CN106986936A - UCNPs‑Ab1With GNRs Ab2And preparation method thereof, the detection method of alpha-fetoprotein - Google Patents

UCNPs‑Ab1With GNRs Ab2And preparation method thereof, the detection method of alpha-fetoprotein Download PDF

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CN106986936A
CN106986936A CN201710176515.0A CN201710176515A CN106986936A CN 106986936 A CN106986936 A CN 106986936A CN 201710176515 A CN201710176515 A CN 201710176515A CN 106986936 A CN106986936 A CN 106986936A
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gnrs
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陈红旗
高妮
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Anhui Normal University
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    • G01N33/57488—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites involving compounds identifable in body fluids
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    • G01—MEASURING; TESTING
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    • G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
    • G01N2333/4701—Details
    • G01N2333/471—Pregnancy proteins, e.g. placenta proteins, alpha-feto-protein, pregnancy specific beta glycoprotein

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Abstract

The invention discloses a kind of UCNPs Ab1With GNRs Ab2And preparation method thereof, the detection method of alpha-fetoprotein, the UCNPs Ab1Preparation method include：1) alkali, water, oleic acid, C1 C3 alcohol, manganese source, ytterbium source, yttrium source, erbium source and NaF are stirred, then hydro-thermal reaction obtains OA UCNPs；2) OA UCNPs are scattered in solvent, are subsequently added into PAA aqueous solution progress ligand exchange reaction and obtain PAA UCNPs；3) in the presence of EDC and NHS, PAA UCNPs is scattered in MES cushioning liquid and oscillation incubation, separation and washing, PBS cushioning liquid is then dissolved in, added 1 two vibrations of antibody, be subsequently added tri- vibrations of BSA and obtain UCNPs Ab1.The detection method is by using UCNPs Ab1With GNRs Ab2Detection alpha-fetoprotein causes it to have sensitivity high, and detection limit is low, the advantages of stability and excellent selectivity.

Description

UCNPs-Ab1And GNRs-Ab2And preparation method thereof, the detection method of alpha-fetoprotein
Technical field
The present invention relates to compound analysis, in particular it relates to UCNPs-Ab1And GNRs-Ab2And preparation method thereof, first tire egg White detection method.
Background technology
Alpha-fetoprotein (AFP) is a kind of cancer embryo glycoprotein, and relative molecular mass is about 68000, by fetus liver and yolk Capsule is synthesized, and the rise for a long time of its concentration in serum is considered as having certain with liver cancer, nasopharyngeal carcinoma and epithelial tumor of ovary Relation.At present, AFP is considered as preferable tumor marker, therefore, early detection AFP to the prevention of above-mentioned disease, diagnosis and Treatment has very important clinical meaning.
In the numerous AFP created detection method, electrochemical assay is the main method for detecting AFP.But, it is existing With the presence of detection method：Limit for height is detected, sensitivity is low, experimental procedure is complicated, takes time and effort, the problems such as instrument price is expensive.
The content of the invention
It is an object of the invention to provide a kind of UCNPs-Ab1And GNRs-Ab2And preparation method thereof, the detection side of alpha-fetoprotein Method, the detection method is by using UCNPs-Ab1And GNRs-Ab2Detection alpha-fetoprotein causes it to have sensitivity high, detection limit It is low, the advantages of stability and excellent selectivity, and UCNPs-Ab1And GNRs-Ab2Preparation method there is mild condition raw material The advantage being easy to get.
To achieve these goals, the invention provides a kind of UCNPs-Ab1Preparation method, the preparation method includes：
1) alkali, water, oleic acid, C1-C3 alcohol, manganese source, ytterbium source, yttrium source, erbium source and NaF are stirred, then hydro-thermal is anti- , OA-UCNPs (OA-NaYF should be centrifugally separating to obtain4:Yb, Er/Mn UCNPs)；
2) OA-UCNPs is scattered in solvent, is subsequently added into PAA (polyacrylic acid) aqueous solution progress ligand exchange anti- , PAA-UCNPs (UCNPs of carboxyl modified) should be centrifugally separating to obtain；
3) in the presence of EDC (carbodiimide) and NHS (n-hydroxysuccinimide), PAA-UCNPs is scattered in MES Simultaneously oscillation incubation, separation and washing, is then dissolved in PBS cushioning liquid, adds anti-in cushioning liquid (ethyl sulfonic acid cushioning liquid) 1 two vibrations of body, are subsequently added three vibrations of BSA (bovine serum albumin(BSA)) with closing activity site, are obtained after centrifugation washing UCNPs-Ab1(NaYF4:The couplet of Yb, Er/Mn UCNPs- antibody 1).
Present invention also offers a kind of UCNPs-Ab1, the UCNPs-Ab1It is prepared by above-mentioned preparation method.
Present invention provides a kind of GNRs-Ab2Preparation method, the preparation method includes：
1) by CTAB (cetyl trimethylammonium bromide), Jin Yuan, NaBH4Carry out haptoreaction to obtain GNRs kinds with water Sub- liquid；
2) will be by CTAB, Jin Yuan, ascorbic acid and AgNO3Carry out haptoreaction and obtain growth-promoting media；
3) GNRs seed liquors and growth-promoting media are subjected to haptoreaction, are centrifugally separating to obtain CTAB-GNRs；
4) CTAB-GNRs is dissolved in the water formation CTAB-GNRs aqueous solution, then by MUDA- ethanol solutions and CTAB-GNRs The aqueous solution carries out carboxyl modified processing, is then centrifuged for, handles that MUDA-GNRs (GNRs of carboxylated) is made；
5) in the presence of EDC and NHS, by MUDA-GNRs be scattered in MES cushioning liquid and oscillation incubation, separation and Washing, is then dissolved in PBS cushioning liquid, adds 2 two vibrations of antibody, is subsequently added tri- vibrations of BSA with closing activity position Point, GNRs-Ab is obtained after centrifugation washing2(couplet of GNRs- antibody 2).
Invention further provides a kind of GNRs-Ab2Preparation method, the GNRs-Ab2Pass through above-mentioned preparation method It is prepared.
Invention still further provides a kind of detection method of alpha-fetoprotein, the detection method includes：
1) by PBS cushioning liquid, UCNPs-Ab1And GNRs-Ab2Mixed liquor is formed, is then added mixed liquor known dense The AFP solution of degree is carried out being reacted to give sample solution, and sample solution then is added into the PBS cushioning liquid containing potassium peroxydisulfate Middle progress electrochemical luminescence energy transfer reaction simultaneously detects electrochemical luminescence intensity, finally using the concentration of AFP solution as abscissa, Electrochemical luminescence intensity is ordinate drawing curve or evaluation work curvilinear equation；
2) by AFP solution to be checked according to step 1) method measure electrochemical luminescence intensity, then according to working curve or Person's working curve equation calculates the concentration of AFP solution to be checked.
In the above-mentioned technical solutions, as shown in figure 11, the present invention prepares UCNPs-Ab to specific principle1During：First Prepare OA-NaYF4:Turn nano-particle (OA represents oleic acid) on Yb, Er/Mn UCNPs, then with PAA and OA-NaYF4:Yb, Er/ Mn UCNPs modify upper carboxyl by ligand exchange method improves its water-soluble and biocompatibility, then, adds antibody 1, profit NaYF is formed with the reaction between carboxyl-amino4:The couplet of Yb, Er/Mn UCNPs- antibody 1 is used as energy donor.
Meanwhile, the present invention prepares GNRs-Ab2During (GNRs represents gold nanorods)：First by GNRs seed liquors with CTAB-GNRs (gold nanorods of CTAB modifications) is made in growth-promoting media, then modifies GNRs with MUDA, then connects antibody 2 and be made GNRs-Ab2It is used as energy acceptor.
Finally, will be to containing UCNPs-Ab1And GNRs-Ab2System in add AFP after, pass through the specificity of antigen-antibody With reference to, electrochemical luminescence energy transfer phenomenon occurs for the distance furthered therebetween, meanwhile, NaYF4:Yb, Er/Mn UCNPs ECL be quenched degree to add AFP concentration it is related, realize the quantitative detection to AFP.
Other features and advantages of the present invention will be described in detail in subsequent embodiment part.
