CN103543260A - Method for detecting biomacromolecule based on magnetic separation-quantum dot immunofluorescence sensing and reagent preparation method - Google Patents
Method for detecting biomacromolecule based on magnetic separation-quantum dot immunofluorescence sensing and reagent preparation method Download PDFInfo
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- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
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- G01N33/536—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase
- G01N33/542—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase with steric inhibition or signal modification, e.g. fluorescent quenching
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- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
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- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
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- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
- G01N33/588—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with semiconductor nanocrystal label, e.g. quantum dots
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- G01N2446/00—Magnetic particle immunoreagent carriers
- G01N2446/80—Magnetic particle immunoreagent carriers characterised by the agent used to coat the magnetic particles, e.g. lipids
Abstract
The invention provides a method for detecting a biomacromolecule based on magnetic separation-quantum dot immunofluorescence sensing and a reagent preparation method. The method is characterized by comprising the following steps: with superparamagnetic nano beads MBs as carriers, coupling the carriers with an antibody Ab1 of a biomacromolecule to be detected to prepare a capturing antibody MB-Ab1; coupling a quantum dot with an antibody Ab2 of the biomacromolecule to be detected to prepare a quantum dot fluorescence probe QDs-Ab2; enriching the biomacromolecule Ag to be detected in a test sample by using the MB-Ab1 to prepare a serial compound MB-Ab1-Ag; reacting the MB-Ab1-Ag with the QDs-Ab2 to generate an MB-Ab1-Ag-Ab2-QDs and carrying out magnetic separation to separate the MB-Ab1-Ag-Ab2-QDs from a liquid-phase QDs-Ab2 immunofluorescence probe; and measuring QDs-Ab2 fluorescence intensity X in a liquid phase and indirectly measuring C by utilizing an inverse relation represented between the concentration C of the biomacromolecule to be detected and the X. The method is simple and convenient to operate, good in stability and high in detection sensitivity.
Description
Technical field
The present invention relates to a kind of macromolecular method of detection of biological, relate in particular to a kind of magnetic separation-quantum dot immune fluorescent sensing detection method and related reagent preparation and using method based on indirect signal analytical model.
Background technology
Communicable animals and plants pathogenic bacteria and virus are key monitoring objects in inspection and quarantining for import/export work; these are had to communicable harmful organism and carry out fast, detect accurately, easily, to protect mankind health and life security, the guarantee gateway of a country safely, maintaining social stability has great importance.
Detect at present these pathogenic bacteria and mainly comprise the methods such as separated cultivation detection, immunology detection, molecular biology with viral detection method.Isolated culture method simply, is easily gone, but due to low, the consuming time length of its detection sensitivity, cannot reach quarantine request.Immunoassay has simply, quick, low cost and other advantages, be suitable for extensive sample examination, but its remolding sensitivity is lower, so its use is also subject to certain restrictions.Molecular biology method detection sensitivity is higher, and specificity is better, but needs expensive instrument and higher professional technique, so its application is subject to certain limitation.Therefore, Check and Examination of Port quarantine be badly in need of setting up a kind of fast, the method for Sensitive Detection pathogenic bacteria and virus.
Immunomagnetic isolation technology based on super suitable nanometer magnetic bead has quick, the simple and efficient advantage of immunoreactive high degree of specificity and magnetic resolution, be current most widely used magnetic separation technique, be widely used in recent years in the research of the aspect, field such as enrichment, clinical diagnosis, food security fast detecting, environmental monitoring of tumour cell separation, enrichment, target protein and polypeptide.In immune magnetic enrichment reaction, the identification of immunomagnetic beads and object is carried out in aaerosol solution, it is a kind of immune response form of homogeneous phase, therefore can combine with other a lot of technology, build the analytical approach of series of new, for example magnetic separation-fluorescence immune analysis method, magnetic separation-chemiluminescence analysis method, magnetic enrichment-micro-fluidic chip technology, magnetic separation-surface plasma resonance biological sensor, the methods such as the brilliant micro-balance biology sensor of magnetic enrichment-quartz.Due to the development of immune magnetic separation technique, greatly promoted the development of Correlation Analysis Technique, its application potential is constantly excavated out.
