CN106353500A - Multi-tumor marker label-free chemiluminescent imaging immunosensor preparation and analysis method - Google Patents
Multi-tumor marker label-free chemiluminescent imaging immunosensor preparation and analysis method Download PDFInfo
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Abstract
The invention provides a copper sulfide nanozyme based label-free chemiluminescent imaging immunosensor preparation and analysis method. The method comprises the following steps: preparing a 4*12 immunosensor array on a jettisonable epoxysilanized glass slide surface through a screen printing technology, and dispensing copper sulfide nanoparticles dispersed in chitosan in micropores of the array; and further dispensing streptavidin in the micropores of the array, then modifying streptavidin functionalized copper sulfide nanoparticle surfaces with a biotinylated antibody, and sealing to prepare the label-free chemiluminescent immunosensor. The label-free chemiluminescent immunosensor can implement simultaneous detection of 48 samples, improves the defects of long analysis time, high working capacity, high reagent consumption and the like in a single-component label-free chemiluminescent immunoassay mode, realizes cheap, quick, high-flux and high-sensitive combined detection of multiple tumor markers, and is suitable for early large-scale screening of tumors, thus having very important application value and practical meaning.
Description
Technical field
The present invention relates to field of immunology and chemiluminescence analytical field are and in particular to a kind of no mark of multi-tumor marker
The preparation of note chemiluminescence imaging immunosensor and analysis method.
Background technology
The early diagnosiss of tumor and treatment are the keys improving tumor patient survival rate.In human serum, tumor markerses contains
Amount and the developmental stage of tumor have close contacting, its to the Differential Diagnosiss of tumor, auxiliary diagnosis, observe curative effect, monitoring is answered
Send out and evaluation of the prognosis has very high using value.Therefore, detection reliable sensitive to tumor markerses be tumor screening and
The key estimated.However, many data confirm, the histological types of different tumors or same tumor both may have common
Tumor markerses, it is also possible to there be different tumor markerses, are therefore found only for a certain tumor, and it are special to have 100%
" preferable " tumor markerses of property and sensitivity are very difficult, clinically in order to improve accuracy and the reliability of lesion detection
Property, it is all often to detect a group mark thing related to target tumor, be only capable of detecting that a kind of one pack system of tumor markerses is divided
Analysis method can not meet the demand of clinical diagnosises, therefore urgently develops multi-component immunity analytical new technique.
While multicomponent analysis technology can realize various ingredients in single analysis process or closely detect have simultaneously
Analysis throughput is high, sample consumption is few, required time is short, the low advantage of analysis cost.Multi-component immunity analytical method is main at present
There are spatial discrimination and multi-tracer both of which.In multi-tracer pattern, need multiple labels are marked on albumen, exist
Complicated, laborious, time-consuming, the defect such as antibody after labelling or antigen molecule biological activity reduce of markers step.Multiple labels
It is frequently necessary to different optimum analysis conditions, such as the optimum ph value of various labelling enzymic catalytic reactions, temperature etc. are different from, therefore
Some conditions of can only compromising are thus lead to poor analytical performance.Further, since chemiluminescent detection does not have selectivity, multiple
Interfere between label or the overlap of chemiluminescence signal also can make analytical performance be deteriorated.
Spatial discrimination pattern is most study, most widely used analytical model in current multi-component immunity analytical pattern, its
By making a distinction each component by the position difference of luminous generation, then entered with array detector (such as cid, ccd etc.)
Row detection, thus obtain corresponding constituent content.Theoretically, as long as substrate is sufficiently large or array is sufficiently small, integrated
Change degree high it is possible to a large amount of arrays are built on substrate, to reach the purpose detecting a large amount of samples simultaneously.Therefore it is based on array
The spatial discrimination pattern of method has high analyte flux and many detection objects, and analyze speed is fast, and the advantages of sample consumption is few, satisfaction is faced
The actual demand of bed application, is the main trend of multi-component immunity analytical.
Patent application " a kind of unmarked chemiluminescence immunoassay sensor based on nanometer analogue enztme and preparation and analysis side
Method " (number of patent application: 201510919955.1) replace native enzyme by using nanometer analogue enztme, improve traditional chemical and light
In immunoassay, natural enzyme stability is poor, the shortcomings of easily affected by environment so that the stability of chemical luminous system building and
Sensitivity is significantly improved, but this once can only based on the unmarked chemiluminescence immunoassay sensor of nanometer analogue enztme
Carry out one-component immunoassay, it is long to there is analysis time, the amount of labour is big, reagent consumes the defects such as many, this greatly limits this
The clinical practice of sensor.The therefore unmarked chemiluminescence strategy based on nanometer analogue enztme for the present invention, constructs further and exempts from
Epidemic disease sensor array is it is achieved that while Diagnostic Value of Several Serum Tumor Markers, high throughput testing is it is adaptable to the extensive sieve of tumor early stage
Look into, there is very important using value and practical significance.
