CN110441294A - One kind wrapping up Co based on ferritin3O4The preparation method of the biosensor of core-shell structure - Google Patents
One kind wrapping up Co based on ferritin3O4The preparation method of the biosensor of core-shell structure Download PDFInfo
- Publication number
- CN110441294A CN110441294A CN201910787983.0A CN201910787983A CN110441294A CN 110441294 A CN110441294 A CN 110441294A CN 201910787983 A CN201910787983 A CN 201910787983A CN 110441294 A CN110441294 A CN 110441294A
- Authority
- CN
- China
- Prior art keywords
- solution
- concentration
- ferritin
- added
- parathyroid hormone
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
- G01N21/76—Chemiluminescence; Bioluminescence
-
- 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
- G01N27/28—Electrolytic cell components
- G01N27/30—Electrodes, e.g. test electrodes; Half-cells
- G01N27/308—Electrodes, e.g. test electrodes; Half-cells at least partially made of carbon
-
- 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
- G01N27/28—Electrolytic cell components
- G01N27/30—Electrodes, e.g. test electrodes; Half-cells
- G01N27/327—Biochemical electrodes, e.g. electrical or mechanical details for in vitro measurements
- G01N27/3275—Sensing specific biomolecules, e.g. nucleic acid strands, based on an electrode surface reaction
-
- 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
- G01N27/28—Electrolytic cell components
- G01N27/30—Electrodes, e.g. test electrodes; Half-cells
- G01N27/327—Biochemical electrodes, e.g. electrical or mechanical details for in vitro measurements
- G01N27/3275—Sensing specific biomolecules, e.g. nucleic acid strands, based on an electrode surface reaction
- G01N27/3278—Sensing specific biomolecules, e.g. nucleic acid strands, based on an electrode surface reaction involving nanosized elements, e.g. nanogaps or nanoparticles
Abstract
The present invention relates to one kind to wrap up Co based on ferritin3O4The preparation method of the biosensor of core-shell structure belongs to novel nano-material field and biosensor technique field；The present invention is based on electrogenerated chemiluminescence ECL technologies, wrap up Co for the first time with ferritin3O4Core-shell structure covalent cross-linking N-(4- ammonia butyl)-N- ethyl different luminol ABEI as signal source utilizes Co using the immobilized antibody molecule of the outstanding biocompatibility of ferritin3O4Detection signal is effectively amplified in the excellent catalytic action that ECL between ABEI and hydrogen peroxide reacts, it proposes and a kind of prepares biosensor preparation method simple, highly sensitive, that energy consumption of reaction is low, and it is applied to the actual sample detection of parathyroid hormone, detection limit is down to 13 fg/mL, the range of linearity is wide to 50 fg/mL-100 ng/mL, high sensitivity, favorable reproducibility have biggish potential using value.
Description
Technical field
The invention belongs to novel nano-material fields and biosensor technique field.
Background technique
The research hotspot for being intersected by a variety of subjects such as biology, chemistry, medicine, electronic technology and being risen as one,
Electrogenerated chemiluminescence (ECL) immuno analytical method is the combination of electrochemistry, chemiluminescence and immuno analytical method, is had
The advantages that at low cost, selective good, high sensitivity, analysis speed are fast, are easy to automate, are miniaturized and are integrated, it is extensive
Applied to fields such as disease marker analysis, Food Safety Analysis, Analysis on Environment Contamination.
Parathyroid hormone is a kind of polypeptide containing 84 amino acid secreted by chief cell.In serum
The variation of parathyroid hormone level can directly indicate disease of parathyroid glands.For example, hyperparathyroidism can lead to first shape
The excess generation of other glandular hormone.Closely related with cardiovascular and chronic renal disease and cancer generation, it is thin by tumour
The growth and differentiation of born of the same parents participates in route of metastasis, especially when breast cancer and prostate cancer are transferred to bone, parathyroid hormone in serum
Plain concentration is significantly raised.Therefore, in blood of human body parathyroid hormone detection for monitor osteoporosis, parathyroid gland disease
Disease, malignant tumour related hypercalcemia and the therapeutic effect of cancer patient are of great significance.Up to the present, only fluorescence
Several analysis methods such as method, electrochemical analysis, chromatography are developed.Because parathormone half-life period is very short,
Develop a kind of simple, fast and accurately measuring method, the instant inspection for parathormone (especially parathormone in art)
Measuring tool is significant.
Summary of the invention
Technical assignment of the invention first is that in order to make up the deficiency of existing detection technique, make full use of ferritin to wrap up
Co3O4Core-shell structure outstanding biocompatibility and electro catalytic activity promote N-(4- ammonia butyl based on the structure for the first time)-N- second
The principle of base different luminol ABEI electrogenerated chemiluminescence proposes a kind of preparation method of novel biosensor, sensor behaviour
Make simple, at low cost, signal response rapidly, substantially reduces the time of detection, save considerably human and material resources, financial resources.
