CN110133246A - A kind of homogeneous immunoassay method on individual particle is horizontal - Google Patents
A kind of homogeneous immunoassay method on individual particle is horizontal Download PDFInfo
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
- G01N21/47—Scattering, i.e. diffuse reflection
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/62—Detectors specially adapted therefor
- G01N30/74—Optical detectors
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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
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
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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
- G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
- G01N33/585—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with a particulate label, e.g. coloured latex
- G01N33/587—Nanoparticles
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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
- G01N33/58—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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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N2030/022—Column chromatography characterised by the kind of separation mechanism
- G01N2030/027—Liquid chromatography
Abstract
A kind of homogeneous immunoassay method on individual particle is horizontal, including, capture antibody is modified on gold nano grain CA-AuNP and quantum dot DA-QD respectively with detection antibody；Sample is added in CA-AuNP and DA-QD mixed liquor, concussion is incubated for a period of time, forms the interlayer structure of CA-AuNP- antigen-DA-QD；It takes the solution of preparation to drip on glass slide, glass slide is placed under dark field microscope and is imaged；It irradiates glass slide for a period of time, the scattering strength of gold particle is recorded in irradiation process；Plasma resonance energy transfer occurs for gold particle and quantum dot in interlayer structure, leads to the reduction of gold particle scattering strength.The CA-AuNP scattering strength and numbers of particles for not forming interlayer structure are distributed as normal distribution, are worth on the basis of the desired value of the normal distribution；The gold particle number below and above a reference value is counted, the two ratio is calculated, antigen concentration is quantified with the ratio.This method is homogeneous immunoassay, simple and fast, few using sample size, is imaged in individual particle level, and detection limit is low, detection sensitivity is high.
Description
Technical field
The present invention relates to biochemistries and technical field of medical detection, and in particular to homogeneously exempts from a kind of individual particle is horizontal
Epidemic disease analysis method.
Background technique
Immunoassay is one of most popular technology in biochemistry and medicine detection.It is to utilize antigen and antibody
Specific binding carrys out one kind point of qualitative, quantitative antigen (can be the substances such as drug, hormone, protein, microorganism) or antibody
Analysis method；Generally using antigen-antibody conjugate or free antibodies content as the detection technique of quantitative basis.
Immunoassay that is, heterogeneous immunoassay and is homogeneously exempted from according to whether needing the separation application can be divided into two classes
Epidemic disease analysis.The former again detects each component after needing first to separate certain component of antigen-antibody reaction mixed liquor, high sensitivity, inspection
It is low to survey limit；But its operating procedure is more, and process is complicated, time-consuming, laborious.The latter is without separating each group in antigen-antibody reaction mixed liquor
Point, a step is completed, and has many advantages, such as that easy to operate, the time is short, easy realization automation；But relative to heterogeneous immunoassay,
Phase immunoassay sensitivity is poor.
Some homogeneous immunoassay methods, including Resonance Energy Transfer, dynamic scattering are reported in recent years document
Method, resonance scattering correlation spectrum, magnetic relaxation, chemoluminescence method etc..The problem of these methods, which is to detect, limits not low enough, complicated body
Reliability is not high enough in system, sample usage amount is larger.Therefore, develop highly sensitive homogeneous immunoassay method in a kind of micro blood
It has a very important significance.
Summary of the invention
The object of the present invention is to provide the homogeneous immunoassay methods in a kind of individual particle level, with the gold in interlayer structure
Gold particle scattering strength is reduced to quantitative basis caused by plasma resonance energy transfer occurs for particle and quantum dot, quickly,
Tumor markers concentration in sensitive, accurate detection blood.
For achieving the above object, technical solution of the present invention is specific as follows:
A kind of homogeneous immunoassay method on individual particle is horizontal, which comprises the following steps:
S1: capture antibody and detection antibody are modified respectively on gold nano grain CA-AuNP and quantum dot DA-QD；
S2: sample is added in CA-AuNP and DA-QD mixed liquor, and concussion is incubated for a period of time, forms CA-AuNP-
The interlayer structure of antigen-DA-QD；
S3: the solution for taking S2 to prepare is dripped on glass slide, and glass slide is placed under dark field microscope and is imaged；
S4: irradiation glass slide for a period of time, records the scattering strength of gold particle in irradiation process；Gold in interlayer structure
Plasma resonance energy transfer occurs for particle and quantum dot, leads to the reduction of gold particle scattering strength, does not form interlayer structure
CA-AuNP scattering strength and numbers of particles are distributed as normal distribution, are worth on the basis of the desired value of the normal distribution, statistic procedure
Below and above the gold particle number of a reference value in S4, the two ratio is calculated；
S5: by gold particle quantity and the gold particle ratio of number for being higher than a reference value in step S4 lower than a reference value and anti-
Original content association.
