CN103575896A - High sensitive and jettisonable multicomponent chemiluminescent imaging immunosensor - Google Patents
High sensitive and jettisonable multicomponent chemiluminescent imaging immunosensor Download PDFInfo
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Abstract
The invention relates to a high sensitive and jettisonable multicomponent chemiluminescent imaging immunosensor. The immunosensor is prepared by: constructing a 4*12 array on a silanized glass slide by a screen printing technique, and coating the array points with different capture antibodies to construct a jettisonable multicomponent immune sensor array; immobilizing biotinylated capture DNA and multiple G-quadruplex sequence repeat signal DNA on gold nanoparticle surfaces simultaneously, combining G-quadruplex signal DNA with heme to form DNA enzyme so as to prepare a multilayer DNA enzyme functionalized gold nanoparticle probe; and based on sandwich immunoassay, forming a sandwich immune complex on the sensor array, carrying out biotin-avidin reaction, labeling different immune complexes with the multilayer DNA enzyme functionalized gold nanoparticle probe; and making use of the peroxidase characteristics of DNA enzyme to catalyze reaction between chemiluminescent substrate H2O2-luminol to obtain a sensitive chemiluminescent signal, thus realizing high sensitive image immunoassay of a variety of protein. The immunosensor has the advantages of simple design, low cost, high sensitivity, high throughput and good repeatability, etc., and has certain clinical application value.
Description
One, technical field
The present invention is a kind of highly sensitive disposable multicomponent chemical luminescence imaging immunosensor, immunoassay when relating in a plurality of samples multiple component to be measured, by building disposable polycomponent immune sensing array, the enzyme functionalized golden nanometer particle probe of design novel multi-layer DNA, carry out chemiluminescence signal amplification, for the highly sensitive image immunoassay of multiple proteins.
Two, background technology
Immunoassay, as a kind of high selectivity and highly sensitive analytical approach, has obtained increasingly extensive application in fields such as environmental monitoring, clinical diagnosis, food securities.According to the difference of immunoassay step, can be divided into out-phase immunoassay and homogeneous immunoassay, the latter is widely used because can obtain higher sensitivity.Chemiluminescence immune assay is combined chemical luminescent detecting technology with immunoassay, have the advantages such as equipment is simple, highly sensitive, analysis speed is fast, the range of linearity is wide, emitted luminescence intensity measurement is noiseless.In practical application area, often need to measure the content of various ingredients in complex sample, and more and more need high-sensitive analytical approach to improve the accurate detection to low abundance components.In most immunologic detection method, selection realizes signal as horseradish peroxidase, alkaline phosphatase load on nano particle by native enzyme and amplifies, but native enzyme is usually because thereby the catalytic activity that small conformation change just affects them causes going bad, the preparation of native enzyme and purifying are also very consuming time and expensive in addition.
Measure the content of various ingredients in complex system, adopt traditionally parallel single component analytic approach, but this method required time is long, reagent consumption is many and workload is large more, and nonideal analytical model.The detection that realizes various ingredients in single analysis process simultaneously or roughly the same time has that analysis throughput is high, required time is short, sample consumption less, the outstanding advantages such as analysis cost is low.Chemiluminescence immunoassay sensing pattern based on spatial discrimination pattern has obtained in multi-component immunity analytical field paying close attention to widely in recent years, this pattern is the fixing corresponding immunoreagent of different components to be measured of zones of different at immune reactor, the immune response of different component is occurred in the different spatial of reactor simultaneously, then with array detector, carry out signals collecting, when realizing a plurality of component, detect.In recent years, the research of protein detection array has caused very large concern, yet protein array adopts photoetching technique or dot matrix techniques to be prepared from more, and price is relatively costly; In addition, because chemiluminescence detection signal is insensitive, protein array often need be equipped with sensitive array detection instrument.
Three, summary of the invention
The object of the invention is: zygomite reticulated printing and chemical coating technique, build disposable polycomponent immune sensing array, design the enzyme functionalized golden nanometer particle probe of a kind of multilayer DNA simultaneously and carry out chemiluminescence signal amplification, propose a kind of highly sensitive disposable multicomponent chemical luminescence imaging immunosensor and corresponding immune analysis method.
The disposable multicomponent chemical luminescence imaging immunosensor that the present invention proposes as shown in Figure 1, produces system (3) by disposable polycomponent immune sensing array (1), the enzyme functionalized golden nanometer particle probe of multilayer DNA (2) and signal and forms.Prepare by the following technical programs:
1) disposable polycomponent immune sensing array is according to spatial discrimination pattern, on microslide, stamp 4 * 12 silanization array, at every 1 row, drip a kind of a certain amount of capture antibody, epoxy radicals covalent bond on the amino of antibody and silanization microslide, reaction finishes post-flush, then drips confining liquid, and sealing finishes post-flush, dry up, make and can catch the array of totally 4 kinds of target antigens (Fig. 2);
2) the enzyme functionalized golden nanometer particle probe of multilayer DNA is the signal dna of the seizure DNA of fixed biologically elementization and multiple G-tetra-serobila sequences repetitions and making simultaneously on golden nanometer particle surface, and the latter is in conjunction with protoheme formation DNA enzyme, as shown in Figure 3;
3) signal generation system is to utilize the peroxidase activity cataluminescence substrate H of DNA enzyme
2o
2reaction between-luminol, produces sensitive chemiluminescence signal.
The principle of work of sensor:
1) on each sensing point surface of disposable 4 * 12 polycomponent immune sensing arrays, drip detected sample, carry out incubation; After flushing, on each sensing point surface, drip corresponding biotinylation two anti-, carry out sandwich immunoassay reaction;
2) after rinsing, drip Streptavidin carry out incubation on each sensor array surface, then rinse again, and drip the enzyme functionalized golden nanometer particle probe of multilayer DNA, reaction finishes post-flush;
3) on sensor array surface, drip chemical luminous substrate, by CCD, carry out chemiluminescence image detection;
4) chemiluminescence signal of each sensor and the concentration of its corresponding detected components become positive correlation, thereby usable criterion solution obtains working curve, obtain the concentration of different proteins in sample (antigen), highly sensitive image immunoassay when realizing in several samples multiple proteins.
Above-mentioned analytical approach, confining liquid is the 0.01M phosphate buffer containing 5% bovine serum albumin(BSA) (BSA), pH7.4; Washing fluid is the 0.01M phosphate buffer containing 0.05% Tween-20, pH7.4.
The present invention compared with prior art, has following characteristics:
The present invention is in conjunction with multicomponent chemical luminescent image immune detection means, the enzyme functionalized golden nanometer particle probe of design multilayer DNA carries out chemiluminescence signal amplification, by disposable polycomponent immune sensing array, a kind of highly sensitive multicomponent chemical luminescent image immuno-sensing method has been proposed.With respect to existing detection system, there is following characteristics:
(1) sensor is to take the disposable polycomponent immune sensing array that silanization microslide is substrate, prepare simple, with low cost, simultaneously because this array has 4 * 12 array sensing points, can carry out the immunoassay of 48 samples simultaneously, have advantages of that flux is high, stability and favorable reproducibility.
(2) area load novel in design has the nano-probe of DNA enzyme at high proportion, in conjunction with DNA enzyme to H
2o
2the catalytic action of-luminol reaction, carries out signal amplification, improves detection sensitivity, makes it be more suitable for the detection of low abundance proteins.
(3) the nano-probe surface of design is rich in biotin, based on biotin-avidin reaction, can be used as the common beacon probe of four kinds of protein sandwich immunoassay reactions.
(4) this detection system detects by chemiluminescence signal, does not need additional light source, and equipment and operation are all very simple.
Four, accompanying drawing explanation
Fig. 1. highly sensitive multicomponent chemical luminescent image immunoassay schematic diagram
Fig. 2. the preparation of immunosensor and sandwich immunoassay reaction, beacon association reaction and chemiluminescence signal produce and gather schematic diagram
Fig. 3. the golden nanometer particle probe that multilayer DNA is enzyme functionalized prepare schematic diagram
Five, embodiment
Embodiment 1: by reference to the accompanying drawings 3, and the preparation of the golden nanometer particle probe that multilayer DNA is enzyme functionalized
The golden nanometer particle of synthetic 13nm particle diameter, the DNA single chain of two kind of 5 ' terminal modified sulfydryl (being comprised: contain the signal dna strand that multiple G-tetra-serobila sequences repeat, seizure DNA single chain with the 3 ' terminal modified biotin) according to certain ratio, by gold-mercapto key, load on golden nanometer particle.Concrete synthetic details is as follows: in the solution of gold nanoparticles of 1mL 13nm particle diameter, add 40 μ L 1 μ M biotinylations to catch DNA single chain and 24 μ L 100 μ M signal dna strands, the ratio that catches DNA and signal dna is 1: 60, stirs 16 hours under room temperature.Then, in above-mentioned reaction solution, dropwise add the 0.01M PBS solution that contains 2M NaCl, make the ultimate density of NaCl in reaction solution reach 0.1M.Subsequently, the 0.01M PBS solution that adds 0.1mL to contain 0.1M KCl in above-mentioned reaction solution, reacts 2 hours.Centrifugally remove unconjugated DNA single chain, the DNA functionalization golden nanometer particle of preparation is distributed in the 0.01M PBS that contains 0.1M KCl.Finally, the DNA functionalization golden nanometer particle of above-mentioned preparation is reacted 1.5 hours with excessive protoheme at 4 ℃, centrifugally remove unconjugated protoheme, make the enzyme functionalized golden nanometer particle probe of multilayer DNA, be dispersed in 1mL 0.01M PBS, and be kept under 4 ℃ of conditions stand-by.
Embodiment 2: by reference to the accompanying drawings 2, and the preparation of immunosensor and sandwich immunoassay reaction, probe association reaction and chemiluminescence signal produce and gatherer process
The silanization array of serigraphy 4 * 12 on microslide, drips the certain density seizure antibody of 1.5 μ L on each array point surface of every 1 row, makes it under 4 ℃ of 100% damp condition, adsorb and spend the night; Epoxy radicals covalent bond on the amino of antibody and silanization microslide, reaction finishes post-flush, after drying, at each sensor surface, drips confining liquid, seals after 1 hour and rinses with washing fluid, after drying, makes and can catch the array of totally 4 kinds of target antigens.
On immune sensing array point surface, drip sample and carry out incubation, in conjunction with determined antigen, then washing fluid washes away sample and drips biotinylated antibody and carries out incubation, form sandwich immunoassay compound, wash away unconjugated biotinylated antibody, then drip Avidin and carry out incubation, biotin and Avidin in conjunction with after wash away unconjugated Avidin, add subsequently the enzyme functionalized golden nanometer particle probe of the prepared multilayer DNA of embodiment 1, continue incubation.After nano-probe is attached on sandwich immunoassay compound, wash away unconjugated nano-probe, finally on immunosensor surface, drip chemical luminous substrate H
2o
2-luminol, DNA enzymatic hydrogen peroxide oxidation luminol produces chemiluminescence.
Embodiment 3: by reference to the accompanying drawings 2, and with AFP, β-HCG, CA125, CEA is example, and the application of this highly sensitive multicomponent chemical luminescent image immunosensor is described
(1) drip respectively the AFP of 1.5 μ L10 μ g/mL, β-HCG, CA125 and CEA capture antibody are in array silanized surface, the epoxy radicals covalent bond of the amino of antibody and microslide, reaction finishes post-flush, then drips confining liquid on each sensing point surface, and sealing finishes post-flush, dry up, make disposable polycomponent immune sensing array (Fig. 2);
(2) on each sensing point surface of the immune sensing array of making, drip respectively standard antigen or the test serum of 1.5 μ L variable concentrations, room temperature incubation 15 minutes, rinses with washing fluid; On the surface of 1-4 line sensor, drip respectively subsequently 1.5 μ L 2 μ g/mL biotinylation AFP, β-HCG, CA125 and CEA antibody, room temperature incubation was cleaned with washing fluid after 20 minutes;
(3) on each sensing point, drip 1.5 μ L 2 μ g/mL Avidins and carry out biotin-avidin reaction, incubation was cleaned with washing fluid after 15 minutes, drip again the prepared enzyme functionalized golden nanometer particle probe of multilayer DNA of 1.5 μ L embodiment 1, react after 30 minutes and clean with washing fluid;
(4) at sensor surface, add chemical luminous substrate, with CCD, carry out the detection of multicomponent chemical luminescent image, setting screening-mode is that integration is taken, accumulative total 3 times, each one minute time shutter, according to the values of chemiluminescence of record, detect AFP in testing sample, β-HCG, the concentration of CA125 and CEA.
Claims (7)
1. a highly sensitive disposable multicomponent chemical luminescence imaging immunosensor.It is characterized in that this sensor produces system (3) by disposable polycomponent immune sensing array (1), the enzyme functionalized golden nanometer particle probe of multilayer DNA (2) and signal and forms.Wherein (1) is on microslide, to stamp 4 * 12 silanization array, in every 1 row chemical packs, by a kind of capture antibody, makes and can catch the array of totally 4 kinds of target antigens; (2) be on golden nanometer particle surface the seizure DNA of fixed biologically elementization and making in conjunction with the signal dna of the multiple G-tetra-serobila sequences repetitions of protoheme simultaneously; (3) be the peroxidase activity cataluminescence substrate H that utilizes DNA enzyme
2o
2reaction between-luminol, produces sensitive chemiluminescence signal, by data acquisition system (DAS) (4) adaptive immune sensed image signal.
2. sensor according to claim 1, is characterized in that described disposable polycomponent immune sensing array is according to spatial discrimination pattern, by the 4 row silanization arrays that stamp, and coated capture antibody 1-4 and making respectively on microslide.
3. sensor according to claim 1, it is characterized in that the described enzyme functionalized golden nanometer particle probe of multilayer DNA is by fixing multiple G-tetra-serobila sequences repeat at high proportion signal dna and biotinylated seizure DNA simultaneously on golden nanometer particle surface, and utilize G-tetra-serobila DNA to be combined formation DNA enzyme with protoheme and make.
4. sensor according to claim 1, is characterized in that the described enzyme functionalized golden nanometer particle probe of multilayer DNA has peroxidase activity, can cataluminescence substrate H
2o
2reaction between-luminol, obtains sensitive chemiluminescence signal.
5. sensor according to claim 1, it is characterized in that the described enzyme functionalized golden nanometer particle probe of multilayer DNA contains biotinylated seizure DNA, can utilize biotin-avidin reaction that probe is attached on the sandwich immunoassay compound forming in sensor array, thereby carry out highly sensitive multicomponent chemical luminescent image immunoassay, its concrete analysis step is as follows:
(1) on each sensing point surface of disposable polycomponent immune sensing array, drip detected sample, carry out incubation; After rinsing, drip corresponding biotinylation two anti-, carry out sandwich immunoassay reaction;
(2) after rinsing, drip Streptavidin carry out incubation on each sensor array surface, then rinse again, and drip the enzyme functionalized golden nanometer particle probe of multilayer DNA, reaction finishes post-flush;
(3) on sensor array surface, drip chemical luminous substrate, by CCD, carry out chemiluminescence image detection;
(4) utilize standard solution to obtain working curve, obtain the concentration of different proteins in sample (antigen).
6. analytical approach according to claim 5, is characterized in that the 1-12 row of sensor array can drip respectively 1-12 different samples, detects the concentration of each component in the different samples of 1-12 kind simultaneously.
7. analytical approach according to claim 5, is characterized in that washing fluid is the 0.01M phosphate buffer containing 0.05% Tween-20, pH7.4.
