodiment 14 process of " the anti-human AFP monoclonal antibody of another plant of mouse of Pt nanoparticle label "；
5. detection process
(1) identical with the process of 5 (1) in embodiment 1；
(2) identical with the process of 5 (2) in embodiment 1；
(3) mode of the mixture Magneto separate after reaction in step (2) is washed into the Pt nanometer that reaction is not participated in removal
The anti-human AFP monoclonal antibody of another plant of mouse of particle marker is then dispersed in citric acid-sodium citrate (LM) buffer；
(4) step (3) is dispersed in the detection of the substance migration ICP-MS in LM buffer, magnetic microsphere surface combines greatly
Pt nano particle on the AFP immune complex of amount can generate strong pulse signal simultaneously, and the detector in instrument captures magnetic
The pulse signal for the Pt on a large amount of AFP immune complexs that microsphere surface combines, a large amount of AFP that magnetic microsphere surface combines are immune
The content of APF has positive correlation in the quantity and sample of the pulse signal of Pt nano particle on compound, passes through AFP standard
Curve calculates the content that can obtain AFP in sample.
6. the foundation of standard curve
Configuration concentration is 0,5,10,50,150, the AFP calibration object of 600ng/mL is for establishing AFP standard curve, detection spirit
Sensitivity is 5ng/mL, and detection range is 5~600ng/mL, and testing result is as shown in table 2 after washing, and testing result is as shown in Figure 3.
Table 2
| AFP calibration object (ng/mL) | 0 | 5 | 10 | 50 | 150 | 600 |
| Pt pulse signal quantity (cps) | 1124 | 12032 | 25064 | 103861 | 321265 | 1518227 |
(1) PBS buffer solution
(2) citric acid-sodium citrate buffer solution (LM)
(3) confining liquid
(4) sodium carbonate buffer (CB)
Sodium carbonate 4.33g
Sodium bicarbonate 2.96g
Purified water is settled to 1000mL；
(5) phosphate buffer solution (PB)
Sodium dihydrogen phosphate 0.99g
Disodium hydrogen phosphate 5.16g
Purified water is settled to 1000mL；
(6) cleaning solution
Although above the present invention is described in detail with a general description of the specific embodiments,
On the basis of the present invention, it can be made some modifications or improvements, this will be apparent to those skilled in the art.Cause
This, these modifications or improvements, fall within the scope of the claimed invention without departing from theon the basis of the spirit of the present invention.
Claims (10)
1. a kind of immune response pulse detection method based on analytical technique of mass spectrum, which is characterized in that include the following steps：
1) anti-by one plant of the determinand of a label in the sample containing determinand, a pair of of substance with pathoklisis
Another strain antibody of body and the determinand of small nanoparticle label is added in reaction tank, is incubated for 1-120min, forms one end mark
Remember one immune complex for thering is small nano particle, the other end to be marked in a pair of of substance with pathoklisis；
2) another label in a pair of of substance with pathoklisis is then added into the mixture after step 1) reaction
Big nanosphere, label one in step 1) in a pair of of substance with pathoklisis of immune complex one end and
It marks and is specifically bound on big nanosphere surface with another in special sexual compatibility a pair of substance, formed big
Nanosphere outer surface combines the immune complex in a large amount of steps 1)；
3) finally the mixture after reaction in step 2) is added directly into necessary instrument, big nanosphere surface combines big
In small nano particle on amount immune complex characteristic metal element and the small nanometer on another strain antibody of reaction is not participated in
Characteristic metal element in grain can generate flash strength signal simultaneously, and the detector in necessary instrument captures in small nano particle
The pulse signal of characteristic metal element and by filtering out feature in small nano particle on another strain antibody for not participating in reaction
The pulse strength signal of metallic element is special in the small nano particle on the immune complex that big nanosphere surface combines to obtain
Levy the signal of metallic element；Characteristic metal element produces in the small nano particle on immune complex that big nanosphere surface combines
The content of raw pulse signal quantity and determinand has positive correlation, can be obtained by the calculating of the standard curve of determinand
The content of determinand in sample.
2. immune response pulse detection method as described in claim 1, which is characterized in that the necessary instrument is microwave etc.
Gas ions torch mass spectrograph or icp ms；The immune response is homogeneous or heterogeneous；It is described to receive greatly
The material of meter Wei Qiu include alkali metal, alkaline-earth metal, lanthanide series metal, actinide metals, transition metal, main group metal or metalloid but
Be not limited to above-mentioned metallic microspheres, perhaps for the nanosphere of above-mentioned metallic compound be perhaps silicon dioxide microsphere or comprising
Polyethylene, polystyrene type, polyvinyl fluoride, organosilicon, melamine, polyvinyl chloride, polylactic acid, epoxy resin, phenolic resin,
Polyesters, polyacrylonitrile, polyacrylic acid, polyamide-based, chitosan class, cellulose family, polyaniline compound, carbene class, poly- (L- paddy ammonia
Acid), polyimides, polypyrrole, beta cyclo dextrin polymer microballoon but be not limited to above-mentioned polymer microballoon, or be above-mentioned any two
The microballoon of core/shell structure or doped structure that kind or two or more substances are formed；And the partial size of big nanosphere is 100nm
~100 μm.
