CN105504314B - Alginic acid cadmium, marine alga lead plumbate and copper alginate nano particle and preparation method thereof and the application in electro-chemistry immunity probe is prepared - Google Patents
Alginic acid cadmium, marine alga lead plumbate and copper alginate nano particle and preparation method thereof and the application in electro-chemistry immunity probe is prepared Download PDFInfo
- Publication number
- CN105504314B CN105504314B CN201410488196.3A CN201410488196A CN105504314B CN 105504314 B CN105504314 B CN 105504314B CN 201410488196 A CN201410488196 A CN 201410488196A CN 105504314 B CN105504314 B CN 105504314B
- Authority
- CN
- China
- Prior art keywords
- alginic acid
- marine alga
- lead plumbate
- acid cadmium
- copper alginate
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
Abstract
A kind of application the invention discloses alginic acid cadmium, marine alga lead plumbate and copper alginate nano particle and preparation method thereof and in electro-chemistry immunity probe is prepared.The present invention can produce distinguishable electrochemical signals by the way that crossslinked sodium alginate is prepared for nano level alginic acid cadmium, marine alga lead plumbate and copper alginate spheric granules respectively with cadmium ion, lead ion or copper ion in microemulsion.The characteristics of preparation method of the present invention is that preparation condition is gentle, is that can be directly used for mark without external signal material.The electrochemical immunosensor prepared using this alginic acid cadmium, marine alga lead plumbate and copper alginate nanoparticle label different antibodies, is detected while can realizing not three target of subregion.A kind of alginic acid cadmium, marine alga lead plumbate and the copper alginate nano particle immunological probe of the present invention, and the electrochemical immunosensor prepared by it will be with a wide range of applications in immune sensing field.
Description
Technical field
It is 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 immune preparing
Application in probe, belongs to electrochemical sensor field, and it can 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 big, and the death rate is high, is to perplex one of problem of the mankind.Research shows, early to find early control
Treatment can effectively improve cancer cure rate while reducing the death rate.The early screening of cancer is primarily directed to tumor-marker
Thing.Tumor markers is generally directly produced by embryonic tissue and tumor tissues, and its change in concentration can react the dynamic of tumour
Change, and (Joseph A.Ludwig, John N.Weinstein, Biomarkers relevant with tumorous size and clinical stages
in Cancer Staging,Prognosis and Treatment Selection,Nat.Rev.Cancer,2005,5,
845–856.).A kind of rise of tumor markers concentration may be relevant with kinds cancer, and a kind of cancer can secrete a variety of swollen
Tumor markers.Therefore, in order to avoid the detection of single tumor markers causes " false positive ", accuracy rate of diagnosis is improved, reduction is surveyed
Examination expense, while detecting that several tumor markerses are the hot issues studied at present.
Electrochemical immunoanalytical technology is high, the Miniaturized detection tumor markers of a kind of quick, economy, sensitivity
Analysis method (Bhaskara V.Chikkaveeraiah, Ashwinkumar A.Bhirde, Nicole Y.Morgan, Henry
S.Eden,Xiaoyuan Chen,Electrochemical Immunosensors for Detection of Cancer
Protein Biomarkers,ACS Nano,2012,6,6546–6561.).Multicomponent is realized while the electro-chemistry immunity detected
Analytical technology mainly has following two.A kind of is the array approach based on position resolution, and the method usually requires special to lead to more
Road working electrode, such as sputtering sedimentation electrode or pyrolytic graphite electrode, these electrode materials are disposable material, cause to test into
This height simultaneously produces a large amount of discarded objects.Another is the Multi-probe labeling method differentiated based on mark, i.e., anti-at different two grades
Different electro-chemical activity semiochemicalses are marked on body, interlayer type electrochemical sensing interface is built, it is real directly on an electrode
Detected while existing Diagnostic Value of Several Serum Tumor Markers.The advantage of the latter is that electrode can effectively reduce cost savings with Reusability
Resource.
Building the conventional electroactive substance of electrochemical immunosensor has ferrocene to birds of the same feather flock together compound, thionine, toluidines
The organic dyestuff class such as blue, Prussian blue, the polymerization species such as quinone, phenol, ether, because this kind of material is difficult to antibody directly in conjunction with progress
Mark, it usually needs bonding hydrophilic functional groups are modified, or as supporter composite are made by nano material, indirectly
Labelled antibody, preparation process is complicated.Cu2+、Zn2+、Pb2+、Cd2+Deng transition metal-type, the oxidation-reduction electrode electricity with feature
Gesture, is often used as electroactive semiochemicals in Stripping Voltammetry method, but needs strong acid dissolution, and plating mercury film is enriched with electrode,
Process is cumbersome.
