CN105504314A - Nanoparticles of cadmium alginate, lead alginate and copper alginate, and preparation method thereof, and applications of nanoparticles of cadmium alginate, lead alginate and copper alginate in preparation of electrochemical immunoassay probes - Google Patents
Nanoparticles of cadmium alginate, lead alginate and copper alginate, and preparation method thereof, and applications of nanoparticles of cadmium alginate, lead alginate and copper alginate in preparation of electrochemical immunoassay probes Download PDFInfo
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- CN105504314A CN105504314A CN201410488196.3A CN201410488196A CN105504314A CN 105504314 A CN105504314 A CN 105504314A CN 201410488196 A CN201410488196 A CN 201410488196A CN 105504314 A CN105504314 A CN 105504314A
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Abstract
The present invention discloses nanoparticles of cadmium alginate, lead alginate and copper alginate, and a preparation method thereof, and applications of the nanoparticles of the cadmium alginate, the lead alginate and the copper alginate in preparation of electrochemical immunoassay probes. According to the present invention, cadmium ions, lead ions or copper ions are respectively subjected to cross-linking with sodium alginate to prepare the nano-scale nanoparticles of the cadmium alginate, the lead alginate and the copper alginate and generate distinguished electrochemical signals; the preparation method is characterized in that the preparation conditions are mild, and the product can be directly used for labeling without additional signal substances; with the electrochemical immunosensor prepared by using the nanoparticles of the cadmium alginate, the lead alginate and the copper alginate to label different antibodies, the simultaneous detection on three targets without partition can be achieved; and the nanoparticle immunoassay probes of the cadmium alginate, the lead alginate and the copper alginate, and the electrochemical immunosensor prepared from the nanoparticles of the cadmium alginate, the lead alginate and the copper alginate have wide application prospects in the immune sensing field.
Description
Technical field
The present invention relates to the preparation method of a kind of alginic acid cadmium, marine alga lead plumbate and copper alginate nano particle and preparing the application in immunological probe, belong to electrochemical sensor field, it may be used for immunoassay and detection.
Background technology
Cancer is the general designation of a class malignant tumour.China has 3,000,000 people to be diagnosed as cancer every year, and this numeral is also rising year by year.The treatment difficulty of cancer is large, and mortality ratio is high, is one of difficult problem of the puzzlement mankind.Research shows, early finds that early treatment effectively can improve cancer curative ratio and reduce mortality ratio simultaneously.The early screening of cancer mainly for be tumor markers.Tumor markers is directly produced by embryonic tissue and tumor tissues usually, its change in concentration can react the dynamic change of tumour, and (JosephA.Ludwig relevant to tumorous size and clinical stages, JohnN.Weinstein, BiomarkersinCancerStaging, PrognosisandTreatmentSelection, Nat.Rev.Cancer, 2005,5,845 – 856.).A kind of rising of tumor-marker substrate concentration may be relevant with kinds cancer, and a kind of cancer can secrete Diagnostic Value of Several Serum Tumor Markers.Therefore, in order to avoid the detection of single tumor markers causes " false positive ", improve accuracy rate of diagnosis, reduce testing expense, detect the hot issue that several tumor markers is research at present simultaneously.
Electrochemical immunoanalytical technology is a kind of analytical procedure (BhaskaraV.Chikkaveeraiah of quick, economic, highly sensitive, Miniaturized detection tumor markers, AshwinkumarA.Bhirde, NicoleY.Morgan, HenryS.Eden, XiaoyuanChen, ElectrochemicalImmunosensorsforDetectionofCancerProteinB iomarkers, ACSNano, 2012,6,6546 – 6561.).Realize the electrochemical immunoanalytical technology that polycomponent detects simultaneously and mainly contain following two kinds.Be the array approach that position-based is differentiated, this method needs special hyperchannel working electrode usually, and as sputtering sedimentation electrode or pyrolytic graphite electrode etc., these electrode materialss are disposable material, cause testing cost high and produce a large amount of waste.Another kind is the Multi-probe labeling method differentiated based on mark, namely in different secondary antibody, marks different electrochemical activity semiochemicalses, builds sandwich-like electrochemical sensing interface, detects while directly realizing Diagnostic Value of Several Serum Tumor Markers on an electrode.The advantage of the latter is that electrode can Reusability, effectively can reduce costs and economize on resources.