Content of the invention
The present invention propose a kind of preparation of the unmarked chemiluminescence imaging immunosensor based on nanometer analogue enztme and
Analysis method, for detecting while Diagnostic Value of Several Serum Tumor Markers.
The present invention passes through screen printing technique and 4 × 12 immune sensings is obtained in the slide surface of disposable epoxy silane
Array, it comprises 48 detection site, you can to detect to 48 samples simultaneously, and will be scattered in the copper sulphide nano of shitosan
Particle drop coating is in array micropore；Again by biotinylated antibody modification in the copper sulphide nano particles of streptavidin functionalization
Surface, is obtained this unmarked chemiluminescence immunoassay sensor after closing；There is formation after specific immune response with antigen
Immune complex can effectively hinder chemical luminous substrate spreading and suppressing mimetic enzyme catalysis chemistry to send out to copper sulphide nano particles
Light, causes weakening of chemiluminescence signal intensity, and chemiluminescence signal is collected by charge-coupled detector(CCD) ccd.
In order to achieve the above object, technical scheme is as follows:
The invention provides a kind of preparation of the unmarked chemiluminescence imaging immunosensor of multi-tumor marker and analysis
Method, comprises the following steps:
(1) use screen process press to make 4 × 12 sensor arrays in the slide surface of epoxy silane, form 48 battle arrays
Row micropore；
(2) by copper sulphide nano particles ultrasonic disperse in distilled water, copper sulfide suspension and the bodies such as chitosan solution are taken
Long-pending mixing, ultrasonic disperse is uniform；Take above-mentioned mixed solution drop coating in array micropore, reaction is until dry at room temperature；
(3) take the uniform drop coating of streptavidin solution in array micropore, after reacting under room temperature, 4 DEG C of left overnight are straight
To drying, subsequently rinsed with phosphate buffer, dry up in nitrogen atmosphere；
(4) take multiple biotinylated antibody to continue drop coating in the array micropore of sensor array, after reacting under room temperature, use
Phosphate buffered solution is rinsed, and dries up in nitrogen atmosphere；
(5) by confining liquid, in uniform drop coating and array micropore, close unnecessary avtive spot, after closing at 4 DEG C, use phosphorus
Hydrochlorate buffer solution rinses, and dries up in nitrogen atmosphere；
(6) sample solution with multiple analysis antigens is taken to add in array micropore, online incubation；
(7) take the phosphate buffered solution (pbst) containing 0.05%tween-20 to rinse immuno-array, remove unreacted
Immunoreagent, dries up in nitrogen atmosphere；
(8) chemical luminous substrate is taken to instill in array micropore；
(9) chemiluminescence array detector collection chemiluminescence signal.
Further, in step (1), 4 × 12 immune sensing arrays are as shown in figure 1, white portion is epoxy silane
Array micropore, a diameter of 2mm, Distances Between Neighboring Edge Points are 4mm；Black portions are the microscope slide around array micropore, and are printed with and have
The insulating paint of hydrophobic interaction.Further, the copper sulphide nano particles consumption described in step (2) is 1.0-3.0mg, and shell gathers
The concentration of sugar juice is 1.0-2.0wt%.
Further, the concentration of the streptavidin solution described in step (3) is 20-50 μ g/ml.
Further, the concentration of the biotinylated antibody described in step (4) is 2-10 μ g/ml.
Further, the confining liquid described in step (5) is 1.0-5.0% bovine serum albumen solution.
Further, the time of the described incubation of step (6) is 20-40min.
Further, the chemiluminescence array detector described in step (9) is charge-coupled detector(CCD) ccd, dynamic integral
Time is 5-10min.
Further, the amount of reagent instilling in array micropore described in step (2) (3) (4) (5) (6) (8) is 4-8 μ l.