Sodium alginate is by two kinds of construction units of α-L- guluronic acids (G units) and beta-D-mannuronic acid (M units)
Unbranched block copolymer is constituted, is a kind of natural polysaccharide.Its good biocompatibility, and with good hydrophily,
And containing substantial amounts of functional group such as carboxyl and hydroxyl, it is usually used in the embedding of the bioactive substances such as albuminous cell.Na on G units+Occur with bivalent metal ion after ion exchange, the accumulation of G groups is cross-linked into network structure, occur irreversible gel and be turned into
With.The microballoon of alginic acid cross-linked bivalent ion can be prepared using this effect.Preparing the method for Alginate microparticles at present mainly has：
Drip, emulsion process, solvent evaporated method, high-voltage electrostatic liquid droplet and spray drying process.Alginate microparticles prepared by these methods
All in micron level, uniform particle sizes, good sphericity, the Alginate microparticles of Nano grade are prepared at present, are still to be solved
Problem.
The content of the invention
An object of the present invention is to provide a kind of alginic acid cadmium, marine alga lead plumbate and copper alginate nanoparticle and its system
Preparation Method；
The second object of the present invention is to provide a kind of by the sour cadmium of the novel alga, marine alga lead plumbate and copper alginate nanometer
Alginic acid cadmium, marine alga lead plumbate and copper alginate nanoparticle immunological probe that microballoon is prepared；
The third object of the present invention is to provide a kind of by the immune spy of alginic acid cadmium, marine alga lead plumbate and copper alginate nanoparticle
The electrochemical immunosensor that pin is prepared.
The above-mentioned purpose of the present invention is realized by following technological means：
The present invention utilizes bivalent metal ion Cd in microemulsion2+、Pb2+Or Cu2+Respectively with sodium alginate cross-linking, formed
Alginic acid cadmium, marine alga lead plumbate or copper alginate nanoparticle.This alginic acid cadmium, marine alga lead plumbate and copper alginate nanoparticle,
Directly immunological probe can be made by coupled antibody with after glutaraldehyde activated, bivalent metal ion therein produces the distinguishable of feature
Electrochemical signals peak, the antibody of the redox peaks of metal ion and mark sets up one-to-one relation, peak height and detection
Antigen concentration it is relevant, so as to be monitored while realizing three kinds of tumor markerses.
A kind of alginic acid cadmium, marine alga lead plumbate or the copper alginate nanoparticle of the present invention, it is characterised in that by with lower section
Method is prepared：
(1) 1-3g Qulas are led to, 0.5-1.5g n-hexyl alcohols, 4-6g normal octanes, 1-2mL sodium alginate aqueous solutions are mixed
30-90min is prepared into uniform microemulsion 1；
(2) 1-3g Qulas are led to, 0.5-1.5g n-hexyl alcohols, 4-6g normal octanes, with 1-2mL CdCl2The aqueous solution, Pb (NO3)2
The aqueous solution or CuCl2The aqueous solution mixes 30-90min and is prepared into uniform microemulsion 2；
(3) microemulsion 1 is added dropwise in microemulsion 2, at room temperature stirring reaction 1-6h, product centrifugation, precipitation spend from
Sub- water cleaning, produces alginic acid cadmium, marine alga lead plumbate or copper alginate nanoparticle.
In the present invention, it is preferred to, the mass percent concentration of the aqueous solution of the sodium alginate described in step (1) is 1-
2%.
In the present invention, it is preferred to, the CdCl described in step (2)2The aqueous solution concentration be 0.1-2.0M, Pb (NO3)2
The aqueous solution concentration be 0.1-2.0M, CuCl2The aqueous solution concentration be 0.1-2.0M.
The inventive method is simple, and prepares the alginic acid cadmium, marine alga lead plumbate and copper alginate microballoon of nano-scale, has
There is the electrochemical signals peak of distinguishable metal ion.Therefore it is micro- using the alginic acid cadmium, marine alga lead plumbate and copper alginate nanometer
Immunological probe prepared by ball prepares electrochemical immunosensor, it is possible to achieve detected while three kinds of tumor markerses.
Therefore, further, the invention also provides described alginic acid cadmium, marine alga lead plumbate or copper alginate nanoparticle
Application in alginic acid cadmium, marine alga lead plumbate or copper alginate nanoparticle immunological probe is prepared.
A kind of alginic acid cadmium, marine alga lead plumbate or the copper alginate nanoparticle immunological probe of the present invention, it is characterised in that logical
Following methods are crossed to prepare：
(1) above-described alginic acid cadmium, marine alga lead plumbate or copper alginate nanoparticle are washed with deionized, divided
Dissipate；
(2) alginic acid cadmium, marine alga lead plumbate or copper alginate nanoparticle are handled into activation amino with glutaraldehyde water solution, from
The heart, precipitation is washed with deionized, disperseed；
(3) at room temperature, it is the alginic acid cadmium, marine alga lead plumbate or copper alginate nanoparticle of activation is even with antibody-solutions respectively
Connection, is centrifuged off supernatant；
(4) precipitation be washed with deionized, it is scattered after, obtained alginic acid cadmium, marine alga lead plumbate or copper alginate nanometer is micro-
Ball immunological probe is in 4 DEG C of storages.