Brief description of the drawings
Accompanying drawing is, for providing a further understanding of the present invention, and to constitute a part for specification, with following tool Body embodiment is used to explain the present invention together, but is not construed as limiting the invention.In the accompanying drawings：
Figure 1A is the statistical chart for detecting the up-conversion luminous intensity in example 1；
Figure 1B is to detect the ECL Luminance-voltage curve maps in example 1；
Fig. 2A is detected in example 2 undoped with Mn2+OA-NaYF4:The TEM figures of Yb, Er UCNPs up-conversions；
Fig. 2 B are the TEM figures for detecting OA-UCNPs in example 2；
Fig. 3 is the elementary analysis figure for detecting OA-UCNPs in example 3；
Fig. 4 is the TEM figures for detecting CTAB-GNRs in example 4；
Fig. 5 is absorption spectrum and electrochemical luminescence spectrogram in detection example 5；
Fig. 6 A are UCNPs-Ab in detection example 61It is coupled the electrochemical luminescence intensity system of the system of the concentration change of liquid solution Meter figure；
Fig. 6 B are GNRs-Ab in detection example 62It is coupled the electrochemical luminescence intensity statistics of the system of the concentration change of liquid solution Figure；
Fig. 6 C are the electrochemical luminescence intensity statistics figures for detecting the system that the oscillation incubation time changes in example 6；
Fig. 7 A are the ECL intensity statistics figures for the system after the change of the composition of ECL detection architectures in example 6 that detects；
Fig. 7 B are to detect statistical chart of the ECL intensity to AFP concentration in example 6；
Fig. 8 is the statistical chart for detecting OA-UCNPs progress electrochemical luminescence intensive analysis in example 7；
Fig. 9 A are to detect the electrochemical luminescence detected intensities statistical chart in example 8 in embodiment 1；
Fig. 9 B are statistical chart of the ECL intensity on the basis of Fig. 9 A to AFP concentration；
Figure 10 is the result statistical chart of the Interference Detection detection of application examples 2；
Figure 11 is the schematic diagram of electrochemiluminescsystem system of the present invention.
Embodiment
The embodiment to the present invention is described in detail below.It should be appreciated that described herein specific Embodiment is merely to illustrate and explain the present invention, and is not intended to limit the invention.
The invention provides a kind of UCNPs-Ab1Preparation method, the preparation method includes：
1) by alkali, water, oleic acid, C1-C3 alcohol, manganese source, ytterbium source, yttrium source, erbium source and NaF, (order of addition of each material can With change) be stirred, then hydro-thermal reaction, be centrifugally separating to obtain OA-UCNPs (OA-NaYF4:Yb, Er/Mn UCNPs)；
2) OA-UCNPs is scattered in solvent, is subsequently added into PAA (polyacrylic acid) aqueous solution progress ligand exchange anti- , PAA-UCNPs (UCNPs of carboxyl modified) should be centrifugally separating to obtain；
3) in the presence of EDC (carbodiimide) and NHS (n-hydroxysuccinimide), PAA-UCNPs is scattered in MES Simultaneously oscillation incubation, separation and washing, is then dissolved in PBS cushioning liquid, adds anti-in cushioning liquid (ethyl sulfonic acid cushioning liquid) 1 two vibrations of body, are subsequently added three vibrations of BSA (bovine serum albumin(BSA)) with closing activity site, are obtained after centrifugation washing UCNPs-Ab1(NaYF4:The couplet of Yb, Er/Mn UCNPs- antibody 1).
In the step 1 of above-mentioned preparation method) in, the consumption of each material can be selected in wide scope, but in order that Obtain obtained UCNPs-Ab1Can be as more excellent energy donor, and then cause UCNPs-Ab1And GNRs-Ab2As body System has more excellent sensitivity, detection limit, stability and selectivity, it is preferable that in step 1) in, the alkali relative to 0.3g, The consumption of water is 5-15mL, and the consumption of oleic acid is 2-8mL, and the consumption of alcohol is 5-15mL, and the consumption of manganese source is 0.01-0.1g, yttrium The consumption in source is 0.1-0.3g, and the consumption in ytterbium source is 0.05-0.11g, and the consumption in erbium source is 5-10mg, and NaF consumptions are 0.05- 0.3g。
In the step 1 of above-mentioned preparation method) in, the condition of stirring can be selected in wide scope, but in order that Obtained UCNPs-Ab1Can be as more excellent energy donor, and then cause UCNPs-Ab1And GNRs-Ab2As system With more excellent sensitivity, detection limit, stability and selectivity, it is preferable that in step 1) in, stirring at least meets following bar Part：Whipping temp is 15-35 DEG C, and mixing time is 5-20min.
In the step 1 of above-mentioned preparation method) in, catalytic condition can be selected in wide scope, but in order to So that obtained UCNPs-Ab1Can be as more excellent energy donor, and then cause UCNPs-Ab1And GNRs-Ab2As System has more excellent sensitivity, detection limit, stability and selectivity, it is preferable that in step 1) in, haptoreaction is at least full It is enough lower condition：Reaction temperature is 180-220 DEG C, and the reaction time is 6-10h.
In the step 1 of above-mentioned preparation method) in, erbium source, manganese source, ytterbium source, yttrium source and alcohol species can be in wide scope Interior selection, but in order that obtain obtained UCNPs-Ab1Can be as more excellent energy donor, and then cause UCNPs-Ab1With GNRs-Ab2As system have more excellent sensitivity, detection limit, stability and selectivity, it is preferable that erbium source be selected from five At least one of nitric hydrate erbium, erbium oxide, anhydrous erbium chloride and eight hydrated sulfuric acid erbiums, manganese source be selected from four chloride hydrate manganese, At least one of anhydrous Manganese chloride, anhydrous manganous sulfate, Manganous sulfate monohydrate, ytterbium source are selected from ytterbium chloride, five water ytterbium nitrates, oxidation At least one of ytterbium and ytterbium carbonate, yttrium source in yttrium nitrate, yittrium oxide, six aqua oxidation yttriums and yttrium nitrate at least one Person, alcohol is selected from least one of methanol, ethanol and propyl alcohol.
In the step 2 of above-mentioned preparation method) in, the consumption of each material can be selected in wide scope, but in order that Obtain obtained UCNPs-Ab1Can be as more excellent energy donor, and then cause UCNPs-Ab1And GNRs-Ab2As body System has more excellent sensitivity, detection limit, stability and selectivity, it is preferable that in step 2) in, the OA- relative to 30mg The consumption of polyacrylic acid is 0.1-0.3g in UCNPs, the PAA aqueous solution, and the volume of the PAA aqueous solution is 5-10mL, the capacity of solvent For 3-5mL.
In the step 2 of above-mentioned preparation method) in, the condition of ligand exchange reaction can be selected in wide scope, but It is in order that obtaining obtained UCNPs-Ab1Can be as more excellent energy donor, and then cause UCNPs-Ab1And GNRs-Ab2 As system have more excellent sensitivity, detection limit, stability and selectivity, it is preferable that in step 2) in, ligand hand over Change reaction and at least meet following condition：Reaction temperature is 20-40 DEG C, and the reaction time is 18-30h.
In the step 2 of above-mentioned preparation method) in, the species of solvent can be selected in wide scope, but in order that Obtained UCNPs-Ab1Can be as more excellent energy donor, and then cause UCNPs-Ab1And GNRs-Ab2As system With more excellent sensitivity, detection limit, stability and selectivity, it is preferable that in step 2) in, solvent be selected from chloroform, At least one of dichloromethane, hexamethylene and benzene.
In the step 2 of above-mentioned preparation method) in, the weight average molecular weight of polyacrylic acid can be selected in wide scope, But in order that obtain obtained UCNPs-Ab1Can be as more excellent energy donor, and then cause UCNPs-Ab1And GNRs- Ab2As system have more excellent sensitivity, detection limit, stability and selectivity, it is preferable that the weight of polyacrylic acid is equal Molecular weight is 1000-3000.
In the step 3 of above-mentioned preparation method) in, the consumption of each material can be selected in wide scope, but in order that Obtain obtained UCNPs-Ab1Can be as more excellent energy donor, and then cause UCNPs-Ab1And GNRs-Ab2As body System has more excellent sensitivity, detection limit, stability and selectivity, it is preferable that in step 3) in, relative to 2.5mg's PAA-UCNPs, the consumption of antibody 1 is that 0.05-0.2 μ g, BSA consumption are 1-5mg, and EDC consumption is 1-5mg, NHS consumption For 1-5mg.
In the step 3 of above-mentioned preparation method) in, the consumption and pH of MES cushioning liquid and PBS cushioning liquid can be in width In the range of select, but in order that obtained UCNPs-Ab1Can be as more excellent energy donor, and then cause UCNPs-Ab1And GNRs-Ab2As system have more excellent sensitivity, detection limit, stability and selectivity, it is preferable that In step 3) in, the PAA-UCNPs relative to 2.5mg, the consumption of MES cushioning liquid and PBS cushioning liquid is each stood alone as 1.5-3mL；Also, the pH of MES cushioning liquid is each independently 6.4-8.4.