Invention achieves following beneficial effect:
The unmarked chemiluminescence analyses strategy based on copper sulphide nano analogue enztme for the present invention, constructs a kind of multicomponent and exempts from
The sensor of epidemic disease analysis, is achieved by chemiluminescence imaging and detects multiple detection tumor markerses simultaneously.The present invention passes through silk
Net printing technology is obtained 4 × 12 immune sensing arrays in the slide surface of disposable epoxy silane, you can with to 48 samples
Product detect simultaneously, and will be scattered in the copper sulphide nano particles drop coating of shitosan in array micropore；Again will be biotinylated anti-
Body modifies the copper sulphide nano particles surface in streptavidin functionalization, and this unmarked chemiluminescence immunoassay is obtained after closing
Sensor array.Additionally, the present invention also constructs a kind of unmarked chemiluminescence imaging immune sensing array of multi-tumor marker
Analysis method.The present invention has the advantage that
(1) present invention successfully constructs immune sensing array it is achieved that spatial discrimination multi-component immunity analytical, improves list
Component immunoassay can only detect a sample every time, has that length analysis time, the amount of labour be big, reagent consumes the defects such as many.This
The constructed immune sensing array of invention can detect 48 analysis samples simultaneously, have high analyte flux and many detection objects, divide
The advantages of speed is fast for analysis, sample consumption is few, meets the actual demand of clinical practice it is adaptable to the extensive examination of tumor early stage.
(2) only need to add 4-8 μ l immunoreagent in each array micropore of the present invention, greatly reduce reagent dosage, section
Save analysis cost.Additionally, the diffusion time to interface for the array micropore reduction immunoreagent of small size, accelerate immunoreagent
To the mass transport process at interface, thus shortening the time of immunoassay, improve the speed of analysis.
(3) sensor that the present invention builds has high susceptiveness, Stability and veracity, this is because copper sulfide has
Big specific surface area and stronger absorption property, in conjunction with the enlarge-effect of biotin-streptavidin system, make sensor array circle
Substantial amounts of antibody has been modified on face.There is the sensing interface of good biocompatibility, not only maintain the biological activity of antibody, and
And the antibody layer of stable homogeneous covers on copper sulphide nano particles signals layer, immunity can be formed again by efficient capture target antigen
Compound, thus hindering the diffusion to signals layer for the chemical luminous substrate, the realization of the unmarked analysis strategy that is highly advantageous to.
Brief description
Fig. 1 is making and the immunoassay schematic diagram of immunosensor of the present invention.
Fig. 2 detects the linearity curve of afp and cea standard sample for immunosensor of the present invention simultaneously.
In figure, 1. Piranha acid (h2so4:h2o2=7:3, v/v)；2.gptms (γ-glycidoxypropyl trimethoxy silicon
Alkane)；3. copper sulphide nano particles；4. biotinylated antibody；5. Streptavidin；6. antigen；7. chemical luminous substrate；8. electricity
Lotus coupled detector ccd.
Specific embodiment
In order to illustrate technical scheme and technical purpose, below in conjunction with the accompanying drawings and specific embodiment is to the present invention
It is described further.
Embodiment 1
A kind of unmarked chemiluminescence imaging immunosensor, for detecting alpha-fetoprotein (afp) and carcinoembryonic antigen simultaneously
(cea), the preparation of this immunosensor and analysis method comprise the following steps:
(1) screen process press is used to make 4 × 12 sensor arrays in the slide surface of epoxy silane；
(2) by 2.0mg copper sulphide nano particles ultrasonic disperse in distilled water, copper sulfide suspension and 1.0wt% shell are taken
Polysaccharide solution equal-volume mixes, and ultrasonic disperse is uniform；Take the above-mentioned mixed solution drop coating of 5 μ l in array micropore, at room temperature instead
Should be up to drying；
(3) take the uniform drop coating of 5 μ l 50 μ g/ml streptavidin solution in array micropore, after reacting under room temperature, at 4 DEG C
Standing overnight until drying, subsequently being rinsed with phosphate buffer, drying up in nitrogen atmosphere；
(4) the biotinylated afp antibody of 5 μ l 1 μ g/ml and 2 μ g/ml biotinylated cea antibody difference drop coating are taken in biography
In two column array micropores of sense array, after reacting under room temperature, rinsed with phosphate buffered solution, dry up in nitrogen atmosphere；
(5) by 5 μ l 1.0% serum albumin solution, in uniform drop coating array micropore, close unnecessary avtive spot, 4
After closing at DEG C, rinsed with phosphate buffered solution, dry up in nitrogen atmosphere, this unmarked immune sensing array is obtained；
(6) take the sample that 5 μ l carry afp and cea antigen to add in array micropore, incubate 25min online；
(7) pbst rinses immuno-array, removes unreacted immunoreagent, dries up in nitrogen atmosphere；
(8) 5 μ l chemical luminous substrate [luminol (5mm)-pip (0.6mm)-h are taken2o2(4mm)] instill in array micropore；
(9) charge-coupled detector(CCD) ccd collection chemiluminescence signal, the dynamic integral time is 400s.