The two of technical assignment of the invention are to provide the purposes of the biosensor, which can quickly detect first
Glandular hormone by shape, has the advantages that high sensitivity, high specificity, favorable reproducibility, and detection is limited to 13 fg/mL, the range of linearity 50
fg/mL-50 ng/mL。
To achieve the above object, The technical solution adopted by the invention is as follows:
Claim
1. one kind wraps up Co based on ferritin3O4The preparation method of the biosensor of core-shell structure, which is characterized in that including
Following steps:
(1) glass-carbon electrode that diameter is 4 mm is successively used into 1.0 μm, 0.3 μm, 0.05 μm of aluminum oxide polishing powder polishing treatment,
It is clean with ultrapure water；
(2) the golden hydridization graphite alkene aeroge marked in 6 μ L of glassy carbon electrode surface drop coating, the primary antibody that concentration is 2 ~ 4 mg/mL
Solution is sensing substrate, places it in 37 °C and dries；
(3) 3 μ L are added dropwise, the bovine serum albumin solution that mass fraction is 1 ~ 3%, it is living with the non-specificity on enclosed-electrode surface
Property site, rinse electrode surface with the phosphate buffer solution PBS of pH 7.4, place it in 4 °C and dry；
(4) standard solution of 6 μ L parathyroid hormones or the solution of parathyroid hormone of unknown concentration is added dropwise, hatches under 37 °C
0.5 ~ 2 h rinses electrode surface with the phosphate buffer solution PBS of pH 7.4, places it in 4 °C and dry；
(5) 6 μ L are added dropwise, the ferritin that the secondary antibody that concentration is 2 ~ 4 mg/mL marks wraps up Co3O4Solution places it in 37 °
C dries, and rinses electrode surface with the phosphate buffer solution PBS of pH 7.4, places it in 4 °C and dry, sensor has constructed
Finish.
2. as described in claim 1 a kind of based on ferritin package Co3O4The preparation of the biosensor of core-shell structure
Method, which is characterized in that the golden hydridization graphite alkene airsetting sol solution of the primary antibody label is prepared according to the following steps:
Take the HAuCl of 4 ~ 6 mL 1%4Solution and 100 mL ultrapure waters are added to the three neck round bottom flask of 250 mL of clean dried
In, under constant stirring, the sodium citrate solution that drop is slowly added to 10 mL thereto, mass fraction is 1%, it is heated to reflux 15 ~
35 min stop heating after solution colour eventually becomes claret, through supercooling and gold nanoparticle are obtained by filtration；
The graphite alkene solution that 20 mL concentration are 1.5 ~ 3.5 mg/mL is prepared, under uniform stirring, 3 ~ 5 mL Jenners are added
Rice corpuscles vibrates 6 h, and with ultrapure water 8 h of dialysis, the golden hydridization graphite alkene gas of 3D structure can be obtained after freeze-drying later
Gel；
The golden hydridization graphite alkene airsetting sol solution of 2 mg/mL is prepared, 100 ~ 300 μ L are added, the first shape that concentration is 10 mg/mL
Other glandular hormone primary antibody solution, 6 ~ 18 h of oscillation hatching, are distributed to the phosphate-buffered of 1 mL pH 7.4 under 4 °C after centrifugation
The golden hydridization graphite alkene airsetting sol solution that primary antibody label is obtained in solution, is placed under 4 °C and stores for future use.
3. as described in claim 1 a kind of based on ferritin package Co3O4The preparation of the biosensor of core-shell structure
Method, which is characterized in that the ferritin of the secondary antibody label wraps up Co3O4Solution is prepared according to the following steps:
Logical N is carried out to 25 mL, the apoferritin solution that concentration is 50 ~ 150 μ g/mL with capillary2It handles, it will after 1 h
The solution carries out water bath with thermostatic control, and temperature is maintained at 65 DEG C, and adjusts pH to 9.5 with the sodium hydroxide solution of 0.1 mol/L, with
25 mL of addition, the cabaltous nitrate hexahydrate that concentration is 0.05 mol/L, after stirring 1 h, 0.4 ~ 1.2 mL, concentration, which is added, is
The hydrogenperoxide steam generator of 10 mmol/L simultaneously reacts 5 min of holding, 10 mL is added, the sodium citrate solution that concentration is 10 mmol
It goes chelating not enter free cobalt ions inside apoferritin, by centrifugation, dialysis, after purification, obtains the iron that secondary antibody marks
Protein encapsulation Co3O4Solution；
2 mL ferritins are taken to wrap up Co3O40.5 ~ 2.5 mL is added, the N- (4- ammonia butyl)-that concentration is 10 mmol/L in solution
The glutaraldehyde solution of N- ethyl different luminol solution and 50 ~ 150 uL, mass fraction 50%, mixing is added 100 after centrifuge separation
~ 300 μ L, the parathyroid hormone two corresponding anti-solution that concentration is 10 mg/mL, 6 ~ 18 h of oscillation hatching under 4 °C, after centrifugation
It is distributed to the ferritin package Co that secondary antibody label is obtained in the phosphate buffer solution of 1 mL pH 7.43O4Solution is placed in 4 °C
Under store for future use.
4. the inspection that the biosensor of preparation method preparation as described in claim 1 is used for parathyroid hormone levels
It surveys.
