CN105891465A - Method for diagnosis of circulating antigen of schistosomiasis japonica - Google Patents
Method for diagnosis of circulating antigen of schistosomiasis japonica Download PDFInfo
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- CN105891465A CN105891465A CN201410628016.7A CN201410628016A CN105891465A CN 105891465 A CN105891465 A CN 105891465A CN 201410628016 A CN201410628016 A CN 201410628016A CN 105891465 A CN105891465 A CN 105891465A
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Abstract
The invention relates to two fields of biomedicine and nanomaterials, and in particular, relates to preparation of a gold nanorod sensor and construction of a method for detection of a circulating antigen of schistosomiasis japonica (Chinese mainland strain). A solid carrier-ITO slide is combined with gold nanorods and then is combined with a schistosomiasis japonica 26-28Kd single-chain antibody to construct the detection sensor, and the detection sensor is used for diagnosis of the circulating antigen of schistosomiasis japonica, and change of extinction peaks is observed by a UV scanning sensor to determine whether schistosoma japonicum infection exists or not.
Description
Technical field
The present invention relates to biomedical and nanomaterial science two aspect content, be specifically related to the preparation of gold nanorods sensor and to Schistosoma japonicum
The structure of (Schistosomiasis Japonica) (Chinese mainland strain) circulating antigen detection method.Its invention uses solid phase carrier-ITO slide and gold
Nanometer rods combines, then builds detection sensor by connection for Schistosoma japonicum 26-28Kd single-chain antibody, and carries out Detecting Schistosome Circulating Antigen
Diagnosis, whether the change being observed its delustring peak by UV scanning sensor determines schistosoma japonicum infection.
Background technology
Schistosomicide (Schistosomiasis) is always the public health difficult problem that the Chinese government faces.Along with the strong control to epidemic situation, Endemic Area is felt
Dye rate is gradually reduced, and gradient of infection substantially reduces.Meanwhile, also sensitivity to schistosomicide low-grade infection diagnosing patient method proposes higher wanting
Ask.Circulating antigen of schistosome (CAg, circulating antigen) can act also as worm alive post as the important symbol of schistosomicide Current Infection
Raw mark and the performance assessment criteria of patient effect, due to SjCAg in blood or body fluid presented in denier, only relatively
Smart detector system can detect.
Solid phase gold nanorods sensor is can to form surface plasma effect at the excellent major axis of gold nanorods with collective's concussion of the longitudinal axis based on free electron
Should, the optical characteristics of this effect shows as the change of nanometer rods draw ratio, may result in surface plasmon resonance (LSPR, Localized
Surface plasmon resonance) the notable change at delustring peak.
Summary of the invention
A kind of method that it is an object of the invention to provide diagnosing Japanese circulating antigen of schistosome.Specifically include procedure below:
1. the preparation of gold nanorods: use seed mediated growth method, prepare gold nanorods.
The pretreatment of 2.ITO slide: first ITO slide distilled water is cleaned up, be then placed in the Piranha washing liquid boiled (formula volume ratio is:
Concentrated sulphuric acid: hydrogen peroxide=3: 1), after cooling down completely, take out, clean, dry up.
3.ITO slide hydroxylating: ITO slide good for pretreatment is put in the Piranha washing liquid cooled down (formula volume ratio is: concentrated sulphuric acid: hydrogen peroxide
=3: 1), then heating in water bath is to 65-75 DEG C, 40-50 minute, after cooling down completely, takes out, cleans, dry up.
4. the most hydroxylated ITO slide is immersed in PSS and PAH solution respectively, 25-30 DEG C, 30-40 minute, takes out, clean, dry up.
5. being immersed in having modified ITO slide in 4 in the gold nanorods solution prepared, 25-30 DEG C is soaked 15-19 hour, takes out, and cleans, dries up.
6. being put into by the ITO slide having combined gold nanorods in 5 in ScFv (Schistosoma japonicum 26-28KD single-chain antibody) solution, place 4 DEG C, 7 is little
Time, take out, clean, dry up.
7. the solid phase sensor built in 6 is immersed in respectively in positive rabbit anteserum and the negative rabbit anteserum of schistosomicide, 4 DEG C, places 30 minutes
-4 hours, take out, clean, dry up.
8. the solid phase sensor with seroreaction is placed in its extinction peaks of UV spectrophotometer measuring.
Circulating antigen of schistosome content in blood is extremely low, and traditional detection method is difficult to detect it, and present invention solid phase based on gold nanorods passes
Sensor, the reaction of the circulating antigen of trace with single-chain antibody can be spread out of by it by optical signalling, by observing the change of its extinction peaks, as with the moon
During property seroreaction, its extinction peaks is not changed in；And when reacting with positive serum, extinction peaks has the red shift of 14nm and 40nm, it was demonstrated that this
Sensor can be sensitive detection distinguish schistosomicide positive and negative serum.
Fig. 1 is the UV scanning figure of gold nanorods of the present invention；
Fig. 2 is gold nanorods electron-microscope scanning figure；
Fig. 3 is the UV scanning figure that gold nanorods is combined with slide A；
Fig. 4 is the UV scanning figure that sensors A assembles with ScFv；
Fig. 5 is the UV scanning figure that slide A is combined with positive serum 30 minutes；
Fig. 6 is the UV scanning figure that slide A is combined with positive serum 2 hours；
Fig. 7 is the UV scanning figure that gold nanorods is combined with slide B；
Fig. 8 is the UV scanning figure that sensor B Yu ScFv assembles；
Fig. 9 is the UV scanning figure that slide B is combined with negative serum 30 minutes；
Figure 10 is the UV scanning figure that slide B is combined with negative serum 2 hours；
Figure 11. gold nanorods and slide assemble the UV scanning figure of sensor C；
The UV scanning figure that Figure 12 sensor C Yu ScFv assembles；
Figure 13. sensor B is combined the UV scanning figure of 30 minutes with negative serum；
Figure 14. sensor C is combined the UV scanning figure of 4 hours with negative serum.
Detailed description of the invention
Embodiment 1
1. the preparation of gold nanorods: using seed mediated growth method, preparation draw ratio is the gold nanorods of 3.2.As shown in Figure 1 and Figure 2
The pretreatment of 2.ITO slide: first ITO slide distilled water is cleaned up, be then placed in the Piranha washing liquid boiled (formula volume ratio is:
Concentrated sulphuric acid: hydrogen peroxide=3: 1), after cooling down completely, take out, clean, dry up.
3.ITO slide hydroxylating: ITO slide good for pretreatment is put in the Piranha washing liquid cooled down (formula volume ratio is: concentrated sulphuric acid: hydrogen peroxide
=3: 1), then heating in water bath is to 65 DEG C, after cooling down completely, takes out, cleans, dry up.
4. the most hydroxylated ITO slide is immersed in PSS and PAH solution respectively, 25 DEG C, 30 minutes, takes out, clean, dry up.
5. being immersed in having modified ITO slide in 4 in the gold nanorods solution prepared, 25 DEG C are soaked 15 hours, take out, and clean, dry up.Warp
UV spectrophotometer measuring, its extinction peaks is respectively 689nm, as shown in Figure 3.
6. being put into by the ITO slide having combined gold nanorods in 5 in ScFv (Schistosoma japonicum 26-28KD single-chain antibody) solution, place 4 DEG C, 7 is little
Time, take out, clean, dry up, by UV spectrophotometer measuring, its extinction peaks is respectively 713nm.As shown in Figure 4.
7. the solid phase sensor built in 6 is immersed in the positive rabbit anteserum of schistosomicide respectively, 4 DEG C, places 30 minutes and 2 hours respectively,
Take out, clean, dry up.
8. the solid phase sensor with seroreaction being placed in UV spectrophotometer measuring, its extinction peaks is respectively 727nm, 753nm such as Fig. 5, such as Fig. 6
Shown in.
Embodiment 2
1. the preparation of gold nanorods: using seed mediated growth method, preparation draw ratio is the gold nanorods of 3.2.As shown in Figure 1 and Figure 2
The pretreatment of 2.ITO slide: first ITO slide distilled water is cleaned up, be then placed in the Piranha washing liquid boiled (formula volume ratio is:
Concentrated sulphuric acid: hydrogen peroxide=3: 1) after cooling down completely, take out, clean, dry up.
3.ITO slide hydroxylating: ITO slide good for pretreatment is put in the Piranha washing liquid cooled down (formula volume ratio is: concentrated sulphuric acid: hydrogen peroxide
=3: 1), then heating in water bath is to 70 DEG C, after cooling down completely, takes out, cleans, dry up.
4. the most hydroxylated ITO slide is immersed in PSS and PAH solution respectively, 28 DEG C, 30 minutes, takes out, clean, dry up.
5. being immersed in having modified ITO slide in 4 in the gold nanorods solution prepared, 28 DEG C are soaked 17 hours, take out, and clean, dry up.Warp
UV spectrophotometer measuring, its extinction peaks be 683nm. as shown in Figure 7.
6. being put into by the ITO slide having combined gold nanorods in 5 in ScFv (Schistosoma japonicum 26-28KD single-chain antibody) solution, place 4 DEG C, 7 is little
Time, take out, clean, dry up, by UV spectrophotometer measuring, its extinction peaks is 712nm, as shown in Figure 8.
7. the solid phase sensor built in 6 is immersed in respectively the negative rabbit anteserum of schistosomicide, 4 DEG C, places 30 minutes and 2 hours respectively,
Take out, clean, dry up.
8. the solid phase sensor with seroreaction is placed in UV spectrophotometer measuring, its extinction peaks be 723nm, 723nm. as shown in Figure 9, Figure 10.
Embodiment 3
1. the preparation of gold nanorods: using seed mediated growth method, preparation draw ratio is the gold nanorods of 3.2.As shown in Figure 1 and Figure 2
The pretreatment of 2.ITO slide: first ITO slide distilled water is cleaned up, be then placed in the Piranha washing liquid boiled (formula is: concentrated sulphuric acid:
Hydrogen peroxide=3: 1) after cooling down completely, take out, clean, dry up.
3.ITO slide hydroxylating: ITO slide good for pretreatment is put into (formula is: concentrated sulphuric acid: hydrogen peroxide=3: 1) in the Piranha washing liquid cooled down,
Then heating in water bath is to 75 DEG C, after cooling down completely, takes out, cleans, dry up.
4. the most hydroxylated ITO slide is immersed in PSS and PAH solution respectively, 30 DEG C, 30 minutes, takes out, clean, dry up.
5. being immersed in having modified ITO slide in 4 in the gold nanorods solution prepared, 25 DEG C are soaked 19 hours, take out, and clean, dry up.Warp
UV spectrophotometer measuring, its extinction peaks is 660nm.As shown in figure 11.
6. being put into by the ITO slide having combined gold nanorods in 5 in ScFv (Schistosoma japonicum 26-28KD single-chain antibody) solution, place 4 DEG C, 7 is little
Time, take out, clean, dry up, by UV spectrophotometer measuring, its extinction peaks is respectively 724nm.As shown in figure 12
7. the solid phase sensor built in 6 is immersed in the negative rabbit anteserum of schistosomicide respectively, 4 DEG C, places 30 minutes, 2 hours, take
Go out, clean, dry up.
8. the solid phase sensor with seroreaction being placed in UV spectrophotometer measuring, its extinction peaks is respectively 723nm, 723nm.Figure 13, Figure 14
Shown in.
Claims (1)
1. the method for a diagnosing Japanese circulating antigen of schistosome, it is characterised in that comprise the following steps:
A.. the preparation of gold nanorods: use seed mediated growth method, prepare gold nanorods；
The pretreatment of B.ITO slide: first cleaned up by ITO slide distilled water, is then placed in the Piranha washing liquid boiled, and formula volume ratio is:
Concentrated sulphuric acid: hydrogen peroxide=3: 1, after cooling down completely, takes out, cleans, dry up；
C.ITO slide hydroxylating: being put in the Piranha washing liquid cooled down by ITO slide good for pretreatment, then heating in water bath is to 65-75 DEG C, 40-50
Minute, after cooling down completely, take out, clean, dry up；
D. the most hydroxylated ITO slide is immersed in respectively in kayexalate (PSS) and polyallylamine hydrochloride (PAH) solution, 25-30 DEG C,
30-40 minute, take out, clean, dry up；
E. being immersed in having modified ITO slide in D in the gold nanorods solution prepared, 25-30 DEG C is soaked 15-19 hour, takes out, and cleans, blows
Dry；
F. putting in ScFv solution by the ITO slide having combined gold nanorods in E, i.e. Schistosoma japonicum 26-28KD single-chain antibody, place 4 DEG C, 7 is little
Time, take out, clean, dry up；
G. the solid phase sensor built in F is immersed in respectively in positive rabbit anteserum and the negative rabbit anteserum of schistosomicide, 4 DEG C, places 30 minutes
-4 hours, take out, clean, dry up；
H. the solid phase sensor with seroreaction is placed in its extinction peaks of UV spectrophotometer measuring；
Finally, the existence of Detecting Schistosome Circulating Antigen is detected whether by observing the change of its extinction value.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110579612A (en) * | 2019-10-17 | 2019-12-17 | 桂林理工大学 | Surface plasma resonance immunoassay method for embedded Au nano-film electrode |
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| CN101158686A (en) * | 2007-11-07 | 2008-04-09 | 湖南大学 | Electrochemical sensor used for detecting blood fluke, preparation method and applications |
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| US7610074B2 (en) * | 2004-01-08 | 2009-10-27 | The Board Of Trustees Of The University Of Illinois | Multi-functional plasmon-resonant contrast agents for optical coherence tomography |
| CN103409517A (en) * | 2013-07-24 | 2013-11-27 | 福建省亚明食品有限公司 | Biosensing chip capable of quickly detecting EHEC (Enterohemorrhagic Escherichia coli) O157:H7 |
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2014
- 2014-11-11 CN CN201410628016.7A patent/CN105891465A/en active Pending
Patent Citations (5)
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| US7610074B2 (en) * | 2004-01-08 | 2009-10-27 | The Board Of Trustees Of The University Of Illinois | Multi-functional plasmon-resonant contrast agents for optical coherence tomography |
| CN1811444A (en) * | 2006-01-25 | 2006-08-02 | 汪世平 | Production and application for schistosoma japonica piezoelectric quality immune sensor |
| CN101429247A (en) * | 2007-11-05 | 2009-05-13 | 何卓 | Oriental schistosomiasis resistant natural numerator vaccine specific single-chain antibody |
| CN101158686A (en) * | 2007-11-07 | 2008-04-09 | 湖南大学 | Electrochemical sensor used for detecting blood fluke, preparation method and applications |
| CN103409517A (en) * | 2013-07-24 | 2013-11-27 | 福建省亚明食品有限公司 | Biosensing chip capable of quickly detecting EHEC (Enterohemorrhagic Escherichia coli) O157:H7 |
Non-Patent Citations (3)
| Title |
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| HAOWEN HUANG等: "Sensitive and simultaneous detection of different disease markers using multiplexed gold nanorods", 《ANALYTICA CHIMICA ACTA》 * |
| 孙恩杰等: "《纳米生物学》", 30 September 2010 * |
| 黄劭文: "基于金纳米棒局域表面等离子体共振构建生物传感器的研究", 《中国优秀硕士学位论文全文数据库工程科技I辑》 * |
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| CN110579612A (en) * | 2019-10-17 | 2019-12-17 | 桂林理工大学 | Surface plasma resonance immunoassay method for embedded Au nano-film electrode |
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Inventor after: Zhou Yunfei Inventor after: Wang Shiping Inventor after: He Xin Inventor before: He Xin Inventor before: Wang Shiping Inventor before: Zhou Yunfei |
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Application publication date: 20160824 |