CN105842451B - Method based on quantum dot fluorescence immune detection DNMT1 - Google Patents
Method based on quantum dot fluorescence immune detection DNMT1 Download PDFInfo
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- CN105842451B CN105842451B CN201610324656.8A CN201610324656A CN105842451B CN 105842451 B CN105842451 B CN 105842451B CN 201610324656 A CN201610324656 A CN 201610324656A CN 105842451 B CN105842451 B CN 105842451B
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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
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
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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
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6486—Measuring fluorescence of biological material, e.g. DNA, RNA, cells
Abstract
The present invention provides a kind of method based on quantum dot fluorescence immune detection DNMT1, including with Fe3O4Nano particle is solid phase carrier, surface is prepared by the immunomagnetic beads of DNMT1 monoclonal antibody covalent modifications, by DNMT1 standard items or testing sample using immunomagnetic beads capture and concentration and separation, then added into the product of gained after separation by quantum dot-labeled DNMT1 polyclonal antibodies, antibody is set to be combined with determined antigen, use multi-functional microplate reader to excite lower carry out fluoroscopic examination with 270 nm, regression equation is set up according to the relation of fluorescence intensity and DNMT1 standard items or testing sample concentration.This method step is simple and the range of linearity that detect by the regression equation that this method is set up to DNMT1 is wide, test limit is low, precision, the degree of accuracy is high and has good specificity.
Description
Technical field
The present invention relates to detection technique field, specifically, it relate to a kind of based on quantum dot fluorescence immune detection DNMT1
Method.
Background technology
In detection field, it is often necessary to which all kinds of antigens or antibody are qualitatively or quantitatively detected.In the prior art,
Through deriving panimmunity response analysis method based on " double antibodies sandwich ", such as：Radioimmunology, ELISA, chemistry
Luminescence method, time-resolved fluorescence method and fluorescent immune method etc., available for pathogenic microorganism is determined, determine the specific proteins of human body
Amount detection to disease so that carry out auxiliary diagnosis or monitoring etc., and purposes is widely.This kind of immune response analysis method it is logical
Chang Zuofa is：Will capture antibody be fixed on solid phase carrier, then with antigen (target protein) react, after washing again with labelled antibody
Reaction, washing adds substrate detection optical signal or directly detects fluorescence signal.Although the automation of the above method is eliminated manually
That washs is loaded down with trivial details, but there is the shortcomings of separative efficiency of solid phase carrier is relatively low, accuracy of detection is not high.
In order to solve the problem of above is present, people are seeking a kind of preferable technical solution always.
The content of the invention
The purpose of the present invention is that in view of the shortcomings of the prior art, so as to provide one kind using nanometer solid phase particles as carrier, have
There are high separative efficiency, accuracy of detection height and the method based on quantum dot fluorescence immune detection DNMT1 with high degree of specificity.
To achieve these goals, the technical solution adopted in the present invention is：One kind is based on quantum dot fluorescence immune detection
DNMT1 method, specific steps include：
A kind of surface is provided by the Fe of DNMT1 monoclonal antibody covalent modifications3O4Nano immune magnetic particle suspension liquid, note
It is used as Fe3O4@McAb；
A kind of surface is provided by quantum dot-labeled DNMT1 polyclonal antibodies, is denoted as QDs@PcAb；
By the Fe after being diluted through CB buffer solutions3O4@McAb with diluted through PBS after DNMT1 standard samples or treat
Survey object and carry out the min of incubation reaction 50 min~60 so that the DNMT1 standard samples or object to be measured are by Fe3O4@McAb
Capture, concentration and separation；
Then by the QDs@PcAb after being diluted through PBST buffer solutions and by Fe3O4@McAb captures, the institute of concentration and separation
State DNMT1 standard samples or object to be measured and carry out the min of the min of incubation reaction 110~130 so that the QDs@PcAb with it is described
DNMT1 standard samples or object to be measured are combined；After being diluted through PBST buffer solutions, determined on multi-functional microplate reader
270 nm excite the fluorescence intensity of lower reaction product；
According to the relation between the fluorescence intensity of detection and the DNMT1 standard samples or target concentration to be measured, segmentation is built
Vertical detection DNMT1 regression equation.
It should be noted that in above steps, CB represent carbonate buffer solution, PBS represent phosphate buffer solution,
PBST represents the phosphate buffer solution of addition Tween-20 nonionic surfactants.
Based on above-mentioned, the method based on quantum dot fluorescence immune detection DNMT1, in addition to use ELISA method detection
The Fe provided3O4The step of@McAb bioactivity.