In the present invention, it is preferred to, the mass percent concentration of glutaraldehyde used is 5-15% in step (2).
Further, the invention also provides described alginic acid cadmium, marine alga lead plumbate and copper alginate nanoparticle are exempted from
Application of the epidemic disease probe in electrochemical immunosensor is prepared.Prepare three kinds of antibody of coupling respectively according to the method described in the present invention
Alginic acid cadmium, marine alga lead plumbate and copper alginate nanoparticle immunological probe, by obtained alginic acid cadmium, marine alga lead plumbate and marine alga
Sour copper nanoparticle immunological probe is fixed on the surface of electrode, obtains that three kinds of tumor markerses can be carried out simultaneously to detect simultaneously
Electrochemical immunosensor.
In a particular embodiment of the present invention, as example, giving one kind can be while realizes to tumor markers
The electrochemical immunosensor that CEA, AFP and PSA are detected, it is characterised in that be prepared by the following method and obtain：
(1) by the alginic acid cadmium described in claim any one of 1-3, marine alga lead plumbate or copper alginate nanoparticle spend from
It is sub- water washing, scattered；
(2) by alginic acid cadmium, marine alga lead plumbate and copper alginate nanoparticle handle activation amino with glutaraldehyde water solution, from
The heart, precipitation is washed with deionized, disperseed；
(3) at room temperature, by the alginic acid cadmium of activation, marine alga lead plumbate and copper alginate nanoparticle respectively with antitumor mark
Thing CEA, AFP or PSA antibody-solutions coupling, are centrifuged off supernatant；
(4) precipitation be washed with deionized, it is scattered after, obtained alginic acid cadmium, marine alga lead plumbate and copper alginate nanometer are micro-
Ball immunological probe is in 4 DEG C of storages.
(5) by immune response by obtained alginic acid cadmium, marine alga lead plumbate and copper alginate nanoparticle immunological probe are solid
The surface of electrode is scheduled on, is produced.
The present invention by being coupled alpha-fetoprotein respectively by alginic acid cadmium, marine alga lead plumbate and copper alginate nanoparticle
(AFP), after the antibody of carcinomebryonic antigen (CEA) and PSA (PSA), be prepared for alginic acid cadmium, marine alga lead plumbate and
Copper alginate nanoparticle immunological probe, by immune response by obtained alginic acid cadmium, marine alga lead plumbate and copper alginate nanometer
Microballoon immunological probe is fixed on the surface of electrode to have obtained a kind of electrochemical immunosensor, is passed using the electro-chemistry immunity
Sensor is detected while can realizing to alpha-fetoprotein, carcinomebryonic antigen and PSA, and has relatively low inspection
Rising limit, wider detection range and good reappearance.
Experiment is proved：Examined simultaneously using alginic acid cadmium, marine alga lead plumbate and copper alginate nanoparticle electro-chemistry immunity probe
Alpha-fetoprotein (AFP), carcinomebryonic antigen (CEA) and the PSA (PSA) of various concentrations are surveyed, it detects the range of linearity
It is 0.01-100ng/mL.Detection limit is respectively 0.01ng/mL, 0.0086ng/mL and 0.0075ng/mL.
Brief description of the drawings
Fig. 1 is the transmission electron microscope picture of (A) alginic acid cadmium nanoparticle prepared by one embodiment of the invention, (B) alginic acid
The transmission electron microscope picture of lead nanoparticle, the transmission electron microscope picture of (C) copper alginate nanoparticle, (D) alginic acid cadmium nanometer is micro-
The scanning electron microscopic picture of ball, the scanning electron microscopic picture of (E) marine alga lead plumbate nanoparticle, the scanning of (F) copper alginate nanoparticle
Electron microscopic picture；
Fig. 2 is to be detected simultaneously not using alginic acid cadmium, marine alga lead plumbate and the copper alginate nanoparticle immunological probe prepared
With AFP, CEA and PSA of concentration differential pulse voltammetry figure (A), electric current and AFP log concentrations (B), electric current and CEA concentration pair
The linear relationship chart of number (C), electric current and PSA log concentrations (D).As can be seen from the figure the immunosensor to AFP, CEA and
The PSA detection range of linearity is 0.01-100ng/mL.Its detection limit is respectively, 0.01ng/mL, 0.0086ng/mL and
0.0075ng/mL。
Embodiment
The invention will now be further described with reference to specific embodiments, advantages of the present invention and feature will be with description and
It is apparent.But embodiment is only exemplary, does not constitute any limitation to the scope of the present invention.Those skilled in the art should
It should be appreciated that, the details and form of technical solution of the present invention can be repaiied without departing from the spirit and scope of the invention
Change or replace, but these modifications and replacement are each fallen within protection scope of the present invention.