5. the detection of parathyroid hormone levels as claimed in claim 4, which is characterized in that operating procedure is as follows:
(1) parameter setting: the photomultiplier tube high pressure of ultraweak electrogenerated chemiluminescence instrument is set as 600 V, electrochemical workstation
Cyclic voltammetry scan potential range is set as 0 ~ 0.6 V, and sweep speed is set as 0.1 V/s；
(2) test: using silver/silver chloride electrode as reference electrode, platinum electrode is to electrode, the sensing that in the above way prepares
Device is working electrode, and electroluminescent chemistry hair is carried out in phosphate buffer solution of 10 mL containing 45 ~ 75 mmol/L hydrogen peroxide
Optical tests obtain corresponding electrochemiluminescence signal intensity when hatching various concentration parathyroid hormone, draw working curve,
Its detection is limited to 13 fg/mL, 50 fg/mL-50 ng/mL of the range of linearity；
(3) it is tested the Electrochemiluminescsensor sensor for the parathyroid hormone actual sample for hatching unknown concentration to obtain phase
The signal strength answered calculates the parathyroid hormone levels that can be obtained in the reagent sample according to working curve accordingly.
Beneficial achievement of the invention
(1) Co is wrapped up based on ferritin for the first time3O4Core-shell structure promotes the principle of different luminol ABEI electrogenerated chemiluminescence to propose
A kind of novel reliable immune sensing technology.Since ferritin wraps up Co3O4Core-shell structure has excellent electrocatalysis characteristic,
Efficient catalytic can be carried out to hydrogen peroxide and decompose generation superoxide anion and hydroxyl radical free radical, provided for the ECL excitation of ABEI
Sufficient coreaction free radical, realizes effective amplification of ECL signal；
(2) present invention compensates for the problem that existing electrochemical measuring technique is complicated for operation, sensitivity is low, reappears difference, by the technology
Applied to the sample detection of parathyroid hormone, detection is limited to 36 fg/mL, and 100 fg/mL-50 ng/mL of the range of linearity has
Fast response time high sensitivity, reappears, prepares simple, at low cost, environmentally protective advantage.
Specific embodiment
Present invention will be further explained below with reference to specific examples, it should be appreciated that these embodiments are merely to illustrate the present invention
Rather than it limits the scope of the invention.
Embodiment 1. is a kind of to wrap up Co based on ferritin3O4The preparation method of the biosensor of core-shell structure, feature
It is, comprising the following steps:
(1) glass-carbon electrode that diameter is 4 mm is successively used into 1.0 μm, 0.3 μm, 0.05 μm of aluminum oxide polishing powder polishing treatment,
It is clean with ultrapure water；
(2) the golden hydridization graphite alkene airsetting sol solution marked in 6 μ L of glassy carbon electrode surface drop coating, the primary antibody that concentration is 2 mg/mL
To sense substrate, places it in 37 °C and dry；
(3) 3 μ L are added dropwise, the bovine serum albumin solution that mass fraction is 1 %, with the nonspecific activity on enclosed-electrode surface
Site rinses electrode surface with the phosphate buffer solution PBS of pH 7.4, places it in 4 °C and dry；
(4) standard solution of 6 μ L parathyroid hormones or the solution of parathyroid hormone of unknown concentration is added dropwise, hatches under 37 °C
0.5 h rinses electrode surface with the phosphate buffer solution PBS of pH 7.4, places it in 4 °C and dry；
(5) 6 μ L are added dropwise, the ferritin that the secondary antibody that concentration is 2 mg/mL marks wraps up Co3O4Solution places it in 37 °C and dries in the air
It is dry, electrode surface is rinsed with the phosphate buffer solution PBS of pH 7.4,4 °C is placed it in and dries, sensor building finishes.
Embodiment 2. is a kind of to wrap up Co based on ferritin3O4The preparation method of the biosensor of core-shell structure, feature
It is, comprising the following steps:
(1) glass-carbon electrode that diameter is 4 mm is successively used into 1.0 μm, 0.3 μm, 0.05 μm of aluminum oxide polishing powder polishing treatment,
It is clean with ultrapure water；
(2) the golden hydridization graphite alkene airsetting sol solution marked in 6 μ L of glassy carbon electrode surface drop coating, the primary antibody that concentration is 3 mg/mL
To sense substrate, places it in 37 °C and dry；
(3) 3 μ L are added dropwise, the bovine serum albumin solution that mass fraction is 2 %, with the nonspecific activity on enclosed-electrode surface
Site rinses electrode surface with the phosphate buffer solution PBS of pH 7.4, places it in 4 °C and dry；
(4) standard solution of 6 μ L parathyroid hormones or the solution of parathyroid hormone of unknown concentration is added dropwise, hatches under 37 °C
1.5 h rinse electrode surface with the phosphate buffer solution PBS of pH 7.4, place it in 4 °C and dry；
(5) 6 μ L are added dropwise, the ferritin that the secondary antibody that concentration is 3 mg/mL marks wraps up Co3O4Solution places it in 37 °C and dries in the air
It is dry, electrode surface is rinsed with the phosphate buffer solution PBS of pH 7.4,4 °C is placed it in and dries, sensor building finishes.