The conventional electroactive substance building electrochemical immunosensor has ferrocene base polymer, thionine, toluidine blue, the organic dye class such as Prussian blue, the polymer class such as quinone, phenol, ether, because this kind of material is difficult to directly be incorporated into row labels with antibody, usually bonding hydrophilic functional groups is needed to carry out modification, or make matrix material by nano material as supporter, indirect labelling antibody, preparation process is complicated.Cu
2+, Zn
2+, Pb
2+, Cd
2+deng transition metal-type, the characteristic redox electrode electromotive force of tool, is used as electroactive semiochemicals in Stripping Voltammetry method of being everlasting, but needs strong acid dissolution, and on electrode, plate the enrichment of mercury film, and process is loaded down with trivial details.
Sodium alginate forms unbranched segmented copolymer by α-L-guluronic acid (G unit) and beta-D-mannuronic acid (M unit) two kinds of structural units, is a kind of natural polysaccharide.Its good biocompatibility, and there is good wetting ability, and containing a large amount of functional groups as carboxyl and hydroxyl, be usually used in the embedding of the biologically active substances such as albuminous cell.Na on G unit
+after divalent-metal ion generation ion-exchange, G group is piled up and is cross-linked into reticulated structure, irreversible gelification occurs.Utilize this effect can prepare the microballoon of Lalgine cross-linked bivalent ion.The method preparing Alginate microparticles at present mainly contains: instillation, emulsion process, solvent evaporated method, high-voltage electrostatic liquid droplet and spray-drying process.Alginate microparticles prepared by these methods, all in micron level, prepares the Alginate microparticles of uniform particle sizes, good sphericity, Nano grade at present, is still have a difficult problem to be solved.
Summary of the invention
An object of the present invention is to provide a kind of Lalgine cadmium, marine alga lead plumbate and copper alginate Nano microsphere and preparation method thereof;
Two of object of the present invention is to provide a kind of the alginic acid cadmium, marine alga lead plumbate and the copper alginate Nano microsphere immunological probe that are prepared by described novel alga acid cadmium, marine alga lead plumbate and copper alginate Nano microsphere;
Three of object of the present invention is to provide a kind of electrochemical immunosensor prepared by alginic acid cadmium, marine alga lead plumbate and copper alginate Nano microsphere immunological probe.
Above-mentioned purpose of the present invention is realized by following technique means:
The present invention utilizes divalent-metal ion Cd in microemulsion
2+, Pb
2+or Cu
2+respectively with sodium alginate cross-linking, form Lalgine cadmium, marine alga lead plumbate or copper alginate Nano microsphere.This Lalgine cadmium, marine alga lead plumbate and copper alginate Nano microsphere, directly immunological probe can be made by coupled antibody after glutaraldehyde activated, divalent-metal ion wherein produces the distinguishable electrochemical signals peak of feature, the redox peak of metal ion and the antibody of mark set up relation one to one, peak height is relevant with the concentration of the antigen of detection, thus monitors while realizing three kinds of tumor markerses.
A kind of Lalgine cadmium of the present invention, marine alga lead plumbate or copper alginate Nano microsphere, is characterized in that preparing by the following method:
(1) by 1-3g Triton, 0.5-1.5g n-hexyl alcohol, 4-6g octane, 1-2mL sodium alginate aqueous solution mix and blend 30-90min is prepared into uniform microemulsion 1;
(2) by 1-3g Triton, 0.5-1.5g n-hexyl alcohol, 4-6g octane, with 1-2mLCdCl
2the aqueous solution, Pb (NO
3)
2the aqueous solution or CuCl
2aqueous solution stirs 30-90min and is prepared into uniform microemulsion 2;
(3) dropwise add in microemulsion 2 by microemulsion 1, stirred at ambient temperature reaction 1-6h, product is centrifugal, and precipitate with deionized water is cleaned, and obtains Lalgine cadmium, marine alga lead plumbate or copper alginate Nano microsphere.