Embodiment 1：
1st, the preparation of alginic acid cadmium, marine alga lead plumbate or copper alginate nanoparticle
(1) 2.0g Qulas are led to, 1.0g n-hexyl alcohols, 3.0g normal octanes, 1.5mL 1.5% (w/w) sodium alginate is water-soluble
Liquid mixes 45min and is prepared into uniform microemulsion 1.
(2) 2.0g Qulas are led to, 1.0g n-hexyl alcohols, 3.0g normal octanes, the CdCl with 0.5M 1.5mL2The aqueous solution, Pb
(NO3)2The aqueous solution or CuCl2The aqueous solution mixes 45min and is prepared into uniform microemulsion 2.
(3) microemulsion 1 is added dropwise in microemulsion 2, at room temperature stirring reaction 4h, product centrifugation, precipitation deionization
Water is cleaned, and produces alginic acid cadmium, marine alga lead plumbate or copper alginate nanoparticle；The alginic acid cadmium that transmission electron microscope observing is obtained, sea
The pattern of Lead alginate and copper alginate nanoparticle is as shown in Figure 1A, 1B and 1C.Alginic acid cadmium that scanning electron microscopic observation is obtained,
The pattern of marine alga lead plumbate and copper alginate nanoparticle is as shown in Fig. 1 D, 1E and 1F.
2nd, prepare immunological probe using alginic acid cadmium, marine alga lead plumbate and copper alginate nanoparticle and its immune performance is real
Example：
2.1 are detected sample：Human serum
2.2 method
(1) alginic acid cadmium, marine alga lead plumbate and copper alginate nanoparticle are washed with deionized and disperseed；
(2) by alginic acid cadmium, marine alga lead plumbate and copper alginate nanoparticle respectively with 10% (w/w) glutaraldehyde water solution
Processing activation amino, centrifugation, precipitation is washed with deionized, disperseed；
(3) at room temperature, 1mL is contained to the solution point of the alginic acid cadmium, marine alga lead plumbate and copper alginate nanoparticle of activation
Not with 100 μ L, 1mg/mL AFP, CEA and PSA antibody coupling, supernatant is centrifuged off；
(4) precipitation addition 1mL 5% bovine serum albumin(BSA) is closed 1 hour, and to eliminate non-specific adsorption, centrifugation is removed
Supernatant.
(5) precipitation is washed with deionized, and obtained alginic acid cadmium, marine alga lead plumbate and copper alginate nanoparticle is immune to be visited
Pin is distributed in 1mL deionized waters, and obtained alginic acid cadmium, marine alga lead plumbate and copper alginate immunological probe are in 4 DEG C of storages.
(6) after glass-carbon electrode is modified with graphene-gold nano-material, the μ g mL of 20 μ L of drop coating 200-1AFP, CEA and PSA
Capture antibody mixed solution, in 4 DEG C of incubated overnights, be not coupled anti-is washed with 0.01M pH=7.3 phosphate buffer
Body.Then, the μ L of drop coating 80 on electrode, 5% bovine serum albumin solution is closed 1 hour, to eliminate non-specific adsorption.Electricity
Pole 0.01M, pH=7.3 phosphate buffer are incubated after excessive bovine serum albumin(BSA) is rinsed with human serum at 37 DEG C to be exceeded
40min.Unreacted human serum uses 0.01M, and pH=7.3 phosphate buffer is washed, at 37 DEG C, with being mixed with equal proportion
Three kinds of alginic acid cadmiums, marine alga lead plumbate and the copper alginate nanoparticle immunological probe closed was incubated more than 40 minutes.Then, it is immunized and passes
Sense electrode 0.01M, pH=7.3 phosphate buffer wash excessive immunological probe.After the completion of immune response, in 0.2M,
In pH=5.0 hac buffer, current responsing signal is detected with difference voltammetry.
Parallel testing experiment is done using conventional ELISA method simultaneously, to illustrate using the inventive method and traditional ELISA side
The matching degree of method measurement result.
3rd, result
Testing result to blood serum sample is as shown in table 1 below：
Table 1
Embodiment 2：
1st, the preparation of alginic acid cadmium, marine alga lead plumbate or copper alginate nanoparticle
(1) 2.5g Qulas are led to, 1.5g n-hexyl alcohols, 3.5g normal octanes, 2.0mL 1.5% (w/w) sodium alginate is water-soluble
Liquid mixes 45min and is prepared into uniform microemulsion 1.
(2) 2.5g Qulas are led to, 1.5g n-hexyl alcohols, 3.5g normal octanes, the CdCl with 1.0M 2.0mL2The aqueous solution, Pb
(NO3)2The aqueous solution or CuCl2The aqueous solution mixes and is prepared within 1 hour uniform microemulsion 2.
(3) microemulsion 1 is added dropwise in microemulsion 2, at room temperature stirring reaction 5h, product centrifugation, precipitation deionization
Water is cleaned, and produces alginic acid cadmium, marine alga lead plumbate or copper alginate nanoparticle.