There is provided a kind of Fe based on above-mentioned3O4The step of@McAb, includes：
（1）Cleaning：By Fe3O4Nano granule suspension uses concentration for 0.01 after abandoning supernatant processing through ultrasound, Magneto separate
Mol/L~0.015 mol/L, pH balance the min of 2 min~5 to it for 6.0 MES buffer solutions, then carry out Magneto separate and abandon
Obtain the first sediment clearly.Wherein MES represents morpholino b acid buffer solution；
（2）Activation：Concentration is sequentially added into first sediment for mg/mL EDC solutions of 8 mg/mL~10 and dense
Spend and be vortexed the min of 10 min of reaction~15 for the mg/mL NHS solution of 8 mg/mL~11, Magneto separate, abandon supernatant processing after adopt
It is washed with MES buffer solutions 3 times, then carry out again Magneto separate, abandon supernatant processing obtain the second sediment.Wherein, EDC is represented
(1- (3- dimethylamino-propyls) -3- ethyl-carbodiimide hydrochlorides solution, NHS represent n-hydroxysuccinimide solution, MES
Represent morpholino b acid buffer solution, mg/mL and represent every milliliter of milligram；
（3）Buffer system is changed：Second sediment is washed 3~4 times times using PBS, Magneto separate is obtained
3rd sediment；
（4）Coupling：DNMT1 monoclonal antibodies are added into the 3rd sediment and PBS carries out vortex reaction
1.5h~2h, through Magneto separate, abandon supernatant processing after it is cleaned 2 times using PBS, Magneto separate, abandon supernatant and obtain the 4th
Sediment；
（5）Closing：BSA confining liquids are added into the 4th sediment, progress, which is vortexed, reacts 0.5h~1h, Magneto separate,
Abandon after supernatant processing, using PBST buffer solution for cleaning 3~4 times, Magneto separate, abandon supernatant and obtain the 4th i.e. Fe of sediment3O4@McAb；
Wherein BSA confining liquids represent bovine serum albumin(BSA) confining liquid；
（6）Preserve：In coupled product Fe3O4PBS is added in@McAb, light shake shakes up, and 4 DEG C save backup.
Based on above-mentioned, in the method based on quantum dot fluorescence immune detection DNMT1, provided using ELISA method detection
The Fe3O4The step of@McAb bioactivity, includes：
（1）By through Fe that concentration is the CB buffer solutions dilution that 45 mmol/L~50 mmol/L, pH are 9.0~9.63O4@
McAb is added in ELISA Plate, Magneto separate, using ELISA Plate described in PBST buffer solution for cleaning；Wherein mmol/L representatives mM are every
Rise；
（2）PBS compound concentration is used for the ng/mL of 300 ng/mL~400 DNMT1 standard items, and is added
Enter in above-mentioned ELISA Plate, the min of 80 min~90 is incubated in insulating box, then carry out Magneto separate processing and use PBST buffer solutions
Clean the ELISA Plate；Wherein ng/mL represents nanograms per milliliter；
（3）DNMT1 polyclonal antibodies are diluted 400~500 times using confining liquid, then added it with every 100 microlitres of hole
Enter into the ELISA Plate, the min of 80 min~90 is incubated in 35 DEG C~37 DEG C insulating boxs, then carry out Magneto separate processing and adopt
With ELISA Plate described in PBST buffer solution for cleaning；
（4）The goat anti-rabbit igg for being marked HRP with confining liquid dilutes 4000~5000 times, with 100 microlitres of every hole by its
It is added in the ELISA Plate, the min of 80 min~90 is incubated in 35 DEG C~37 DEG C insulating boxs, then carries out Magneto separate processing simultaneously
Using ELISA Plate described in PBST buffer solution for cleaning；
（5）TMB developer A liquid and B liquid are mixed in equal volume, added it in the ELISA Plate, 35 DEG C~37 DEG C are kept away
The min of the min of light incubation reaction 15~20；After chromogenic reaction, H is added into the ELISA Plate2SO4Terminate liquid, and immediately in enzyme mark
The absorbance OD in each hole is detected on instrument, wherein, Detection wavelength is 450 nm.
Carried based on above-mentioned, the method based on quantum dot fluorescence immune detection DNMT1, in addition to using sandwich method detection
The step of QDs@PcAb supplied fluorescence intensity and antibody activity.
Based on above-mentioned, the method based on quantum dot fluorescence immune detection DNMT1, it is characterised in that provide a kind of described
The step of QDs@PcAb, includes：
PH is added in PE pipes for 7.0~7.4 borate buffer solution, and adds concentration into the PE pipes successively and is
The QDs and concentration of every liter of the micromole of 0.5 every liter of micromole~1 is the mg/mL of 0.5 mg/mL~1 DNMT1 polyclonal antibodies,
Add every time and vibration mixing is both needed to after above-mentioned substance；Wherein, mg/mL represents every milliliter of milligram；
Then the EDC solution that concentration is the mg/mL of 8 mg/mL~10, the whirlpool at 18 DEG C~25 DEG C are added into the PE pipes
The PBS containing 0.5% mass percent BSA confining liquids is added in rotation reaction 1.5h~2h, the backward PE pipes of reaction end to buffer
Liquid, be vortexed reaction 20 min~30 min at 18 DEG C~25 DEG C；
Gained reaction product is dissolved in BS buffer solutions after removing EDC through centrifugation, ultrafiltration, 4 DEG C of preservations, so as to be made described
QDs@PcAb。
Based on above-mentioned, in the method based on quantum dot fluorescence immune detection DNMT1, detect what is provided using sandwich method
The step of fluorescence intensity and antibody activity of the QDs@PcAb, includes：To be 45 mmol/L~50 mmol/L, pH through concentration
QDs@PcAb for 9.0~9.6 CB buffer solutions dilution are added in the hole elisa Plates of black 96, and constant temperature incubates 80 min~90
Min, then carries out Magneto separate and using the hole elisa Plates of black 96 described in PBST buffer solution for cleaning, then adds PBST micro- in many work(
Fluorescence intensity is surveyed on orifice plate readout instrument.