Embodiment 3. is a kind of to wrap up Co based on ferritin3O4The preparation method of the biosensor of core-shell structure, feature
It is, comprising the following steps:
(1) glass-carbon electrode that diameter is 4 mm is successively used into 1.0 μm, 0.3 μm, 0.05 μm of aluminum oxide polishing powder polishing treatment,
It is clean with ultrapure water；
(2) the golden hydridization graphite alkene airsetting sol solution marked in 6 μ L of glassy carbon electrode surface drop coating, the primary antibody that concentration is 4 mg/mL
To sense substrate, places it in 37 °C and dry；
(3) 3 μ L are added dropwise, the bovine serum albumin solution that mass fraction is 3 %, with the nonspecific activity on enclosed-electrode surface
Site rinses electrode surface with the phosphate buffer solution PBS of pH 7.4, places it in 4 °C and dry；
(4) standard solution of 6 μ L parathyroid hormones or the solution of parathyroid hormone of unknown concentration is added dropwise, hatches under 37 °C
0.5 ~ 2 h rinses electrode surface with the phosphate buffer solution PBS of pH 7.4, places it in 4 °C and dry；
(5) 6 μ L are added dropwise, the ferritin that the secondary antibody that concentration is 4 mg/mL marks wraps up Co3O4Solution places it in 37 °C and dries in the air
It is dry, electrode surface is rinsed with the phosphate buffer solution PBS of pH 7.4,4 °C is placed it in and dries, sensor building finishes.
The golden hydridization graphite alkene airsetting sol solution of the label of primary antibody described in embodiment 4., prepares according to the following steps:
Take the HAuCl of 4 mL 1%4Solution and 100 mL ultrapure waters are added in the three neck round bottom flask of 250 mL of clean dried；
Under constant stirring, the sodium citrate solution that drop is slowly added to 10 mL thereto, mass fraction is 1%, is heated to reflux 15 min,
Stop heating after solution colour eventually becomes claret；Through supercooling and gold nanoparticle is obtained by filtration；
The graphite alkene solution that 20 mL concentration are 1.5 mg/mL is prepared, under uniform stirring, 3 mL gold nanoparticles, oscillation is added
The golden hydridization graphite alkene aeroge of 3D structure can be obtained with ultrapure water 8 h of dialysis in 6 h after freeze-drying later；
The golden hydridization graphite alkene airsetting sol solution of 2 mg/mL is prepared, 100 μ L are added, the parathyroid hormone that concentration is 10 mg/mL
Plain primary antibody solution, 6 h of oscillation hatching, are distributed in the phosphate buffer solution of 1 mL pH 7.4 after centrifugation and obtain under 4 °C
The golden hydridization graphite alkene airsetting sol solution of primary antibody label, is placed under 4 °C and stores for future use.
The golden hydridization graphite alkene airsetting sol solution of the label of primary antibody described in embodiment 5., prepares according to the following steps:
Take the HAuCl of 5 mL 1%4Solution and 100 mL ultrapure waters are added in the three neck round bottom flask of 250 mL of clean dried；
Under constant stirring, the sodium citrate solution that drop is slowly added to 10 mL thereto, mass fraction is 1%, is heated to reflux 25 min,
Stop heating after solution colour eventually becomes claret；Through supercooling and gold nanoparticle is obtained by filtration；
The graphite alkene solution that 20 mL concentration are 2.5 mg/mL is prepared, under uniform stirring, 4 mL gold nanoparticles, oscillation is added
The golden hydridization graphite alkene aeroge of 3D structure can be obtained with ultrapure water 8 h of dialysis in 6 h after freeze-drying later；
The golden hydridization graphite alkene airsetting sol solution of 2 mg/mL is prepared, 200 μ L are added, the parathyroid hormone that concentration is 10 mg/mL
Plain primary antibody solution, 12 h of oscillation hatching, are distributed in the phosphate buffer solution of 1 mL pH 7.4 after centrifugation and obtain under 4 °C
The golden hydridization graphite alkene airsetting sol solution of primary antibody label, is placed under 4 °C and stores for future use.
The golden hydridization graphite alkene airsetting sol solution of the label of primary antibody described in embodiment 6., prepares according to the following steps:
Take the HAuCl of 6 mL 1%4Solution and 100 mL ultrapure waters are added in the three neck round bottom flask of 250 mL of clean dried；
Under constant stirring, the sodium citrate solution that drop is slowly added to 10 mL thereto, mass fraction is 1%, is heated to reflux 35 min,
Stop heating after solution colour eventually becomes claret；Through supercooling and gold nanoparticle is obtained by filtration；
The graphite alkene solution that 20 mL concentration are 3.5 mg/mL is prepared, under uniform stirring, 5 mL gold nanoparticles, oscillation is added
The golden hydridization graphite alkene aeroge of 3D structure can be obtained with ultrapure water 8 h of dialysis in 6 h after freeze-drying later；
The golden hydridization graphite alkene airsetting sol solution of 2 mg/mL is prepared, 300 μ L are added, the parathyroid hormone that concentration is 10 mg/mL
Plain primary antibody solution, 18 h of oscillation hatching, are distributed in the phosphate buffer solution of 1 mL pH 7.4 after centrifugation and obtain under 4 °C
The golden hydridization graphite alkene airsetting sol solution of primary antibody label, is placed under 4 °C and stores for future use.