In the present invention, preferably, the mass percent concentration of the aqueous solution of the sodium alginate described in step (1) is 1-2%.
In the present invention, preferably, the CdCl described in step (2)
2the concentration of the aqueous solution be 0.1-2.0M, Pb (NO
3)
2the concentration of the aqueous solution be 0.1-2.0M, CuCl
2the concentration of the aqueous solution be 0.1-2.0M.
The inventive method is simple, and prepares the Lalgine cadmium of nano-scale, marine alga lead plumbate and copper alginate microballoon, has the electrochemical signals peak of distinguishable metal ion.Therefore the immunological probe using this Lalgine cadmium, marine alga lead plumbate and copper alginate Nano microsphere to prepare prepares electrochemical immunosensor, detects while can realizing three kinds of tumor markerses.
Therefore, further, the invention allows for described Lalgine cadmium, marine alga lead plumbate or copper alginate Nano microsphere and prepare the application in Lalgine cadmium, marine alga lead plumbate or copper alginate Nano microsphere immunological probe.
A kind of Lalgine cadmium of the present invention, marine alga lead plumbate or copper alginate Nano microsphere immunological probe, is characterized in that preparing by the following method:
(1) by above-described Lalgine cadmium, marine alga lead plumbate or copper alginate Nano microsphere deionized water wash, dispersion;
(2) Lalgine cadmium, marine alga lead plumbate or copper alginate Nano microsphere is amino, centrifugal with glutaraldehyde water solution process activation, precipitate with deionized water washing, dispersion;
(3) under room temperature, by the Lalgine cadmium of activation, marine alga lead plumbate or copper alginate Nano microsphere respectively with antibody-solutions coupling, centrifugal removing supernatant liquor;
(4), after precipitate with deionized water washing, dispersion, the Lalgine cadmium obtained, marine alga lead plumbate or copper alginate Nano microsphere immunological probe are in 4 DEG C of storages.
In the present invention, preferably, in step (2), the mass percent concentration of glutaraldehyde used is 5-15%.
Further, the invention allows for described Lalgine cadmium, marine alga lead plumbate and copper alginate Nano microsphere immunological probe and prepare the application in electrochemical immunosensor.Prepare the Lalgine cadmium of coupling three kinds of antibody, marine alga lead plumbate and copper alginate Nano microsphere immunological probe according to the method described in the present invention respectively, obtained Lalgine cadmium, marine alga lead plumbate and copper alginate Nano microsphere immunological probe are fixed on the surface of electrode, obtaining can simultaneously to the electrochemical immunosensor that three kinds of tumor markerses detect simultaneously.
In a particular embodiment of the present invention, as example, give a kind of electrochemical immunosensor that simultaneously can realize that tumor markers CEA, AFP and PSA are detected, it is characterized in that preparing by the following method:
(1) by the Lalgine cadmium described in any one of claim 1-3, marine alga lead plumbate or copper alginate Nano microsphere deionized water wash, dispersion;
(2) by Lalgine cadmium, marine alga lead plumbate and copper alginate Nano microsphere glutaraldehyde water solution process activation amino, centrifugal, precipitate with deionized water washing, dispersion;
(3) under room temperature, by the Lalgine cadmium of activation, marine alga lead plumbate and copper alginate Nano microsphere respectively with the antibody-solutions coupling of antitumor mark CEA, AFP or PSA, centrifugal removing supernatant liquor;
(4), after precipitate with deionized water washing, dispersion, the Lalgine cadmium obtained, marine alga lead plumbate and copper alginate Nano microsphere immunological probe are in 4 DEG C of storages.
(5) by the Lalgine cadmium that immune response will obtain, marine alga lead plumbate and copper alginate Nano microsphere immunological probe are fixed on the surface of electrode, to obtain final product.