Based on a kind of above-mentioned, described method based on quantum dot fluorescence immune detection DNMT1, in addition to by above-mentioned each step
In rapid the step of the optimization of each parameter, the regression equation for detecting DNMT1 is then set up according to Optimal Parameters, wherein, in DNMT1 marks
Quasi- product concentration is that in the range of the ng/mL of 5.0 ng/mL~1500, the regression equation set up is Y=0.00775X+20.77161,
Wherein, it is DNMT1 standard concentrations that Y, which represents fluorescent value RFU, X, and correlation coefficient r is 0.9873；It is in DNMT1 standard concentrations
In the range of the ng/mL of 0.1 ng/mL~5.0, this method detection DNMT1 regression equation is Y=0.02779X+1.29858, wherein
Y represents lgRFU, and X represents lgDNMT1, and correlation coefficient r is 0.9877.
Based on above-mentioned, the method based on quantum dot fluorescence immune detection DNMT1, the minimum inspection to DNMT1 concentration
Survey is limited to 0.1 ng/mL.
Of the invention to have prominent substantive distinguishing features and significant progress compared with the prior art, specifically, the present invention will
Fe3O4The high fluorescent of the rapid separation of nano particle, the high degree of specificity of immune response and quantum dot is organically combined
Come, according to the relation between the fluorescence intensity of detection and the DNMT1 standard samples or target concentration to be measured, detection is set up in segmentation
DNMT1 regression equation, so as to calculate the content of DNMT1 in sample using this method, method and step is simple and is built by this method
The range of linearity that vertical regression equation is detected to DNMT1 is wide, test limit is low, precision, the degree of accuracy are high and with good special
Property.
Brief description of the drawings
Fig. 1 is the Fe of the present invention3O4@McAb bioactivity testing result figure.
Fig. 2 is QDs@PcAb of the present invention fluorescence intensity and antibody activity testing result figure.
Fig. 3 is the Fe of different extension rates of the invention3O4With reaction system fluorescence intensity graph of a relation during@McAb capture antigens.
Fig. 4 is QDs@PcAb complex concentrations of the present invention and reaction system fluorescence intensity graph of a relation.
Fig. 5 is the immune buffer solution species of the present invention and reaction system fluorescence intensity graph of a relation.
Fig. 6 is insolubilized antibody capture antigenic action time of the present invention and reaction system fluorescence intensity graph of a relation.
Fig. 7 is the action time of QDs@PcAb of the present invention and antigen to reaction system fluorescence intensity graph of a relation.
Fig. 8 is the low concentration DNMT1 examination criteria curve synoptic diagrams that the present invention is set up.
Fig. 9 is the high concentration DNMT1 examination criteria curve synoptic diagrams that the present invention is set up.
Embodiment
Below by embodiment, technical scheme is described in further detail.
Reagent type used is as follows in the present invention：DNMT1 antigens（OriGene）, DNMT1 monoclonal antibodies（Win in Beijing
Ao Long Immune Technology Corp.）, DNMT1 polyclonal antibodies（ABclonal）, Fe3O4Nano particle（Self-control）, CdSe/ZnS amounts
Sub- point（Wuhan Jia Yuan quantum dots company）, people DNA methylation transferase 3a ELISA kits, people's DNA methylation transferase 3b
ELISA kit（Upper thing Science and Technology Ltd. of Haikang nangzan）, the goat anti-rabbit igg of HRP marks, bovine serum albumin(BSA)（BSA,
Beijing Suo Laibao Science and Technology Ltd）, other reagents are to analyze pure, experimental water Milli-Q ultra-pure waters（Resistivity is more than
18.2 MΩ·cm）.
Instrument used is as follows in the present invention：Multi-functional microplate reader（SpectraMax M2e, the U.S.）, electric heating perseverance
Warm incubator（DHG-9080 types, the upper grand experimental facilities Co., Ltd of Nereid）, numerical control supersonic cleaning device（KQ-300DE types, elder brother
Ultrasonic instrument Co., Ltd of mountain city）, adjustable micro sample adding appliance（Eppendorf, Germany）, XH-C type vortex blending instruments（Jintan City
Guo Wang laboratory apparatus factory）, refrigerated centrifuge（Centrifuge 5424 R, Eppendorf）.
