CN110108624B - Method for preparing nano single particle by functionalized phospholipid and detection of nano single particle - Google Patents
Method for preparing nano single particle by functionalized phospholipid and detection of nano single particle Download PDFInfo
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- 229910052757 nitrogen Inorganic materials 0.000 claims description 6
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- 238000004140 cleaning Methods 0.000 claims description 5
- 229940079593 drug Drugs 0.000 claims description 5
- KTWOOEGAPBSYNW-UHFFFAOYSA-N ferrocene Chemical compound [Fe+2].C=1C=C[CH-]C=1.C=1C=C[CH-]C=1 KTWOOEGAPBSYNW-UHFFFAOYSA-N 0.000 claims description 5
- 229910052697 platinum Inorganic materials 0.000 claims description 5
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- 125000004057 biotinyl group Chemical group [H]N1C(=O)N([H])[C@]2([H])[C@@]([H])(SC([H])([H])[C@]12[H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C(*)=O 0.000 description 5
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- B01J13/00—Colloid chemistry, e.g. the production of colloidal materials or their solutions, not otherwise provided for; Making microcapsules or microballoons
- B01J13/02—Making microcapsules or microballoons
- B01J13/04—Making microcapsules or microballoons by physical processes, e.g. drying, spraying
- B01J13/043—Drying and spraying
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
- G01N15/10—Investigating individual particles
- G01N15/1031—Investigating individual particles by measuring electrical or magnetic effects
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
- G01N27/416—Systems
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Abstract
The invention relates to a method for preparing nano single particles by functionalized phospholipid and detection of the nano single particles. The preparation process of the functionalized liposome nano single particle comprises the following steps: respectively dissolving phospholipid DOPC and functionalized phospholipid Biotinyl PE in chloroform, mixing according to a certain proportion, blowing the solvent with nitrogen gas, and forming a layer of uniform thin phospholipid membrane at the bottom of the container; vacuum drying to remove chloroform, adding PBS buffer solution for hydration, shaking, mixing, standing for a period of time, and then using an ultrasonic cell disruptor to carry out ultrasonic treatment until the solution is clarified, thus obtaining the functionalized liposome nano single particles (Biotin-liposome) with uniform size. The liposome single particle with poor electrochemical activity can be detected by utilizing the specific combination of the liposome single particle and an Avidin-SH modified gold electrode and an electrochemical technology.
Description
Technical Field
The invention belongs to the field of chemistry, and particularly relates to a method for preparing nano single particles by functionalized phospholipid and detection of the nano single particles.
Background
Liposomes (liposomes) were first formally discovered by A.D.Bangham, UK in 1956 (BanghamAD, Standish MM, Watkins JC.J.Mole.biol.,1965,13: 238-. Liposomes are closed vesicles composed of lipid bilayers, and lipid molecules are amphiphilic molecules composed of a polar hydrophilic head and a non-polar hydrophobic tail. When the lipid molecules are dispersed in water to form liposomes, the hydrophobic ends of the lipid molecules are directed inward and the hydrophilic ends are on the surface of the bilayer. It is noteworthy that lipids and proteins are the major components that make up the biofilm, and thus liposomes are also an important model biofilm system. Liposomes have been found to have a wide range of applications in the fields of cosmetics, food and medicine due to their unique structure. Particularly, as the phospholipid molecules forming the liposome have amphipathy, the inside of the phospholipid molecules can be wrapped with water-soluble and fat-soluble medicines, and the composition of the phospholipid and the human cell membrane is similar, the liposome becomes a very effective gene and medicine carrier and is successfully used for gene and medicine delivery.
There are several methods for preparing liposome vesicles, such as extrusion, sonication, hydration swelling, and electroformation. For small unilamellar vesicles and large unilamellar vesicles, a certain amount of phospholipid is generally dissolved in chloroform or a chloroform/methanol mixed solution, nitrogen is used for blowing the solvent dry, trace residual solvent is removed under vacuum, a layer of dry phospholipid membrane is obtained on the glass surface, buffer solution is added for hydration swelling of the phospholipid membrane, and multi-chamber liposome vesicles with different sizes can be obtained by shaking or stirring. By using an extrusion method or an ultrasonic method, multichamber liposome vesicles with different sizes can be converted into large unilamellar vesicles or small unilamellar vesicles. For the giant unilamellar vesicles, the lamellar phospholipid membrane obtained on the glass surface can be put into an aqueous solution for hydration, and the lamellar phospholipid membrane is naturally expanded to obtain the giant vesicles, or the giant vesicles are formed under the condition of an external alternating current field.
The functions and properties of biological nanoparticles, such as proteins, vesicles, liposomes, viruses, etc., are closely related to their morphology, size, charge density, and surface chemistry. Therefore, the development of research methods for single particle detection and analysis is of great significance for understanding the relationship between particle structure and performance. Currently, the methods and techniques available for particle detection mainly include electron microscopy and spectroscopy. As the former, a Scanning Electron Microscope (SEM) and a Transmission Electron Microscope (TEM) are more common. However, such methods are currently difficult to directly apply to real-time detection of single particles in a solution state. Dynamic Light Scattering (DLS), while useful for in situ real-time detection of particles in solution, gives statistical results for particles throughout the system, fails to provide specific characteristics for individual particles, and the presence of agglomerated large particles or impurities in the system interferes with the detection results (Edwards MA, German SR, Dick JE, Bard AJ, White hs. acsnano,2015,9: 12274-. With the development of electroanalytical chemistry and micro-nano processing technologies, single-particle electrochemical detection technology has been rapidly developed in recent years. Since the method can accurately detect the properties (such as surface charge, geometric size and surface chemistry) of single particles, the method has a strong application prospect in single particle analysis (ZHao LJ, QianRC, Ma W, Tian H, Long YT.anal.chem.2016, 88: 8375-.
Disclosure of Invention
One of the purposes of the invention is to provide a method for preparing nano single particles by functionalized phospholipid.
The method for preparing the nano single particle by the functionalized phospholipid comprises the following steps:
1) respectively dissolving phospholipid DOPC and functionalized phospholipid Biotinyl PE in an organic solvent to obtain a phospholipid DOPC solution and a functionalized phospholipid Biotinyl PE solution, mixing the two solutions, and removing the solvent in the mixed system to obtain a thin phospholipid membrane;
2) adding PBS buffer solution into the thin phospholipid membrane obtained in the step 1), uniformly mixing, standing, hydrating and swelling, and performing ultrasonic dispersion to obtain a solution of functionalized liposome nano single particles (Biotin-liposome).
In the step 1) of the method, the organic solvent may be chloroform;
the concentration of DOPC in the phospholipid DOPC solution may be 8X 10-4mol/L；
The concentration of the Biotinyl PE in the functionalized phospholipid Biotinyl PE solution can be 2 x 10-4mol/L；
In the mixed system, the concentration ratio of phospholipid DOPC to functionalized phospholipid Biotinyl PE can be 1:1-6:1, specifically 1:1, 2:1, 4:1 or 6: 1;
the operation of removing the solvent in the mixed system comprises the following steps: blowing the solvent in the system by using nitrogen;
the method can further comprise the operation of drying the thin phospholipid membrane to remove residual solvent before the step 2), wherein the drying can be carried out in a vacuum drying oven; the drying time can be 6-12h, specifically 6h, 9h, 12h and the like.
In the step of hydrating and swelling the thin phospholipid membrane, the standing time can be 10-60min, specifically 30 min;
the operation of ultrasonic dispersion is as follows: performing ultrasonic treatment by using an ultrasonic cell disruption instrument until the solution is clarified; wherein the ultrasonic cell disruption instrument is purchased from Ningbo Xinzhi Biotech limited, model number: SCIENTZ-IID;
the ultrasound parameters were set as follows: the power is 70%, the time is 10s on, the time is 10s off, and the ultrasonic time is 10-40min, specifically 10min and 20 min.
In the solution of the functionalized liposome single nanoparticle (Biotin-liposome), the concentration of the liposome single nanoparticle can be 500nM-5mM, and specifically can be 1 mM.
The method can further comprise the operation of separating the functionalized liposome single nanoparticle from the solution of the functionalized phospholipid single nanoparticle (Biotin-liposome).
The method specifically comprises the following steps: and separating out the functionalized liposome nano single particles by adopting membrane filtration.
The pore diameter of the membrane is 50-200 nm.
The functionalized liposome nano single particle prepared by the method also belongs to the protection scope of the invention.
Another object of the present invention is to provide a method for detecting the functionalized liposome nano-single particles.
The method for detecting the functionalized liposome nano single particle provided by the invention comprises the following steps: a three-electrode system is constructed by using an Avidin-SH modified gold microelectrode as a working electrode, and detection of functionalized liposome nano single particles is realized through an electrochemical technology.
The specific operation of the detection is as follows:
the method comprises the steps of taking an Avidin-SH modified gold microelectrode Au-S-Avidin as a working electrode, an Ag/AgCl electrode as a reference electrode, a platinum electrode as a counter electrode to construct a three-electrode system, taking methyl alcohol ferrocene as an electrochemical probe, detecting a time-current curve after adding functionalized liposome nanoparticles with certain concentration at different potentials, observing and analyzing an experimental result, and obtaining information such as the particle size, surface charge, collision frequency with the electrode and the like of the nanoparticles.
The Avidin-SH modified gold microelectrode Au-S-Avidin is prepared by a method comprising the following steps: cleaning a gold microelectrode, then carrying out electrochemical activation in a sulfuric acid solution, and then cleaning with secondary water; and soaking the cleaned gold microelectrode in an Avidin-SH solution to obtain an Avidin-SH modified gold microelectrode (Au-S-Avidin).
The cleaning is to clean the gold microelectrode by using ethanol and secondary water in sequence;
the sulfuric acid solution may specifically be a 0.5M sulfuric acid solution.
The soaking time can be 6-48h, and specifically can be 12 h; the temperature can be 4 ℃;
the modified electrode was washed 3 times with secondary water to remove non-immobilized molecules before performing the electrochemical test.
The Avidin-SH modified gold microelectrode (Au-S-Avidin) is stored at 4 ℃ when not used.
The different potential may be any potential of-0.5V to 0.5V.
The certain concentration refers to the concentration of the liposome nano single particles in the detection solution, and can be specifically any value between 500 mu M and 1 pM.
The invention provides a simple method for preparing single-size functionalized liposome nanoparticles, and the functionalized phospholipid Biotinyl PE and phospholipid DOPC can be used for preparing the liposome single nanoparticles; the detection of the liposome single particle with poor electrochemical activity is realized through the specific combination of the Biotin-liposome and the gold electrode modified by the Avidin-SH and the electrochemical technology. To our knowledge, this is the first time to detect single particles by specific recognition of Avidin and Biotin and collision of Biotin functionalized single particles with electrodes.
Drawings
Fig. 1 is a dynamic light scattering particle size distribution diagram of liposome single nanoparticles prepared in example 1 of the present invention.
Fig. 2 is an SEM representation of the adsorption of liposomal single nanoparticles onto the surface of an electrode prepared in example 1 of the present invention.
Fig. 3 is a time-current curve of modified gold electrode detection of liposomal single nanoparticles prepared in example 1.
FIG. 4 is a schematic diagram of the principle of detecting liposome nano single particles by a gold microelectrode.
Detailed Description
The present invention will be described below with reference to specific examples, but the present invention is not limited thereto.
The experimental methods used in the following examples are all conventional methods unless otherwise specified; reagents, materials and the like used in the following examples are commercially available unless otherwise specified.
The phospholipid DOPC used in the examples described below was obtained from Innovation technologies, Inc. of Chikay, Beijing under the trade name 1, 2-dioleoyl-sn-glycerol-3-phoschooline, powder (Avanti);
biotinyl PE is available from Seisan Rexi Biotech under the trade name 1, 2-dioleoyl-sn-glycerol-3-phosphoethanomine-N- (biotin) (sodium salt), powder (Avanti).
Example 1 preparation of Liposome Nano-Single particles by functionalized Phospholipids and detection thereof
1) Preparing liposome nano single particles by functionalized phospholipid:
dissolving purchased phospholipid in chloroform, mixing according to a certain proportion, and the concentration ratio of phospholipid DOPC to Biotinyl PE after mixing is 4: 1; blowing the solvent with nitrogen to form a layer of uniform thin phospholipid membrane at the bottom of the container;
drying the obtained thin phospholipid membrane in a vacuum drying oven for 12h, then adding 2mL PBS buffer solution for hydration, shaking, uniformly mixing, standing for 30min, and performing ultrasonic treatment with a cell disruptor for 20min to uniformly disperse the thin phospholipid membrane (power is 70%, the time is 10s on, and the time is 10s off) to obtain a solution containing liposome nano single particles with uniform size (the concentration of the liposome nano single particles is 1 mM);
2) preparing a modified electrode:
pretreatment of the gold microelectrode: washing the electrode with ethanol and secondary water in sequence, electrochemically activating the electrode in 0.5M sulfuric acid solution for 30min, and washing the electrode with secondary water;
preparing a modified electrode: and soaking the cleaned electrode in Avidin-SH solution, and modifying for 12h at 4 ℃ in a refrigerator, wherein the sulfydryl is modified on the surface of the gold microelectrode.
All modified electrodes were washed 3 times with water before electrochemical testing to remove non-immobilized molecules, and were stored at 4 ℃ when not in use.
3) The modified electrode is used for detecting liposome nano single particles:
and (2) taking the Au-S-Avidin microelectrode obtained in the step 2) as a working electrode, taking an Ag/AgCl electrode as a reference electrode, taking a platinum electrode as a counter electrode to construct a three-electrode system, taking methyl alcohol ferrocene as an electrochemical probe, detecting a time-current curve after adding a liposome nanoparticle with a certain concentration (40 mu M, the concentration after adding the solution of the liposome nanoparticle prepared in the step 1) into a blank solution) at a potential of 0.5V, and observing and analyzing experimental results to obtain information such as the particle size, the surface charge, the collision frequency with the electrode and the like of the nanoparticle.
FIG. 1 shows a dynamic light scattering particle size distribution of the prepared liposome single nanoparticle, from which it can be seen that the diameter of the nanoparticle is about 100 nm.
FIG. 2 is a Scanning Electron Microscope (SEM) image of the modified gold microelectrode Au-S-Avidin, after a period of time of interaction with liposome single nanoparticles through specific recognition, the single nanoparticles are adsorbed on the surface of the electrode.
FIG. 3 is a time-current curve of modified gold microelectrode Au-S-Avidin for detecting liposome single nanoparticle. As can be seen from fig. 3: after the functionalized liposome nanoparticles collide with the electrodes, peak signals and step signals are mainly generated.
FIG. 4 is a schematic diagram of the principle of detecting liposome nano single particles by a modified gold microelectrode.
Example 2 preparation of Liposome Nano-Single particles by functionalized phospholipid and detection thereof
1) Preparing liposome single nano particles by functionalized phospholipid:
dissolving purchased phospholipid in chloroform, mixing according to a certain proportion, and the concentration ratio of phospholipid DOPC to Biotinyl PE after mixing is 2: 1; blowing the solvent with nitrogen to form a layer of uniform thin phospholipid membrane at the bottom of the container;
drying the obtained thin phospholipid membrane in a vacuum drying oven for 9h, then adding 2mL PBS buffer solution for hydration, shaking and uniformly mixing, standing for 30min, and performing ultrasonic treatment for 10min by using a cell disruptor to uniformly disperse the solution to obtain a solution containing liposome nano single particles with uniform size (the concentration of the liposome nano single particles is 1 mM);
2) preparing a modified electrode:
pretreatment of the gold microelectrode: washing the electrode with ethanol and secondary water in sequence, electrochemically activating the electrode in 0.5M sulfuric acid solution, and washing the electrode with secondary water;
preparing a modified electrode: and (3) soaking the cleaned electrode in Avidin-SH solution, placing the electrode in a refrigerator for modification at 4 ℃ for 24 hours, and modifying the sulfydryl on the surface of the gold microelectrode.
All modified electrodes were washed 3 times with water before electrochemical testing to remove non-immobilized molecules, and were stored at 4 ℃ when not in use.
3) The modified electrode is used for detecting liposome nano single particles:
and (3) taking the Au-S-Avidin microelectrode obtained in the step 2) as a working electrode, taking an Ag/AgCl electrode as a reference electrode, taking a platinum electrode as a counter electrode to construct a three-electrode system, taking methyl alcohol ferrocene as an electrochemical probe, detecting a time-current curve after liposome nano single particles with a certain concentration are added under the potential of 0V, and observing and analyzing an experimental result.
Example 3 preparation of Liposome Nano-Single particles by functionalized Phospholipids and detection thereof
1) Preparing liposome single nano particles by functionalized phospholipid:
dissolving purchased phospholipid in chloroform, mixing according to a certain proportion, and the concentration ratio of phospholipid DOPC to Biotinyl PE after mixing is 6: 1; blowing the solvent with nitrogen to form a layer of uniform thin phospholipid membrane at the bottom of the container;
drying the obtained thin phospholipid membrane in a vacuum drying oven for 6h, then adding 2mL PBS buffer solution for hydration, shaking and uniformly mixing, standing for 30min, and performing ultrasonic treatment by using a cell disruptor for 40min to uniformly disperse the solution to obtain a solution containing liposome nano single particles with uniform size (the concentration of the liposome nano single particles is 1 mM);
2) preparing a modified electrode:
pretreatment of the gold microelectrode: washing the electrode with ethanol and secondary water in sequence, electrochemically activating the electrode in 0.5M sulfuric acid solution, and washing the electrode with secondary water;
preparing a modified electrode: and soaking the cleaned electrode in Avidin-SH solution, and modifying for 12h at 4 ℃ in a refrigerator, wherein the sulfydryl is modified on the surface of the gold microelectrode.
All modified electrodes were washed 3 times with water before electrochemical testing to remove non-immobilized molecules, and were stored at 4 ℃ when not in use.
3) The modified electrode is used for detecting liposome nano single particles:
and (3) taking the Au-S-Avidin microelectrode obtained in the step 2) as a working electrode, taking an Ag/AgCl electrode as a reference electrode, taking a platinum electrode as a counter electrode to construct a three-electrode system, taking methyl alcohol ferrocene as an electrochemical probe, detecting a time-current curve after liposome nano single particles with a certain concentration are added under a potential of-0.5V, and observing and analyzing an experimental result.
Lipid is one of main components forming a biological membrane, phospholipid molecules of the liposome have amphiphilicity, water-soluble and fat-soluble medicines can be wrapped in the phospholipid molecules, and the phospholipid and a cell membrane have similar compositions, so that the liposome becomes a very effective gene and medicine carrier and is successfully used for gene and medicine transfer. By utilizing the good biocompatibility of the liposome and the cell membrane, the functionalized liposome single particle (or the functionalized liposome single particle after encapsulating the drug) can be incubated with the cell to study the activity state of the functionalized liposome single particle in the cell and the influence of the addition of the functionalized liposome single particle on some physiological states of the cell. The electrochemical technology is utilized to realize the detection of the distribution, the shape and the like of the liposome/drug-loaded liposome nanoparticles in cells, and the like, thereby providing help for disease observation, diagnosis and treatment.
Claims (8)
1. A method for detecting functionalized liposome nano single particles, wherein the functionalized liposome nano single particles are prepared by the following method:
1) respectively dissolving phospholipid DOPC and 1, 2-dioleoyl-sn-propanetriyl-3-phosphatidylethanolamine-N-biotin sodium salt in an organic solvent to obtain a phospholipid DOPC solution and a 1, 2-dioleoyl-sn-propanetriyl-3-phosphatidylethanolamine-N-biotin sodium salt solution, mixing the two solutions, and removing the solvent in the mixed system to obtain a thin phospholipid membrane;
2) adding a PBS buffer solution into the thin phospholipid membrane obtained in the step 1), uniformly mixing, standing, hydrating and swelling, and performing ultrasonic dispersion to obtain a solution of functionalized liposome nano single particles;
the method for detecting the functionalized liposome nano single particle comprises the following steps: and constructing a three-electrode system by using an Avidin-SH modified gold microelectrode as a working electrode, and realizing the detection of the functionalized liposome nano single particle by an electrochemical technology.
2. The method for detecting functionalized liposomal nanomonoparticles of claim 1, wherein:
the method for preparing the functionalized liposome nano single particle comprises the following steps of 1), wherein the organic solvent is chloroform;
the concentration of DOPC in the phospholipid DOPC solution is 8 x 10-4mol/L；
The concentration of the 1, 2-dioleoyl-sn-propanetriyl-3-phosphatidylethanolamine-N-biotin sodium salt in the 1, 2-dioleoyl-sn-propanetriyl-3-phosphatidylethanolamine-N-biotin sodium salt solution is 2 multiplied by 10-4mol/L；
In the mixed system, the concentration ratio of phospholipid DOPC to 1, 2-dioleoyl-sn-propanetriyl-3-phosphatidylethanolamine-N-biotin sodium salt is 1:1-6: 1;
the operation of removing the solvent in the mixed system comprises the following steps: blowing the solvent in the system by using nitrogen;
before step 2), further comprising an operation of drying the thin phospholipid membrane to remove residual solvent,
the drying is carried out in a vacuum drying oven; the drying time is 6-12 h.
3. The method for detecting functionalized liposomal nanomonoparticles of claim 1, wherein: the method for preparing the functionalized liposome nano single particle comprises the following steps of 2), in the step of hydrating and swelling the thin phospholipid membrane, standing for 10-60 min;
the operation of ultrasonic dispersion is as follows: performing ultrasonic treatment by using an ultrasonic cell disruption instrument until the solution is clarified;
the ultrasound parameters were set as follows: the power is 70%, the time is 10s on, the time is 10s off, and the ultrasonic time is 10-40 min;
in the solution of the functionalized liposome nano single particles, the concentration of the liposome nano single particles is 500nM-5 mM.
4. The method for detecting functionalized liposomal nanomonoparticles of claim 1, wherein: the method for preparing the functionalized liposomal nanomonoparticles further comprises the operation of separating the functionalized liposomal nanomonoparticles from the solution of the functionalized liposomal nanomonoparticles.
5. The method for detecting functionalized liposomal nanomonoparticles of claim 4, wherein: the operation is as follows: separating the functionalized liposome nano single particles by adopting membrane filtration;
the pore diameter of the membrane is 50-200 nm.
6. The method of claim 1, wherein: the detection is as follows: and (3) taking an Avidin-SH modified gold microelectrode Au-S-Avidin as a working electrode, an Ag/AgCl electrode as a reference electrode, a platinum electrode as a counter electrode to construct a three-electrode system, methanol ferrocene as an electrochemical probe, detecting a time-current curve after adding functionalized liposome nano single particles with certain concentration at different potentials, and observing and analyzing experimental results.
7. The method of claim 6, wherein: the Avidin-SH modified gold microelectrode Au-S-Avidin is prepared by a method comprising the following steps: cleaning a gold microelectrode, then carrying out electrochemical activation in a sulfuric acid solution, and then cleaning with secondary water; and (3) soaking the cleaned gold microelectrode in an Avidin-SH solution to obtain the gold microelectrode.
8. Use of the method of any one of claims 1-7 for detecting the intracellular distribution, morphology and effects of liposome/drug loaded liposome nanoparticles on certain physiological states of a cell.
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Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1553188A (en) * | 2003-06-06 | 2004-12-08 | 克 宋 | Microarray signal amplifying method |
| CN1875113A (en) * | 2003-10-29 | 2006-12-06 | 新加坡科技研究局 | Method for detecting analytes by means of an analyte/polymeric activator bilayer arrangement |
| CN102307885A (en) * | 2008-10-14 | 2012-01-04 | 江陵原州大学校产学协力团 | Method for introducing functional group to surface of material |
| CN102539494A (en) * | 2012-01-12 | 2012-07-04 | 福建医科大学 | Amperometric DNA (deoxyribonucleic acid) electrochemical sensor based on protein controlled assembling interface |
| CN106754860A (en) * | 2016-11-22 | 2017-05-31 | 上海纳米技术及应用国家工程研究中心有限公司 | A kind of method to interface Rapid Modification Avidin based on liposome |
| CN106891018A (en) * | 2017-03-05 | 2017-06-27 | 北京化工大学 | A kind of nanoporous gold grain and preparation method thereof |
| CN109060917A (en) * | 2018-08-15 | 2018-12-21 | 天津中医药大学 | A kind of aptamer electrochemical sensor and its preparation method and application detecting enteropathogenic E.Coli |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| SE0004334D0 (en) * | 2000-11-24 | 2000-11-24 | Sahltech Ab | Electron spectroscopy |
| JP5048100B2 (en) * | 2010-04-28 | 2012-10-17 | シャープ株式会社 | Peptide having binding property to adiponectin, and analysis method and analyzer using the peptide |
-
2019
- 2019-05-08 CN CN201910380258.1A patent/CN110108624B/en active Active
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1553188A (en) * | 2003-06-06 | 2004-12-08 | 克 宋 | Microarray signal amplifying method |
| CN1875113A (en) * | 2003-10-29 | 2006-12-06 | 新加坡科技研究局 | Method for detecting analytes by means of an analyte/polymeric activator bilayer arrangement |
| CN102307885A (en) * | 2008-10-14 | 2012-01-04 | 江陵原州大学校产学协力团 | Method for introducing functional group to surface of material |
| CN102539494A (en) * | 2012-01-12 | 2012-07-04 | 福建医科大学 | Amperometric DNA (deoxyribonucleic acid) electrochemical sensor based on protein controlled assembling interface |
| CN106754860A (en) * | 2016-11-22 | 2017-05-31 | 上海纳米技术及应用国家工程研究中心有限公司 | A kind of method to interface Rapid Modification Avidin based on liposome |
| CN106891018A (en) * | 2017-03-05 | 2017-06-27 | 北京化工大学 | A kind of nanoporous gold grain and preparation method thereof |
| CN109060917A (en) * | 2018-08-15 | 2018-12-21 | 天津中医药大学 | A kind of aptamer electrochemical sensor and its preparation method and application detecting enteropathogenic E.Coli |
Non-Patent Citations (5)
| Title |
|---|
| An immunosensor for haemoglobin based on impedimetric properties of a new mixed self-assembled monolayer;S. Hleli 等;《Materials Science and Engineering: C》;20051207;第26卷(第2-3期);第322-327页 * |
| Electrochemical Detection of Single Phospholipid Vesicle Collisions at a Pt Ultramicroelectrode;Estelle Lebegue 等;《Langmuir》;20151008;第31卷(第42期);第11734-11739页 * |
| Immobilization in surface-tethered lipid vesicles as a new tool for single biomolecule spectroscopy;Erez Boukobza 等;《The journal of physical chemistry B》;20011102;第105卷(第48期);第12165-12170页 * |
| New Electrochemical Methods;Christopher Batchelor-McAuley 等;《Analatical Chemistry》;20111021;第84卷(第2期);第669-684页 * |
| 铂电极表面生物素-亲合素固载ssDNA的电化学基因传感器研究;王保珍;《万方》;20060116;第1-10页 * |
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