Quantum dot is the detection in fluorescence imaging, food and environment middle peasant residue of veterinary drug, relevant pathogenic bacteria and virus as a kind of novel fluorescent/phosphorescent probe, the fields such as biochip multiplex detection have obtained application widely, it has many superior optical properties: fluorescence intensity is stable, and anti-photobleaching is strong; Have an elementary excitation, the advantage of polynary transmitting, is a kind of desirable polynary marker material; Excitation spectrum bandwidth, emission spectrum are symmetrical and narrow, and emission wavelength and its interrelation of particle size, can effectively overcome the overlapping weakness of organic fluorescence probe emission spectrum emission spectrum wider and easy and acceptor, applicable multivariate detection.
At present, there is Many researchers that the advantage of the fluorescence property of quantum dot excellence and immunomagnetic isolation technology is combined, built magnetic separation-Fluoro-Immnnosensor, this sensor is compared with traditional ELISA, there is sample pre-treatments few, analysis speed is fast, highly sensitive, can realize high flux, multiplex detection, in fields such as food and Environmental security, inspection and quarantine, has obtained application widely.But there is the problem that needs solution in traditional magnetic separation-quantum dot sensor, the fluorescence signal of traditional magnetic separation-quantum dot immune sensor passes through Ag-Ab specific reaction in conjunction with upper quantum dot immune probe from immunomagnetic beads, this analytical model has two shortcomings: first, itself has fluorescence immunomagnetic beads, can cause fluorescence background to disturb; Second, the diameter of quantum dot is than the little order of magnitude of immunomagnetic beads, and after Ear Mucosa Treated by He Ne Laser Irradiation is come, the fluorescence of the quantum dot on the other surface of magnetic bead cannot be excited effectively, this phenomenon, just as the phenomenon at " daytime and night ", therefore causes effective fluorescence signal loss.Background fluorescence interference problem for the first from superparamagnetic pearl, has researcher by regulating the position of the fluorescence emission peak of quantum dot, avoids the fluorescence peak position of immunomagnetic beads itself, substantially can overcome the problem of fluorescence background value.But for the solution of Second Problem, also there is no relevant report at present.In order to overcome above-mentioned shortcoming, expand the range of application of this fluorescent optical sensor, this chapter intends adopting a kind of novel indirect signal analytical model, under the certain condition of the quantum dot immune probe total amount adding, detection is not combined in the fluorescence intensity of the quantum dot on immunomagnetic beads just can instead release the content of object in sample.
Summary of the invention
In the present invention, we have built a kind of universality, fast, pathogenic bacteria and viral detection for sensitive homogeneous phase immunosensor, this immunosensor is based on magnetic separation-quantum dot indirect fluorescent signal analysis pattern.In indirect signal analytical model, the fluorescence intensity of remaining quantum dot immune probe is as fluorescence signal, thereby avoid from super suitable nanometer magnetic bead autofluorescence interference problem, the problem that adopts indirect signal analytical model not only can avoid background fluorescence to disturb, and can improve effective fluorescence signal value, and then the sensitivity of raising method.Indirect signal analytical model does not need again to hang molten immunomagnetic beads-quantum dot immune compound in addition, thereby has simplified experimental implementation block diagram 1.
Hypersensitive QDs-MB immunosensor based on indirect signal analytical model is as follows for the detecting step of biomacromolecule:
1. the preparation of super suitable nano immune magnetic bead MB-Ab1 (1)
The preparation of 1.1 phosphate buffered solution PBS: sodium hydrogen phosphate 0.2mol L
-1with sodium dihydrogen phosphate 0.2mol L
-1according to a certain amount of ratio, mix, be mixed with 0.01mol L
-1phosphate buffered solution, pH=7.0-7.4.
The preparation of 1.2 confining liquids: be 0.01mol L by volumetric molar concentration
-1, pH==7.4 PBS dissolve bovine serum albumin (BSA) to make its weight percent concentration be 0.1-1%; After dissolving, the preferred weight percent concentration of bovine serum albumin is 0.1%-1%.
The preparation of 1.3 cleansing solutions (PBST): be 0.01mol L by concentration
-1, in pH=7.4PBS solution, adding mass concentration is 0.05%Tween-20
The activation of 1.4 nanometer magnetic beads: the super suitable nanometer magnetic bead that to get 1-5mg particle diameter be 100-2000nm, have carboxyl is in 1.5mL centrifuge tube, then separated by magnetic, remove supernatant and obtain super suitable nanometer magnetic bead, then add 100-1000 μ L phosphate buffered solution, be placed in and put into the vortex oscillator 10s that vibrates after the ultrasonic 30s of ultrasonic cleaner, add 1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride that N-hydroxy thiosuccinimide that 5-20 μ L mass concentration is 50mg/mL and 5-20 μ L mass concentration are 50mg/mL, shake 20min; Separated by magnetic, remove remaining activator and accessory substance, then wash with the PBS buffer solution of pH=7.4, wash 2-3 time, then be dissolved in the above-mentioned PBS buffer solution of 100-1000 μ L, obtain the super suitable nanometer magnetic bead suspending liquid of activation.