The ferritin of the label of secondary antibody described in embodiment 7. wraps up Co3O4Solution is prepared according to the following steps:
Logical N is carried out to 25 mL, the apoferritin solution that concentration is 50 μ g/mL with capillary2It handles, by the solution after 1 h
Water bath with thermostatic control is carried out, temperature is maintained at 65 DEG C, and adjusts pH to 9.5 with the sodium hydroxide solution of 0.1 mol/L, is added therewith
After stirring 1 h, 0.4 mL is added, the mistake that concentration is 10 mmol/L in 25 mL, the cabaltous nitrate hexahydrate that concentration is 0.05 mol/L
Hydrogen peroxide solution simultaneously reacts 5 min of holding, 10 mL is added, the sodium citrate solution that concentration is 10 mmol goes chelating not enter
Free cobalt ions inside iron ferritin obtains the ferritin package Co of secondary antibody label by centrifugation, dialysis, after purification3O4It is molten
Liquid；
2 mL ferritins are taken to wrap up Co3O40.5 mL is added, N- (4- ammonia butyl)-N- ethyl that concentration is 10 mmol/L in solution
The glutaraldehyde solution of different luminol solution and 50 uL, mass fraction 50%, 100 μ L, concentration 10 are added in mixing after centrifuge separation
The parathyroid hormone two corresponding anti-solution of mg/mL, 6 h of oscillation hatching, are distributed to the phosphoric acid of 1 mL pH 7.4 under 4 °C after centrifugation
The ferritin package Co of secondary antibody label is obtained in salt buffer solution3O4Solution is placed under 4 °C and stores for future use.
The ferritin of the label of secondary antibody described in embodiment 8. wraps up Co3O4Solution is prepared according to the following steps:
Logical N is carried out to 25 mL, the apoferritin solution that concentration is 100 μ g/mL with capillary2It handles, by the solution after 1 h
Water bath with thermostatic control is carried out, temperature is maintained at 65 DEG C, and adjusts pH to 9.5 with the sodium hydroxide solution of 0.1 mol/L, is added therewith
After stirring 1 h, 0.8 mL is added, the mistake that concentration is 10 mmol/L in 25 mL, the cabaltous nitrate hexahydrate that concentration is 0.05 mol/L
Hydrogen peroxide solution simultaneously reacts 5 min of holding, 10 mL is added, the sodium citrate solution that concentration is 10 mmol goes chelating not enter
Free cobalt ions inside iron ferritin obtains the ferritin package Co of secondary antibody label by centrifugation, dialysis, after purification3O4It is molten
Liquid；
2 mL ferritins are taken to wrap up Co3O41.5 mL are added, N- (4- ammonia butyl)-N- ethyl that concentration is 10 mmol/L in solution
The glutaraldehyde solution of different luminol solution and 100 uL, mass fraction 50%, 200 μ L are added in mixing after centrifuge separation, concentration is
The parathyroid hormone two corresponding anti-solution of 10 mg/mL, 12 h of oscillation hatching, are distributed to 1 mL pH's 7.4 under 4 °C after centrifugation
The ferritin package Co of secondary antibody label is obtained in phosphate buffer solution3O4Solution is placed under 4 °C and stores for future use.
The ferritin of the label of secondary antibody described in embodiment 9. wraps up Co3O4Solution is prepared according to the following steps:
Logical N is carried out to 25 mL, the apoferritin solution that concentration is 150 μ g/mL with capillary2It handles, by the solution after 1 h
Water bath with thermostatic control is carried out, temperature is maintained at 65 DEG C, and adjusts pH to 9.5 with the sodium hydroxide solution of 0.1 mol/L, is added therewith
After stirring 1 h, 1.2 mL are added, the mistake that concentration is 10 mmol/L in 25 mL, the cabaltous nitrate hexahydrate that concentration is 0.05 mol/L
Hydrogen peroxide solution simultaneously reacts 5 min of holding, 10 mL is added, the sodium citrate solution that concentration is 10 mmol goes chelating not enter
Free cobalt ions inside iron ferritin obtains the ferritin package Co of secondary antibody label by centrifugation, dialysis, after purification3O4It is molten
Liquid；
2 mL ferritins are taken to wrap up Co3O42.5 mL are added, N- (4- ammonia butyl)-N- ethyl that concentration is 10 mmol/L in solution
The glutaraldehyde solution of different luminol solution and 150 uL, mass fraction 50%, 300 μ L are added in mixing after centrifuge separation, concentration is
The parathyroid hormone two corresponding anti-solution of 10 mg/mL, 18 h of oscillation hatching, are distributed to 1 mL pH's 7.4 under 4 °C after centrifugation
The ferritin package Co of secondary antibody label is obtained in phosphate buffer solution3O4Solution is placed under 4 °C and stores for future use.
The technology is used for the detection of parathyroid hormone levels by embodiment 10., and operating procedure is as follows:
(1) parameter setting: the photomultiplier tube high pressure of ultraweak electrogenerated chemiluminescence instrument is set as 600 V, electrochemical workstation
Cyclic voltammetry scan potential range is set as 0 ~ 0.6 V, and sweep speed is set as 0.1 V/s；
(2) test: using silver/silver chloride electrode as reference electrode, platinum electrode is to electrode, the sensing that in the above way prepares
Device is working electrode, and electrogenerated chemiluminescence survey is carried out in phosphate buffer solution of 10 mL containing 45 mmol/L hydrogen peroxide
Examination obtains corresponding electrochemiluminescence signal intensity when hatching various concentration parathyroid hormone, draws working curve, inspection
Rising limit is 13 fg/mL, 50 fg/mL-50 ng/mL of the range of linearity；
(3) it is tested the Electrochemiluminescsensor sensor for the parathyroid hormone actual sample for hatching unknown concentration to obtain phase
The signal strength answered calculates the parathyroid hormone levels that can be obtained in the reagent sample according to working curve accordingly.