The present invention passes through Lalgine cadmium, marine alga lead plumbate and copper alginate Nano microsphere distinguish coupling alpha-fetoprotein (AFP), after the antibody of carcinomebryonic antigen (CEA) and prostate specific antigen (PSA), prepare Lalgine cadmium, marine alga lead plumbate and copper alginate Nano microsphere immunological probe, by immune response by obtained Lalgine cadmium, marine alga lead plumbate and copper alginate Nano microsphere immunological probe are fixed on the surface of electrode thus obtain a kind of electrochemical immunosensor, use this electrochemical immunosensor can realize alpha-fetoprotein, detect while carcinomebryonic antigen and prostate specific antigen, and there is lower detection limit, wider sensing range and good circulation ratio.
Experiment proves: utilize Lalgine cadmium, marine alga lead plumbate and copper alginate Nano microsphere electro-chemistry immunity probe to detect the alpha-fetoprotein (AFP) of different concns, carcinomebryonic antigen (CEA) and prostate specific antigen (PSA) simultaneously, and its detection linearity range is 0.01-100ng/mL.Detection limit is respectively 0.01ng/mL, 0.0086ng/mL and 0.0075ng/mL.
Accompanying drawing explanation
Fig. 1 is the transmission electron microscope picture of (A) Lalgine cadmium Nano microsphere prepared by one embodiment of the invention, (B) the transmission electron microscope picture of marine alga lead plumbate Nano microsphere, (C) the transmission electron microscope picture of copper alginate Nano microsphere, (D) scanning electron microscopic picture of Lalgine cadmium Nano microsphere, (E) scanning electron microscopic picture of marine alga lead plumbate Nano microsphere, the scanning electron microscopic picture of (F) copper alginate Nano microsphere;
Fig. 2 be utilize the Lalgine cadmium of preparation, differential pulse voltammetry figure (A) that marine alga lead plumbate and copper alginate Nano microsphere immunological probe detect AFP, CEA and PSA of different concns simultaneously, electric current and AFP log concentration (B), electric current and CEA log concentration (C), electric current and PSA log concentration (D) linear relationship chart.As can be seen from the figure the detection linearity range of this immunosensor to AFP, CEA and PSA is 0.01-100ng/mL.Its detection limit is respectively, 0.01ng/mL, 0.0086ng/mL and 0.0075ng/mL.
Embodiment
Further describe the present invention below in conjunction with specific embodiment, advantage and disadvantage of the present invention will be more clear along with description.But embodiment is only exemplary, does not form any restriction to scope of the present invention.It will be understood by those skilled in the art that and can modify to the details of technical solution of the present invention and form or replace down without departing from the spirit and scope of the present invention, but these amendments and replacement all fall within the scope of protection of the present invention.
Embodiment 1:
1, the preparation of Lalgine cadmium, marine alga lead plumbate or copper alginate Nano microsphere
(1) by 2.0g Triton, 1.0g n-hexyl alcohol, 3.0g octane, the sodium alginate aqueous solution mix and blend 45min of 1.5mL1.5% (w/w) is prepared into uniform microemulsion 1.
(2) by 2.0g Triton, 1.0g n-hexyl alcohol, 3.0g octane, with the CdCl of 0.5M1.5mL
2the aqueous solution, Pb (NO
3)
2the aqueous solution or CuCl
2aqueous solution stirs 45min and is prepared into uniform microemulsion 2.
(3) dropwise add in microemulsion 2 by microemulsion 1, stirred at ambient temperature reaction 4h, product is centrifugal, and precipitate with deionized water is cleaned, and obtains Lalgine cadmium, marine alga lead plumbate or copper alginate Nano microsphere; The Lalgine cadmium that transmission electron microscope observing obtains, the pattern of marine alga lead plumbate and copper alginate Nano microsphere is as shown in Figure 1A, 1B and 1C.The pattern of the Lalgine cadmium that scanning electron microscopic observation obtains, marine alga lead plumbate and copper alginate Nano microsphere is as shown in Fig. 1 D, 1E and 1F.