2nd, prepare immunological probe using alginic acid cadmium, marine alga lead plumbate and copper alginate nanoparticle and its immune performance is real
Example：
2.1 are detected sample：Human serum
2.2 method
(1) alginic acid cadmium, marine alga lead plumbate and copper alginate nanoparticle are washed with deionized and disperseed；
(2) by alginic acid cadmium, marine alga lead plumbate and copper alginate nanoparticle respectively with 12% (w/w) glutaraldehyde water solution
Processing activation amino, centrifugation, precipitation is washed with deionized, disperseed；
(3) at room temperature, 1mL is contained to the solution point of the alginic acid cadmium, marine alga lead plumbate and copper alginate nanoparticle of activation
Not with 100 μ L, 1mg/mL AFP, CEA and PSA antibody coupling, supernatant is centrifuged off；
(4) precipitation addition 1mL 5% bovine serum albumin(BSA) is closed 1 hour, and to eliminate non-specific adsorption, centrifugation is removed
Supernatant.
(5) precipitation is washed with deionized, and obtained alginic acid cadmium, marine alga lead plumbate and copper alginate nanoparticle is immune to be visited
Pin is distributed in 1mL deionized waters, and obtained alginic acid cadmium, marine alga lead plumbate and copper alginate immunological probe are in 4 DEG C of storages.
(6) after glass-carbon electrode is modified with graphene-gold nano-material, the μ g mL of 20 μ L of drop coating 200-1AFP, CEA and PSA
Capture antibody mixed solution, in 4 DEG C of incubated overnights, be not coupled anti-is washed with 0.01M pH=7.3 phosphate buffer
Body.Then, the μ L of drop coating 80 on electrode, 5% bovine serum albumin solution is closed 1 hour, to eliminate non-specific adsorption.Electricity
Pole 0.01M, pH=7.3 phosphate buffer are incubated after excessive bovine serum albumin(BSA) is rinsed with human serum at 37 DEG C to be exceeded
40min.Unreacted human serum uses 0.01M, and pH=7.3 phosphate buffer is washed, at 37 DEG C, with being mixed with equal proportion
Three kinds of alginic acid cadmiums, marine alga lead plumbate and the copper alginate nanoparticle immunological probe closed was incubated more than 40 minutes.Then, it is immunized and passes
Sense electrode 0.01M, pH=7.3 phosphate buffer wash excessive immunological probe.After the completion of immune response, in 0.2M,
In pH=5.0 hac buffer, current responsing signal is detected with difference voltammetry.
Parallel testing experiment is done using conventional ELISA method simultaneously, to illustrate using the inventive method and traditional ELISA side
The matching degree of method measurement result.
3rd, result
Testing result to blood serum sample is as shown in table 2 below,：
Table 2
Embodiment 3：
1st, the preparation of alginic acid cadmium, marine alga lead plumbate or copper alginate nanoparticle
(1) 1.5g Qulas are led to, 0.5g n-hexyl alcohols, 2.5g normal octanes, 1.0mL 1.5% sodium alginate aqueous solution mixing
Stirring 45min is prepared into uniform microemulsion 1.
(2) 1.5g Qulas are led to, 0.5g n-hexyl alcohols, 2.5g normal octanes, the CdCl with 0.75M 1.0mL2The aqueous solution, or Pb
(NO3)2The aqueous solution or CuCl2The aqueous solution mixes 30min and is prepared into uniform microemulsion 2.
(3) microemulsion 1 is added dropwise in microemulsion 2, at room temperature stirring reaction 3h, product centrifugation, precipitation deionization
Water is cleaned, and produces alginic acid cadmium, marine alga lead plumbate and copper alginate nanoparticle.
2nd, prepare immunological probe using alginic acid cadmium, marine alga lead plumbate and copper alginate nanoparticle and its immune performance is real
Example：
2.1 are detected sample：Human serum
2.2 method
(1) alginic acid cadmium, marine alga lead plumbate and copper alginate nanoparticle are washed with deionized and disperseed；
(2) by alginic acid cadmium, marine alga lead plumbate and copper alginate nanoparticle respectively with 8% (w/w) glutaraldehyde water solution
Processing activation amino, centrifugation, precipitation is washed with deionized, disperseed；
(3) at room temperature, 1mL is contained to the solution point of the alginic acid cadmium, marine alga lead plumbate and copper alginate nanoparticle of activation
Not with 100 μ L, 1mg/mL AFP, CEA and PSA antibody coupling, supernatant is centrifuged off；
(4) precipitation addition 1mL 5% bovine serum albumin(BSA) is closed 1 hour, and to eliminate non-specific adsorption, centrifugation is removed
Supernatant.
(5) precipitation is washed with deionized, and obtained alginic acid cadmium, marine alga lead plumbate and copper alginate nanoparticle is immune to be visited
Pin is distributed in 1mL deionized waters, and obtained alginic acid cadmium, marine alga lead plumbate and copper alginate immunological probe are in 4 DEG C of storages.