Further, the gold nano grain in the step S1 includes gold nanosphere, gold nanorods, gold nano star and does not advise
Gold nano grain then；Incubation time in the step S2 is 10~100 minutes；The step S3 is specifically included: being taken 1.8 micro-
The solution for rising S2 preparation is dripped on glass slide, and glass slide is placed under dark field microscope and is imaged；When irradiation in the step S4
Between be 2~30 minutes；The ratio for being associated as obtaining using step S4 in the step S5 is ordinate, with the logarithm of antigen concentration
Standard curve or standard addition method are obtained for abscissa.
The present invention also provides above-mentioned analysis methods in simple solution in prostate-specific antigen PSA quantitative detection
Using.
The present invention also provides application of the above-mentioned analysis method in blood-serum P SA quantitative detection.
Compared with prior art, beneficial effects of the present invention:
Homogeneous immunoassay of the invention, without fixed, separation, cleaning；It is few using sample size, it only needs several micro-
It rises；
Sample to be tested is added in CA-AuNP and DA-QD mixed liquor by analysis method of the invention, is immunoreacted,
Then using dark field microscope as detection platform, the scattering strength of sampling observation gold particle, with gold particle in interlayer structure and quantum
Point occurs plasma resonance energy transfer and gold particle scattering strength is caused to be reduced to quantitative basis, and there is detection to limit low, detection
High sensitivity advantage can reach fM rank；It can be used for the quantitative detection of tumor markers in simple solution, it can also be used in serum
The quantitative detection of tumor markers.
Detailed description of the invention
Fig. 1 is the schematic illustration of the analysis method in the embodiment of the present invention 1；
When Fig. 2 is that plasma resonance energy transfer does not occur for the gold particle and quantum dot in the embodiment of the present invention 1, gold particle
Scatter intensity distribution figure；
Fig. 3 is after plasma resonance energy transfer occurs for the gold particle and quantum dot in the embodiment of the present invention 1, and gold particle dissipates
Penetrate intensity distribution；
Fig. 4 be the embodiment of the present invention 2 in simple solution in PSA quantitation curves；
Fig. 5 is that the standard addition method in the embodiment of the present invention 3 measures PSA content in serum.
Specific embodiment:
Following will be combined with the drawings in the embodiments of the present invention, and technical solution in the embodiment of the present invention carries out clear, complete
Site preparation description, it is clear that the described embodiment is only a part of the embodiment of the present invention, instead of all the embodiments.Based on this
Embodiment in invention, every other reality obtained by those of ordinary skill in the art without making creative efforts
Example is applied, shall fall within the protection scope of the present invention.
Embodiment 1
As shown in Figure 1, the homogeneous immunoassay method in a kind of individual particle level, includes the following steps:
It 1), respectively will capture antibody and detection antibody modification to gold particle (CA-AuNP) according to determinand (antigen) property
With the surface quantum dot (DA-QD)；
2) sample is added in CA-AuNP and DA-QD mixed liquor, is incubated for 10-100 minutes, form CA-AuNP- antigen-
DA-QD interlayer structure；
3) 1.8 microlitres of solution drops is taken in above-mentioned system to be imaged on glass slide, being placed under dark field microscope；
4) glass slide 2-30min is irradiated, the scattering strength of gold particle is recorded in irradiation process；
5) plasma resonance energy transfer occurs for the gold particle in interlayer structure and quantum dot, leads to gold particle scattering strength
It reduces, as shown in Figures 2 and 3.Fig. 2 show gold particle and when plasma resonance energy transfer does not occur for quantum dot, gold particle
Scatter intensity distribution figure.Gold particle scattering strength is single symmetrical Gaussian Profile peak, and peak height is between 880-920.It dissipates
Gold particle number of the intensity less than 880 is penetrated, it is roughly the same greater than 920 gold particle number with scattering strength.Fig. 3 show gold
After with quantum dot plasma resonance energy transfer occurs for grain, gold particle scatter intensity distribution figure.Scatter intensity distribution occurs two
Peak, a peak peak height is between 880-920, and another peak peak height is between 800-840；It can be seen that gold of the scattering strength less than 880
Grain number mesh is greater than 920 gold particle number more than scattering strength.Reason is that plasma resonance energy occurs for gold particle and quantum dot
Amount transfer leads to the reduction of gold particle scattering strength.