3. immune response pulse detection method as claimed in claim 2, which is characterized in that the alkali metal is lithium Li, sodium
Na, potassium K, rubidium Rb, caesium Se or francium Fr；The alkaline-earth metal is beryllium Be, magnesium Mg, calcium Ca, strontium Sr, barium Ba or radium Ra；The group of the lanthanides
Metal be 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 metals are actinium Ac, thorium Th, protactinium Pa, uranium U, neptunium Np, plutonium Pu, americium Am, mend Cm, berkelium, californium Cf, einsteinium Es, fermium with cramps
Fm, mendelevium Md, nobelium No or lawrencium Lr；The transition metal be scandium Sc, titanium Ti, vanadium V, chromium Cr, manganese Mn, iron Fe, cobalt Co, nickel, 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 be aluminium Al, gallium Ga, indium In, tin Sn, thallium Tl, lead Pb, bismuth Bi, Uut,
Uuq, Uup or Uuh；The metalloid is boron, silicon Si, germanium Ge, arsenic As, antimony Sb, tellurium Te or polonium Po.
4. immune response pulse detection method as described in claim 1, which is characterized in that the material of the small nano particle
Comprising alkali metal, alkaline-earth metal, lanthanide series metal, actinide metals, transition metal, main group metal, metalloid but it is not limited to above-mentioned gold
The microballoon of category, perhaps for the nano particle of above-mentioned metallic compound or be above-mentioned metal material and silica or polymer
The core/shell structure of the formation perhaps nano particle of doped structure or be above-mentioned metallic compound and silica or polymer
The core/shell structure of formation or the nano particle of doped structure；And the partial size of small nano particle is 0.1nm~800nm.
5. immune response pulse detection method as claimed in claim 4, which is characterized in that the alkali metal is lithium Li, sodium
Na, potassium K, rubidium Rb, caesium Se or francium Fr；The alkaline-earth metal is beryllium Be, magnesium Mg, calcium Ca, strontium Sr, barium Ba or radium Ra；The group of the lanthanides
Metal be 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 metals are actinium Ac, thorium Th, protactinium Pa, uranium U, neptunium Np, plutonium Pu, americium Am, mend Cm, berkelium, californium Cf, einsteinium Es, fermium with cramps
Fm, mendelevium Md, nobelium No or lawrencium Lr；The transition metal be scandium Sc, titanium Ti, vanadium V, chromium Cr, manganese Mn, iron Fe, cobalt Co, nickel, 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 be aluminium Al, gallium Ga, indium In, tin Sn, thallium Tl, lead Pb, bismuth Bi, Uut,
Uuq, Uup or Uuh；The metalloid is boron, silicon Si, germanium Ge, arsenic As, antimony Sb, tellurium Te or polonium Po.
6. pulse detection method is immunoreacted as described in claim 1, in the big nanosphere and small nano particle
Metallic element is not same metal simultaneously；And the big nanosphere partial size is greater than small nano particle.
7. immune response pulse detection method as described in claim 1, which is characterized in that described with pathoklisis
A pair of of substance is biotin and streptavidin, biotin and Avidin, fluorescein and anti-fluorescein, antibody and specific binding
The secondary antibody of this antibody；
And a pair of of substance with pathoklisis and big nanosphere, determinand a strain antibody between connection side
Formula is chemical coupling or is physical absorption；Another strain antibody connection type of small nano particle and determinand be chemical coupling or
Person's physical absorption.
8. immune response pulse detection method as described in claim 1, which is characterized in that a strain antibody of the determinand
It is directly connected to by way of chemical coupling or physical absorption on the surface of big nanosphere, it is special not affine by having
A pair of of substance of property is attached；
Pass through the bridge of a pair of of substance with pathoklisis between the small nano particle and another strain antibody of determinand
Effect is attached, and the connection type between small nano particle of one in a pair of of substance with pathoklisis is that chemistry is even
Connection or physical absorption, another company between another strain antibody of determinand in a pair of of substance with pathoklisis
The mode of connecing is chemical coupling or is physical absorption.
9. such as the described in any item immune response pulse detection methods of claim 1-8, which is characterized in that in step 2) and step
It is rapid 3) between further include wash to remove with cleaning solution by the mixture after step 2) reaction not participating in the small nano particle mark of reaction
Another strain antibody of the determinand of note, the step of then dispersion with citric acid-sodium citrate buffer solution；It is saved in the step 3)
The process of characteristic metal element pulse signal in the small nano particle on another strain antibody for not participating in reaction is gone to filter out.
10. immune response pulse detection method as claimed in claim 9, which is characterized in that the washing is using centrifugation point
From or the mode that staticly settles；Or when the material of big nanosphere is magnetic Fe3O4、γ-Fe2O3, Pt, Ni or Co microballoon,
It or is magnetic Fe3O4、γ-Fe2O3, Pt, Ni or Co and inorganic matter or the organic matter core/shell structure or doped structure that are formed
Microballoon when, then it is described washing by the way of Magneto separate.
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