(6) after glass-carbon electrode is modified with graphene-gold nano-material, the μ g mL of 20 μ L of drop coating 200-1AFP, CEA and PSA
Capture antibody mixed solution, in 4 DEG C of incubated overnights, be not coupled anti-is washed with 0.01M pH=7.3 phosphate buffer
Body.Then, the μ L of drop coating 80 on electrode, 5% bovine serum albumin solution is closed 1 hour, to eliminate non-specific adsorption.Electricity
Pole 0.01M, pH=7.3 phosphate buffer are incubated after excessive bovine serum albumin(BSA) is rinsed with human serum at 37 DEG C to be exceeded
40min.Unreacted human serum uses 0.01M, and pH=7.3 phosphate buffer is washed, at 37 DEG C, with being mixed with equal proportion
Three kinds of alginic acid cadmiums, marine alga lead plumbate and the copper alginate nanoparticle immunological probe closed was incubated more than 40 minutes.Then, it is immunized and passes
Sense electrode 0.01M, pH=7.3 phosphate buffer wash excessive immunological probe.After the completion of immune response, in 0.2M,
In pH=5.0 hac buffer, current responsing signal is detected with difference voltammetry.
Parallel testing experiment is done using conventional ELISA method simultaneously, to illustrate using the inventive method and traditional ELISA side
The matching degree of method measurement result.
3rd, result
Testing result to blood serum sample is as shown in table 3 below：
Table 3
Embodiment 4：The detection of the alginic acid cadmium, marine alga lead plumbate and copper alginate nanoparticle immunological probe of the present invention is linear
The measure of scope and detection limit
1st, the preparation of alginic acid cadmium, marine alga lead plumbate and copper alginate nanoparticle immunological probe：
Method according to embodiment 1 prepares alginic acid cadmium, marine alga lead plumbate and copper alginate nanoparticle immunological probe.
2nd, immunosensor building process：
20μL 200μg·mL-1AFP, CEA and PSA capture antibody mixed solution repaiied under the conditions of 4 DEG C in porous platinum
Incubated overnight on the glass-carbon electrode of decorations.Electrode surface unnecessary antibody 0.1M, pH=7.3 PBS fall.Then, use
150 μ L, 2% bovine serum albumin solution is closed 1 hour, to eliminate non-specific adsorption.Electrode 0.1M, pH=7.3's
PBS excess bovine serum albumin(BSA)s with concentration are respectively 0.01,0.05,0.1,0.5,1,5,10,50,100ngmL after rinsing-1's
AFP, CEA or PSA antigen mixed solution are incubated more than 40min at 37 DEG C.Unreacted antigen uses 0.1M, pH=7.3's
PBS falls, at 37 DEG C, and three kinds of alginic acid cadmiums, marine alga lead plumbate and the copper alginate nanoparticle mixed with equal proportion is exempted from
Epidemic disease probe was incubated more than 40 minutes.Then, immune spy of immune sensing electrode 0.1M, the pH=7.3 PBS to excess
Pin.After the completion of immune response, with 0.1M, pH=6.5 PBS is detection liquid, and current responsing signal is detected with square wave voltammetry.
3rd, result
Fig. 2 is that alginic acid cadmium, marine alga lead plumbate and copper alginate nanoparticle immunological probe detect various concentrations simultaneously
AFP, CEA and PSA differential pulse voltammetry figure (A), electric current and AFP log concentrations (B), electric current and CEA log concentrations (C), electricity
The linear relationship chart of stream and PSA log concentrations (D).
As can be seen from the figure the immunosensor is 0.01-100ng/ to AFP, CEA and PSA the detection range of linearity
mL.Its detection limit is respectively, 0.01ng/mL, 0.0086ng/mL and 0.0075ng/mL.
Claims (10)
1. a kind of alginic acid cadmium, marine alga lead plumbate or copper alginate nanoparticle, it is characterised in that be prepared by the following method and obtain：
(1) 1-3g Qulas are led to, 0.5-1.5g n-hexyl alcohols, 4-6g normal octanes, 1-2mL sodium alginate aqueous solutions mix 30-
90min is prepared into uniform microemulsion 1；
(2) 1-3g Qulas are led to, 0.5-1.5g n-hexyl alcohols, 4-6g normal octanes, the CdCl with 1-2mL2The aqueous solution, Pb (NO3)2Water
Solution or CuCl2The aqueous solution mixes 30-90min and is prepared into uniform microemulsion 2；
(3) microemulsion 1 is added dropwise in microemulsion 2, at room temperature stirring reaction 1-6h, product centrifugation, precipitation deionized water
Cleaning, produces alginic acid cadmium, marine alga lead plumbate or copper alginate nanoparticle.
2. alginic acid cadmium as claimed in claim 1, marine alga lead plumbate or copper alginate nanoparticle, it is characterised in that：Step (1)
The mass percent concentration of the aqueous solution of described sodium alginate is 1-2%.