The antibody of 1.5 identification biomacromolecules and the super suitable nanometer magnetic bead coupling having activated: get the above-mentioned activation microsphere suspension liquid of 80-100 μ L in centrifuge tube, add 0.1-0.5mg Ab1 (protein content: 5-10mg/mL), shake 0.5-2h, by magnetic separation, remove unreacted reactant, again magnetic bead is dissolved in to the above-mentioned confining liquid of 500-10001 μ L, react half an hour, then separated by magnetic, remove supernatant, add 200-1000 μ L cleansing solution PBST again to dissolve, the 30s that vibrates in vortex oscillator carries out magnetic separation again, this washing step repeats 2 times, use again 1000 μ L PBS solution weight molten, obtain magnetic immunity affine enrichment reagent MB-Ab1 (1), be stored in 4 ℃ of refrigerators.
2. quantum dot immune fluorescent probe QDs-Ab
2(2) preparation
2.1 quantum dots (QDs) activation: getting 200-500 μ L finishing has the quantum dot of carboxyl to join in 1.5mL centrifuge tube, be placed in and put into the vortex oscillator 10s that vibrates after the ultrasonic 30s of ultrasonic cleaner, add the EDC that NHS that 5-20 μ L mass concentration is 5mg/mL and 5-20 μ L mass concentration are 10mg/mL, shake 10-20min activates again.
Large molecular antibody (the Ab of 2.2 antibiont
2) with the coupling of QDs: by 0.1-0.3mg Ab
2(protein content 5-10mg/mL) joins in the QDs suspending liquid that above-mentioned activation is good, shake 0.5-2h, add again the above-mentioned confining liquid of 500-1000 μ L to react again 0.5h, then the ultra-filtration centrifuge tube that is 100kd with molecular weight is the centrifugal 15min of 8000g at centrifugal force, remove lower clear liquid, then use the molten QDs-Ab of PBS solution weight of 100-500 μ L
2, centrifugal 15min under the condition that the ultra-filtration centrifuge tube that is 100kd with molecular weight is 8000g at centrifugal force, this step repeats 2-3 time, finally uses the heavy molten QDs-Ab of PBS solution (0.5%BSA) of 100-500 μ L
2, prepare QDs-Ab
2fluorescence immunoassay probe (2).
3. separation-the quantum dot fluorescence of the magnetic based on indirect signal analytical model immune analysis method detecting step
3.1 standard curve making
3.1.1MB-Ab1 (1) affine enrichment: get the above-mentioned MB-Ab that has optimized concentration of 100 μ L
1(1) biomacromolecule (Ag) (3) standard series of solution and 1000 μ L variable concentrations gradients is blended in respectively in 1.5mL centrifuge tube, vortex 10-15min under room temperature, through magnetic separator frame magnetic after separating, by the above-mentioned phosphate buffered solution of 100 μ L, redissolve, make the MB-Ab1 compound (MB-Ab1-Ag) that series is caught Ag.
3.1.2 immune response: get respectively the above-mentioned serial MB-Ab1-Ag immune complex of 100 μ L in 96 porocyte culture plates, add respectively 100 μ L to optimize the QDs-Ab of concentration
2fluorescence immunoassay probe (2) reacts 15min at 37 ℃, and magnetic is separated, obtains respectively MB-Ab1-Ag-Ab
2-QDs immune complex (4) and remaining QDs-Ab
2fluorescence immunoassay probe (2).
3.1.3 quantitative fluorescence analysis: utilize Multifunction fluorescent microplate reader to respectively to MB-Ab
1-Ag-QDs immune complex (4) and remaining QDs-Ab
2the fluorescence intensity of fluorescence immunoassay probe (2) is measured, take fluorescence intensity as ordinate, the concentration of biomacromolecule object is horizontal ordinate, sets up respectively the typical curve of (indirect signal analytical model and direct signal analytical model) under two kinds of unlike signal analytical models.