The technology is used for the detection of parathyroid hormone levels by embodiment 11., and operating procedure is as follows:
(1) parameter setting: the photomultiplier tube high pressure of ultraweak electrogenerated chemiluminescence instrument is set as 600 V, electrochemical workstation
Cyclic voltammetry scan potential range is set as 0 ~ 0.6 V, and sweep speed is set as 0.1 V/s；
(2) test: using silver/silver chloride electrode as reference electrode, platinum electrode is to electrode, the sensing that in the above way prepares
Device is working electrode, and electrogenerated chemiluminescence survey is carried out in phosphate buffer solution of 10 mL containing 65 mmol/L hydrogen peroxide
Examination obtains corresponding electrochemiluminescence signal intensity when hatching various concentration parathyroid hormone, draws working curve, inspection
Rising limit is 13 fg/mL, 50 fg/mL-50 ng/mL of the range of linearity；
(3) it is tested the Electrochemiluminescsensor sensor for the parathyroid hormone actual sample for hatching unknown concentration to obtain phase
The signal strength answered calculates the parathyroid hormone levels that can be obtained in the reagent sample according to working curve accordingly.
The technology is used for the detection of parathyroid hormone levels by embodiment 12., and operating procedure is as follows:
(1) parameter setting: the photomultiplier tube high pressure of ultraweak electrogenerated chemiluminescence instrument is set as 600 V, electrochemical workstation
Cyclic voltammetry scan potential range is set as 0 ~ 0.6 V, and sweep speed is set as 0.1 V/s；
(2) test: using silver/silver chloride electrode as reference electrode, platinum electrode is to electrode, the sensing that in the above way prepares
Device is working electrode, and electrogenerated chemiluminescence survey is carried out in phosphate buffer solution of 10 mL containing 75 mmol/L hydrogen peroxide
Examination obtains corresponding electrochemiluminescence signal intensity when hatching various concentration parathyroid hormone, draws working curve, inspection
Rising limit is 13 fg/mL, 50 fg/mL-50 ng/mL of the range of linearity；
(3) it is tested the Electrochemiluminescsensor sensor for the parathyroid hormone actual sample for hatching unknown concentration to obtain phase
The signal strength answered calculates the parathyroid hormone levels that can be obtained in the reagent sample according to working curve accordingly.
Claims (5)
1. one kind wraps up Co based on ferritin3O4The preparation method of the biosensor of core-shell structure, which is characterized in that including with
Lower step:
(1) glass-carbon electrode that diameter is 4 mm is successively used into 1.0 μm, 0.3 μm, 0.05 μm of aluminum oxide polishing powder polishing treatment,
It is clean with ultrapure water；
(2) the golden hydridization graphite alkene aeroge marked in 6 μ L of glassy carbon electrode surface drop coating, the primary antibody that concentration is 2 ~ 4 mg/mL
Solution is sensing substrate, places it in 37 °C and dries；
(3) 3 μ L are added dropwise, the bovine serum albumin solution that mass fraction is 1 ~ 3%, it is living with the non-specificity on enclosed-electrode surface
Property site, rinse electrode surface with the phosphate buffer solution PBS of pH 7.4, place it in 4 °C and dry；
(4) standard solution of 6 μ L parathyroid hormones or the solution of parathyroid hormone of unknown concentration is added dropwise, hatches under 37 °C
0.5 ~ 2 h rinses electrode surface with the phosphate buffer solution PBS of pH 7.4, places it in 4 °C and dry；
(5) 6 μ L are added dropwise, the ferritin that the secondary antibody that concentration is 2 ~ 4 mg/mL marks wraps up Co3O4Solution places it in 37 °C
It dries, rinses electrode surface with the phosphate buffer solution PBS of pH 7.4, place it in 4 °C and dry, sensor building finishes.
2. as described in claim 1 a kind of based on ferritin package Co3O4The preparation method of the biosensor of core-shell structure,
It is characterized in that, the golden hydridization graphite alkene airsetting sol solution of the primary antibody label, prepares according to the following steps:
Take the HAuCl of 4 ~ 6 mL 1%4Solution and 100 mL ultrapure waters are added to the three neck round bottom flask of 250 mL of clean dried
In, under constant stirring, the sodium citrate solution that drop is slowly added to 10 mL thereto, mass fraction is 1%, it is heated to reflux 15 ~
35 min stop heating after solution colour eventually becomes claret, through supercooling and gold nanoparticle are obtained by filtration；
The graphite alkene solution that 20 mL concentration are 1.5 ~ 3.5 mg/mL is prepared, under uniform stirring, 3 ~ 5 mL Jenners are added
Rice corpuscles vibrates 6 h, and with ultrapure water 8 h of dialysis, the golden hydridization graphite alkene gas of 3D structure can be obtained after freeze-drying later
Gel；
The golden hydridization graphite alkene airsetting sol solution of 2 mg/mL is prepared, 100 ~ 300 μ L are added, the first shape that concentration is 10 mg/mL
Other glandular hormone primary antibody solution, 6 ~ 18 h of oscillation hatching, are distributed to the phosphate-buffered of 1 mL pH 7.4 under 4 °C after centrifugation
The golden hydridization graphite alkene airsetting sol solution that primary antibody label is obtained in solution, is placed under 4 °C and stores for future use.