Using the gold particle ratio of number below and above peak height as quantitative basis, Fig. 4 show prostate in simple solution
The quantitation curves of specific antigen (PSA).When signal-to-noise ratio is 3, the detection limit of PSA reaches 0.4fM.Fig. 5 show standard
Addition method measures PSA content in serum.This analysis method step in homogeneous phase solution is disposably completed, solid without antibody embedding
Fixed, free antibodies separation carries out antigen detection using the interlayer structure in step 2), and detection limit molar concentration, which reaches, flies rank of rubbing
(fM).It can be used for the quantitative detection of PSA in simple solution, it can also be used to the quantitative detection of PSA in serum.
The detection of PSA in 2 simple solution of embodiment
A. gold particle, single stranded DNA 1, DNA 2, PBS, 4M sodium chloride are mixed rapidly according to a certain percentage, it immediately will mixing
Liquid is placed in -20 DEG C of freeze overnights.The final concentration of phosphoric acid in mixed liquor, sodium chloride, gold particle, single stranded DNA 1 and DNA 2 is respectively
4.2mM, 155mM, 196pM, 1.7 μM and 1.7 μM.Then at 18 DEG C, with 13000rpm revolving speed, by natural thaw to room temperature
Mixed liquor is centrifuged 8min, after removing supernatant, PBS is added and suspends again gold particle.Repeated washing twice, removes excessive DNA.Most
Afterwards at 18 DEG C, with 2500rpm revolving speed, suspension centrifugation 2min is taken into supernatant, to remove gold particle aggregate.2 μ L are fresh
The 10mg/mL EDC (dissolution of 155mM sodium chloride) of preparation is added in above-mentioned gold particle (70nm) after purification, room temperature shake
It swings, mix 20 minutes.With 155mM NaCl solution centrifuge washing gold particle 1 time, excessive EDC is removed.Immediately by gold particle with
PSA captures antibody (CA) and BSA and mixes according to 1:96:384 ratio, is incubated for 3 hours at room temperature.Addition tris-HCl (4mM,
PH7.4) in Yu Shangshu reaction mixture, reaction is terminated；With PBS (first with after 13000rpm with 2500rpm) centrifuge washing gold
Grain, removes excessive protein and gold particle aggregate, the capture antibody (CA-AuNP) of obtained gold particle label.
B. detection antibody, which is reacted by its primaquine group with the carboxyl of quantum dot, is fixed on quantum dot surface.Mix 20 μ L
PSA detects the quantum dot solution after antibody (DA, 1.2mg/ml) and 10 μ L activation.After being protected from light incubation 4 hours, using exclusion chromatography
The quantum dot of column purification antibody modification.Chromatographic condition is as follows: column size 250*4.6mm*mm, filler Superose6,
Mobile phase is 50mM NaHCO3, pH 9.0；Flow velocity is 0.1mL/min, and sampling volume is 15 μ L, Detection wavelength 224nm.It receives
Collect quantum dot-labeled detection antibody (DA-QD) fraction, places it in 4 degree of refrigerators and save backup.
C. will after purification DA-QD and CA-AuNP by concentration ratio 1:1 mix after, the PSA of various concentration is added, be incubated at room temperature
30 minutes, form CA-AuNP-PSA-DA-QD interlayer structure；
D. above-mentioned solution 1.8L is taken, drop is observed, shooting in glass slide, upper dark field microscope imaging.
E. quantitative experiment: the dark-field imaging figure under various concentration antigen that d is obtained is handled with imageJ, is calculated
Below and above the gold particle ratio of number of peak height under PSA various concentration.Using the logarithm of PSA concentration as abscissa, lower than and
Gold particle ratio of number higher than peak height is that ordinate is mapped (Fig. 4), in a linear relationship within the scope of 0.32-200fM, linear phase
Relationship number is 0.9958.
Test the detection of PSA in 3 serum
A. gold particle, single stranded DNA 1, DNA 2, PBS, 4M sodium chloride are mixed rapidly according to a certain percentage, it immediately will mixing
Liquid is placed in -20 DEG C of freeze overnights.The final concentration of phosphoric acid in mixed liquor, sodium chloride, gold particle, single stranded DNA 1 and DNA 2 is respectively
4.2mM, 155mM, 196pM, 1.7 μM and 1.7 μM.Then at 18 DEG C, with 13000rpm revolving speed, by natural thaw to room temperature
Mixed liquor is centrifuged 8min, after removing supernatant, PBS is added and suspends again gold particle.Repeated washing twice, removes excessive DNA.Most
Afterwards at 18 DEG C, with 2500rpm revolving speed, suspension centrifugation 2min is taken into supernatant, to remove gold particle aggregate.2 μ L are fresh
The 10mg/mL EDC (dissolution of 155mM sodium chloride) of preparation is added in above-mentioned gold particle (70nm) after purification, room temperature shake
It swings, mix 20 minutes.With 155mM NaCl solution centrifuge washing gold particle 1 time, excessive EDC is removed.Immediately by gold particle with
PSA captures antibody (Capture antibody, CA) and BSA and mixes according to 1:96:384 ratio, is incubated for 3 hours at room temperature.Add
Enter in tris-HCl (4mM, pH7.4) Yu Shangshu reaction mixture, terminates reaction；With PBS (first with after 13000rpm with
2500rpm) centrifuge washing gold particle removes excessive protein and gold particle aggregate, the capture of obtained gold particle label
Antibody (CA-AuNP).