In the step 3 of above-mentioned preparation method) in, the condition of oscillation incubation, secondary vibration and three vibrations can be in width In the range of select, but in order that obtained UCNPs-Ab1Can be as more excellent energy donor, and then cause UCNPs-Ab1And GNRs-Ab2As system have more excellent sensitivity, detection limit, stability and selectivity, it is preferable that In step 3) in, oscillation incubation at least meets following condition：Vibration temperature is 15-35 DEG C, and duration of oscillation is 1.5-2.5h；It is secondary Vibration at least meets following condition：Vibration temperature is 15-35 DEG C, and duration of oscillation is 12-30h；Three vibrations at least meet following Condition：Vibration temperature is 15-35 DEG C, and duration of oscillation is 0.5-3h.
In the step 3 of above-mentioned preparation method) in, the specific species of antibody 1 can be selected in wide scope, but in order to So that obtained UCNPs-Ab1Can be as more excellent energy donor, and then cause UCNPs-Ab1And GNRs-Ab2As System has more excellent sensitivity, detection limit, stability and selectivity, it is preferable that in step 3) in, antibody 1 is trade mark 1A6 Antibody.
Present invention also offers a kind of UCNPs-Ab1, the UCNPs-Ab1It is prepared by above-mentioned preparation method.
Present invention provides a kind of GNRs-Ab2Preparation method, the preparation method includes：
1) by CTAB, Jin Yuan, NaBH4Carry out haptoreaction to obtain GNRs seed liquors with water；
2) will be by CTAB, Jin Yuan, ascorbic acid and AgNO3Carry out haptoreaction and obtain growth-promoting media；
3) GNRs seed liquors and growth-promoting media are subjected to haptoreaction, are centrifugally separating to obtain CTAB-GNRs；
4) CTAB-GNRs is dissolved in the water formation CTAB-GNRs aqueous solution, then by MUDA- ethanol solutions (MUDA and ethanol The mixed solution of solution) carboxyl modified processing is carried out with the CTAB-GNRs aqueous solution, it is then centrifuged for, handles that MUDA-GNRs is made (GNRs of carboxylated)；
5) in the presence of EDC and NHS, by MUDA-GNRs be scattered in MES cushioning liquid and oscillation incubation, separation and Washing, is then dissolved in PBS cushioning liquid, adds 2 two vibrations of antibody, is subsequently added tri- vibrations of BSA with closing activity position Point, GNRs-Ab is obtained after centrifugation washing2(couplet of GNRs- antibody 2).
In the step 1 of above-mentioned preparation method) in, the consumption of each material can be selected in wide scope, but in order that Obtain obtained GNRs-Ab2Can be as more excellent energy acceptor, and then cause UCNPs-Ab1And GNRs-Ab2As system With more excellent sensitivity, detection limit, stability and selectivity, it is preferable that in step 1) in, the CTAB relative to 0.36g, Jin Yuan consumption is 0.5-1.5mg, NaBH4Consumption be 0.1-0.5mg, the consumption of water is 9-12g.
In the step 1 of above-mentioned preparation method) in, Jin Yuan specific species can be selected in wide scope, but in order to So that obtained GNRs-Ab2Can be as more excellent energy acceptor, and then cause UCNPs-Ab1And GNRs-Ab2As body System has more excellent sensitivity, detection limit, stability and selectivity, it is preferable that Jin Yuan is gold chloride.
In the step 1 of above-mentioned preparation method) in, catalytic actual conditions can be selected in wide scope, still In order that obtaining obtained GNRs-Ab2Can be as more excellent energy acceptor, and then cause UCNPs-Ab1And GNRs-Ab2As System have more excellent sensitivity, detection limit, stability and selectivity, it is preferable that in step 1) in, haptoreaction is at least Meet following condition：Reaction temperature is 20-40 DEG C, and the reaction time is 1-5min.
In the step 2 of above-mentioned preparation method) in, the consumption of each material can be selected in wide scope, but in order that Obtain obtained GNRs-Ab2Can be as more excellent energy acceptor, and then cause UCNPs-Ab1And GNRs-Ab2As system With more excellent sensitivity, detection limit, stability and selectivity, it is preferable that in step 2) in, the CTAB relative to 0.36g, Jin Yuan consumption is 1-3mg, and the consumption of ascorbic acid is 0.5-1.5mg, AgNO3Consumption be 0.033-0.048g, the use of water Measure as 9-12g.
In the step 2 of above-mentioned preparation method) in, catalytic actual conditions can be selected in wide scope, still In order that obtaining obtained GNRs-Ab2Can be as more excellent energy acceptor, and then cause UCNPs-Ab1And GNRs-Ab2As System have more excellent sensitivity, detection limit, stability and selectivity, it is preferable that in step 2) in, haptoreaction is at least Meet following condition：Reaction temperature is 20-40 DEG C, and the reaction time is 1-5min.
In the step 3 of above-mentioned preparation method) in, the consumption of each material can be selected in wide scope, but in order that Obtain obtained GNRs-Ab2Can be as more excellent energy acceptor, and then cause UCNPs-Ab1And GNRs-Ab2As system With more excellent sensitivity, detection limit, stability and selectivity, it is preferable that in step 3) in, the growth relative to 11mL Liquid, the consumption of seed liquor is 5-20 μ L.
In the step 3 of above-mentioned preparation method) in, catalytic actual conditions can be selected in wide scope, still In order that obtaining obtained GNRs-Ab2Can be as more excellent energy acceptor, and then cause UCNPs-Ab1And GNRs-Ab2As System have more excellent sensitivity, detection limit, stability and selectivity, it is preferable that in step 3) in, haptoreaction is at least Following conditioned response temperature is met for 20-30 DEG C, the reaction time is 20-60min.
In the step 4 of above-mentioned preparation method) in, the consumption of each material can be selected in wide scope, but in order that Obtain obtained GNRs-Ab2Can be as more excellent energy acceptor, and then cause UCNPs-Ab1And GNRs-Ab2As system With more excellent sensitivity, detection limit, stability and selectivity, it is preferable that in step 4) in, relative to 2.4pmol's MUDA consumption is 2-7mg in CTAB-GNRs, MUDA- ethanol solution, and the consumption of water is 5-10mL, the body of MUDA- ethanol solutions Product is 100-1000 μ L.
In the step 4 of above-mentioned preparation method) in, the condition of carboxyl modified processing can be selected in wide scope, still In order that obtaining obtained GNRs-Ab2Can be as more excellent energy acceptor, and then cause UCNPs-Ab1And GNRs-Ab2As System have more excellent sensitivity, detection limit, stability and selectivity, it is preferable that in step 4) in, carboxyl modified processing Condition be：The ultrasonically treated 1-3h at 20-60 DEG C.
In the step 5 of above-mentioned preparation method) in, the consumption of raw material can be selected in wide scope, but in order that Obtained GNRs-Ab2Can be as more excellent energy acceptor, and then cause UCNPs-Ab1And GNRs-Ab2As system tool Have more excellent sensitivity, detection limit, stability and selectivity, it is preferable that in step 5) in, the MUDA- relative to 24pmol GNRs, the consumption of antibody 2 is that 0.05-0.2 μ g, BSA consumption are 1-5mg, and EDC consumption is 1-5mg, and NHS consumption is 1- 5mg。
In the step 5 of above-mentioned preparation method) in, the consumption and pH of MES cushioning liquid and PBS cushioning liquid can be in width In the range of select, but in order that obtained GNRs-Ab2Can be as more excellent energy acceptor, and then cause UCNPs-Ab1And GNRs-Ab2As system have more excellent sensitivity, detection limit, stability and selectivity, it is preferable that In step 5) in, the MUDA-GNRs relative to 24pmol, the consumption of MES cushioning liquid and PBS cushioning liquid is each stood alone as 1.5-3mL；Also, the pH of MES cushioning liquid is each independently 6.4-8.4.
In the step 5 of above-mentioned preparation method) in, the condition of oscillation incubation, secondary vibration and three vibrations can be in width In the range of select, but in order that obtained GNRs-Ab2Can be as more excellent energy acceptor, and then cause UCNPs-Ab1And GNRs-Ab2As system have more excellent sensitivity, detection limit, stability and selectivity, it is preferable that In step 5) in, oscillation incubation at least meets following condition：Vibration temperature is 15-35 DEG C, and duration of oscillation is 1.5-2.5h；It is secondary Vibration at least meets following condition：Vibration temperature is 15-35 DEG C, and duration of oscillation is 12-30h；Three vibrations at least meet following Condition：Vibration temperature is 15-35 DEG C, and duration of oscillation is 0.5-3h.
In the step 5 of above-mentioned preparation method) in, the specific species of antibody 2 can be selected in wide scope, but in order to So that obtained GNRs-Ab2Can be as more excellent energy acceptor, and then cause UCNPs-Ab1And GNRs-Ab2As body System has more excellent sensitivity, detection limit, stability and selectivity, it is preferable that in step 5) in, antibody 2 is trade mark 2A5's Antibody.