3. as described in claim 1 a kind of based on ferritin package Co3O4The preparation method of the biosensor of core-shell structure,
It is characterized in that, the ferritin of the secondary antibody label wraps up Co3O4Solution is prepared according to the following steps:
Logical N is carried out to 25 mL, the apoferritin solution that concentration is 50 ~ 150 μ g/mL with capillary2It handles, it should after 1 h
Solution carries out water bath with thermostatic control, and temperature is maintained at 65 DEG C, and adjusts pH to 9.5 with the sodium hydroxide solution of 0.1 mol/L, therewith
25 mL are added, the cabaltous nitrate hexahydrate that concentration is 0.05 mol/L, after stirring 1 h, 0.4 ~ 1.2 mL, concentration 10 is added
The hydrogenperoxide steam generator of mmol/L simultaneously reacts 5 min of holding, 10 mL is added, the sodium citrate solution that concentration is 10 mmol goes chela
It closes and does not enter free cobalt ions inside apoferritin, by centrifugation, dialysis, after purification, obtain the ferritin that secondary antibody marks
Wrap up Co3O4Solution；
2 mL ferritins are taken to wrap up Co3O40.5 ~ 2.5 mL is added, the N- (4- ammonia butyl)-that concentration is 10 mmol/L in solution
The glutaraldehyde solution of N- ethyl different luminol solution and 50 ~ 150 uL, mass fraction 50%, mixing is added 100 after centrifuge separation
~ 300 μ L, the parathyroid hormone two corresponding anti-solution that concentration is 10 mg/mL, 6 ~ 18 h of oscillation hatching under 4 °C, after centrifugation
It is distributed to the ferritin package Co that secondary antibody label is obtained in the phosphate buffer solution of 1 mL pH 7.43O4Solution is placed in 4 °C
Under store for future use.
4. the detection that the biosensor of preparation method preparation as described in claim 1 is used for parathyroid hormone levels.
5. the detection of parathyroid hormone levels as claimed in claim 4, which is characterized in that operating procedure is as follows:
(1) parameter setting: the photomultiplier tube high pressure of ultraweak electrogenerated chemiluminescence instrument is set as 600 V, electrochemical workstation
Cyclic voltammetry scan potential range is set as 0 ~ 0.6 V, and sweep speed is set as 0.1 V/s；
(2) test: using silver/silver chloride electrode as reference electrode, platinum electrode is to electrode, the sensing that in the above way prepares
Device is working electrode, and electroluminescent chemistry hair is carried out in phosphate buffer solution of 10 mL containing 45 ~ 75 mmol/L hydrogen peroxide
Optical tests obtain corresponding electrochemiluminescence signal intensity when hatching various concentration parathyroid hormone, draw working curve,
Its detection is limited to 13 fg/mL, 50 fg/mL-50 ng/mL of the range of linearity；
(3) it is tested the Electrochemiluminescsensor sensor for the parathyroid hormone actual sample for hatching unknown concentration to obtain phase
The signal strength answered calculates the parathyroid hormone levels that can be obtained in the reagent sample according to working curve accordingly.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910787983.0A CN110441294B (en) | 2019-08-26 | 2019-08-26 | Co-coated based on ferritin3O4Preparation method of biosensor with core-shell structure |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910787983.0A CN110441294B (en) | 2019-08-26 | 2019-08-26 | Co-coated based on ferritin3O4Preparation method of biosensor with core-shell structure |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN110441294A true CN110441294A (en) | 2019-11-12 |
| CN110441294B CN110441294B (en) | 2021-10-26 |
Family
ID=68437574
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201910787983.0A Active CN110441294B (en) | 2019-08-26 | 2019-08-26 | Co-coated based on ferritin3O4Preparation method of biosensor with core-shell structure |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN110441294B (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111751435A (en) * | 2020-06-22 | 2020-10-09 | 济南大学 | Preparation method and application of ferroportin-based encapsulated tetraphenylethylene immunosensor |
| CN111766287A (en) * | 2020-06-22 | 2020-10-13 | 济南大学 | Ferro-ferritin-based packaging Ru (bpy)32+Preparation method and application of immunosensor |
| CN114047178A (en) * | 2021-11-03 | 2022-02-15 | 济南大学 | Preparation and application of functionalized Zn-Co bimetal core-shell ZIF-9@ ZIF-8 composite material |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102778453A (en) * | 2012-08-08 | 2012-11-14 | 济南大学 | Manufacture method and application of silver hybridization SBA-15 electrochemical luminescence immunosensor |
| CN104677892A (en) * | 2015-03-27 | 2015-06-03 | 济南大学 | Method for preparing electrochemiluminescence biological sensing interface based on loaded graphite phase carbon nitride and application thereof |
| US20150247816A1 (en) * | 2014-03-03 | 2015-09-03 | The Florida International University Board Of Trustees | Label-free electrochemical biosensor |
| CN109212197A (en) * | 2018-08-09 | 2019-01-15 | 迪瑞医疗科技股份有限公司 | Parathormone kit and preparation method thereof |
-
2019