3.2 Specimen Determination
With actual sample alternate standard sample to be measured, by the operation steps of 3.1 standard curve making, to test, the fluorescence intensity recording according to actual sample is derived the content of biomacromolecule in actual sample (Ag) (3) from above-mentioned typical curve.
Accompanying drawing explanation
Fig. 1: the magnetic separation-quantum dot fluorescence immune analysis method schematic diagram based on indirect signal analytical model
Fig. 2. the optimization of immunomagnetic beads concentration (a) Direct Analysis pattern (b) indirect analysis pattern
Fig. 3. optimization (a) Direct Analysis pattern (b) the indirect analysis pattern of quantum dot fluorescence immunological probe concentration
Fig. 4. the sensitivity of the withered bacterium of detection corn under indirect signal analytical model (a) and direct signal analytical model (b)
1 is magnetic immunity affine enrichment reagent (MB-Ab1); 2 is quantum dot fluorescence immunological probe (QDs-Ab
2); 3 is biomacromolecule (Ag); 4 is MB-Ab1-Ag-Ab
2-QDs immune complex
Embodiment
The present invention is further described in detail by following examples.
Embodiment 1: the preparation of analog sample
Take that to detect corn Fusarium oxysporum content in sample be example.
Get 15 corn seed samples in 50mL centrifuge tube, with the NaCl0 soaking disinfection of 30mL3-5%, process 5min-15min, use 10mL aseptic water washing 3 times.With 40mL PBS buffer solution (0.01mol/L, pH7.4), soak, 4 ℃ are spent the night.Soak solution is moved into centrifuge tube, with the centrifugal 10min of 1000r/min, get supernatant and move into other centrifuge tube, with the centrifugal 15min of 10000r/mmin, abandoning supernatant, suspends and precipitates with the above-mentioned PBS buffer solution of 100mL, is prepared into corn seed matrix liquid.With this matrix liquid, as diluent preparing, become the different content corn bacterium (10 that withers
7, 10
6, 10
5, 10
4, 10
3, 10
2, 10 and 0cfu/mL) serial positive.Do not add the withered bacterium of corn, by aforesaid operations step, prepare negative sample.
Embodiment 2: the preparation of immunomagnetic beads reagent
Take that to detect corn Fusarium oxysporum content in sample be example.
1. the preparation of phosphate buffered solution: sodium hydrogen phosphate (0.2mol L
-1) and sodium dihydrogen phosphate (0.2mol L
-1) according to (Na
2hPO
4/ NaH
2pO
4=4: 1 ratio) mix, be mixed with 0.01mol L
-1phosphate buffered solution, pH=7.4.
2. the preparation of confining liquid: be 0.01mol L by volumetric molar concentration
-1, PH=7.4 PBS dissolve bovine serum albumin (BSA) to make its weight divide in vain specific concentration be 0.5%.
3. the activation of super suitable nanometer magnetic bead: get 2mg particle diameter and be the super suitable nanometer magnetic bead of 1000nm carboxyl in 1.5mL centrifuge tube, be placed in and put into the vortex oscillator 10s that vibrates after the ultrasonic 30s of ultrasonic cleaner, with the separation of magnetic separator frame, obtain solid-state super suitable nanometer magnetic bead, then at every turn with the resuspended super suitable nanometer magnetic bead of phosphate buffer of 1000 μ L pH=7.4, add the NHS that EDC that 10 μ L mass concentrations are 50mg/mL and 10 μ L mass concentrations are 50mg/ml, shake 20min activates; Activator and the accessory substance not reacted removed in magnetic separation, by 500 μ L volumetric molar concentrations, is then 0.01mol L
-1, pH=7.4 PBS buffer solution again dissolve, obtain the suspending liquid of super suitable nanometer magnetic bead of activation.
4. the super suitable nanometer magnetic bead coupling of corn Fusarium oxysporum antibody and activation: get the above-mentioned activation microsphere suspension liquid of 100 μ L in centrifuge tube, add 0.3mg corn Fusarium oxysporum polyclonal antibody (protein content: 10mg/mL), shake 1 hour, to outwell waste liquid after the centrifugal 6min of 8000rpm centrifugal force, be dissolved in again the above-mentioned confining liquid of 600 μ L, the microballoon that is fixed corn Fusarium oxysporum polyclonal antibody obtains magnetic enrichment reagent (1), is stored in 4 ℃ of refrigerators.