Invention further provides a kind of GNRs-Ab2Preparation method, the GNRs-Ab2Pass through above-mentioned preparation method It is prepared.
Invention still further provides a kind of detection method of alpha-fetoprotein, the detection method includes：
1) by PBS cushioning liquid, UCNPs-Ab1And GNRs-Ab2Mixed liquor is formed, is then added mixed liquor known dense The AFP solution of degree is carried out being reacted to give sample solution, and sample solution then is added into the PBS cushioning liquid containing potassium peroxydisulfate Middle progress electrochemical luminescence energy transfer reaction simultaneously detects electrochemical luminescence intensity, finally using the concentration of AFP solution as abscissa, Electrochemical luminescence intensity is ordinate drawing curve or evaluation work curvilinear equation；
2) by AFP solution to be checked according to step 1) method measure electrochemical luminescence intensity, then according to working curve or Person's working curve equation calculates the concentration of AFP solution to be checked.
In above-mentioned detection method, the consumption of each material can be selected in wide scope, but in order that obtain the detection Method has more excellent sensitivity, detection limit, stability and selectivity, it is preferable that relative to 6.54 μ g UCNPs-Ab1, GNRs-Ab2Consumption be 5 × 10-3-1.0×10-3Pmol, the consumption of detection sample is 10-30 μ L and the middle AFP of detection sample Concentration be 2-5000ng/mL.
In above-mentioned detection method, the consumption of PBS cushioning liquid and the concentration of potassium sulfate can be selected in wide scope Select, but in order that obtaining the detection method with more excellent sensitivity, detection limit, stability and selectivity, it is preferable that it is relative In 20-50 μ L sample solution, the consumption of the PBS cushioning liquid containing potassium peroxydisulfate is 3-8mL, and the concentration of potassium peroxydisulfate is 0.05-0.2mol/L；
In above-mentioned detection method, the condition of electrochemical luminescence energy transfer reaction can be selected in wide scope, but It is in order that obtaining the detection method with more excellent sensitivity, detection limit, stability and selectivity, it is preferable that electrochemistry is sent out Light energy transfer reaction at least meets following condition：20-30 DEG C of reaction temperature, the reaction time is 100-120min.
On the basis of the above, working curve equation has differences under different Detection wavelengths, but in order that must have More excellent linear relationship, it is preferable that working curve equation is y=2.88 × 103lgx-2.17×103, wherein, y is electrochemistry Luminous intensity quenching value, x is AFP concentration.
In above-mentioned detection method, the pH of PBS cushioning liquid can be selected in wide scope, but in order that obtain the inspection Survey method has more excellent sensitivity, detection limit, stability and selectivity, it is preferable that the pH of PBS cushioning liquid is 6.4- 8.4。
In addition, in above-mentioned detection method, the source of AFP solution to be checked can be selected in wide scope, but from reality Considered with property, it is preferable that AFP solution to be checked is human serum.
Finally, in the case where AFP solution to be checked is human serum, the source mode on human serum can also be in width In the range of select, but in order to simplify processing procedure, it is preferable that in step 2) before, detection method also includes：By human body blood Liquid is centrifuged, and takes upper strata to obtain human serum after purification.
The present invention will be described in detail by way of examples below.In following examples, antibody 1 is northern biotechnology The commercially available trade mark 1A6 of research institute antibody, antibody 2 is the commercially available trade mark 2A5 of northern biotechnology research institute antibody.
Embodiment 1
1)OA-UCNPs(OA-NaYF4:Yb, Er/Mn UCNPs) preparation：
First, the NaOH for weighing 300mg is placed in 50mL small beakers, add 1.50mL ultra-pure waters, 5.00mL oleic acid, 10.0mL ethanol, sequentially adds 0.500mL MnCl after stirring2Solution (0.600mol/L), 1.00mL Y (NO3)3Solution (0.500mol/L), 0.900mL YbCl3Solution (0.200mol/L) and 0.100mL Er (NO3)3Solution (0.200mol/L). Then, 2.00mL NaF solution (2.00mol/L) and the gentle agitation 15min at 25 DEG C is added dropwise, 50mL reactions are transferred to In kettle, 8h is reacted at 200 DEG C.Naturally cool to after 25 DEG C, centrifugation (10000rmp rotating speeds, centrifuge 5min), with ultra-pure water and Ethanol cyclic washing several times after isolated OA-NaYF4:Yb, Er/Mn UCNPs.
2)OA-NaYF4:PAA-UCNPs is made in Yb, Er/Mn UCNPs carboxyl modified：
30.0mg OA-UCNPs are dissolved in 4.00mL chloroforms OA-UCNPs solution is made, by 200mg polyacrylic acid (polyacrylic acid weight average molecular weight be 1800) is dissolved in 8.00mL) polyacrylic acid solution is made in ultra-pure water；Then, by OA-UCNPs Chloroform soln is added drop-wise in polyacrylic acid solution dropwise, and 24h is stirred under the conditions of 37 DEG C, by the absorption of polyacrylic acid, OA-UCNPs enters water layer from chloroform layer；Finally, product is isolated in centrifugation (10000rmp rotating speeds centrifuge 5min), is used in combination Ultra-pure water and ethanol washing obtain PAA-UCNPs.
3) by 2.50mgPAA-UCNPs be dissolved in 2.00mLMES cushioning liquid (10.0mmol/L, pH be 6.4) in, ultrasonic shape Into uniform solution, 3mg EDC and 3mg NHS are subsequently added into, the oscillation incubation 2h at 25 DEG C is centrifuged, washing；By obtained production Product add 2.00mL PBS cushioning liquid (10.0mmol/L, pH be 7.4) in, add 0.1 μ g antibody 1 and vibrated at 25 DEG C 24h, then adding BSA closed systems, (200 μ L, 0.300 weight % purposes are to close the position that UCNPs surfaces are not combined by antibody 1 Point), continue to centrifuge after vibrating 0.5h at 25 DEG C, washing obtains UCNPs-Ab1。
4) CTAB-GNRs preparation：
First, by 5.00mL CTAB solution (0.200mol/L) solution and 5.00mL HAuCl4Solution (5.00 × 10- 4Mol/L) solution is mixed in the case where not stopping stirring, rapidly joins the NaBH of 0.600mL brand-news4Solution (1.00 × 10-2Mol/L it is) molten Liquid, solution becomes yellowish-brown by colourless, obtains GNRs seed liquors after 2min is stirred at 27 DEG C, deposits in 27 DEG C of water-baths.
Then, by 5.00mL CTAB solution (0.200mol/L), 0.260mL AgNO3Solution (4.00 × 10-3Mol/L), 5.00mL HAuCl4Solution (1.00 × 10-3Mol/L) mix successively, 70.0 μ L ascorbic acid solutions (7.88 × 10-2mol/L) It is added dropwise under agitation, solution becomes colourless by yellow and obtains growth-promoting media, and growth-promoting media is also placed in reacting in 27 DEG C of water-baths 0.5h。
After the completion of prepared by GNRs seed liquors and growth-promoting media, draw 12.0 μ L seed liquors with liquid-transfering gun and add 11mL growth-promoting medias In, rock uniform, continue the water-bath 45min at 27 DEG C, solution is in bluish violet, and centrifugation, washing obtains CTAB-GNRs.
5) GNRs modification is to obtain MUDA-GNRs：
Taking 10.0mL CTAB-GNRs solution centrifugals, (10000rmp rotating speeds centrifuge 5min, the CTAB- containing 2.40pmol GNRs), abandoning supernatant, is redissolved in 10.0mL ultra-pure waters, is then slowly added to 1.00mL MUDA- ethanol solutions (20.0mmol/L) obtains mixed liquor, by the mixture at 50 DEG C ultrasound 0.5h, then the continual ultrasonic 2h at 25 DEG C；Finally Centrifugation obtains MUDA-GNRs, and MUDA-GNRs is redissolved in 10.0mL ultra-pure waters.
6)GNRs-Ab2The preparation of couplet：
3mg EDC and 3mg NHS are added into 2mL GNRs-MES solution, and (2.40pmol MUDA-GNRs and MES bufferings are molten The mixed liquor of liquid), 2h is vibrated at 30 DEG C, is centrifuged, washing；Then product is dissolved in 2.00mL PBS cushioning liquid (10.0mmol/L, pH are 7.4), to add 0.1 μ g antibody 2 and vibrate 24h at 30 DEG C；Finally, BSA closed systems (200 μ are added L, 0.300 weight %, it is therefore an objective to close the site that GNRs surfaces are not combined by antibody 2) continue to vibrate 0.5h at 30 DEG C, from The heart, washing obtains the couplet of GNRs- antibody 2.
