CN102053161A - Disposable multi-channel electrochemical immunosensor with high sensitivity - Google Patents
Disposable multi-channel electrochemical immunosensor with high sensitivity Download PDFInfo
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- CN102053161A CN102053161A CN2009102334167A CN200910233416A CN102053161A CN 102053161 A CN102053161 A CN 102053161A CN 2009102334167 A CN2009102334167 A CN 2009102334167A CN 200910233416 A CN200910233416 A CN 200910233416A CN 102053161 A CN102053161 A CN 102053161A
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Abstract
The invention relates to a disposable multi-channel electrochemical immunosensor with high sensitivity. A prussian blue composite, nanogold and a capture antibody are assembled layer by layer on a disposable printing electrode array to obtain a multi-channel immunosensor. Enzyme and secondary antibody in great proportion are assembled on a carbon nano tube loading gold nanoparticles and a novel glucose oxidase functional nano composite probe is designed for sandwich immunoassay. The nano composite probe is combined with the multi-channel immunosensor to realize double signal amplification and high sensitive immunodetection of protein. The prussian blue, as electronic transmission media, catalyzes and reduces hydrogen peroxide generated by glucose oxidation under the catalysis of glucose oxidase on oxygen so as to obtain current signal. The method avoids the interference of dissolved oxygen in the detection solution and deoxidization is not required in process of amperometric detection. The invention has the advantages of wide detection concentration range, good repeatability, accurate results and the like and has a certain clinical application value.
Description
One, technical field
The present invention is a kind of highly sensitive disposable multi-channel electrochemical immunosensor.It amplifies and specific recognition by the signal of glucose oxidase enzyme functionalized nano compound probe, and the disposable multi-channel electrochemical immunosensor in conjunction with by the preparation of screen printing electrode array carries out the high sensitive electrochemical immunoassay.
Two, background technology
Immunoassay has obtained increasingly extensive application as a kind of high selectivity and high-sensitivity analysis method in fields such as clinical diagnosis, environmental monitoring, food securities.Immunoassay is divided into homogeneous phase and out-phase immunoassay, and the latter is widely used because can obtain higher sensitivity.With based on radiometric analysis, fluorescence, chemiluminescence, electrochemiluminescence, the immune analysis method of analysis technology such as surface plasma resonance is compared with traditional enzyme-linked immune analytic method, and it is cheap that electrochemical immunoanalytical has an instrument, easy and simple to handle, distinct advantages such as sensitivity height.Have great importance in the diagnosis that is determined at tumour of tumor markers and the early screening.Various electrochemical immunosensors, especially ampere immunity sensor obtain extensive studies and application in the detection of tumor markers.Yet, in tumor screening, more and more need high-sensitive analytical approach to improve accurate detection to low-abundance protein.In traditional electrochemical immunoanalytical method, use single enzyme labeling thing to carry out signal conduction and detection mostly, sensitivity for analysis improves limited.
In clinical diagnosis, many tumor markerses be not only with a certain disease association, the different tumours or the different tissues of tumour of the same race have common tumor markers, and a kind of tumour also often all has multiple relevant tumor markers.Yet traditional electrochemical immunoanalytical method is merely able to one-component is measured mostly, and each analysis process is only measured wherein a kind of component concentration.This analytical model required time is long, and reagent consumption is many, and workload is big, and detection efficiency is lower.Characteristics such as detecting the kinds of tumors mark simultaneously, to have a detection time shorter, and step is simplified, and sample size is less, and detection efficiency is higher, and cost is few, thus significant in medical diagnosis on disease and tumor screening.Electrochemical immunosensor based on electrod-array has obtained in the multi-component immunity analytical field paying close attention to widely in recent years.Usually a shared contrast electrode and one are to electrode for this type of immune sensing array, and a plurality of working electrode forms array, measures different components on different working electrodes.Yet, these electrod-arrays adopt technology preparations such as photoengraving more, price is comparatively expensive, and common electrochemical detection method is based upon horseradish peroxidase catalysis peroxidating reduction, be vulnerable to detect the interference of dissolved oxygen DO in the solution, need to adopt method deoxygenations such as logical nitrogen, it is very inconvenient to operate, and also is unfavorable for the further microminiaturization of detection system.
Three, summary of the invention
The objective of the invention is: prepare the multi-channel electrochemical sensor with the screen printing electrode array, utilize glucose oxidase enzyme functionalized nano compound probe to carry out two electrochemical signals and amplify, set up a kind of highly sensitive multi-channel electrochemical immunologic detection method that can be under the dissolved oxygen DO existence condition.
The present invention is achieved through the following technical solutions:
The Prussian blue compound of layer assembly, nm of gold and different capture antibodies make disposable hyperchannel immunosensor on the array that prints electrode.To be prepared into the nano-complex probe on a high proportion of glucose oxidase and the two anti-carbon nano-tube that are assembled in the load gold nano particle by " one kettle way ".After carrying out sandwich incubation of two steps on the hyperchannel immunosensor, sensor surface can be in conjunction with the glucose oxidase enzyme functionalized nano compound relevant with different determined antigen content.After dripping detection solution, but captive glucose oxidase catalysis dissolved oxygen DO oxidizing glucose produces hydrogen peroxide, hydrogen peroxide further reduces at electrode surface under fixing Prussian blue mediator effect, produces electrochemical signals, carries out quantitative measurement by differential pulse voltammetry.
Above-mentioned glucose oxidase enzyme functionalized nano compound is under certain condition and consumption, makes by laminated assembling technology; The hyperchannel immunosensor also is successively fixing Prussian blue compound and a golden nanometer particle on the screen printing electrode array, and further assembles capture antibody and make.
Detecting solution is the 0.05M phosphate solution (containing 0.1M KCl supporting electrolyte) that contains 10mM glucose; Each step of sandwich immunoassay is all used the unnecessary component of pH 7.0 phosphate washing fluid flush awaies that contains 0.05% Tween-20; Seal non-specific site with the BSA confining liquid that contains 0.05% Tween-20 behind the fixed trapped antibody.
Based on the disposable multi-channel electrochemical immunosensor of ultra-high sensitive that glucose oxidase enzyme functionalized nano compound dual signal is amplified, its concrete analysis step is as follows:
(1) by the method for layer assembly, utilize polyelectrolyte PDDA load one deck golden nanometer particle on carboxylic carbon nano-tube, and then a step a high proportion of glucose oxidase of assembling and the two anti-nano-complex probes (Fig. 1) that are prepared into as the trace labelling thing.
(2) utilizing micropipettor accurately to pipette a certain amount of Prussian blue compound modifies in working electrode surface, pipette a certain amount of golden nanometer particle again and assemble, make hyperchannel immunosensor (Fig. 2) with golden nanometer particle at the fixing different capture antibody of different working electrode surfaces at last.
(3) with the unnecessary capture antibody in washing fluid flush away immunosensor surface, stand-by behind the non-specific site of confining liquid shutoff.
(4) drip sample on immunosensor surface and carry out incubation,, continue incubation and form the sandwich immunoassay compound in conjunction with further washing fluid flush away sample and drip the nano-complex probe behind the determined antigen.
(5) drip detection solution on the immunosensor surface,, carry out multi-channel electrochemical with differential pulse voltammetry and detect by the sandwich immunoassays pattern.
(6) obtain the concentration of different proteins the sample (antigen) from working curve.
The formation of the highly sensitive disposable multi-channel electrochemical immune detection system that amplifies based on glucose oxidase enzyme functionalized nano compound dual signal:
The structural principle of this multi-channel detection system as shown in Figure 3, on the multi-channel electrochemical immunosensor, by the sandwich immunoassay reaction of two steps, in conjunction with glucose oxidase enzyme functionalized nano compound probe, drip to detect solution then in sensor surface, the glucose oxidase enzymatic dissolved oxygen DO oxidizing glucose of catching on the corresponding working electrode produces hydrogen peroxide and since a high proportion of enzyme/two anti-existence, and the catalytic action of enzyme, produced the dual signal amplification.Hydrogen peroxide reduction under Prussian blue mediator effect fixing on the working electrode produces electric current, detects this electric current by differential pulse voltammetry and can realize highly sensitive quantitative test.
This detection system is measured the principle of kinds of tumors mark:
When containing some protein (tumor markers) in the testing sample, the capture antibody generation Ag-Ab specific immunity combination corresponding in the incubation process of these tumor markerses to be measured (antigen) with sensor surface, further carry out incubation, on sensor, form corresponding double-antibody sandwich compound with the nano-complex probe.Dropping contains the detection solution of glucose, but the glucose oxidase oxygen catalytic oxidation glucose of combination on the immune complex produces hydrogen peroxide, and the electrochemical reduction that is fixed on the Prussian blue mediator catalyzing hydrogen peroxide on the sensor produces current signal, corresponding tumor-marker substrate concentration becomes positive correlation in this signal and the testing sample, can obtain the concentration of several tumor markerses to be measured simultaneously by working curve.
The present invention compared with prior art has following characteristics:
The present invention is in conjunction with the multi-channel electrochemical detection means, utilize the nano-complex probe of preparation to carry out the electrochemical signals amplification, set up a kind of high-sensitive multi-channel electrochemical immunologic detection method by disposable multi-channel electrochemical immunosensor as the trace labelling thing.Have now relatively in detection system, have following characteristics:
(1) detect by electrochemical means, do not need expensive instrument and equipment, simple to operate.
(2) to be based upon with the screen printing electrode array be on the disposable multi-channel electrochemical immunosensor that makes up of substrate to whole Electrochemical Detection, measures when can once carry out a plurality of tumor markers, detects the flux height, with low cost.
(3) design a kind of nano-complex probe of novelty, can realize that as the trace labelling thing of sandwich immunoassays dual signal amplifies, improved detection sensitivity greatly, be suitable for the detection of low abundance proteins (tumor markers).
(4) Prussian blue electron mediator is fixed on sensor surface, can eliminates the cross interference between the working electrode.
(5) utilize glucose oxidase as the trace labelling enzyme, can get rid of of the interference of traditional horseradish peroxidase effectively as the dissolved oxygen DO that exists in the trace labelling enzyme detection architecture, testing process need not deoxygenation, and is easy to operate, helps the further microminiaturization of detection system.
Four, description of drawings
Fig. 1. the preparation synoptic diagram of glucose oxidase enzyme functionalized nano probe complex
Fig. 2. the preparation of immunosensor and sandwich immunoassays process synoptic diagram
Fig. 3. sensor array lists hyperchannel immunoassay synoptic diagram
(a) nylon6 chips (b) Yin Mo (c) graphite auxiliary electrode (d) Ag/AgCl contrast electrode (e) working electrode 1 (f) working electrode 2 (g) insulation course
Five, embodiment
Embodiment 1: in conjunction with the accompanying drawings 1, and the preparation of glucose oxidase enzyme functionalized nano probe
With multi-walled carbon nano-tubes 3: 1H
2SO
4/ HNO
3In sonicated four hours, be washed to neutrality, dry carboxylic carbon nano-tube.Then, the carbon nano-tube that 0.75mg is pretreated is scattered among the 0.20% polyelectrolyte PDDA that 1.5mL contains 0.5MNaCl, and ultrasonic 30 minutes, high speed centrifugation discarded unnecessary PDDA, washed to obtain the PDDA functionalized carbon nano-tube three times.This PDDA functionalized carbon nano-tube is scattered in the 9.0mL 13-nm gold size nano particle, stirring reaction 20 minutes, centrifugal, wash the carbon mano-tube composite that obtains lilac load gold nano particle for three times.Next, this compound is scattered in the 2.5mL 50mM pH 9.0Tris-HCl solution, adding 1.9mL 2mg/mL glucose oxidase and 75 μ L 0.5mg/mL two are anti-, evenly stirring reaction is 3 hours, centrifugal 15 minutes of 3500rpm, abandoning supernatant is with the phosphate buffer washing of pH 7.0, after centrifugal three times it is scattered in the washing fluid that 500 μ L contain 0.2%BSA and preserves, promptly get required glucose oxidase enzyme functionalized nano compound probe.Use 5 times of dilutions of washing fluid before using.
Embodiment 2: in conjunction with the accompanying drawings 2, and the preparation of disposable hyperchannel immunosensor
Under room temperature, continuous stirring state, contain 6.25mM FeCl to 16mL
2, in the solution of 0.4% polyelectrolyte PDDA and 0.15% shitosan, slowly drip 4mL 25mM K
3Fe[(CN)
6] solution, solution gradually becomes mazarine, thereby generates required Prussian blue compound.Use the dilute sulfuric acid of 0.1mol/L in 1.3V constant potential pre-oxidation treatment 120s the screen printing electrode array, clean and dry the back drips 1 μ L on working electrode Prussian blue compound, room temperature is placed and was dried back room temperature absorption 13-nm golden nanometer particle 6 hours, successively use washing fluid, the phosphate buffer of pH 7.0 is cleaned, and dries.Working electrode surface in different Prussian blue compounds/golden nanometer particle modification, drip the different capture antibody of 0.5 μ L 0.5mg/mL respectively, absorption is spent the night under 4 ℃ of 100% damp condition, clean with washing fluid, dry the back and drip confining liquid sealing 60min, dry behind the liquid and promptly get required disposable hyperchannel immunosensor.
Embodiment 3: in conjunction with the accompanying drawings 3, and the hyperchannel immune analysis method
(1) on the hyperchannel immunosensor of making, drip the standard antigen or the test serum of 10 μ L variable concentrations, room temperature incubation 40min cleans with washing fluid;
(2) the nano-complex probe incubation reaction 40min of 5 times of dilutions of dropping 10 μ L cleans with washing fluid;
(3) drip the detection solution contain 10mM glucose, carry out differential pulse voltammetry in 0.30V to-0.20V potential range and measure, pulse-response amplitude is 50mV, and pulse width is 50ms.Electrochemical signals according to record obtains the working curve of different components to be measured, and component to be measured is carried out hyperchannel measure simultaneously.
(4) this hyperchannel immunosensor is disposable use.
Embodiment 4: with two kinds of important tumor markers: cancer embryo's albumen (CEA) and alpha-fetoprotein (AFP) are example, and the application of this hyperchannel immunosensor is described
Used glucose oxidase and two resists: anticancer embryonated egg of mouse monoclonal white (anti-CEA) and mouse monoclonal anti alpha-fetoprotein (anti-AFP) antibody are assembled into respectively on the carbon nano-tube of load gold nano particle, with BSA sealing residual activity site, make two kinds of nano-complex probes, as the tracer antibody of immunoassay.Mouse monoclonal anti-CEA capture antibody is fixed on the working electrode 1 of sensor array, mouse monoclonal anti-AFP capture antibody is fixed on the working electrode 2 of sensor array, with CEA and AFP standard antigen potpourri or blood serum sample incubation 40 minutes, further anti-nano-complex probe mixed solution incubation 40 minutes with containing anti-CEA and anti-AFP two.Drip the detection liquid that contains 10mM glucose at last, carry out differential pulse voltammetry and measure, collect the gained electrochemical signals.Successively detect the signal of series of standards solution, obtain the working curve of CEA and AFP respectively, utilize the electrochemical signals of this working curve and test sample gained again, obtain the concentration of two kinds of tumor markerses in the clinical blood sample.
Claims (7)
1. the present invention relates to the glucose oxidase enzyme functionalized nano compound probe of a kind of highly sensitive disposable multi-channel electrochemical immunosensor and a kind of novelty.The nano-complex probe resists assembling on the carbon nano-tube of golden nanometer particle load by glucose oxidase and two and makes.The multi-channel electrochemical immunosensor is made by the layer assembly on disposable screen printing electrode array of Prussian blue compound, nm of gold and capture antibody.Glucose oxidase at high proportion on the nano-complex probe and enzymic catalytic reaction can dual amplification sandwich immunoassays detection signal.The effective electron transfer mediator catalytic reduction of Prussian blue conduct enzymatic product hydrogen peroxide produces current signal.
2. detection system according to claim 1 is characterized in that described multi-channel electrochemical immunosensor makes on disposable serigraphy array.
3. detection system according to claim 1 is characterized in that described disposable multi-channel electrochemical immunosensor makes by the layer assembly of Prussian blue compound, gold size nano particle and monoclonal capture antibody.