Embodiment 3: quantum dot fluorescence probe (QDs-Ab
2) preparation of (2)
Take that to detect corn Fusarium oxysporum content in sample be example.
1.QDs surface activated carboxylic: get 100 μ L QDs solution in 1.5mL centrifuge tube, be placed in and put into the vortex oscillator 10s that vibrates after the ultrasonic 30s of ultrasonic cleaner, add the NHS that EDC that 10 μ L mass concentrations are 10mg/mL and 5 μ L mass concentrations are 10mg/mL, shake 15min activates again;
2.QDs-Ab
2the preparation of fluorescence probe (2): 0.3mg corn Fusarium oxysporum monoclonal antibody (protein content 5mg/mL) is joined in the QDs solution that above-mentioned activation got well, at room temperature react 2h, and then add the PBS buffer solution that 50 μ L BSA concentration are 1%, capping 0.5h, then transfer in the ultra-filtration centrifuge tube that molecular weight is 100kd, centrifugal 15min under the centrifugal force of 8000g, the antibody of removing lower clear liquid and not reacting completely, then use the resuspended QDs-Ab of above-mentioned PBS buffer solution 200 μ L
2fluorescence immunoassay probe, then in same ultra-filtration centrifuge tube centrifugal 15min (4 ℃), this step repeats 2 times, the BSAPBS solution that is finally 0.1% by the concentration of 500 μ L is resuspended, is kept in 4 ℃ of refrigerators.
Embodiment 4: the large Molecular Detection of bacterium living beings
Take that to detect corn Fusarium oxysporum content in sample be example.
1. the drafting of typical curve
1) enrichment of corn Fusarium oxysporum
Get respectively withered bacterium (3) standard series (10 of corn
7, 10
6, 10
5, 10
4, 10
3, 10
2, 10 and 0cfu/mL) in 1.5mL centrifuge tube, add the MB-Ab1 (1) that 100 μ L particle diameters are 1000nm, eddy current rotation 10min, abandons supernatant after magnetic separation, with the above-mentioned PBS buffer solution redissolution of 100 μ L, obtains MB-Ab1-Ag immune complex.
2) optimization of immune condition
The optimization of MB-Ab (1) concentration: immunomagnetic beads concentration has important impact to the non-specific adsorption in the bioaccumulation efficiency of object and immune response in the method, in this patent, its concentration is optimized, selected a kind of bioaccumulation efficiency high, non-specific little concentration is as best concentration.
QDs-Ab
2the optimization of fluorescence immunoassay probe (2) concentration: the fluorescence immunoassay probe of QDs-Ab has vital effect to the sensitivity of method and stability, QDs-Ab fluorescence immunoassay probe (2) concentration is too high, cause remaining fluorescence probe too much, the sensitivity that causes method, the concentration of fluorescence probe is too low, by causing, in the sample of high concentration, can not effectively catch fluorescence probe, cause the range of linearity of method to narrow down.This patent is optimized its concentration.
3) step of immune condition
Get respectively above-mentioned MB-Ab1-Ag immune complex in 96 porocyte culture plates, add respectively 100 μ L QDs-Ab1 fluorescence immunoassay probes (2), in 37 ℃ of reaction 15min, obtain MB-Ab1-Ag-QDs-Ab
2compound (4) and there is no the QDs-Ab of complete reaction
2fluorescence immunoassay probe (2).
4) fluorescence signal test
By Multifunction fluorescent microplate reader, the fluorescence intensity in said sample is measured, be take quantum dot fluorescence intensity as ordinate, the corn Fusarium oxysporum content in sample of take is horizontal ordinate, is depicted as typical curve.
Instrument parameter is as follows: fluorescence exciting wavelength is 300nm, and emission wavelength is 621nm.
Fig. 2. the optimization of immunomagnetic beads concentration (a) Direct Analysis pattern (b) indirect analysis pattern
Fig. 3. optimization (a) Direct Analysis pattern (b) the indirect analysis pattern of quantum dot fluorescence immunological probe concentration
2. actual sample detects
Analog sample prepared by the embodiment 1 of take is example, by above-mentioned steps operation, according to the relation between fluorescence intensity in typical curve and analyte concentration, records the withered bacterial concentration of corn in sample.
Fig. 4. the sensitivity of the withered bacterium of detection corn under indirect signal analytical model (a) and direct signal analytical model (b)
Embodiment 5: the large Molecular Detection of viral organism
It is example that the H9N2 subtype avian influenza virus of take detects.