The present invention provides a kind of method based on quantum dot fluorescence immune detection DNMT1, and specific steps include：
A kind of surface is provided by the Fe of DNMT1 monoclonal antibody covalent modifications3O4Nano immune magnetic particle suspension liquid, note
It is used as Fe3O4@McAb；And the Fe provided using ELISA method detection3O4@McAb bioactivity；
There is provided a kind of by quantum dot-labeled DNMT1 Anti-TNF-α liquid solutions, be denoted as QDs PcAb；And using sandwich
The fluorescence intensity and antibody activity for the QDs@PcAb that method detection is provided；
The Fe3O4@McAb are added in the orifice plate of black 96 after being diluted through CB buffer solutions, and add buffered through PBS thereto
The DNMT1 antigens of liquid dilution carry out constant temperature and breed the min of 50 min~60；Then added into the orifice plate of black 96 through PBST
The QDs PcAb after buffer solution dilution carry out the min of incubation reaction 110 min~130, the most backward orifice plate of black 96
Middle addition PBST, fluorescence intensity is determined on multi-functional microplate reader；Then the fluorescence obtained according to the parameter after optimization
Detection DNMT1 regression equation is set up in relation between intensity and the DNMT1 standard samples or target concentration to be measured, segmentation.
Wherein, it is both needed to carry out the orifice plate of black 96 Magneto separate and the processing of PBST board-washings after each incubation reaction.Set up detection DNMT1
Regression equation, wherein, be the recurrence side set up in the range of the ng/mL of 5.0 ng/mL~1500 in DNMT1 standard concentrations
Journey is Y=0.00775X+20.77161, wherein, it is DNMT1 standard concentrations that Y, which represents fluorescent value RFU, X, and correlation coefficient r is
0.9873；It is being that in the range of the ng/mL of 0.1 ng/mL~5.0, this method detects DNMT1 recurrence in DNMT1 standard concentrations
Equation is Y=0.02779X+1.29858, and wherein Y represents lgRFU, and X represents lgDNMT1, and correlation coefficient r is 0.9877.
Wherein there is provided a kind of Fe3O4The step of@McAb, includes：
（1）Cleaning：By Fe3O4Nano granule suspension uses concentration for 0.01 after abandoning supernatant processing through ultrasound, Magneto separate
Mol/L~0.015 mol/L, pH balance the min of 2 min~5 to it for 6.0 MES buffer solutions, then carry out Magneto separate and abandon
The first sediment is obtained clearly, wherein MES represents morpholino b acid buffer solution；
（2）Activation：Concentration is sequentially added into first sediment for mg/mL EDC solutions of 8 mg/mL~10 and dense
Spend and be vortexed the min of 10 min of reaction~15 for 8 mg/mL~11mg/mL NHS solution, Magneto separate, abandon supernatant processing after adopt
It is washed with MES buffer solutions 3 times, then carry out again Magneto separate, abandon supernatant processing obtain the second sediment, wherein, EDC represent
(1- (3- dimethylamino-propyls) -3- ethyl-carbodiimide hydrochlorides solution, NHS represent n-hydroxysuccinimide solution, MES
Represent morpholino b acid buffer solution；
（3）Buffer system is changed：Second sediment is washed 3 times using PBS, Magneto separate obtains the 3rd and sunk
Starch；
（4）Coupling：DNMT1 monoclonal antibodies are added into the 3rd sediment and PBS carries out vortex reaction
1.5h~2h, through Magneto separate, abandon supernatant processing after it is cleaned 2 times using PBS, Magneto separate, abandon supernatant and obtain the 4th
Sediment；
（5）Closing：BSA confining liquids are added into the 4th sediment, progress, which is vortexed, reacts 0.5h~1h, Magneto separate,
Abandon after supernatant using PBST buffer solution for cleaning 3 times, Magneto separate, abandon supernatant and obtain the 4th i.e. Fe of sediment3O4@McAb, wherein BSA
Confining liquid represents bovine serum albumin(BSA) confining liquid；
（6）Preserve：In coupled product Fe3O4PBS is added in@McAb, light shake shakes up, and 4 DEG C save backup.
The Fe provided using ELISA method detection3O4@McAb bioactivity, testing result in figure as shown in figure 1, show
Show that DNMT1 blank control groups have higher background value, but less than experimental group absorbance, while provable in Fe3O4Nano particle
Surface has successfully modified DNMT1 monoclonal antibodies and has not influenceed antibody activity.Specific detecting step includes：
（1）By through Fe that concentration is the CB buffer solutions dilution that 45 mmol/L~50 mmol/L, pH are 9.0~9.63O4@
McAb is added in ELISA Plate, is 100 microlitres per hole addition, Magneto separate is clapped using ELISA Plate described in PBST buffer solution for cleaning
It is dry standby；
（2）PBS compound concentration is used for the ng/mL of 300 ng/mL~400 DNMT1 standard items, and is added
Enter in above-mentioned ELISA Plate, be 100 microlitres per hole addition, the min of 80 min~90 is incubated in insulating box, Magneto separate is then carried out
Handle and use ELISA Plate described in PBST buffer solution for cleaning；
（3）DNMT1 polyclonal antibodies are diluted 400~500 times using confining liquid, then added it with every 100 microlitres of hole
Enter into the ELISA Plate, the min of 80 min~90 is incubated in 35 DEG C~37 DEG C insulating boxs, then carry out Magneto separate processing and adopt
With ELISA Plate described in PBST buffer solution for cleaning；
（4）The goat anti-rabbit igg for being marked HRP with confining liquid dilutes 4000~5000 times, with 100 microlitres of every hole by its