2, Lalgine cadmium, marine alga lead plumbate and copper alginate Nano microsphere is utilized to prepare immunological probe and immune performance example thereof:
2.1 detected samples: human serum
2.2 method
(1) Lalgine cadmium, marine alga lead plumbate and copper alginate Nano microsphere deionized water wash disperse;
(2) Lalgine cadmium, marine alga lead plumbate and copper alginate Nano microsphere are used the glutaraldehyde water solution process activation of 10% (w/w) amino, centrifugal respectively, precipitate with deionized water washing, dispersion;
(3) under room temperature, solution 1mL being contained the Lalgine cadmium of activation, marine alga lead plumbate and copper alginate Nano microsphere respectively with AFP, CEA and PSA antibody coupling of 100 μ L, 1mg/mL, centrifugal removing supernatant liquor;
(4) precipitate the bovine serum albumin adding 1mL5% and close 1 hour, to eliminate non-specific adsorption, centrifugal segregation supernatant liquor.
(5) precipitate with deionized water washing, the Lalgine cadmium obtained, marine alga lead plumbate and copper alginate Nano microsphere immunological probe are distributed in 1mL deionized water, and obtained Lalgine cadmium, marine alga lead plumbate and copper alginate immunological probe are in 4 DEG C of storages.
(6), after glass-carbon electrode Graphene-gold nano-material is modified, painting 20 μ L200 μ gmL is dripped
-1the capture antibody mixing solutions of AFP, CEA and PSA, in 4 DEG C of incubated overnight, wash the antibody of non-coupling with the phosphoric acid buffer of 0.01MpH=7.3.Then, electrode drips painting 80 μ L, the bovine serum albumin solution of 5% closes 1 hour, to eliminate non-specific adsorption.Excessive bovine serum albumin is rinsed rear human serum by the phosphoric acid buffer of electrode 0.01M, pH=7.3 hatches more than 40min at 37 DEG C.Unreacted human serum uses the phosphoric acid buffer of 0.01M, pH=7.3 to wash, and at 37 DEG C, the three kinds of Lalgine cadmiums mixed with equal proportion, marine alga lead plumbate and copper alginate Nano microsphere immunological probe were hatched more than 40 minutes.Then, the phosphoric acid buffer of immune sensing electrode 0.01M, pH=7.3 washes excessive immunological probe.After immune response completes, 0.2M, pH=5.0 hac buffer in, with difference voltammetry detect current responsing signal.
Adopt conventional ELISA method to do Parallel testing test, so that the matching degree adopting the inventive method and conventional ELISA method measurement result to be described simultaneously.
3, result
As shown in table 1 below to the detected result of serum sample:
Table 1
Embodiment 2:
1, the preparation of Lalgine cadmium, marine alga lead plumbate or copper alginate Nano microsphere
(1) by 2.5g Triton, 1.5g n-hexyl alcohol, 3.5g octane, the sodium alginate aqueous solution mix and blend 45min of 2.0mL1.5% (w/w) is prepared into uniform microemulsion 1.
(2) by 2.5g Triton, 1.5g n-hexyl alcohol, 3.5g octane, with the CdCl of 1.0M2.0mL
2the aqueous solution, Pb (NO
3)
2the aqueous solution or CuCl
2aqueous solution stirs and within 1 hour, is prepared into uniform microemulsion 2.
(3) dropwise add in microemulsion 2 by microemulsion 1, stirred at ambient temperature reaction 5h, product is centrifugal, and precipitate with deionized water is cleaned, and obtains Lalgine cadmium, marine alga lead plumbate or copper alginate Nano microsphere.
2, Lalgine cadmium, marine alga lead plumbate and copper alginate Nano microsphere is utilized to prepare immunological probe and immune performance example thereof:
2.1 detected samples: human serum
2.2 method
(1) Lalgine cadmium, marine alga lead plumbate and copper alginate Nano microsphere deionized water wash disperse;
(2) Lalgine cadmium, marine alga lead plumbate and copper alginate Nano microsphere are used the glutaraldehyde water solution process activation of 12% (w/w) amino, centrifugal respectively, precipitate with deionized water washing, dispersion;
(3) under room temperature, solution 1mL being contained the Lalgine cadmium of activation, marine alga lead plumbate and copper alginate Nano microsphere respectively with AFP, CEA and PSA antibody coupling of 100 μ L, 1mg/mL, centrifugal removing supernatant liquor;
(4) precipitate the bovine serum albumin adding 1mL5% and close 1 hour, to eliminate non-specific adsorption, centrifugal segregation supernatant liquor.