- 2019-08-26 CN CN201910787983.0A patent/CN110441294B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102778453A (en) * | 2012-08-08 | 2012-11-14 | 济南大学 | Manufacture method and application of silver hybridization SBA-15 electrochemical luminescence immunosensor |
| US20150247816A1 (en) * | 2014-03-03 | 2015-09-03 | The Florida International University Board Of Trustees | Label-free electrochemical biosensor |
| CN104677892A (en) * | 2015-03-27 | 2015-06-03 | 济南大学 | Method for preparing electrochemiluminescence biological sensing interface based on loaded graphite phase carbon nitride and application thereof |
| CN109212197A (en) * | 2018-08-09 | 2019-01-15 | 迪瑞医疗科技股份有限公司 | Parathormone kit and preparation method thereof |
Non-Patent Citations (2)
| Title |
|---|
| SHIJIE LI ET AL: "A novel polydopamine electrochemiluminescence organic nanoparticle-based biosensor for parathyroid hormone detection", 《TALANTA》 * |
| 朱利国 等: "甲状旁腺激素荧光免疫层析定量检测技术的研制及应用研究", 《现代免疫学》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111751435A (en) * | 2020-06-22 | 2020-10-09 | 济南大学 | Preparation method and application of ferroportin-based encapsulated tetraphenylethylene immunosensor |
| CN111766287A (en) * | 2020-06-22 | 2020-10-13 | 济南大学 | Ferro-ferritin-based packaging Ru (bpy)32+Preparation method and application of immunosensor |
| CN114047178A (en) * | 2021-11-03 | 2022-02-15 | 济南大学 | Preparation and application of functionalized Zn-Co bimetal core-shell ZIF-9@ ZIF-8 composite material |
| CN114047178B (en) * | 2021-11-03 | 2023-12-22 | 济南大学 | Preparation and application of functionalized Zn-Co bimetallic core-shell ZIF-9@ZIF-8 composite material |
Also Published As
| Publication number | Publication date |
|---|---|
| CN110441294B (en) | 2021-10-26 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN103116023B (en) | ECL (electrochemiluminescence) immunosensor for detecting tumor markers and preparation method and applications thereof | |
| CN110441294A (en) | One kind wrapping up Co based on ferritin3O4The preparation method of the biosensor of core-shell structure | |
| CN106442994B (en) | A kind of preparation method and application of the electrochemical immunosensor based on Ag@Au nano composite materials | |
| CN106596969B (en) | A kind of preparation of electrochemiluminescimmunosensor immunosensor, product, detection and application | |
| CN102980925B (en) | Sandwich type electrochemical immunosensor, preparation method and application thereof | |
| CN102818893B (en) | Preparation and application of Au-Pd core-shell material constructed lung cancer tumor marker immunosensor | |
| CN109839501B (en) | Electrochemiluminescence immunosensor for measuring circulating tumor cells and preparation method and application thereof | |
| CN108982630A (en) | A kind of preparation method and application of the electrochemical immunosensor of interlayer type detection prostate-specific antigen | |
| CN104880456B (en) | A kind of based on GO/MWCNTs-COOH/Au@CeO2the preparation method and application of the electrochemiluminescence immunosensor built | |
| CN104459132B (en) | A kind of is preparation method and the application of the cancer of pancreas immunosensor of label based on golden electro-deposition and Au@Ag/CuO-GS | |
| CN104297479B (en) | The preparation of detection tumor markers electrochemiluminescimmunosensor immunosensor and application | |
| CN110455786A (en) | One kind being based on CeO2@SnS2Promote the preparation method of luminol Electrochemiluminescsensor sensor | |
| CN110441295A (en) | One kind is based on ferritin encapsulation Ir (ppy)3Biosensor preparation method | |
| CN110231335A (en) | A kind of preparation method and application based on ferritin three-dimensional structure immunosensor | |
| CN108051491B (en) | It is a kind of for detecting the electrochemical immunosensor of LAG-3 albumen | |
| CN109507256B (en) | Unmarked electrochemiluminescence aptamer sensor for detecting carcino-embryonic antigen and preparation method and using method thereof | |
| CN105954339B (en) | A kind of preparation method and application of the interlayer type immunosensor based on CeO2@Cu2O/Au@Pt | |
| CN109613244B (en) | Preparation method and application of Ag @ Pt-CuS labeled immunosensor | |
| CN108896638B (en) | Preparation method and application of immunosensor based on titanium dioxide doped graphene loaded sea cucumber-like gold-palladium core-shell nanoparticles | |
| CN104931698A (en) | Preparation method and application of NP-NiGd@Au-based gastric cancer marker gold nano-cluster electrogenerated chemiluminescence sensor | |
| CN106198699B (en) | Prepare two kinds of secondary antibody conjugates and its at the same the method that detects alpha-fetoprotein and carcinomebryonic antigen | |
| CN110554027A (en) | preparation method and application of immunosensor for promoting gold nanocluster electroluminescent response based on iron oxide array coreaction | |
| CN105842460A (en) | Preparation method of electro-chemiluminescence immunosensor based on silver-hybridized bismuth sulfide | |
| CN106093396A (en) | A kind of preparation method and application of immunosensor based on Au GQD@PtPd | |
| CN110441528B (en) | Mo based on core-shell structure2Construction of C @ C nanosphere cardiac troponin I immunosensor |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |