CN1226614C - Nano immunological biosensor - Google Patents

Nano immunological biosensor Download PDF

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Publication number
CN1226614C
CN1226614C CN 03127127 CN03127127A CN1226614C CN 1226614 C CN1226614 C CN 1226614C CN 03127127 CN03127127 CN 03127127 CN 03127127 A CN03127127 A CN 03127127A CN 1226614 C CN1226614 C CN 1226614C
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electrode
gold
antibody
gold nanocrystals
phosphate buffer
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CN 03127127
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CN1523346A (en
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李景虹
王美佳
孙春燕
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Changchun Institute of Applied Chemistry of CAS
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Changchun Institute of Applied Chemistry of CAS
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Abstract

In the present invention, a sulfhydryl compound, such as p-aminothiophenol, mercaptoethylamine, etc., is chemically modified on the surface of a gold electrode. The characteristic that a sulfhydryl end of a sulfhydryl compound can be combined with a gold electrode in the mode of a chemical bond is used; consequently, the sulfhydryl end is fixed on the gold electrode, and an amino group at the other end of the sulfhydryl compound is exposed on the electrode surface. Under certain conditions, the exposed amino group can be combined with gold nanometer crystal by a chemical bond, and consequently, the gold nanometer crystal is fixed on the surface of the gold electrode. The surface of the gold nanometer crystal has charges so that strong adsorption action on antibodies is generated in the neutral or physiology pH environment; consequently, the antibodies are firmly combined on the surface of the gold nanometer crystal, which means that the antibodies are fixed on the surface of the gold electrode. As a result, a nanometer immunity biosensor is prepared.

Description

The preparation method of nano immune biology sensor
Technical field
The present invention relates to the preparation method of nano immune biology sensor.
Background technology
The interaction that utilizes chemical method to detect antigen and antibody is the developing direction of present many researchs.Highly sensitive sensing technology and specific immune response are combined, be called immunosensor, be applied to aspects such as medical science diagnosis and treatment, environmental pollution monitoring and food analysis in order to the biology sensor of monitoring antigen and antibody response.The principle of work of immunosensor is similar with traditional immunity test, all belongs to the solid-phase immunity method of testing, promptly antigen or antibody is fixed on the solid support surface and comes antibody or antigen in the test sample.In nearly 15 years of immunodiagnosis field development, people have been placed on the binding kinetics aspect that antibody is fixed on the solid support surface to more interest.Biological sample is fixed on electrode surface, and the interaction of biology is converted into the purpose that electric signal reaches the detection of biological activity.How structure biology sensor, immunosensor, bioelectronics equipment there is extremely important actual application value at the electrode surface immobilizing biological samples.And the life-span of biology sensor often is decided by the situation of combination between antibody and the transducer.In the work in the past, (Richad, C. such as Richad; Korri-Youssoufi, H.; Yassar, A.Synt.Met., 2001,121,1261.) synthesized a kind of conjugated polymers body, as the connector of antibody and transducer, this conjugated polymers body can also carry out directly chemistry replacement, for example can be connected with ferrocene, crown ether, DNA group etc. by substitution reaction.Ben-Dov etc. (Ben-Dov, I., Willner, I., Zisman, E.Anal.Chem.1997,69,3506) adopt the maleimide monofilm to fix antibody, but this sensor is disposable, can not reuse.
Summary of the invention
The objective of the invention is to propose a kind of preparation method of nano immune biology sensor.
The present invention is at gold electrode surfaces chemical modification sulfhydryl compound, as mercaptoaniline, mercaptoethylmaine etc.Utilize sulfhydryl compound the sulfydryl end can with the mode combination of gold electrode with chemical bond, the sulfydryl end is fixed on the gold electrode and the amino of the other end is exposed at electrode surface.Under certain condition Luo Lu amino can with gold nanocrystals with the chemical bond combination, thereby gold nanocrystals is fixed on gold electrode surfaces.The surface of gold nanocrystals has electric charge, produces strong suction-operated with antibody under the environment of neutrality or physiology pH, makes the surface of antibody strong bonded at gold nanocrystals, so just makes antibody be fixed on gold electrode surfaces, makes the nano immune biology sensor.
The exempt from service preparation of biology sensor of nanometer comprises following several steps:
One gold nanocrystals is fixing gold electrode surfaces:
1) gold electrode of handling cleaning well immerses and to contain in the solution of sulfhydryl compound, at room temperature soaks 1~10 hour, takes out the back and cleans up and dry up with high pure nitrogen with a large amount of high purity waters;
2) to put into the pH value be 3~10 gold nanocrystals solution to electrode, in the dark assembled 1~20 hour, takes out the back and cleans up with high purity water;
Two antibody fixing on the gold nanocrystals modified electrode:
3) electrode that will modify gold nanocrystals is put into the phosphate buffer solution that 0.5ml contains 0.1mg/ml antibody, and pH is 6~9, respectively 4 ℃ and 37 ℃ of following soaked overnight;
4) electrode with soaked overnight takes out the phosphate buffer solution of back with 0.02M, pH is 7.4, wash off combine stable inadequately antibody with gold nanocrystals after, bovine serum albumin(BSA) (BSA) effect 60 minutes of putting into 0.5mg/ml, and be that 7.4 phosphate buffer solution washes with 0.02MpH.
Three immune detection:
Electrode is put into the antigen of variable concentrations, and 37 ℃ of effects 30 minutes down, taking out the back is that 7.4 phosphate buffer solution washes with 0.02M pH, detects with electrochemical impedance spectroscopy.The result shows that this method has good sensitivity, and when detecting hepatitis B virus surface antigen, the concentration range of linearity is 0.5~200 μ g/1, detects and is limited to 50ng/1.With other method mutually specific sensitivity improve a lot.
The nano immune biology sensor preparation method of the present invention's preparation is simple, utilizes the gold nanocrystals sessile antibody, has kept the activity of antibody to the full extent.And this sensor can reuse, and is the product that nanometer technology combines with immunological technique.Utilize electrochemical method to detect simultaneously, can obtain very high sensitivity.
Description of drawings
Fig. 1 adopts the 4-mercaptoaniline that gold nanocrystals is fixed on the gold electrode surfaces synoptic diagram.The surface of visible gold nanocrystals has electric charge among the figure, can produce strong suction-operated with antibody under the environment of neutrality or physiology pH, makes the surface of antibody strong bonded at gold nanocrystals, so just makes antibody be fixed on gold electrode surfaces.Detect the antigen of variable concentrations with the gold electrode of sessile antibody.
Accompanying drawing 2 is the curve maps with the hepatitis B virus surface antigen of sensor variable concentrations of the present invention, detect with AC impedance, the result shows that this method has good sensitivity, and the concentration range of linearity that detects hepatitis B virus surface antigen is 0.5~200 μ g/l, detects and is limited to 50ng/1.
Embodiment
Embodiment 1:
One gold nanocrystals is fixing gold electrode surfaces:
1) gold electrode that will handle cleaning well immerses in the 4-mercaptoaniline solution of 0.1M, at room temperature soaks 1 hour, takes out the back and cleans with absolute ethyl alcohol, and physisorption is cleaned up at the 4-of electrode surface mercaptoaniline.Clean up and dry up with a large amount of high purity waters at last with high pure nitrogen.
2) to put into the pH value be 3 gold nanocrystals solution to electrode, in the dark assembled 1 hour, takes out the back and cleans up with high purity water.
Two antibody fixing on the gold nanocrystals modified electrode:
1) electrode that will modify gold nanocrystals is put into the phosphate buffer solution that 0.5ml contains 0.1mg/ml antibody, and pH is 6.And after miniature vortex mixed instrument fully mixes 20 minutes, 4 ℃ of following soaked overnight.
2) taking out the back is that 7.4 phosphate buffer solution is washed off with gold nanocrystals and combine stable inadequately antibody with 0.02M pH, and the bovine serum albumin(BSA) (BSA) of putting into 0.5mg/ml acts on 60 minutes, and is that 7.4 phosphate buffer solution washes with 0.02M pH.
Three immune detection:
Electrode is put into the antigen of variable concentrations, and 37 ℃ of effects 30 minutes down, taking out the back is that 7.4 phosphate buffer solution washes with 0.02M pH.Detect with electrochemical impedance spectroscopy.The concentration range of linearity that detects hepatitis B virus surface antigen is 0.5~2 μ g/1.The result shows that antibody activity is affected, and antibody seldom is fixed.
Embodiment 2:
One gold nanocrystals is fixing gold electrode surfaces:
1) gold electrode that will handle cleaning well immerses in the 4-mercaptoaniline solution of 0.1M, at room temperature soaks 4 hours, takes out the back and cleans with absolute ethyl alcohol, and physisorption is cleaned up at the 4-of electrode surface mercaptoaniline.Clean up and dry up with a large amount of high purity waters at last with high pure nitrogen.
2) electrode being put into the pH value is 10 gold nanocrystals solution, in the dark assembles 5 hours, takes out the back and cleans up with high purity water.
Two antibody fixing on the gold nanocrystals modified electrode:
1) electrode that will modify gold nanocrystals is put into the phosphate buffer solution that 0.5ml contains 0.1mg/ml antibody, and pH is 6.And after miniature vortex mixed instrument fully mixes 20 minutes, 37 ℃ of following soaked overnight.
2) taking out the back is that 7.4 phosphate buffer solution is washed off with gold nanocrystals and combine stable inadequately antibody with 0.02M pH, and the bovine serum albumin(BSA) (BSA) of putting into 0.5mg/ml acts on 60 minutes, and is that 7.4 phosphate buffer solution washes with 0.02M pH.
Three immune detection:
Electrode is put into the antigen of variable concentrations, and 37 ℃ of effects 30 minutes down, taking out the back is that 7.4 phosphate buffer solution washes with 0.02M pH.Detect with electrochemical impedance spectroscopy.The concentration range of linearity that detects hepatitis B virus surface antigen is 0.5~1 μ g/1.
Embodiment 3:
One gold nanocrystals is fixing gold electrode surfaces:
1) gold electrode of handling cleaning well immerses in the 4-mercaptoaniline solution of 0.1M, at room temperature soaks 6 hours, takes out the back and cleans with absolute ethyl alcohol, and physisorption is cleaned up at the 4-of electrode surface mercaptoaniline.Clean up and dry up with a large amount of high purity waters at last with high pure nitrogen.
2) electrode being put into the pH value is 8 gold nanocrystals solution, in the dark assembles 10 hours, takes out the back and cleans up with high purity water.
Two antibody fixing on the gold nanocrystals modified electrode:
1) electrode of having modified gold nanocrystals is put into the phosphate buffer solution that 0.5ml contains 0.1mg/ml antibody, and pH is 7.4.And after miniature vortex mixed instrument fully mixes 20 minutes, 37 ℃ of following soaked overnight.
2) taking out the back is that 7.4 phosphate buffer solution is washed off with gold nanocrystals and combine stable inadequately antibody with 0.02M pH, and the bovine serum albumin(BSA) (BSA) of putting into 0.5mg/ml acts on 60 minutes, and is that 7.4 phosphate buffer solution washes with 0.02M pH.
Three immune detection:
Electrode is put into the antigen of variable concentrations, and 37 ℃ of effects 30 minutes down, taking out the back is that 7.4 phosphate buffer solution washes with 0.02M pH.Detect with electrochemical impedance spectroscopy.The concentration range of linearity that detects hepatitis B virus surface antigen is 1~5 μ g/l.
Embodiment 4:
One gold nanocrystals is fixing gold electrode surfaces:
1) gold electrode of the good cleaning of reason immerses in the 4-mercaptoaniline solution of 0.1M, at room temperature soaks 4 hours, takes out the back and cleans with absolute ethyl alcohol, and physisorption is cleaned up at the 4-of electrode surface mercaptoaniline.Clean up and dry up with a large amount of high purity waters at last with high pure nitrogen.
2) electrode being put into the pH value is 3.5 gold nanocrystals solution, in the dark assembles 5 hours, takes out the back and cleans up with high purity water.
Two antibody fixing on the gold nanocrystals modified electrode:
1) electrode that will modify gold nanocrystals is put into the phosphate buffer solution that 0.5ml contains 0.1mg/ml antibody, and pH is 8.And after miniature vortex mixed instrument fully mixes 20 minutes, 4 ℃ of following soaked overnight.
2) taking out the back is that 7.4 phosphate buffer solution is washed off with gold nanocrystals and combine stable inadequately antibody with 0.02M pH, and the bovine serum albumin(BSA) (BSA) of putting into 0.5mg/ml acts on 60 minutes, and is that 7.4 phosphate buffer solution washes with 0.02M pH.
Three immune detection:
Electrode is put into the antigen of variable concentrations, and 37 ℃ of effects 30 minutes down, taking out the back is that 7.4 phosphate buffer solution washes with 0.02M pH.Detect with electrochemical impedance spectroscopy.The concentration range of linearity that detects hepatitis B virus surface antigen is 1~20 μ g/1.The result shows that the antibody fixed amount is little.
Embodiment 5:
One gold nanocrystals is fixing gold electrode surfaces:
1) gold electrode of handling cleaning well immerses in the 4-mercaptoaniline solution of 0.1M, at room temperature soaks 10 hours, takes out the back and cleans with absolute ethyl alcohol, and physisorption is cleaned up at the 4-of electrode surface mercaptoaniline.Clean up and dry up with a large amount of high purity waters at last with high pure nitrogen.
2) electrode being put into the pH value is 3.5 gold nanocrystals solution, in the dark assembles 5 hours, takes out the back and cleans up with high purity water.
Two antibody fixing on the gold nanocrystals modified electrode:
1) electrode that will modify gold nanocrystals is put into the phosphate buffer solution that 0.5ml contains 0.1mg/ml antibody, and pH is 9.And after miniature vortex mixed instrument fully mixes 20 minutes, 37 ℃ of following soaked overnight.
2) taking out the back is that 7.4 phosphate buffer solution is washed off with gold nanocrystals and combine stable inadequately antibody with 0.02M pH, and the bovine serum albumin(BSA) (BSA) of putting into 0.5mg/ml acts on 60 minutes, and is that 7.4 phosphate buffer solution washes with 0.02M pH.
Three immune detection:
Electrode is put into the antigen of variable concentrations, and 37 ℃ of effects 30 minutes down, taking out the back is that 7.4 phosphate buffer solution washes with 0.02M pH.Detect with electrochemical impedance spectroscopy.The concentration range of linearity that detects hepatitis B virus surface antigen is 1~10 μ g/l, and the result shows the antibody fixed amount seldom
Embodiment 6:
One gold nanocrystals is fixing gold electrode surfaces:
1) gold electrode that will handle cleaning well immerses in the 4-mercaptoaniline solution of 0.1M, at room temperature soaks 4 hours, takes out the back and cleans with absolute ethyl alcohol, and physisorption is cleaned up at the 4-of electrode surface mercaptoaniline.Clean up and dry up with a large amount of high purity waters at last with high pure nitrogen.
2) electrode being put into the pH value is 3.5 gold nanocrystals solution, in the dark assembles 5 hours, takes out the back and cleans up with high purity water.
Two antibody fixing on the gold nanocrystals modified electrode:
1) electrode of having modified gold nanocrystals is put into the phosphate buffer solution that 0.5ml contains 0.1mg/ml antibody, and pH is 7.And after miniature vortex mixed instrument fully mixes 20 minutes, 4 ℃ of following soaked overnight.
2) taking out the back is that 7.4 phosphate buffer solution is washed off with gold nanocrystals and combine stable inadequately antibody with 0.02M pH, and the bovine serum albumin(BSA) (BSA) of putting into 0.5mg/ml acts on 60 minutes, and is that 7.4 phosphate buffer solution washes with 0.02M pH.
Three immune detection:
Electrode is put into the antigen of variable concentrations, and 37 ℃ of effects 30 minutes down, taking out the back is that 7.4 phosphate buffer solution washes with 0.02M pH.Detect with electrochemical impedance spectroscopy.The concentration range of linearity that detects hepatitis B virus surface antigen is 0.5~100 μ g/l, detects to be limited to 100ng/l.
Embodiment 7:
One gold nanocrystals is fixing gold electrode surfaces:
1) gold electrode of handling cleaning well immerses in the 4-mercaptoaniline solution of 0.1M, at room temperature soaks 4 hours, takes out the back and cleans with absolute ethyl alcohol, and physisorption is cleaned up at the 4-of electrode surface mercaptoaniline.Clean up and dry up with a large amount of high purity waters at last with high pure nitrogen.
2) electrode being put into the pH value is 3.5 gold nanocrystals solution, in the dark assembles 5 hours, takes out the back and cleans up with high purity water.
Two antibody fixing on the gold nanocrystals modified electrode:
1) electrode of having adornd gold nanocrystals is put into the phosphate buffer solution that 0.5ml contains 0.1mg/ml antibody, and pH is 7.4.And after miniature vortex mixed instrument fully mixes 20 minutes, 4 ℃ of following soaked overnight.
2) taking out the back is that 7.4 phosphate buffer solution is washed off with gold nanocrystals and combine stable inadequately antibody with 0.02M pH, and the bovine serum albumin(BSA) (BSA) of putting into 0.5mg/ml acts on 60 minutes, and is that 7.4 phosphate buffer solution washes with 0.02M pH.
Three immune detection:
Electrode is put into the antigen of variable concentrations, and 37 ℃ of effects 30 minutes down, taking out the back is that 7.4 phosphate buffer solution washes with 0.02M pH.Detect with electrochemical impedance spectroscopy.The result shows that this method has good sensitivity, and the concentration range of linearity that detects hepatitis B virus surface antigen is 0.5~200 μ g/l, detects and is limited to 50ng/l.

Claims (2)

1. the preparation method of a nano immune biology sensor comprises following several steps: a gold nanocrystals is fixing gold electrode surfaces:
1) gold electrode that will handle cleaning well immerses and to contain in the solution of sulfhydryl compound, at room temperature soaks 1~10 hour, takes out the back and cleans up and dry up with high pure nitrogen with a large amount of high purity waters;
2) electrode being put into the pH value is 3~10 gold nanocrystals solution, in the dark assembles 1~20 hour, takes out the back and cleans up with high purity water;
Two antibody fixing on the gold nanocrystals modified electrode:
1) electrode that will modify gold nanocrystals is put into the phosphate buffer solution that 0.5ml contains 0.1mg/ml antibody, and pH is 6~9, respectively 4 ℃ and 37 ℃ of following soaked overnight;
2) electrode with soaked overnight takes out the phosphate buffer solution of back with 0.02M, pH is 7.4, wash off combine stable inadequately antibody with gold nanocrystals after, the bovine serum albumin(BSA) effect 60 minutes of putting into 0.5mg/ml, and be 7.4 phosphate buffer solution flushing with 0.02M pH.
2. the preparation method of nano immune biology sensor according to claim 1 is characterized in that described sulfhydryl compound is mercaptoaniline or mercaptoethylmaine.
CN 03127127 2003-09-03 2003-09-03 Nano immunological biosensor Expired - Fee Related CN1226614C (en)

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CN101339154B (en) * 2007-07-05 2013-03-27 五鼎生物技术股份有限公司 Composite modified electrode test piece
CN101666804B (en) * 2009-09-24 2013-03-27 浙江大学 Immunosensor for detecting haptoglobin content in milk and detection method
CN101957375B (en) * 2010-10-15 2013-03-20 扬州大学 Preparation method of label-free impedance type immunosensor for furaltadone residues and application thereof
CN103207218B (en) * 2012-01-11 2015-07-22 西南大学 Electrochemical immunosensor making method and Streptococcus suis detection method using electrochemical immunosensor
CN103616413A (en) * 2013-12-12 2014-03-05 苏州大学 Gas sensor based on reduction-oxidation graphene and preparation method of gas sensor
CN103675052B (en) * 2013-12-25 2015-11-11 华中师范大学 A kind of electrochemical immunosensor detecting dibutyl phthalate and its preparation method and application
CN104267183B (en) * 2014-10-14 2016-08-31 电子科技大学 A kind of making of the dual-purpose biosensor of photoelectricity that can be used for tumor-marker analyte detection
CN107389923A (en) * 2017-08-01 2017-11-24 甘肃农业大学 A kind of preparation method for exempting from mark type aptamer sensor and the method with the sensor quick detection ochratoxin A
CN111380933B (en) * 2020-03-23 2021-07-20 山东农业大学 Electrochemical immunosensor for detecting bombyx mori nuclear polyhedrosis virus and detection method thereof

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