(5) precipitate with deionized water washing, the Lalgine cadmium obtained, marine alga lead plumbate and copper alginate Nano microsphere immunological probe are distributed in 1mL deionized water, and obtained Lalgine cadmium, marine alga lead plumbate and copper alginate immunological probe are in 4 DEG C of storages.
(6), after glass-carbon electrode Graphene-gold nano-material is modified, painting 20 μ L200 μ gmL is dripped
-1the capture antibody mixing solutions of AFP, CEA and PSA, in 4 DEG C of incubated overnight, wash the antibody of non-coupling with the phosphoric acid buffer of 0.01MpH=7.3.Then, electrode drips painting 80 μ L, the bovine serum albumin solution of 5% closes 1 hour, to eliminate non-specific adsorption.Excessive bovine serum albumin is rinsed rear human serum by the phosphoric acid buffer of electrode 0.01M, pH=7.3 hatches more than 40min at 37 DEG C.Unreacted human serum uses the phosphoric acid buffer of 0.01M, pH=7.3 to wash, and at 37 DEG C, the three kinds of Lalgine cadmiums mixed with equal proportion, marine alga lead plumbate and copper alginate Nano microsphere immunological probe were hatched more than 40 minutes.Then, the phosphoric acid buffer of immune sensing electrode 0.01M, pH=7.3 washes excessive immunological probe.After immune response completes, 0.2M, pH=5.0 hac buffer in, with difference voltammetry detect current responsing signal.
Adopt conventional ELISA method to do Parallel testing test, so that the matching degree adopting the inventive method and conventional ELISA method measurement result to be described simultaneously.
3, result
It is as shown in table 2 below to the detected result of serum sample:
Table 2
Embodiment 3:
1, the preparation of Lalgine cadmium, marine alga lead plumbate or copper alginate Nano microsphere
(1) by 1.5g Triton, 0.5g n-hexyl alcohol, 2.5g octane, the sodium alginate aqueous solution mix and blend 45min of 1.0mL1.5% is prepared into uniform microemulsion 1.
(2) by 1.5g Triton, 0.5g n-hexyl alcohol, 2.5g octane, with the CdCl of 0.75M1.0mL
2the aqueous solution, or Pb (NO
3)
2the aqueous solution or CuCl
2aqueous solution stirs 30min and is prepared into uniform microemulsion 2.
(3) dropwise add in microemulsion 2 by microemulsion 1, stirred at ambient temperature reaction 3h, product is centrifugal, and precipitate with deionized water is cleaned, and obtains Lalgine cadmium, marine alga lead plumbate and copper alginate Nano microsphere.
2, Lalgine cadmium, marine alga lead plumbate and copper alginate Nano microsphere is utilized to prepare immunological probe and immune performance example thereof:
2.1 detected samples: human serum
2.2 method
(1) Lalgine cadmium, marine alga lead plumbate and copper alginate Nano microsphere deionized water wash disperse;
(2) Lalgine cadmium, marine alga lead plumbate and copper alginate Nano microsphere are used the glutaraldehyde water solution process activation of 8% (w/w) amino, centrifugal respectively, precipitate with deionized water washing, dispersion;
(3) under room temperature, solution 1mL being contained the Lalgine cadmium of activation, marine alga lead plumbate and copper alginate Nano microsphere respectively with AFP, CEA and PSA antibody coupling of 100 μ L, 1mg/mL, centrifugal removing supernatant liquor;
(4) precipitate the bovine serum albumin adding 1mL5% and close 1 hour, to eliminate non-specific adsorption, centrifugal segregation supernatant liquor.
(5) precipitate with deionized water washing, the Lalgine cadmium obtained, marine alga lead plumbate and copper alginate Nano microsphere immunological probe are distributed in 1mL deionized water, and obtained Lalgine cadmium, marine alga lead plumbate and copper alginate immunological probe are in 4 DEG C of storages.