7) AFP detection：Glass-carbon electrode (GCE) is polished with 0.30 μm and 0.05 μm of aluminium powder successively, in ethanol and ultrapure Dried naturally as working electrode in atmosphere after ultrasound in water.
By 1.25mg/mLUCNPs-Ab1Being coupled liquid solution, (2-10 μ LmL contain 6.54 μ g μ g UCNPs-Ab1) with 1200pmol/LGNRs-Ab2Being coupled liquid solution (6 μ LmL) addition 2.00mL PBS cushioning liquid, (7.4) 10.0mmol/L, pH are In, it is well mixed, adds the AFP solution of same volume various concentrations, oscillation incubation 102min is used as sample solution at 30 DEG C.
ECL is detected：Using three-electrode system, cushioning liquid is that (0.100mol/L, pH are the cushioning liquid of PBS containing 6.00mL 7.4, contain 0.100mol/L K2S2O8), 30.0 μ L sample solution are inwardly added, electricity is carried out to it with Electrochemial luminescence detecting instrument Chemiluminescence detection, drawing curve, is as a result shown in Fig. 9, wherein, working curve equation is y=2.88 × 103lgx-2.17× 103, wherein y is that value is quenched in electrochemical luminescence intensity, and x is AFP concentration.
Embodiment 2
1)OA-UCNPs(OA-NaYF4:Yb, Er/Mn UCNPs) preparation：
First, the NaOH for weighing 300mg is placed in 50mL small beakers, adds 5mL ultra-pure waters, 2.00mL oleic acid, 5.0mL second Alcohol, sequentially adds 0.500mL MnCl after stirring2Solution (contains 0.01gMnCl2), 1.00mL Y (NO3)3Solution (contains 0.1g Y(NO3)3), 0.900mL YbCl3Solution (contains 0.05YbCl3) and 0.100mL Er (NO3)3Solution (contains 5mg Er (NO3)3).Then, 2.00mL NaF solution (containing 0.05g NaF) and the gentle agitation 15min at 25 DEG C is added dropwise, shifts Into 50mL reactors, 6h is reacted at 180 DEG C.Naturally cool to after 25 DEG C, centrifugation (10000rmp rotating speeds centrifuge 5min), With ultra-pure water and ethanol cyclic washing several times after isolated OA-NaYF4:Yb, Er/Mn UCNPs.
2)OA-NaYF4:PAA-UCNPs is made in Yb, Er/Mn UCNPs carboxyl modified：
30.0mg OA-UCNPs are dissolved in 3.00mL chloroforms OA-UCNPs solution is made, by 100mg polyacrylic acid (polyacrylic acid weight average molecular weight is 1800), which is dissolved in 5.00mL ultra-pure waters, is made polyacrylic acid solution；Then, by OA-UCNPs Chloroform soln is added drop-wise in polyacrylic acid solution dropwise, and 18h is stirred under the conditions of 37 DEG C, by the absorption of polyacrylic acid, OA-UCNPs enters water layer from chloroform layer；Finally, product is isolated in centrifugation (10000rmp rotating speeds centrifuge 5min), is used in combination Ultra-pure water and ethanol washing obtain PAA-UCNPs.
3) by 2.50mgPAA-UCNPs be dissolved in 2.00mLMES cushioning liquid (10.0mmol/L, pH be 6.4) in, ultrasonic shape Into uniform solution, 1mg EDC and 1mg NHS are subsequently added into, the oscillation incubation 2h at 25 DEG C is centrifuged, washing；By obtained production Product add 2.00mL PBS cushioning liquid (10.0mmol/L, pH be 7.4) in, add 0.1 μ g antibody 1 and vibrated at 25 DEG C 24h, then adding BSA closed systems, (200 μ L, contain 1mg BSA, it is therefore an objective to close what UCNPs surfaces were not combined by antibody 1 Site), continue to centrifuge after vibrating 0.5h at 25 DEG C, washing obtains UCNPs-Ab1。
4) CTAB-GNRs preparation：
First, by 5.00mL CTAB solution (0.200mol/L contains 0.36g CTAB) solution and 5.00mL HAuCl4 Solution (contains 0.5mg HAuCl4) solution mixing in the case where not stopping stirring, rapidly join the NaBH of 0.600mL brand-news4Solution (contains 0.1mg NaBH4) solution, solution becomes yellowish-brown by colourless, obtains GNRs seed liquors after 2min is stirred at 27 DEG C, deposits in In 27 DEG C of water-baths.
Then, by 5.00mL CTAB solution (0.200mol/L contains 0.36g CTAB), 0.260mL AgNO3Solution (contain 0.033-0.048g AgNO3), 5.00mL HAuCl4Solution (the HAuCl containing 1mg4) mix successively, 70.0 μ L are anti-bad Hematic acid solution (containing 0.5mg ascorbic acid) is added dropwise under agitation, and solution becomes colourless by yellow and obtains growth-promoting media, growth Liquid is also placed in reacting 0.5h in 27 DEG C of water-baths.
After the completion of prepared by GNRs seed liquors and growth-promoting media, draw 5.0 μ L seed liquors with liquid-transfering gun and add 11mL growth-promoting medias In, rock uniform, continue the water-bath 20min at 20 DEG C, solution is in bluish violet, and centrifugation, washing obtains CTAB-GNRs.
5) GNRs modification is to obtain MUDA-GNRs：
Taking 10.0mL CTAB-GNRs solution centrifugals, (10000rmp rotating speeds centrifuge 5min, the CTAB- containing 2.40pmol GNRs), abandoning supernatant, is redissolved in 10.0mL ultra-pure waters, is then slowly added to 1.00mL MUDA- ethanol solutions and (contains 2mgMUDA) obtain mixed liquor, by the mixture at 50 DEG C ultrasound 0.5h, then the continual ultrasonic 2h at 25 DEG C；Finally centrifuge MUDA-GNRs is obtained, and MUDA-GNRs is redissolved in 10.0mL ultra-pure waters.
6)GNRs-Ab2The preparation of couplet：
1mg EDC and 1mg NHS are added into 1.5mL GNRs-MES solution, and (2.40pmolMUDA-GNRs and MES bufferings are molten The mixed liquor of liquid), 2h is vibrated at 30 DEG C, is centrifuged, washing；Then product is dissolved in 2.00mL PBS cushioning liquid (10.0mmol/L, pH are 7.4), to add 0.05 μ g antibody 2 and vibrate 24h at 30 DEG C；Finally, BSA closed systems are added (0.300 weight %, contains 1mg BSA, it is therefore an objective to close the site that GNRs surfaces are not combined by antibody 2), continue at 30 DEG C 0.5h is vibrated, centrifugation, washing obtains the couplet of GNRs- antibody 2.
7) AFP detection：Glass-carbon electrode (GCE) is polished with 0.30 μm and 0.05 μm of aluminium powder successively, in ethanol and ultrapure Dried naturally as working electrode in atmosphere after ultrasound in water.
By 1.25mg/mL UCNPs-Ab1It is coupled liquid solution and (contains 6.54 μ g UCNPs-Ab1) and 1200pmol/L GNRs-Ab2Coupling liquid solution (6 μ L) add 2.00mLPBS cushioning liquid (10.0mmol/L, pH be 7.4) in, be well mixed, The AFP solution of same volume various concentrations is added, oscillation incubation 102min is used as sample solution at 30 DEG C.
ECL is detected：Using three-electrode system, cushioning liquid is that (0.100mol/L, pH are the cushioning liquid of PBS containing 6.00mL 7.4, contain 0.100mol/L K2S2O8), 30.0 μ L sample solution are inwardly added, electricity is carried out to it with Electrochemial luminescence detecting instrument As a result chemiluminescence detection, drawing curve shows that the working curve that work song is obtained with embodiment 1 is consistent substantially.
Embodiment 3
1)OA-UCNPs(OA-NaYF4:Yb, Er/Mn UCNPs) preparation：
First, the NaOH for weighing 300mg is placed in 50mL small beakers, adds 15mL ultra-pure waters, 8.00mL oleic acid, 15.0mL Ethanol, sequentially adds 0.500mL MnCl after stirring2Solution (contains 0.1g MnCl2), 1.00mL Y (NO3)3Solution (contains There are 0.3g Y (NO3)3), 0.900mL YbCl3Solution (contains 0.11g YbCl3) and 0.100mL Er (NO3)3Solution (contains 10mg Er(NO3)3).Then, 2.00mL NaF solution (containing 0.3g NaF) and the gentle agitation at 25 DEG C is added dropwise 15min, is transferred in 50mL reactors, and 10h is reacted at 220 DEG C.Naturally cool to after 25 DEG C, centrifugation (10000rmp rotating speeds, Centrifuge 5min), with ultra-pure water and ethanol cyclic washing several times after isolated OA-NaYF4:Yb, Er/Mn UCNPs.
2)OA-NaYF4:PAA-UCNPs is made in Yb, Er/Mn UCNPs carboxyl modified：
30.0mg OA-UCNPs are dissolved in 5mL chloroforms OA-UCNPs solution is made, 0.3g polyacrylic acid is (poly- Polyacrylic acid solution is made 1800) to be dissolved in 10mL ultra-pure waters in acrylic acid weight average molecular weight；Then, by the chloromethanes of OA-UCNPs tri- Alkane solution is added drop-wise in polyacrylic acid solution dropwise, and 30h is stirred under the conditions of 37 DEG C, passes through the absorption of polyacrylic acid, OA- UCNPs enters water layer from chloroform layer；Finally, product is isolated in centrifugation (10000rmp rotating speeds, centrifuge 5min), and with ultrapure Water and ethanol washing obtain PAA-UCNPs.
3) by 2.50mgPAA-UCNPs be dissolved in 2.00mLMES cushioning liquid (10.0mmol/L, pH be 6.4) in, ultrasonic shape Into uniform solution, 5mg EDC and 5mg NHS are subsequently added into, the oscillation incubation 2h at 25 DEG C is centrifuged, washing；By obtained production Product add 2.00mL PBS cushioning liquid (10.0mmol/L, pH be 7.4) in, add 0.2 μ g antibody 1 and vibrated at 25 DEG C 24h, then adding BSA closed systems, (200 μ L, contain 5mg BSA, it is therefore an objective to close what UCNPs surfaces were not combined by antibody 1 Site), continue to centrifuge after vibrating 0.5h at 25 DEG C, washing obtains UCNPs-Ab1。
4) CTAB-GNRs preparation：
First, by 5.00mL CTAB solution (0.200mol/L contains 0.36g CTAB) solution and 5.00mL HAuCl4 Solution (contains 1.5mg HAuCl4) solution mixing in the case where not stopping stirring, rapidly join the NaBH of 0.600mL brand-news4Solution (contains 0.5mg NaBH4) solution, solution becomes yellowish-brown by colourless, obtains GNRs seed liquors after 2min is stirred at 27 DEG C, deposits in In 27 DEG C of water-baths.
Then, by 5.00mL CTAB solution (0.200mol/L contains 0.36g CTAB), 0.260mL AgNO3Solution (contain 0.033-0.048g AgNO3), 5.00mL HAuCl4Solution (the HAuCl containing 3mg4) mix successively, 70.0 μ L are anti-bad Hematic acid solution (containing 1.5mg ascorbic acid) is added dropwise under agitation, and solution becomes colourless by yellow and obtains growth-promoting media, growth Liquid is also placed in reacting 0.5h in 27 DEG C of water-baths.
After the completion of prepared by GNRs seed liquors and growth-promoting media, draw 20 μ L seed liquors with liquid-transfering gun and add 11mL growth-promoting medias In, rock uniform, continue the water-bath 60min at 30 DEG C, solution is in bluish violet, and centrifugation, washing obtains CTAB-GNRs.
5) GNRs modification is to obtain MUDA-GNRs：
Taking 10.0mL CTAB-GNRs solution centrifugals, (10000rmp rotating speeds centrifuge 5min, the CTAB- containing 2.4pmol GNRs), abandoning supernatant, is redissolved in 10.0mL ultra-pure waters, is then slowly added to 1.00mL MUDA- ethanol solutions and (contains 7mg MUDA) obtain mixed liquor, by the mixture at 50 DEG C ultrasound 0.5h, then the continual ultrasonic 2h at 25 DEG C；It is last from Gains in depth of comprehension are redissolved in 10.0mL ultra-pure waters to MUDA-GNRs, and by MUDA-GNRs.
6)GNRs-Ab2The preparation of couplet：
5mg EDC and 5mg NHS are added into 3mL GNRs-MES solution, and (2.4pmol/L, MUDA-GNRs and MES buffering are molten The mixed liquor of liquid), 2h is vibrated at 30 DEG C, is centrifuged, washing；Then product is dissolved in 2.00mL PBS cushioning liquid (10.0mmol/L, pH are 7.4), to add 0.2 μ g antibody 2 and vibrate 24h at 30 DEG C；Finally, BSA closed systems (200 μ are added L, contains 5mg BSA, it is therefore an objective to close the site that GNRs surfaces are not combined by antibody 2), continuation vibrates 0.5h at 30 DEG C, from The heart, washing obtains the couplet of GNRs- antibody 2.
7) AFP detection：Glass-carbon electrode (GCE) is polished with 0.30 μm and 0.05 μm of aluminium powder successively, in ethanol and ultrapure Dried naturally as working electrode in atmosphere after ultrasound in water.
By 1.25mg/mL UCNPs-Ab1It is coupled liquid solution and (contains 6.54 μ g UCNPs-Ab1) and 1200pmol/L GNRs-Ab2Coupling liquid solution (6 μ L) add 2.00mLPBS cushioning liquid (10.0mmol/L, pH be 7.4) in, be well mixed, The AFP solution of same volume various concentrations is added, oscillation incubation 102min is used as sample solution at 30 DEG C.
ECL is detected：Using three-electrode system, cushioning liquid is that (0.100mol/L, pH are the cushioning liquid of PBS containing 6.00mL 7.4, contain 0.100mol/L K2S2O8), 30.0 μ L sample solution are inwardly added, electricity is carried out to it with Electrochemial luminescence detecting instrument As a result chemiluminescence detection, drawing curve shows that the working curve that work song is obtained with embodiment 1 is consistent substantially.
Embodiment 4
According to the step 1 of embodiment 1) OA-UCNPs is made, except that MnCl2The concentration of manganese ion is in solution 0.4mol/L。
Embodiment 5
According to the step 1 of embodiment 1) OA-UCNPs is made, except that MnCl2The concentration of manganese ion is in solution 0.5mol/L。
Embodiment 6
According to the step 1 of embodiment 1) OA-UCNPs is made, except that MnCl2The concentration of manganese ion is in solution 0.7mol/L。
Embodiment 8
According to the step 1 of embodiment 1) OA-UCNPs is made, except that MnCl2The concentration of manganese ion is in solution 0.8mol/L。
Detect example 1
The detection of up-conversion luminous intensity：By the trade mark for CHI 660A electrochemical workstation to embodiment 1, real Apply OA-UCNPs in a 4-8 and carry out electrochemical luminescence detection, concrete outcome is shown in Figure 1A, Mn is worked as known in the figure2+Concentration is 0.600mol/L, up-conversion luminous intensity reaches maximum.
The detection of ECL Luminance-voltage curves：Testing result is shown in Figure 1B, wherein, a curves are undoped with Mn in embodiment 12+ NaYF4:Yb, Er ECL Luminance-voltage curves are (undoped with Mn2+NaYF4:Yb according to embodiment 1 step 1) method Prepare, the difference is that MnCl is not used2Solution), b curves are OA-NaYF in embodiment 14:Yb, Er/Mn UCNPs ECL hairs Optical-electronic is buckled line, it can be seen that voltage from 0V scan to -1.9V when, above two UCNPs without ECL signals, And voltage from -1.9V scan to -2.5V when, the ECL peaks of a, b curve all rapidly rise, and highest is reached in -2.5V.
Detect example 2
By the trade mark for JEOL2010 transmission electron microscope to OA-UCNPs in embodiment 1 and undoped with Mn2+'s OA-NaYF4:OA-UCNPs is made in Yb, Er UCNPs up-conversions (according to step 1 of embodiment 1), and different is not used MnCl2Solution) Shape measure, be specifically shown in Fig. 2A and Fig. 2 B, wherein, Fig. 2A is undoped with Mn2+OA-NaYF4:Yb, Er The TEM figures of UCNPs up-conversions, Fig. 2 B are the TEM figures of OA-UCNPs in embodiment 1；From Fig. 2A, do not adulterate Mn2+ When, two kinds of forms are presented in up-conversion, and existing cube has the nanometer rods of hexagonal phase again, but with 30%Mn2+Doping, UCNPs all becomes cube.
Detect example 3
Enter row element point to OA-UCNPs in embodiment 1 using the trade mark for Hitachi S-4800 SEM Analyse (EDS), as a result such as Fig. 3.From the figure 3, it may be seen that NaYF4:Yb, Er/Mn UCNPs are successfully prepared.
Detect example 4
Shape measure is carried out to the CTAB-GNRs in embodiment 1 for HT-7700 transmission electron microscope by the trade mark, Testing result such as Fig. 4.As shown in Figure 4, obtained GNRs particle diameters are more uniform.