It is added in the ELISA Plate, the min of 80 min~90 is incubated in 35 DEG C~37 DEG C insulating boxs, then carries out Magneto separate processing simultaneously
Using ELISA Plate described in PBST buffer solution for cleaning；
（5）TMB developer A liquid and B liquid are mixed in equal volume, the ELISA Plate is added it to every 100 microlitres of hole
In, 35 DEG C~37 DEG C Incubation in dark react 15~20 min；After chromogenic reaction, 50 microlitres are added per hole into the ELISA Plate
Concentration be 2 mol/L H2SO4Terminate liquid, and the absorbance OD in each hole is detected on ELIASA immediately, wherein, Detection wavelength is
450 nm。
Wherein there is provided include the step of a kind of QDs@PcAb：
PH is added in PE pipes for 7.0~7.4 borate buffer solution, and adds concentration into the PE pipes successively and is
The QDs and concentration of every liter of the micromole of 0.5 every liter of micromole~1 is the mg/mL of 0.5 mg/mL~1 DNMT1 polyclonal antibodies,
Add every time and vibration mixing is both needed to after above-mentioned substance；
Then the EDC solution that concentration is the mg/mL of 8 mg/mL~10, the whirlpool at 18 DEG C~25 DEG C are added into the PE pipes
The rotation reaction h of 1.5 h~2, reaction, which terminates to add the PBS containing 0.5% mass percent BSA confining liquids in the backward PE pipes, to be buffered
Liquid, be vortexed reaction 20 min~30 min at 18 DEG C~25 DEG C；
Gained reaction product is dissolved in BS buffer solutions after removing EDC through centrifugation, ultrafiltration, 4 DEG C of preservations, so as to be made described
QDs@PcAb。
The QDs@PcAb provided fluorescence intensity and antibody activity is detected using sandwich method, as a result as shown in Fig. 2 empty
White control is DNMT1 antigen blank, and system fluorescence intensity increases and reduced with QDs@PcAb thinner ratios, but is above blank pair
According to, illustrate that QDs PcAb coupled products are successfully prepared, and the activity of antibody is still present after mark quantum dot.Specific detecting step
Including：It will be added through the QDs@PcAb that concentration is the CB buffer solutions dilution that 45 mmol/L~50 mmol/L, pH are 9.0~9.6
Into the hole elisa Plates of black 96, constant temperature incubates the min of 80 min~90, then carries out Magneto separate and uses PBST buffer solution for cleaning
The hole elisa Plates of black 96, then add PBST and survey fluorescence intensity on multi-functional microplate reader.
As can be seen here, to final detection essence in the method based on quantum dot fluorescence immune detection DNMT1 that the present invention is provided
Spend influence factor relatively more, such as：Fe3O4@McAb thinner ratios, QDs@PcAb thinner ratios, immune response buffer solution species, solid phase
Antibody capture antigenic action time, QDs@PcAb action times etc..
Below with regard to these influence factors, the method based on quantum dot fluorescence immune detection DNMT1 that the present invention is provided is made
It is further elucidated above.
First, Fe3O4@McAb thinner ratios optimize
To obtain Fe3O4The Fe of different extension rates has been investigated in@McAb optimal consumption, experiment3O4@McAb capture antigen
When influence to reaction system fluorescence intensity, as a result as shown in figure 3, reaction system fluorescence intensity first increases with the increase of extension rate
Plus rear rapid reduction, in Fe3O4@McAb thinner ratios are 1：Fluorescence intensity reaches maximum when 50, and its reason is Fe3O4Nano particle
There is quenching effect to the fluorescence of quantum dot, work as Fe3O4During@McAb excessive concentrations, Fe3O4Nano particle is quenched the fluorescence of quantum dot
The effect of going out strengthens, in addition Fe3O4The black of nano particle itself also has masking action to fluorescence.Therefore Fe is selected3O4@McAb's
Optimal thinner ratio is 1：50.
2nd, QDs@PcAb thinner ratios optimize
QDs@PcAb originate as the fluorescence signal of FLISA methods, and its consumption and method sensitivity and linear measurement range etc. are close
Cut is closed, therefore influence of the QDs@PcAb complex concentrations to reaction system fluorescence intensity has been investigated in experiment, as a result such as Fig. 4 institutes
Show, with the increase of QDs@PcAb complex concentrations, reaction system fluorescence intensity gradually tends towards stability after first increasing, in QDs@PcAb
Compound thinner ratio is 1：:Fluorescence intensity is higher when 30, and when continuing to increase QDs@PcAb complex concentrations, system fluorescence is strong
Degree increase is not obvious, therefore the optimal thinner ratio of selection QDs PcAb compounds is 1：30.
3rd, immune response buffer solution species optimizes
Immune buffer solution also has a certain impact to the fluorescence intensity of reaction system, four kinds of conventional buffer solutions of experimental selection
Concentration be 0.01 mol/L, pH 7.4 PBS, concentration be 0.01 mol/L, pH 7.4 PBST, concentration be 0.01 mol/L,
PH 7 BS, concentration is investigated for 0.05 mol/L, pH 9.6 BC, as a result as shown in figure 5, system fluorescence intensity from greatly to
Small corresponding buffer solution is respectively PBST, PBS, BS and CB, and reason is that CB pH of buffer is higher, and the stability to quantum dot may
Affect, PBST with the addition of surfactant Tween-20 compared with PBS, it can improve quantum dot in solution
Dispersiveness, reduce because quantum dot reunite and caused by fluorescent quenching.Therefore it is PBST to select optimal immune response buffer solution.