3. alginic acid cadmium as claimed in claim 1, marine alga lead plumbate or copper alginate nanoparticle, it is characterised in that：Step (2)
Described CdCl2The aqueous solution concentration be 0.1-2.0M, Pb (NO3)2The aqueous solution concentration be 0.1-2.0M, CuCl2Water
The concentration of solution is 0.1-2.0M.
4. the alginic acid cadmium, marine alga lead plumbate or copper alginate nanoparticle described in claim any one of 1-3 are preparing alginic acid
Application in cadmium, marine alga lead plumbate or copper alginate nanoparticle immunological probe.
5. a kind of alginic acid cadmium, marine alga lead plumbate or copper alginate nanoparticle immunological probe, it is characterised in that by the following method
Prepare：
(1) by the alginic acid cadmium described in claim any one of 1-3, marine alga lead plumbate or copper alginate nanoparticle deionized water
Wash, disperse；
(2) alginic acid cadmium, marine alga lead plumbate or copper alginate nanoparticle are handled into activation amino with glutaraldehyde water solution, centrifuged,
Precipitation is washed with deionized, disperseed；
(3) at room temperature, the alginic acid cadmium, marine alga lead plumbate or copper alginate nanoparticle of activation are coupled with antibody-solutions respectively,
It is centrifuged off supernatant；
(4) precipitation be washed with deionized, it is scattered after, obtained alginic acid cadmium, marine alga lead plumbate or copper alginate nanoparticle is exempted from
Epidemic disease probe is in 4 DEG C of storages.
6. alginic acid cadmium as claimed in claim 5, marine alga lead plumbate or copper alginate nanoparticle immunological probe, its feature exist
In：The mass percent concentration of glutaraldehyde used is 5-15% in step (2).
7. the alginic acid cadmium, marine alga lead plumbate or copper alginate nanoparticle immunological probe described in claim any one of 5-6 are in system
Application in standby electrochemical immunosensor.
8. application as claimed in claim 7, it is characterised in that it is not of the same race that the method according to claim 5 prepares coupling respectively
Alginic acid cadmium, marine alga lead plumbate and the copper alginate nanoparticle immunological probe of antibody, by obtained alginic acid cadmium, marine alga lead plumbate and
Copper alginate nanoparticle immunological probe is fixed on the surface of electrode, and obtain can be while detect to three kinds of tumor markerses
Electrochemical immunosensor.
9. a kind of can realize the electrochemical immunosensor detected to tumor markers CEA, AFP and PSA simultaneously, it is special
Levy to be to be prepared by the following method and obtain：
(1) by the alginic acid cadmium described in claim any one of 1-3, marine alga lead plumbate or copper alginate nanoparticle deionized water
Wash, disperse；
(2) by alginic acid cadmium, marine alga lead plumbate and copper alginate nanoparticle handle activation amino with glutaraldehyde water solution, centrifuge,
Precipitation is washed with deionized, disperseed；
(3) at room temperature, by the alginic acid cadmium of activation, marine alga lead plumbate and copper alginate nanoparticle respectively with antitumor mark
CEA, AFP or PSA antibody-solutions coupling, are centrifuged off supernatant；
(4) precipitation be washed with deionized, it is scattered after, obtained alginic acid cadmium, marine alga lead plumbate and copper alginate nanoparticle are exempted from
Epidemic disease probe is in 4 DEG C of storages；
(5) obtained alginic acid cadmium, marine alga lead plumbate and copper alginate nanoparticle immunological probe are fixed on by immune response
The surface of electrode, is produced.
10. the electrochemical immunosensor described in claim 9 is being prepared to tumor markers CEA, AFP and PSA progress simultaneously
Application in the reagent of detection.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410488196.3A CN105504314B (en) | 2014-09-22 | 2014-09-22 | Alginic acid cadmium, marine alga lead plumbate and copper alginate nano particle and preparation method thereof and the application in electro-chemistry immunity probe is prepared |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410488196.3A CN105504314B (en) | 2014-09-22 | 2014-09-22 | Alginic acid cadmium, marine alga lead plumbate and copper alginate nano particle and preparation method thereof and the application in electro-chemistry immunity probe is prepared |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN105504314A CN105504314A (en) | 2016-04-20 |
| CN105504314B true CN105504314B (en) | 2017-10-24 |
Family
ID=55712654
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201410488196.3A Expired - Fee Related CN105504314B (en) | 2014-09-22 | 2014-09-22 | Alginic acid cadmium, marine alga lead plumbate and copper alginate nano particle and preparation method thereof and the application in electro-chemistry immunity probe is prepared |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN105504314B (en) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105990578A (en) * | 2016-04-27 | 2016-10-05 | 厦门百美特生物材料科技有限公司 | Lead carbon compound material as well as preparation method and application thereof |