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Cited By (8)
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| CN104062288A (en) * | 2014-07-09 | 2014-09-24 | 北京师范大学 | Chemiluminiscence-based detection method of naphthylamine compound |
| CN105866105A (en) * | 2016-04-06 | 2016-08-17 | 扬州大学 | Preparation and analysis method for chemiluminiscence imaging immunosensor for detecting multiple chicken cytokines |
| CN106645109A (en) * | 2016-12-29 | 2017-05-10 | Tcl集团股份有限公司 | Method, device and system for detecting hormones |
| CN107976548A (en) * | 2017-11-21 | 2018-05-01 | 湖南工程学院 | Detection fibrin ferment kit based on tetra- serobilas of micro-fluidic chip and G--ferroheme DNA enzymatic and its preparation method and application |
| 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 |
| CN109298187A (en) * | 2018-12-10 | 2019-02-01 | 中国药科大学 | A kind of chemiluminescence immunoassay method measuring c reactive protein |
| CN111551607A (en) * | 2020-05-21 | 2020-08-18 | 福建医锦智能科技有限公司 | Biological array for detection and detection method thereof |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2005085809A1 (en) * | 2004-03-08 | 2005-09-15 | Korea Institute Of Science And Technology | Nanowire light sensor and kit with the same |
| CN101256191A (en) * | 2008-03-07 | 2008-09-03 | 中国科学院上海微***与信息技术研究所 | Method for determining minim proteins based on magnetic pearl and nano gold probe |
| JP2008292425A (en) * | 2007-05-28 | 2008-12-04 | Ricoh Co Ltd | Biosensor |
| CN101833001A (en) * | 2010-04-21 | 2010-09-15 | 黄若磐 | Protein chip kit for detecting inflammatory factors and preparation method thereof |
| CN101943703A (en) * | 2010-06-28 | 2011-01-12 | 首都医科大学 | Nanotechnology-based trace protein detection method |
| CN102539733A (en) * | 2011-03-16 | 2012-07-04 | 北京师范大学 | Visual plastic-based biochip, preparation method and detection method thereof |
-
2012
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Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2005085809A1 (en) * | 2004-03-08 | 2005-09-15 | Korea Institute Of Science And Technology | Nanowire light sensor and kit with the same |
| JP2008292425A (en) * | 2007-05-28 | 2008-12-04 | Ricoh Co Ltd | Biosensor |
| CN101256191A (en) * | 2008-03-07 | 2008-09-03 | 中国科学院上海微***与信息技术研究所 | Method for determining minim proteins based on magnetic pearl and nano gold probe |
| CN101833001A (en) * | 2010-04-21 | 2010-09-15 | 黄若磐 | Protein chip kit for detecting inflammatory factors and preparation method thereof |
| CN101943703A (en) * | 2010-06-28 | 2011-01-12 | 首都医科大学 | Nanotechnology-based trace protein detection method |
| CN102539733A (en) * | 2011-03-16 | 2012-07-04 | 北京师范大学 | Visual plastic-based biochip, preparation method and detection method thereof |
Non-Patent Citations (1)
| Title |
|---|
| 孔德明: "G-四链体-氯化血红素DNA酶在传感器设计中的应用", 《化学进展》, vol. 23, no. 10, 31 October 2011 (2011-10-31), pages 2119 - 2131 * |
Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104062288A (en) * | 2014-07-09 | 2014-09-24 | 北京师范大学 | Chemiluminiscence-based detection method of naphthylamine compound |
| CN104062288B (en) * | 2014-07-09 | 2018-02-02 | 北京师范大学 | A kind of detection method of the naphthylamine compound based on chemoluminescence method |
| CN105866105A (en) * | 2016-04-06 | 2016-08-17 | 扬州大学 | Preparation and analysis method for chemiluminiscence imaging immunosensor for detecting multiple chicken cytokines |
| CN106645109A (en) * | 2016-12-29 | 2017-05-10 | Tcl集团股份有限公司 | Method, device and system for detecting hormones |
| CN107976548A (en) * | 2017-11-21 | 2018-05-01 | 湖南工程学院 | Detection fibrin ferment kit based on tetra- serobilas of micro-fluidic chip and G--ferroheme DNA enzymatic and its preparation method and application |
| 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 |
| CN108333345B (en) * | 2018-02-05 | 2021-05-14 | 扬州大学 | Multi-chicken cytokine chemiluminescence immune analysis method with double-mimic enzyme signal amplification |
| CN109298187A (en) * | 2018-12-10 | 2019-02-01 | 中国药科大学 | A kind of chemiluminescence immunoassay method measuring c reactive protein |
| CN111551607A (en) * | 2020-05-21 | 2020-08-18 | 福建医锦智能科技有限公司 | Biological array for detection and detection method thereof |
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