1. Specification Curve of Increasing
1) enrichment of virus
With H9N2 subtype avian influenza virus inoculated into chick embryo, obtain chick embryo allantoic liquid.With above-mentioned PBS buffer solution, carry out continuous doubling dilution, until be diluted to 2
12doubly.Get respectively the H9N2 subtype avian influenza virus (2 of the different dilution ratios of 1000 μ L
12, 2
11, 2
10, 2
9, 2
8, 2
7, 2
6, 2
5, 2
4, 2
3, 2
2with 2
0) in 1.5mL centrifuge tube, add respectively the MB-Ab1 (1) of 100 μ L, after eddy current rotation concussion 10min, separated by magnetic, abandon supernatant, with the above-mentioned PBS buffer solution of 100 μ L, redissolve, obtain MB-Ab1-Ag immune complex.
2) immune response
Get respectively above-mentioned MB-Ab1-Ag immune complex in 96 porocyte culture plates, add respectively 100 μ LQDs-Ab
2(2),, in 37 ℃ of reaction 15min, obtain MB-Ab1-Ag-QDs compound (4) and the QDs-Ab of complete reaction not
2(2).
3) measurement of fluorescence intensity
By Multifunction fluorescent microplate reader, the fluorescence intensity in said sample is measured, be take quantum dot fluorescence intensity as ordinate, the corn Fusarium oxysporum content in sample of take is horizontal ordinate, is depicted as typical curve.
Instrument parameter is as follows: fluorescence exciting wavelength is 300nm, and emission wavelength is 621nm.
2. actual sample detects
With sample, replace H9N2 subtype avian influenza virus standard series, by above-mentioned steps operation, according to the relation between fluorescence intensity in typical curve and testing concentration, record H9N2 subtype avian influenza virus content in sample.
Research unit, company that bacterium required for the present invention and viral antigen can arrive relevant speciality buy or customization; Required instrument, equipment, reagent all have commercially available.
Claims (4)
1. the method based on magnetic separation-quantum dot immune fluorescent sensing detection biomacromolecule, is characterized in that, with super suitable nanometer magnetic bead MBs, is that carrier is after functional modification and resist the antibody A b of biomacromolecule to be measured
1coupling and make capture antibody MB-Ab
1; By quantum dot after functional modification with the antibody A b of anti-biomacromolecule to be measured
2coupling and make quantum dot fluorescence probe QDs-Ab
2; Utilize MB-Ab
1from sample, specific enrichment biomacromolecule Ag to be measured, makes serial compound MB-Ab
1-Ag; Make MB-Ab
1-Ag and QDs-Ab
2reaction forms MB-Ab
1-Ag-Ab
2-QDs, makes it and liquid phase QDs-Ab through magnetic separation
2immune fluorescent probe is separated; Measure QDs-Ab
2fluorescence intensity X, utilizes the inverse correlation funtcional relationship presenting between biomacromolecule concentration C to be measured and X and realizes sensing detection biomacromolecule concentration to be measured; Its step is,
(1) super suitable nanometer magnetic bead MBs and antibody A b
1coupling:
A. the activation of super suitable nanometer magnetic bead: the super suitable nanometer magnetic bead that to get 1-5mg particle diameter be 100-2000nm, have carboxyl is in 1.5mL centrifuge tube, separated by magnetic, remove supernatant and obtain super suitable nanometer magnetic bead, add 100-1000 μ L phosphate buffered solution, be placed in and put into the vortex oscillator 10s that vibrates after the ultrasonic 30s of ultrasonic cleaner, add 1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride EDC that N-hydroxy thiosuccinimide NHS that 5-20 μ L concentration is 50mg/mL and 5-20 μ L concentration are 50mg/mL, shake 20min; Separated by magnetic, remove remaining activator and accessory substance, then wash with the PBS buffer solution of pH=7.4, wash 2-3 time, then be dissolved in the above-mentioned PBS buffer solution of 100-1000 μ L, obtain the super suitable nanometer magnetic bead suspending liquid of activation.