Detect example 5
Absorption light is carried out for the CTAB-GNRs in UV-4100 ultraviolet-visible spectrophotometer embodiment 1 by the trade mark A curves in spectrum measure, testing result such as Fig. 5.
Electrification is carried out to OA-UCNPs in embodiment 1 for the Weak light investigating instrument of (BPCL-T (- 2-I-C)) by the trade mark B curves in luminescent spectrum measure, testing result such as Fig. 5.
As shown in Figure 5, the maximum absorption band of the CTAB-GNRs in embodiment 1 is at 635nm, with OA- in embodiment 1 UCNPs emission peak has significantly overlapping, is to occur one of ECL-RET necessary condition.
Detect example 6
Recorded using the multi-functional electrochemistry and Electrochemiluminescprocess process system of trade mark MPI-A types to electrochemical luminescence energy The optimization and contrast of transfer reaction condition are measured, as a result such as Fig. 6 A-6B, Fig. 7 A-7B.
Wherein, carried out in Fig. 6 A by the way of embodiment 1, except that change step 7) in UCNPs-Ab1Coupling The concentration of liquid solution, it is UCNPs-Ab as a result to show 1.09 μ g/mL1The optium concentration of couplet.
Carried out in Fig. 6 B by the way of embodiment 1, except that change step 7) in GNRs-Ab2It is coupled liquid solution, As a result it is GNRs-Ab to show 1.20pmol/L2It is coupled the optium concentration of liquid solution.
Carried out in Fig. 6 C by the way of embodiment 1, except that change step 7) in the oscillation incubation time, as a result show It is the optimal oscillation incubation time to show more than 100min.
Carried out in Fig. 7 A by the way of embodiment 1, except that change step 7) ECL detection architectures composition, As a result UCNPs-Ab is shown1Containing K2S2O8PBS cushioning liquid (0.100mol/L K2S2O8, pH be 7.4) in have very strong ECL (a curves).But, when in cushioning liquid be free of K2S2O8When, luminous (g curves) is not observed but, this explanation K2S2O8It is altogether Reactant.And work as in cushioning liquid containing only K2S2O8When (f curves), only observing has faint luminous, illustrates in the present invention UCNPs is luminous as ECL bodies.To UCNPs-Ab1+K2S2O8When being individually added into appropriate GNRs in system, due to inner filtering effect ECL There is a small amount of quenching (b curves), when being individually added into AFP, because the BSA resistance in the closing activity site of addition is larger, ECL also has micro- Amount quenching (c curves).And add AFP and GNRs-Ab simultaneously thereto2When, produce luminescence queenching phenomenon (d curves, e curves)； This is due to the specific binding of antigen-antibody, and furthered the distance between UCNPs and GNRs, occurs ECL-RET, and ECL lights It is quenched,
Carried out in Fig. 7 B by the way of embodiment 1, except that change step 7) in AFP concentration, analyze UCNPs- Ab1The relation between luminous intensity and AFP concentration at 620nm, is as a result illustrated with the increase of AFP concentration, △ IECL (△ IECL=I0- I, I0It is the ECL intensity when being free of AFP in system, I is ECL intensity when AFP is added among system) by It is cumulative big, further demonstrate ECL-RET generation.
Detect example 7
Recorded using the multi-functional electrochemistry and Electrochemiluminescprocess process system of trade mark MPI-A types to OA- in embodiment 1 UCNPs carries out electrochemical luminescence intensive analysis, as a result such as Fig. 8.As shown in Figure 8, cyclical voltages of the OA-UCNPs in continuous 18 circles The lower signal of scanning is still stablized.
Detect example 8
Utilize the electricity in multi-functional electrochemistry and Electrochemiluminescprocess process system the record embodiment 1 of trade mark MPI-A types Chemiluminescence Resonance energy transfer reaction carries out electrochemical luminescence intensity detection, and testing result is as shown in fig. 9 a and fig. 9b.By scheming 9A and Fig. 9 B understand that △ I are quenched in logarithm value and the ECL intensity of AFP concentrationECL(△IECL=I0- I, I0With I be respectively in system not Plus AFP antigens and add AFP antigens ECL intensity) between have preferable linear relationship.
Application examples 1
Electrochemical luminescence Resonance energy transfer detects actual sample：
Serum is purified, and then the method according to embodiment 1 adds to being detected to the AFP in serum, then into serum Enter the AFP of concentration known, determine again.Add to represent to pass through and standard AFP samples, discovery table are added in Standard entertion normal direction system Show after AFP additions, the electrochemical luminescence intensity level measured, according to working curve, the concentration value drawn.Concrete outcome is shown in Table 1, Wherein RSD is relative standard deviation.
Table 1
Application examples 2
Interference Detection：
Carried out according to the method for embodiment 1, except that adding interfering material into electrochemical luminescence detection architecture (HPO4 2-:5.32×106Pg/mL, Fe3+:3.04×105Pg/mL, Ni2+:2.91×105Pg/mL, glycine:1.42× 104Pg/mL, proline:4.32×103Pg/mL, cysteine:2.27×104Pg/mL, galactolipin:3.38×105Pg/mL, Glucose:3.38×107Pg/mL, urea:1.13×107Pg/mL, citric acid:3.60×107Pg/mL, bovine serum albumin： 6.23×106Pg/mL, ferritin:1.88×102Pg/mL, human prostate's antigen:1.88×102pg/mL).According to gained Electrochemical luminescence intensity level, draws block diagram, as a result sees Figure 10, wherein, each English in figure represents glycine respectively：Sweet ammonia Acid；Pro：Proline；Cys：Cysteine；galactose：Galactolipin；glucose：Glucose；urea：Urea；citric acid：Citric acid；BSA:Bovine serum albumin；SF:Ferritin；PSA：Human prostate's antigen；AFP:Alpha-fetoprotein.Can be with by figure Find out, various chaff interferences are on system without influence.The last item block diagram is standard AFP samples, it can be seen that electrochemical luminescence Intensity quenching effects are good.
According to above-mentioned detection example and application examples to the method in embodiment 2-8 or UCNPs-Ab therein1Couplet, GNRs-Ab2Couplet or OA-UCNPs are detected that the result of detection is consistent substantially with the testing result of embodiment 1.
The preferred embodiment of the present invention described in detail above, still, the present invention are not limited in above-mentioned embodiment Detail, in the range of the technology design of the present invention, a variety of simple variants can be carried out to technical scheme, this A little simple variants belong to protection scope of the present invention.
It is further to note that each particular technique feature described in above-mentioned embodiment, in not lance In the case of shield, can be combined by any suitable means, in order to avoid unnecessary repetition, the present invention to it is various can The combination of energy no longer separately illustrates.
In addition, various embodiments of the present invention can be combined randomly, as long as it is without prejudice to originally The thought of invention, it should equally be considered as content disclosed in this invention.

Claims (10)

1. a kind of UCNPs-Ab1Preparation method, it is characterised in that the preparation method includes：
1) alkali, water, oleic acid, C1-C3 alcohol, manganese source, ytterbium source, yttrium source, erbium source and NaF are stirred, then hydro-thermal reaction, from Isolated OA-UCNPs (the OA-NaYF of the heart4:Yb, Er/Mn UCNPs)；
2) OA-UCNPs is scattered in solvent, be subsequently added into PAA (polyacrylic acid) aqueous solution carry out ligand exchange reaction, from The isolated PAA-UCNPs of the heart (UCNPs of carboxyl modified)；
3) in the presence of EDC (carbodiimide) and NHS (n-hydroxysuccinimide), the PAA-UCNPs is scattered in MES Simultaneously oscillation incubation, separation and washing, is then dissolved in PBS cushioning liquid, adds anti-in cushioning liquid (ethyl sulfonic acid cushioning liquid) 1 two vibrations of body, are subsequently added three vibrations of BSA (bovine serum albumin(BSA)) with closing activity site, are obtained after centrifugation washing UCNPs-Ab1(NaYF4:The couplet of Yb, Er/Mn UCNPs- antibody 1).
2. preparation method according to claim 1, wherein, in step 1) in, the alkali relative to 0.3g, the consumption of the water For 5-15mL, the consumption of the oleic acid is 2-8mL, and the consumption of the alcohol is 5-15mL, and the consumption of the manganese source is 0.01- 0.1g, the consumption in the yttrium source is 0.1-0.3g, and the consumption in the ytterbium source is 0.05-0.11g, and the consumption in the erbium source is 5- 10mg, the NaF consumptions are 0.05-0.3g；
Preferably, in step 1) in, the stirring at least meets following condition：Whipping temp is 15-35 DEG C, and mixing time is 5- 20min；
It is highly preferred that in step 1) in, the haptoreaction at least meets following condition：Reaction temperature is 180-220 DEG C, reaction Time is 6-10h；
It is further preferred that the erbium source is in five nitric hydrate erbiums, erbium oxide, anhydrous erbium chloride and eight hydrated sulfuric acid erbiums At least one, the manganese source in four chloride hydrate manganese, anhydrous Manganese chloride, anhydrous manganous sulfate and Manganous sulfate monohydrate at least One, the ytterbium source is selected from least one of ytterbium chloride, five water ytterbium nitrates, ytterbium oxide and ytterbium carbonate, and the yttrium source is selected from nitre At least one of sour yttrium, yittrium oxide, six chloride hydrate yttriums and yttrium phosphate, the alcohol in methanol, ethanol and propyl alcohol extremely Few one.
3. preparation method according to claim 1 or 2, wherein, in step 2) in, the OA- relative to 30mg The consumption of polyacrylic acid is 0.1-0.3g in UCNPs, the PAA aqueous solution, and the volume of the PAA aqueous solution is 5-10mL, institute The capacity for stating solvent is 3-5mL；
Preferably, in step 2) in, the ligand exchange reaction at least meets following condition：Reaction temperature is 20-40 DEG C, instead It is 18-30h between seasonable；
It is highly preferred that in step 2) in, the solvent is selected from least one of chloroform, dichloromethane, hexamethylene and benzene；
More preferably, the weight average molecular weight of the polyacrylic acid is 1000-3000；
It is further preferred that in step 3) in, the PAA-UCNPs relative to 2.5mg, the consumption of the antibody 1 is 0.05- 0.2 μ g, BSA consumption is 1-5mg, and the consumption of the EDC is 1-5mg, and the consumption of the NHS is 1-5mg；
It is further preferred that in step 3) in, the PAA-UCNPs relative to 2.5mg, the MES cushioning liquid and PBS The consumption of cushioning liquid each stands alone as 1.5-3mL；Also, the pH of the MES cushioning liquid is each independently 6.4-8.4；
Still further preferably, in step 3) in, the oscillation incubation at least meets following condition：Vibration temperature is 15-35 DEG C, Duration of oscillation is 1.5-2.5h；The secondary vibration at least meets following condition：Vibration temperature is 15-35 DEG C, and duration of oscillation is 12-30h；Three vibrations at least meet following condition：Vibration temperature is 15-35 DEG C, and duration of oscillation is 0.5-3h；
More still further preferably, in step 3) in, the antibody 1 is trade mark 1A6 antibody.
4. a kind of UCNPs-Ab1, it is characterised in that the UCNPs-Ab1Pass through the system described in any one in claim 1-3 Preparation Method is prepared.
5. a kind of GNRs-Ab2Preparation method, it is characterised in that the preparation method includes：
1) by CTAB, Jin Yuan, NaBH4Carry out haptoreaction to obtain GNRs seed liquors with water；
2) will be by CTAB, Jin Yuan, ascorbic acid and AgNO3Carry out haptoreaction and obtain growth-promoting media；
3) the GNRs seed liquors and growth-promoting media are subjected to haptoreaction, are centrifugally separating to obtain CTAB-GNRs；
4) CTAB-GNRs is dissolved in the water formation CTAB-GNRs aqueous solution, then by MUDA- ethanol solutions and the CTAB-GNRs The aqueous solution carries out carboxyl modified processing, is then centrifuged for, handles that MUDA-GNRs (GNRs of carboxylated) is made；
5) in the presence of EDC and NHS, by the MUDA-GNRs be scattered in MES cushioning liquid and oscillation incubation, separation and Washing, is then dissolved in PBS cushioning liquid, adds 2 two vibrations of antibody, is subsequently added tri- vibrations of BSA with closing activity position Point, GNRs-Ab is obtained after centrifugation washing2(couplet of GNRs- antibody 2).
6. preparation method according to claim 5, wherein, in step 1) in, the CTAB relative to 0.36g, the Jin Yuan Consumption be 0.5-1.5mg, the NaBH4Consumption be 0.1-0.5mg, the consumption of the water is 9-12g；
Preferably, the Jin Yuan is gold chloride.
Preferably, in step 1) in, the haptoreaction at least meets following condition：Reaction temperature is 20-40 DEG C, reaction time For 1-5min；
It is highly preferred that in step 2) in, the CTAB relative to 0.36g, the consumption of the Jin Yuan is 1-3mg, the ascorbic acid Consumption be 0.5-1.5mg, the AgNO3Consumption be 0.033-0.048g, the consumption of the water is 9-12g；
It is further preferred that in step 2) in, the haptoreaction at least meets following condition：Reaction temperature is 20-40 DEG C, instead It is 1-5min between seasonable.
It is further preferred that in step 3) in, the growth-promoting media relative to 11mL, the consumption of the seed liquor is 5-20 μ L；
Still further preferably, in step 3) in, the haptoreaction at least meets following conditioned response temperature for 20-30 DEG C, Reaction time is 20-60min.
7. the preparation method according to claim 5 or 6, wherein, in step 4) in, the CTAB- relative to 2.4pmol MUDA consumption is 2-7mg in GNRs, the MUDA- ethanol solutions, and the consumption of the water is 5-10mL, the MUDA- ethanol The volume of solution is 500-1000 μ L；
Preferably, in step 4) in, the condition of the carboxyl modified processing is：The ultrasonically treated 1-3h at 20-60 DEG C；
It is highly preferred that in step 5) in, the MUDA-GNRs relative to 24pmol, the consumption of the antibody 2 is 0.05-0.2 μ g, the BSA consumption are 1-5mg, and the consumption of the EDC is 1-5mg, and the consumption of the NHS is 1-5mg；
It is highly preferred that in step 5) in, the MUDA-GNRs relative to 24pmol, the MES cushioning liquid is buffered with PBS The consumption of solution each stands alone as 1.5-3mL；Also, the pH of the MES cushioning liquid is each independently 6.4-8.4；
It is further preferred that in step 5) in, the oscillation incubation at least meets following condition：Vibration temperature is 15-35 DEG C, is shaken The time is swung for 1.5-2.5h；The secondary vibration at least meets following condition：Vibration temperature is 15-35 DEG C, and duration of oscillation is 12- 30h；Three vibrations at least meet following condition：Vibration temperature is 15-35 DEG C, and duration of oscillation is 0.5-3h；
It is further preferred that in step 5) in, the antibody 2 is trade mark 2A5 antibody.
8. a kind of GNRs-Ab2Preparation method, it is characterised in that the GNRs-Ab2Pass through any one in claim 5-7 Described preparation method is prepared.
9. a kind of detection method of alpha-fetoprotein, it is characterised in that the detection method includes：
1) by PBS cushioning liquid, the UCNPs-Ab1And GNRs-Ab2Mixed liquor is formed, then adds the mixed liquor Know that the AFP solution of concentration carries out being reacted to give sample solution, the sample solution is then added into the PBS containing potassium peroxydisulfate Electrochemical luminescence energy transfer reaction is carried out in cushioning liquid and electrochemical luminescence intensity is detected, finally with the concentration of AFP solution For abscissa, electrochemical luminescence intensity is ordinate drawing curve or evaluation work curvilinear equation；
2) by AFP solution to be checked according to step 1) method measure electrochemical luminescence intensity, then according to the working curve or Person's working curve equation calculates the concentration of AFP solution to be checked；
Preferably, relative to the 6.54 μ g UCNPs-Ab1, the consumption of the couplet of GNRs- antibody 2 is：5×10-3- 1.0×10-3Pmol, the consumption of detection sample is 10-30 μ L and the middle AFP of detection sample concentration is 2-5000ng/mL；
It is highly preferred that relative to the 20-50 μ L sample solution, the consumption of the PBS cushioning liquid containing potassium peroxydisulfate For 3-8mL, the concentration of the potassium peroxydisulfate is 0.05-0.2mol/L；
It is further preferred that the electrochemical luminescence energy transfer reaction at least meets following condition：Reaction response temperature 20-30 DEG C, the reaction time is 100-120min；
It is further preferred that the working curve equation is 2.88 × 103lgx-2.17×103, wherein, y is electrochemical luminescence Intensity quenching value, x is AFP concentration；
Still further preferably, the pH of the PBS cushioning liquid is 6.4-8.4.
10. detection method according to claim 9, wherein, the AFP solution to be checked is human serum；
Preferably, in step 2) before, the detection method also includes：Blood of human body is centrifuged, takes upper strata to obtain after purification The human serum.
CN201710176515.0A 2017-03-23 2017-03-23 UCNPs‑Ab1With GNRs Ab2And preparation method thereof, the detection method of alpha-fetoprotein Pending CN106986936A (en)

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