B. detection antibody, which is reacted by its primaquine group with the carboxyl of quantum dot, is fixed on quantum dot surface.Mix 20 μ L
PSA detects the quantum dot solution after antibody (Detection antibody, DA, 1.2mg/ml) and 10 μ L activation.It is protected from light incubation 4
After hour, using the quantum dot of exclusion chromatography column purification antibody modification.Chromatographic condition is as follows: column size 250*4.6mm*
Mm, filler are Superose 6, and mobile phase is 50mM NaHCO3, pH 9.0；Flow velocity is 0.1mL/min, and sampling volume is 15 μ
L, Detection wavelength 224nm.Quantum dot-labeled detection antibody (DA-QD) fraction is collected, it is standby to place it in 4 degree of refrigerators preservations
With.
C. will after purification DA-QD and CA-AuNP by concentration ratio 1:1 mix after, be divided into several pieces.It is certain to being added in every part
The serum of amount and the PSA of various concentration are incubated at room temperature 30 minutes, form CA-AuNP-PSA-DA-QD interlayer structure；
D. above-mentioned solution 1.8L is taken, drop is observed, shooting in glass slide, upper dark field microscope imaging.
E. quantitative experiment: the dark-field imaging figure under the various concentration antigen that Image J software processing operation d is obtained calculates
Out be added various concentration PSA when below and above peak height gold particle ratio of number.It is low using the PSA concentration of addition as abscissa
In be higher than peak height gold particle ratio of number be ordinate do figure (Fig. 5).X-axis intercept is the PSA in serum in upper mirror
Final concentration in liquid.It is computed, the concentration of PSA is 7.42nM in serum, consistent with clinical examination value (6.18nM).
Claims (4)
1. the homogeneous immunoassay method in a kind of individual particle level, which comprises the following steps:
S1: capture antibody and detection antibody are modified respectively on gold nano grain CA-AuNP and quantum dot DA-QD；
S2: sample is added in CA-AuNP and DA-QD mixed liquor, and concussion is incubated for a period of time, forms CA-AuNP- antigen-
The interlayer structure of DA-QD；
S3: the solution for taking S2 to prepare is dripped on glass slide, and glass slide is placed under dark field microscope and is imaged；
S4: irradiation glass slide for a period of time, records the scattering strength of gold particle in irradiation process；Gold particle in interlayer structure
Plasma resonance energy transfer occurs with quantum dot, leads to the reduction of gold particle scattering strength；The CA- of interlayer structure is not formed
AuNP scattering strength and numbers of particles are distributed as normal distribution, are worth on the basis of the desired value of the normal distribution, statistic procedure S4
In below and above a reference value gold particle number, calculate both ratio；
S5: will be dense lower than the gold particle quantity of a reference value and the gold particle ratio of number and antigen that are higher than a reference value in step S4
Degree association.
2. analysis method according to claim 1, which is characterized in that the gold nano grain in the step S1 includes Jenner
Rice ball, gold nanorods, gold nano star and irregular gold nano grain；Incubation time in the step S2 is 10~100 points
Clock；The step S3 is specifically included: taking the solution of 1~5 microlitre of S2 preparation to drip on glass slide, it is micro- that glass slide is placed in dark field
It is imaged under mirror；Irradiation time in the step S4 is 2~30 minutes；It is associated as in the step S5 with step S4 acquisition
Ratio is ordinate, obtains standard curve or standard addition method by abscissa of the logarithm of antigen concentration.
3. application of any analysis method of claim 1-2 in simple solution in tumor markers quantitative detection.
4. application of any analysis method of claim 1-2 in blood serum tumor markers quantitative detection.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN111304297A (en) * | 2020-03-23 | 2020-06-19 | 江苏师范大学 | Method for analyzing circulating tumor DNA on single molecule level |
| CN111398592A (en) * | 2020-03-20 | 2020-07-10 | 江苏师范大学 | Homogeneous multi-component immunoassay method based on quantum dot dimer number identification statistics |
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