B. the coupling of the super suitable nanometer magnetic bead of antibody and activation: get the above-mentioned activation nanometer magnetic bead of 80-100 μ L suspending liquid in centrifuge tube, adding protein content is the Ab of 5-10mg/mL
1antibody 0.3-0.5mg, shake 0.5-2h, by magnetic separation, remove unreacted reactant, then magnetic bead is dissolved in to the above-mentioned confining liquid of 500-1000 μ L, react half an hour, by magnetic separation, remove supernatant again, add 200-1000 μ L cleansing solution PBST again to dissolve, the 30s that vibrates in vortex oscillator carries out magnetic separation again, and this washing step repeats 2 times, with the PBS solution of 1000 μ L, redissolve again, obtain the affine enrichment reagent MB-Ab of magnetic immunity
1, be stored in 4 ℃ of refrigerators.
(2) quantum dot QDs and the antibody A b that resists biomacromolecule to be measured
2coupling:
A. the activation of quantum dot QDs: getting 200-500 μ L finishing has the quantum dot of carboxyl to join in 1.5mL centrifuge tube, be placed in and put into the vortex oscillator 10s that vibrates after the ultrasonic 30s of ultrasonic cleaner, adding 10-50 μ L mass concentration is the NHS of 5 mg/mL and 1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride that 10-50 μ L concentration is 10mg/mL again, and shake 10-20min activates.
B. the large molecular antibody Ab of antibiont
2coupling with QDs: by the Ab that is protein content 5-10mg/mL
2antibody 0.2-0.3mg joins in the QDs suspending liquid that above-mentioned activation is good, shake 0.5-2h, add the above-mentioned confining liquid reaction of 500-1000 μ L 0.5h, the ultra-filtration centrifuge tube that is 100kd with molecular weight is centrifugal 15min under 8000g at centrifugal force, remove lower clear liquid, with the molten QDs-Ab of PBS solution weight of 100-500 μ L
2, the ultra-filtration centrifuge tube that is 100kd with molecular weight is centrifugal 15min under 8000g at centrifugal force, this step repeats 2-3 time, finally uses the PBS solution redissolution QDs-Ab of 100-500 μ L
2, make QDs-Ab
2fluorescence immunoassay probe.
(3) enrichment of biomacromolecule Ag to be measured in sample:
A. affine enrichment: get the above-mentioned MB-Ab of 100 μ L
1solution is mixed in 1.5mL centrifuge tube with the biomacromolecule Ag to be measured of 1000 μ L variable concentrations respectively, and vortex 10-15min under room temperature redissolves by the above-mentioned phosphate buffered solution of 100 μ L after magnetic separation, makes serial compound MB-Ab
1-Ag.
(4) biomacromolecule content to be measured in microwell plate fluorescence detector indirect detection sample:
A. immune response: get respectively above-mentioned serial MB-Ab
1-Ag compound 100 μ L, in 96 porocyte culture plates, add respectively the above-mentioned QDs-Ab of 100 μ L
2fluorescence immunoassay probe reacts 15min at 37 ℃, separated through magnetic, makes MB-Ab
1-Ag-Ab
2-QDs immune complex and liquid phase QDs-Ab
2immune fluorescent probe is separated.
B. immunofluorescence quantitative test: utilize Multifunction fluorescent microplate reader respectively to liquid phase QDs-Ab
2immune fluorescent probe carries out fluorometric assay, and its fluorescence intensity X and biomacromolecule concentration C to be measured meet following functional relation, thereby has indirectly recorded biomacromolecule concentration to be measured.
Lg[C]＝AX+B
C: the concentration of determinand;
X: record quantum dot fluorescence intensity;
A and B are constant.
2. the method based on magnetic separation-quantum dot immune fluorescent sensing detection biomacromolecule according to claim 1, is characterized in that, by detecting remaining quantum dot immune fluorescent probe QDs-Ab in immune response
2fluorescence intensity and indirectly recorded biomacromolecule to be measured.
3. the reagent MB-Ab based on magnetic separation-quantum dot immune fluorescent sensing detection biomacromolecule according to claim 1
1preparation method, it is characterized in that, MB particle diameter is 100-2000nm, with NHS and EDC, activates and makes MB and Ab
1coupling and be prepared into MB-Ab
1, MB and Ab
1mol ratio be 1: 10-1: 50.
4. the quantum dot immune fluorescent probe QDs-Ab based on magnetic separation-quantum dot immune fluorescent sensing detection biomacromolecule according to claim 1
2preparation method, it is characterized in that, use NHS and EDC activates afterwards and Ab
2coupling and be prepared into QDs-Ab
2, quantum dot QDs and Ab
2mol ratio be 1: 10-1: 50.
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