4th, insolubilized antibody capture antigenic action is time-optimized
Test and the action time that insolubilized antibody captures determined antigen in sample is investigated, as a result as shown in fig. 6, solid phase
Reaction has reached balance, system fluorescence intensity highest when antibody is 60 min with the antigenic action time, therefore selects optimal antigen
Action time is 60 min.
5th, QDs@PcAb action times optimize
Test and quantum dot-labeled antibody QDs@PcAb and antigen action time investigated, as a result see as shown with 7, with
Time increases, and system fluorescence intensity gradually increases, and 120 min reach to be declined after maximum, therefore during selection QDs@PcAb effect
Between be 120 min.
6th, the foundation of standard curve
DNMT1 standard items are configured to sample of the concentration range for the ng/mL of 0.001 ng/mL~1500 with PBS, according to
Optimal experiment condition after optimization detected, each concentration parallel determination 3 times, and sample well fluorescent value is designated as into RFU.In experiment
It was found that for different DNMT1 concentration ranges, fluorescent value is different from the relation of DNMT1 concentration, therefore, drawn using the method for segmentation
Standard curve.
It is in the range of the ng/mL of 5.0 ng/mL~1500, with DNMT1 standard concentrations in DNMT1 concentration（CDNMT1）For horizontal stroke
Coordinate, fluorescent value RFU is that ordinate draws standard curve as shown in figure 8, in the range of 5.0~1500 ng/mL, this method is examined
The regression equation for surveying DNMT1 is Y=0.00775X+20.77161（Y is RFU, and X is CDNMT1）, coefficient correlationrFor 0.9873.
It is in the range of the ng/mL of 0.1 ng/mL~5.0, to DNMT1 concentration and corresponding fluorescent value in DNMT1 concentration
RFU takes the logarithm, with the logarithm of DNMT1 standard concentrations（lgC）For abscissa, fluorescent value RFU logarithm（lgRFU）For ordinate
Standard curve is drawn as shown in figure 9, in the range of the ng/mL of 0.1 ng/mL~5.0, this method detects DNMT1 regression equation
For Y=0.02779X+1.29858（Y is lgRFU, and X is lgCDNMT1）, coefficient correlationrFor 0.9877.
7th, the establishment of this method testing conditions：
7.1 test limit
Take concentration be respectively 0.001 ng/mL, 0.005 ng/mL, 0.01 ng/mL, 0.05 ng/mL, 0.1 ng/mL,
0.5 ng/mL, 1.0 ng/mL, the DNMT1 samples of 5.0 ng/mL 8 low concentration regions are examined according to above-mentioned detection method
Survey, the results are shown in Table 7.1, while detecting the sample of 6 DNMT1 antigen blank, X is that 16.131, SD is 0.996, obtains X+3SD's
It is worth for 19.119, RFU(0.5)>19.119, therefore the detection of this method is limited to 0.1 ng/mL.
The low concentration DNMT1 sample testing results of table 7.1
7.2 precision
The measurement result of precision is shown in Table 7.2 between plate in plate, for the sample of high, medium and low three concentration, accurate in plate
Spend relative standard deviation（RSD）Scope be 5.45%~11.29%, precision between plateRSDScope for 7.03%~11.25% because
The scope that the detection of this this method is limited to recovery of standard addition in 0.1ng/mL plates is mark-on reclaims between 91.67%~106.50%, plate
The scope of rate is 92.18%~108.50%.
The interior precision between plate of the plate of table 7.2（n=3）
7.3 the degree of accuracy
The measurement result of the rate of recovery in plate between plate is shown in Table 7.3, for the standard items of high, medium and low three concentration, in plate
The scope of recovery of standard addition is that the scope of recovery of standard addition between 91.67%~106.50%, plate is 92.18%~108.50%.
The interior rate of recovery between plate of the plate of table 7.3
7.4 it is specific
Detect that serum composition is complicated because FLISA will be used for DNMT1 in serum, it is desirable to which FLISA has higher to DNMT1
Specificity, therefore the specificity of method is investigated using DNMT3a and DNMT3b similar to DNMT1 structures in serum,
Bring the fluorescent intensity measured into standard curve Y=0.02779X+1.29858（Y is lgRFU, and X is lgCDNMT1）, calculate
To the amount equivalent to DNMT1,7.4 are the results are shown in Table, as can be seen from the table cross reacting rates of the DNMT3a and DNMT3b to FLISA
Respectively 4.0% and 9.4%, cross reacting rate is low, shows that FLISA methods are used to detect that DNMT1 specificity is good.
The FLISA of table 7.4 determines DNMT1 specificity
(8), this method testing result checking test：
The FLISA regression curve equations set up in experiment are applied to the detection of DNMT1 contents in blood serum sample, and will inspection
The ELISA and MELISA that survey result is set up with laboratory are compared, while with the testing result of commercial ELISA kits to three
The method of kind is evaluated.In addition, adding certain density DNMT1 standard items in blood serum sample, carried out with the method set up
Determine, calculate recovery of standard addition.