As shown in Fig. 2 measuring afp the and cea standard sample of variable concentrations, the linear song of prepared afp and cea standard sample
Line.After prepared standard curve, actual for investigating this unmarked chemiluminescence immunoassay sensor based on copper sulphide nano analogue enztme
The reliability of application, has carried out the detection of human serum sample, and (in table 1, the reference value of sample 1-5 is by Jiangsu Province with standard method
Tumour hospital provides, by the Electrogenerated chemiluminescent immunoassay instrument measurement of commercialization) compare, experimental result is as shown in table 1:
Table 1
Embodiment 2
A kind of unmarked chemiluminescence imaging immunosensor is used for detection CA125 (ca125) simultaneously, sugar chain resists
Former 199 (ca199), alpha-fetoprotein (afp) and carcinoembryonic antigen (cea), the preparation of this immunosensor and analysis method include with
Lower step:
(1) screen process press is used to make 4 × 12 sensor arrays in the slide surface of epoxy silane；
(2) by 3.0mg copper sulphide nano particles ultrasonic disperse in distilled water, copper sulfide suspension and 2.0wt% shell are taken
Polysaccharide solution equal-volume mixes, and ultrasonic disperse is uniform；Take the above-mentioned mixed solution drop coating of 6 μ l in array micropore, at room temperature instead
Should be up to drying；
(3) take the uniform drop coating of 6 μ l 100 μ g/ml streptavidin solution in array micropore, after reacting under room temperature, 4 DEG C
Left overnight, until drying, is subsequently rinsed with phosphate buffer, dries up in nitrogen atmosphere；
(4) the biotinylated afp antibody of 6 μ l 1 μ g/ml, the biotinylated cea antibody of 1 μ g/ml, 1 μ g/ml biotin are taken
The ca125 antibody changed and the biotinylated ca199 antibody of 1 μ g/ml, distinguish drop coating in a column array micropore of sensor array,
After reacting under room temperature, rinsed with phosphate buffered solution, dry up in nitrogen atmosphere；
(5) by 6 μ l 1.0% serum albumin solution, uniform drop coating closes unnecessary avtive spot in array micropore,
After closing at 4 DEG C, rinsed with phosphate buffered solution, dry up in nitrogen atmosphere, this unmarked immune sensing array is obtained；
(6) sample that 6 μ l carry ca125, ca199, afp and cea antigen is taken to add in array micropore, online incubation
35min；
(7) pbst rinses immuno-array, removes unreacted immunoreagent, dries up in nitrogen atmosphere；
(8) 6 μ l chemical luminous substrate [luminol (5mm)-pip (0.6mm)-h are taken2o2(4mm)] instill in array micropore；
(9) charge-coupled detector(CCD) ccd collection chemiluminescence signal, the dynamic integral time is 8min.
Ultimate principle, principal character and the advantages of the present invention of the present invention have been shown and described above.The technology of the industry
, it should be appreciated that the present invention is not restricted to the described embodiments, the simply explanation described in above-described embodiment and description is originally for personnel
The principle of invention, without departing from the spirit and scope of the present invention, the present invention also has various changes and modifications, the present invention
Claimed scope is by appending claims, description and its equivalent thereof.