(6), after glass-carbon electrode Graphene-gold nano-material is modified, painting 20 μ L200 μ gmL is dripped
-1the capture antibody mixing solutions of AFP, CEA and PSA, in 4 DEG C of incubated overnight, wash the antibody of non-coupling with the phosphoric acid buffer of 0.01MpH=7.3.Then, electrode drips painting 80 μ L, the bovine serum albumin solution of 5% closes 1 hour, to eliminate non-specific adsorption.Excessive bovine serum albumin is rinsed rear human serum by the phosphoric acid buffer of electrode 0.01M, pH=7.3 hatches more than 40min at 37 DEG C.Unreacted human serum uses the phosphoric acid buffer of 0.01M, pH=7.3 to wash, and at 37 DEG C, the three kinds of Lalgine cadmiums mixed with equal proportion, marine alga lead plumbate and copper alginate Nano microsphere immunological probe were hatched more than 40 minutes.Then, the phosphoric acid buffer of immune sensing electrode 0.01M, pH=7.3 washes excessive immunological probe.After immune response completes, 0.2M, pH=5.0 hac buffer in, with difference voltammetry detect current responsing signal.
Adopt conventional ELISA method to do Parallel testing test, so that the matching degree adopting the inventive method and conventional ELISA method measurement result to be described simultaneously.
3, result
As shown in table 3 below to the detected result of serum sample:
Table 3
Embodiment 4: Lalgine cadmium of the present invention, marine alga lead plumbate and the detection linearity range of copper alginate Nano microsphere immunological probe and the mensuration of detection limit
1, the preparation of Lalgine cadmium, marine alga lead plumbate and copper alginate Nano microsphere immunological probe:
Lalgine cadmium, marine alga lead plumbate and copper alginate Nano microsphere immunological probe is prepared according to the method for embodiment 1.
2, immunosensor building process:
20 μ L200 μ gmL
-1the glass-carbon electrode modified at porous platinum under 4 DEG C of conditions of the capture antibody mixing solutions of AFP, CEA and PSA on incubated overnight.The antibody 0.1M that electrode surface is unnecessary, the PBS of pH=7.3 washes.Then, use 150 μ L, the bovine serum albumin solution of 2% closes 1 hour, to eliminate non-specific adsorption.0.01,0.05,0.1,0.5,1,5,10,50,100ngmL is respectively by concentration after the excessive bovine serum albumin flushing of PBS of electrode 0.1M, pH=7.3
-1aFP, CEA or PSA antigen mixing solutions hatch more than 40min at 37 DEG C.Unreacted antigen uses the PBS of 0.1M, pH=7.3 to wash, and at 37 DEG C, the three kinds of Lalgine cadmiums mixed with equal proportion, marine alga lead plumbate and copper alginate Nano microsphere immunological probe were hatched more than 40 minutes.Then, the PBS of immune sensing electrode 0.1M, pH=7.3 cleans to excessive immunological probe.After immune response completes, with the PBS of 0.1M, pH=6.5 for detecting liquid, detect current responsing signal with square wave voltammetry.
3, result
The linear relationship chart of the differential pulse voltammetry figure (A) that Fig. 2 is Lalgine cadmium, marine alga lead plumbate and copper alginate Nano microsphere immunological probe detect AFP, CEA and PSA of different concns simultaneously, electric current and AFP log concentration (B), electric current and CEA log concentration (C), electric current and PSA log concentration (D).
As can be seen from the figure the detection linearity range of this immunosensor to AFP, CEA and PSA is 0.01-100ng/mL.Its detection limit is respectively, 0.01ng/mL, 0.0086ng/mL and 0.0075ng/mL.
Claims (9)
1. Lalgine cadmium, marine alga lead plumbate or a copper alginate Nano microsphere, is characterized in that preparing by the following method:
(1) by 1-3g Triton, 0.5-1.5g n-hexyl alcohol, 4-6g octane, 1-2mL sodium alginate aqueous solution mix and blend 30-90min is prepared into uniform microemulsion 1;
(2) by 1-3g Triton, 0.5-1.5g n-hexyl alcohol, 4-6g octane, with the CdCl of 1-2mL
2the aqueous solution, Pb (NO
3)
2the aqueous solution or CuCl
2aqueous solution stirs 30-90min and is prepared into uniform microemulsion 2;
(3) dropwise add in microemulsion 2 by microemulsion 1, stirred at ambient temperature reaction 1-6h, product is centrifugal, and precipitate with deionized water is cleaned, and obtains Lalgine cadmium, marine alga lead plumbate or copper alginate Nano microsphere.