| DE102017000896A1 (en) * | 2017-02-01 | 2018-08-02 | Fraunhofer-Gesellschaft zur Förderung der angewandten Forschung e.V. | Selective release system for tumor therapeutics and tumor diagnostics and biosensor for tumor tissue |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1560633A (en) * | 2004-02-19 | 2005-01-05 | 上海交通大学 | Preparation method of quantum point microspheric for bio-medical fluorescence probe |
| EP1680142A2 (en) * | 2003-10-28 | 2006-07-19 | MagnaMedics GmbH | Thermosensitive, biocompatible polymer carriers with a variable physical structure for treatment, diagnosis and analysis |
| WO2012045994A1 (en) * | 2010-10-05 | 2012-04-12 | Iota Nanosolutions Limited | Processes for preparing improved compositions |
| CN103396786A (en) * | 2013-08-06 | 2013-11-20 | 武汉大学 | Reversibly assembled and decomposable fluorescent magnetic nano material and nano biological probe and preparation methods thereof |
-
2014
- 2014-09-22 CN CN201410488196.3A patent/CN105504314B/en not_active Expired - Fee Related
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1680142A2 (en) * | 2003-10-28 | 2006-07-19 | MagnaMedics GmbH | Thermosensitive, biocompatible polymer carriers with a variable physical structure for treatment, diagnosis and analysis |
| CN1560633A (en) * | 2004-02-19 | 2005-01-05 | 上海交通大学 | Preparation method of quantum point microspheric for bio-medical fluorescence probe |
| WO2012045994A1 (en) * | 2010-10-05 | 2012-04-12 | Iota Nanosolutions Limited | Processes for preparing improved compositions |
| CN103396786A (en) * | 2013-08-06 | 2013-11-20 | 武汉大学 | Reversibly assembled and decomposable fluorescent magnetic nano material and nano biological probe and preparation methods thereof |
Non-Patent Citations (3)
| Title |
|---|
| "Effect of Ca2+, Ba2+, and Sr2+ on Alginate Microbeads";Yrr A. Mørch等;《Biomacromolecules》;20060531;第7卷(第5期);第1471-1480页 * |
| "Electrochemical Coding for Multiplexed Immunoassays of Proteins";Guodong Liu等;《Anal. Chem.》;20041201;第76卷(第23期);第7126-7130页 * |
| "功能性海澡酸复合微球的制备研究";刘继伟;《万方数据库》;20140918;全文 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN105504314A (en) | 2016-04-20 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN105588939B (en) | MPO, H FABP and cTnT joint-detections device and preparation method | |
| JP5788330B2 (en) | Organic colored fine particles, diagnostic kit containing the same, and in vitro diagnostic method | |
| CN102967706B (en) | Preparation method and application of flow injection chemiluminiscence immuno sensor for detecting tumor marker | |
| CN103116023A (en) | ECL (electrochemiluminescence) immunosensor for detecting tumor markers and preparation method and applications thereof | |
| Chen et al. | A dual-responsive biosensor for blood lead detection | |
| WO2023202271A1 (en) | Multiple pathogen combined detection apparatus and manufacturing method therefor | |
| CN109001463A (en) | A kind of gastric cancer risk indicator Internet of Things close quantitative testing test paper and preparation method | |
| CN208125741U (en) | A kind of fluorescence micro-fluidic chip detecting Anti-Mullerian hormone | |
| CN104193906A (en) | Photonic crystal microsphere as well as preparation method and application thereof | |
| CN105504314B (en) | Alginic acid cadmium, marine alga lead plumbate and copper alginate nano particle and preparation method thereof and the application in electro-chemistry immunity probe is prepared | |
| US9389162B2 (en) | Detection of analyte using coffee-ring effect | |
| CN108414495A (en) | The preparation method of iron oxide synergic nano silver/graphene oxide SERS substrates | |
| TWI696833B (en) | Organic colored microparticles, diagnostic kits and in vitro diagnostic methods | |
| Fang et al. | A blood compatible, high-efficient sensor for detection of Cr (VI) in whole blood | |
| CN105153440B (en) | A kind of preparation method of dextran microspheres gel | |
| CN106770530B (en) | A kind of preparation method and application of squamous cell carcinoma marker interlayer type immunosensor | |
| CN106370858A (en) | Potential addressing mode-based double tumor marker photoelectric detection method | |
| WO2018186267A1 (en) | Hydrophilic coloring cellulose microparticles | |
| CN111638365B (en) | GDF15 quick quantitative detection device and preparation method thereof | |
| Kharati et al. | Development of a nano biosensor for anti-gliadin detection for Celiac disease based on suspension microarrays | |
| CN109406602B (en) | Preparation method and application of immunosensor based on echinoid hollow silver platinum palladium trimetal nanoparticles | |
| WO2023104059A1 (en) | Circulating tumor cell detection material, detector and detection method | |
| CN104849449B (en) | Application of the enzyme labelled antibody gold nano-probe in diaminobenzidine catalyzed coloration and dark-field imaging | |
| CN110133246A (en) | A kind of homogeneous immunoassay method on individual particle is horizontal | |
| CN109765164A (en) | A kind of absolute quantitation detection method of unicellular middle gold nanorods |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20171024 Termination date: 20210922 |