Claims (9)
1. a kind of preparation of the unmarked chemiluminescence imaging immunosensor of multi-tumor marker and analysis method it is characterised in that
Comprise the following steps:
(1) use screen process press to make 4 × 12 sensor arrays in the slide surface of epoxy silane, form 48 arrays micro-
Hole；
(2) by copper sulphide nano particles ultrasonic disperse in distilled water, copper sulfide suspension is taken to mix with chitosan solution equal-volume
Close, ultrasonic disperse is uniform；Take above-mentioned mixed solution drop coating in array micropore, reaction is until dry at room temperature；
(3) take the uniform drop coating of streptavidin solution in array micropore, after reacting under room temperature, 4 DEG C of left overnight are until dry in the air
Dry, subsequently rinsed with phosphate buffer, dry up in nitrogen atmosphere；
(4) take multiple biotinylated antibody to continue drop coating in the array micropore of sensor, after reacting under room temperature, use phosphate
Buffer solution rinses, and dries up in nitrogen atmosphere；
(5) by uniform for confining liquid drop coating in array micropore, close unnecessary avtive spot, after closing at 4 DEG C, use phosphate
Buffer solution rinses, and dries up in nitrogen atmosphere；
(6) sample solution with multiple analysis antigens is taken to add in array micropore, online incubation；
(7) take the phosphate buffered solution (pbst) containing 0.05%tween-20 to rinse immuno-array, remove unreacted immunity
Reagent, dries up in nitrogen atmosphere；
(8) chemical luminous substrate is taken to instill in array micropore；
(9) chemiluminescence array detector collection chemiluminescence signal.
2. as claimed in claim 1 a kind of preparation of the unmarked chemiluminescence imaging immunosensor of multi-tumor marker and point
Analysis method is it is characterised in that in step (1), in 4 × 12 immune sensing arrays, the microscope slide around array micropore is printed with tool
There is the insulating paint of hydrophobic interaction, the array micro-pore diameter of epoxy silane is 2mm, and Distances Between Neighboring Edge Points are 4mm.
3. as claimed in claim 1 a kind of preparation of the unmarked chemiluminescence imaging immunosensor of multi-tumor marker and point
Analysis method it is characterised in that the copper sulphide nano particles consumption described in step (2) be 1.0-3.0mg, chitosan solution dense
Spend for 1.0-2.0wt%.
4. as claimed in claim 1 a kind of preparation of the unmarked chemiluminescence imaging immunosensor of multi-tumor marker and point
Analysis method is standby it is characterised in that the concentration of the streptavidin solution described in step (3) is 20-50 μ g/ml.
5. as claimed in claim 1 a kind of preparation of the unmarked chemiluminescence imaging immunosensor of multi-tumor marker and point
Analysis method is it is characterised in that the concentration of the biotinylated antibody described in step (4) is 2-10 μ g/ml.
6. as claimed in claim 1 a kind of preparation of the unmarked chemiluminescence imaging immunosensor of multi-tumor marker and point
Analysis method is it is characterised in that the confining liquid described in step (5) is 1.0-5.0% bovine serum albumen solution.
7. as claimed in claim 1 a kind of preparation of the unmarked chemiluminescence imaging immunosensor of multi-tumor marker and point
Analysis method is it is characterised in that the time of the described incubation of step (6) is 20-40min.
8. as claimed in claim 1 a kind of preparation of the unmarked chemiluminescence imaging immunosensor of multi-tumor marker and point
Analysis method it is characterised in that chemiluminescence array detector described in step (9) is charge-coupled detector(CCD) ccd, dynamic integral
Time is 5-10min.
9. as claimed in claim 1 a kind of preparation of the unmarked chemiluminescence imaging immunosensor of multi-tumor marker and point
Analysis method is it is characterised in that the amount of reagent instilling in array micropore described in step (2), (3), (4), (5), (6), (8) is 4-
8μl.
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| CN108303537A (en) * | 2018-01-24 | 2018-07-20 | 扬州大学 | The unmarked chemiluminescence imaging immuno-array sensor of multicomponent based on three-dimensional cage modle Kocide SD analogue enztme |
| CN108333345A (en) * | 2018-02-05 | 2018-07-27 | 扬州大学 | More chicken cell factor chemiluminescence immune analysis methods of dual analog enzyme signal amplification |
| CN111879920A (en) * | 2020-08-06 | 2020-11-03 | 扬州大学 | Multi-component unmarked immunosensor based on single metal Cu-MOF mimic enzyme |
| CN115236063A (en) * | 2022-06-28 | 2022-10-25 | 扬州大学 | Chemiluminescence imaging immunosensor based on copper oxide nanosheet assembled hollow cubic nanoenzyme and preparation method thereof |
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| CN105403696A (en) * | 2015-12-11 | 2016-03-16 | 扬州大学 | Label-free chemiluminescent immunosensor based on nanometer mimic enzyme, and preparation and analysis methods thereof |
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| CN108303537A (en) * | 2018-01-24 | 2018-07-20 | 扬州大学 | The unmarked chemiluminescence imaging immuno-array sensor of multicomponent based on three-dimensional cage modle Kocide SD analogue enztme |
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Application publication date: 20170125 |