2. Lalgine cadmium, marine alga lead plumbate or copper alginate Nano microsphere as claimed in claim 1, is characterized in that: the mass percent concentration of the aqueous solution of the sodium alginate described in step (1) is 1-2%.
3. Lalgine cadmium, marine alga lead plumbate or copper alginate Nano microsphere as claimed in claim 1, is characterized in that: the CdCl described in step (2)
2the concentration of the aqueous solution be 0.1-2.0M, Pb (NO
3)
2the concentration of the aqueous solution be 0.1-2.0M, CuCl
2the concentration of the aqueous solution be 0.1-2.0M.
4. the Lalgine cadmium described in any one of claim 1-3, marine alga lead plumbate or copper alginate Nano microsphere are preparing the application in Lalgine cadmium, marine alga lead plumbate or copper alginate Nano microsphere immunological probe.
5. Lalgine cadmium, marine alga lead plumbate or a copper alginate Nano microsphere immunological probe, is characterized in that preparing by the following method:
(1) by the Lalgine cadmium described in any one of claim 1-3, marine alga lead plumbate or copper alginate Nano microsphere deionized water wash, dispersion;
(2) Lalgine cadmium, marine alga lead plumbate or copper alginate Nano microsphere is amino, centrifugal with glutaraldehyde water solution process activation, precipitate with deionized water washing, dispersion;
(3) under room temperature, by the Lalgine cadmium of activation, marine alga lead plumbate or copper alginate Nano microsphere respectively with antibody-solutions coupling, centrifugal removing supernatant liquor;
(4), after precipitate with deionized water washing, dispersion, the Lalgine cadmium obtained, marine alga lead plumbate or copper alginate Nano microsphere immunological probe are in 4 DEG C of storages.
6. Lalgine cadmium, marine alga lead plumbate or copper alginate Nano microsphere immunological probe as claimed in claim 5, is characterized in that: in step (2), the mass percent concentration of glutaraldehyde used is 5-15%.
7. the Lalgine cadmium described in any one of claim 5-6, marine alga lead plumbate or copper alginate Nano microsphere immunological probe are preparing the application in electrochemical immunosensor, preferably, the coupling not Lalgine cadmium of isoantibody, marine alga lead plumbate and copper alginate Nano microsphere immunological probe is prepared respectively according to the method for claim 5, obtained Lalgine cadmium, marine alga lead plumbate and copper alginate Nano microsphere immunological probe are fixed on the surface of electrode, obtaining can simultaneously to the electrochemical immunosensor that three kinds of tumor markerses detect.
8. can realize the electrochemical immunosensor that tumor markers CEA, AFP and PSA are detected simultaneously, it is characterized in that preparing by the following method:
(1) by the Lalgine cadmium described in any one of claim 1-3, marine alga lead plumbate or copper alginate Nano microsphere deionized water wash, dispersion;
(2) by Lalgine cadmium, marine alga lead plumbate and copper alginate Nano microsphere glutaraldehyde water solution process activation amino, centrifugal, precipitate with deionized water washing, dispersion;
(3) under room temperature, by the Lalgine cadmium of activation, marine alga lead plumbate and copper alginate Nano microsphere respectively with the antibody-solutions coupling of antitumor mark CEA, AFP or PSA, centrifugal removing supernatant liquor;
(4), after precipitate with deionized water washing, dispersion, the Lalgine cadmium obtained, marine alga lead plumbate and copper alginate Nano microsphere immunological probe are in 4 DEG C of storages.
(5) by the Lalgine cadmium that immune response will obtain, marine alga lead plumbate and copper alginate Nano microsphere immunological probe are fixed on the surface of electrode, to obtain final product.
9. electrochemical immunosensor according to claim 8 is preparing the application in the reagent simultaneously detected tumor markers CEA, AFP and PSA.
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