4. detection system according to claim 1 is characterized in that used glucose oxidase enzyme functionalized nano compound probe is by assembling glucose oxidase and two anti-making at high proportion on the carbon nano-tube of load gold nano particle.
5. detection system according to claim 1, it is characterized in that detecting solution is the 0.05M phosphate buffer (containing 0.1M KCl is supporting electrolyte) that contains 10mM glucose, pH 6.5; Washing fluid is the 0.05M phosphate buffer that contains 0.05% Tween-20, and pH 7.0; Confining liquid is 2% bovine serum albumin(BSA) (BSA) that contains 0.05% Tween-20.
6. detection system according to claim 1 is characterized in that described glucose oxidase enzyme functionalized nano compound probe under Prussian blue electron transfer mediator effect, and the amplification detection signal has been avoided the interference of dissolved oxygen DO in the testing process.
7. highly sensitive disposable multi-channel electrochemical immunosensor that amplifies based on glucose oxidase enzyme functionalized nano compound dual signal, its concrete analysis step is as follows:
(1) drips sample on the immunosensor surface and carry out incubation;
(2) after the flushing, further drip the nano-complex probe, flushing again behind the incubation.
(3) drip detection solution,, carry out multi-channel electrochemical with differential pulse voltammetry and detect by the sandwich immunoassays pattern.
(4) obtain the concentration of different proteins the sample (antigen) from working curve.
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| CN102672197A (en) * | 2012-05-29 | 2012-09-19 | 江南大学 | Method for preparing multipurpose isoionic gold nanoparticle film |
| CN102706939A (en) * | 2012-03-20 | 2012-10-03 | 温州大学 | Electrochemical immunosensor for directly detecting IL-6 (interleukin-6) antigen and application thereof |
| CN102944549A (en) * | 2012-11-23 | 2013-02-27 | 清华大学 | Electrogenerated chemiluminescence bacterium sensing method and multi-functional probe |
| CN103995103A (en) * | 2014-06-11 | 2014-08-20 | 中国农业科学院农业质量标准与检测技术研究所 | Method for detecting small molecule compound based on prussian blue bionic marker |
| CN105067685A (en) * | 2015-07-23 | 2015-11-18 | 武汉大学 | Blood multi-component detection test paper and preparation method thereof |
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| CN109444240A (en) * | 2018-11-06 | 2019-03-08 | 湖北师范大学 | A kind of electrochemistry immuno-sensing method established based on Prussian blue electrochemical immunosensor and based on the sensor and application |
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| CN102672197B (en) * | 2012-05-29 | 2013-09-18 | 江南大学 | Method for preparing multipurpose isoionic gold nanoparticle film |
| CN102672197A (en) * | 2012-05-29 | 2012-09-19 | 江南大学 | Method for preparing multipurpose isoionic gold nanoparticle film |
| CN102944549A (en) * | 2012-11-23 | 2013-02-27 | 清华大学 | Electrogenerated chemiluminescence bacterium sensing method and multi-functional probe |
| CN102944549B (en) * | 2012-11-23 | 2015-04-15 | 清华大学 | Electrogenerated chemiluminescence bacterium sensing method and multi-functional probe |
| CN103995103A (en) * | 2014-06-11 | 2014-08-20 | 中国农业科学院农业质量标准与检测技术研究所 | Method for detecting small molecule compound based on prussian blue bionic marker |
| CN105067685A (en) * | 2015-07-23 | 2015-11-18 | 武汉大学 | Blood multi-component detection test paper and preparation method thereof |
| CN106468682B (en) * | 2015-08-17 | 2019-07-26 | 南京理工大学 | A kind of method of electrochemical nucleic acid aptamer sensor detection carcinomebryonic antigen |
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| CN108627559A (en) * | 2018-05-09 | 2018-10-09 | 福州大学 | Immunoassay method based on the Prussian blue self energizing electrochemical sensor structure of aluminium- |
| CN108627559B (en) * | 2018-05-09 | 2019-08-09 | 福州大学 | Immunoassay method based on the Prussian blue self energizing electrochemical sensor building of aluminium- |
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| US11331020B2 (en) | 2020-02-06 | 2022-05-17 | Trustees Of Boston University | Enzyme-based electrochemical nicotine biosensor |
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Application publication date: 20110511 |