8.1 serum samples are collected and processing
Serum sample is taken from the breathing of affiliated hospital of Zhengzhou University first and severe section office.All serum samples are in patient
Gathered from morning under fasted conditions, venous blood samples 3mL is placed in non-anticoagulant tube, 37 DEG C of water-bath 30min, 3000r/min centrifugations
5min, separation serum, which is stored in dry cryopreservation tube, to be sealed, and is placed in -80 DEG C of refrigerators standby.Discard the serum sample of haemolysis.
8.2 DNMT1 content detections in serum
30 blood serum samples are taken, DNMT1 contents in serum, and and commodity are detected with FLISA, ELISA and MELISA method
ELISA kit testing result is contrasted.
8.3 blood serum sample recovery of standard addition and precision
Added respectively in blood serum sample high, medium and low（1000 ng/mL、500 ng/mL、50 ng/mL）Three concentration
DNMT1 standard items, determine the fluorescence intensity or absorbance of mark-on sample, while the fluorescence intensity or extinction of the non-mark-on serum of detection
Degree, each sample parallel determination 3 times calculates recovery of standard addition, while calculating corresponding relative standard deviation according to formula 4.1.
Testing results of the FLISA of table 8.1 to DNMT1 contents in serum
Statistical analysis, FLISA, ELISA and commercial ELISA kits are carried out with more than 21.0 pairs testing results of SPSS
Testing result meet normal distribution, respectively by the testing result of FLISA and commercial ELISA kits, ELISA and commodity
The testing result of ELISA kit carries out paired sampletExamine, as a result the former t=1.913,P=0.233, the latter t=0.256,P=
0.927, two groups of dataPValue is all higher than 0.05, illustrates FLISA, ELISA method and the commercial ELISA kits set up in experiment
To the no significant difference of DNMT1 content detections result in serum.
Therefore, the range of linearity that the regression equation set up by this method is detected to DNMT1 is wide, test limit is low, precision, standard
Exactness is high and with good specificity.
Finally it should be noted that:The above embodiments are merely illustrative of the technical scheme of the present invention and are not intended to be limiting thereof；To the greatest extent
The present invention is described in detail with reference to preferred embodiments for pipe, those of ordinary skills in the art should understand that：Still
The embodiment of the present invention can be modified or equivalent substitution is carried out to some technical characteristics；Without departing from this hair
The spirit of bright technical scheme, it all should cover among claimed technical scheme scope of the invention.
Claims (6)
1. a kind of method based on quantum dot fluorescence immune detection DNMT1, specific steps include：
A kind of surface is provided as steps described below by the Fe of DNMT1 monoclonal antibody covalent modifications3O4Nano immune magnetic particle,
It is designated as Fe3O4@McAb：
Cleaning：By Fe3O4Nano granule suspension through ultrasound, Magneto separate abandon supernatant processing after, use concentration for 0.01 mol/L~
0.015 mol/L, pH balances the min of 2min~5 to it for 6.0 MES buffer solutions, then carries out Magneto separate, abandons supernatant and handle
To the first sediment；
Activation：It is 8 that EDC solution and concentration that concentration is the mg/mL of 8 mg/mL~10 are sequentially added into first sediment
The mg/mL of mg/mL~11 NHS solution be vortexed the min of 10 min of reaction~15, through Magneto separate, abandon supernatant processing after, use
MES buffer solutions are washed 3~4 times to it, then carry out again Magneto separate, abandon supernatant processing obtain the second sediment；
Buffer system is changed：Second sediment is washed 3~4 times times using PBS, Magneto separate obtains the 3rd precipitation
Thing；
Coupling：DNMT1 monoclonal antibodies are added into the 3rd sediment and PBS carries out 1.5 h~2 of reaction that are vortexed
H, through Magneto separate, abandon supernatant processing after it is cleaned 2 times using PBS, Magneto separate, abandon supernatant and obtain the 4th sediment；
Closing：BSA confining liquids are added into the 4th sediment, the reaction h of 0.5 h~1 that is vortexed is carried out, Magneto separate, supernatant are abandoned
After processing, using PBST buffer solution for cleaning 3~4 times, Magneto separate, abandon supernatant and obtain the 4th i.e. Fe of sediment3O4@McAb；
Preserve：In Fe3O4PBS is added in@McAb, light shake shakes up, and 4 DEG C save backup；
A kind of surface is provided as steps described below by quantum dot-labeled DNMT1 polyclonal antibodies, is designated as QDs@PcAb：It is by pH
7.0~7.4 borate buffer solution is added in PE pipes, and it is 0.5 every liter of micromole to add concentration into the PE pipes successively
The QDs and concentration of~1 every liter of micromole is the mg/mL of 0.5 mg/mL~1 DNMT1 polyclonal antibodies, and above-mentioned thing is added every time
Vibration is both needed to after matter to mix；
Then the EDC solution that concentration is the mg/mL of 8 mg/mL~10 is added into the PE pipes, is vortexed at 18 DEG C~25 DEG C anti-
The h of 1.5 h~2 is answered, it is 0.5%~1.0% BSA confining liquids that reaction, which terminates to add in the backward PE pipes containing mass percent,
PBS, be vortexed reaction 20 min~30 min at 18 DEG C~25 DEG C；Gained reaction product removes EDC through centrifugation, ultrafiltration
After be dissolved in BS buffer solutions, 4 DEG C of preservations, so that QDs@PcAb are made；
By the Fe after being diluted through CB buffer solutions3O4@McAb and DNMT1 standard samples or mesh to be measured after being diluted through PBS
Mark thing and carry out the min of incubation reaction 50 min~60 so that the DNMT1 standard samples or object to be measured are by Fe3O4@McAb are caught
Obtain, concentration and separation；
Then by the QDs@PcAb after being diluted through PBST buffer solutions and by Fe3O4@McAb capture, concentration and separation it is described
DNMT1 standard samples or object to be measured carry out the min of the min of incubation reaction 110~130 so that the QDs@PcAb with it is described
DNMT1 standard samples or object to be measured are combined；After being diluted through PBST buffer solutions, determined on multi-functional microplate reader
270 nm excite the fluorescence intensity of lower reaction product；
According to the relation between the fluorescence intensity of detection and the DNMT1 standard samples or target concentration to be measured, inspection is set up in segmentation
Survey DNMT1 regression equation；Wherein, it is institute in the range of the ng/mL of 5.0 ng/mL~1500 in the DNMT1 standard concentrations
The regression equation of foundation is Y=0.00775X+20.77161, wherein, it is DNMT1 standard concentrations, phase that Y, which represents fluorescent value RFU, X,
Relation number r is 0.9873；It is this method detection in the range of the ng/mL of 0.1 ng/mL~5.0 in the DNMT1 standard concentrations
DNMT1 regression equation is Y=0.02779X+1.29858, and wherein Y represents lgRFU, and X represents lgDNMT1, and correlation coefficient r is
0.9877。
2. the method according to claim 1 based on quantum dot fluorescence immune detection DNMT1, it is characterised in that it is also wrapped
The Fe provided using ELISA method detection is provided3O4The step of@McAb bioactivity.
3. the method according to claim 2 based on quantum dot fluorescence immune detection DNMT1, it is characterised in that use
The Fe that ELISA method detection is provided3O4The step of@McAb bioactivity, includes：
（1）By through Fe that concentration is the CB buffer solutions dilution that 45 mmol/L~50 mmol/L, pH are 9.0~9.63O4@McAb add
Enter into ELISA Plate, Magneto separate, patted dry using ELISA Plate described in PBST buffer solution for cleaning standby；
（2）PBS compound concentration is used for the ng/mL of 300 ng/mL~400 DNMT1 standard items, and is added into
State in ELISA Plate, the min of 80 min~90 is incubated in insulating box, then carry out Magneto separate processing and use PBST buffer solution for cleaning
The ELISA Plate；
（3）DNMT1 polyclonal antibodies are diluted 400~500 times using confining liquid, are then added to it with every 100 microlitres of hole
In the ELISA Plate, the min of 80 min~90 is incubated in 35 DEG C~37 DEG C insulating boxs, Magneto separate processing is then carried out and uses
ELISA Plate described in PBST buffer solution for cleaning；
（4）The goat anti-rabbit igg for being marked HRP with confining liquid dilutes 4000~5000 times, is added into every 100 microlitres of hole
Into the ELISA Plate, the min of 80 min~90 is incubated in 35 DEG C~37 DEG C insulating boxs, Magneto separate processing is then carried out and uses
ELISA Plate described in PBST buffer solution for cleaning；
（5）TMB developer A liquid and B liquid are mixed in equal volume, added it in the ELISA Plate, 35 DEG C~37 DEG C lucifuge temperature
Educate 15~20min of reaction；After chromogenic reaction, H is added into the ELISA Plate2SO4Terminate liquid, and detected immediately on ELIASA
The absorbance OD in each hole, wherein, Detection wavelength is 450 nm.
4. the method according to claim 3 based on quantum dot fluorescence immune detection DNMT1, it is characterised in that it is also wrapped
Include the step of QDs@PcAb provided fluorescence intensity and antibody activity is detected using sandwich method.
5. the method according to claim 4 based on quantum dot fluorescence immune detection DNMT1, it is characterised in that using folder
The step of QDs@PcAb that the detection of heart method is provided fluorescence intensity and antibody activity, includes：To be 45 mmol/L through concentration
~50 mmol/L, pH are added in the hole elisa Plates of black 96 for the QDs@PcAb of 9.0~9.6 CB buffer solutions dilution, constant temperature temperature
The min of 80 min~90 is educated, Magneto separate is then carried out and using the hole elisa Plates of black 96, Ran Houjia described in PBST buffer solution for cleaning
Enter PBST and survey fluorescence intensity on many work(microplate readers.
6. the method according to claim 5 based on quantum dot fluorescence immune detection DNMT1, it is characterised in that this method
Lowest detection to DNMT1 concentration is limited to 0.1 ng/mL.
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