CN107741415A - One kind is based on magnetic Nano assembly double check small molecule and method of protein - Google Patents
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Abstract
One kind is based on magnetic Nano assembly double check small molecule and method of protein, belongs to biology sensor detection field.The present invention utilizes the interaction of biotin and Streptavidin, Streptavidin and biotin are modified to the surface in gold-magnetic nanoparticle and golden nanometer particle respectively, and in golden nanometer particle surface modification beacon molecule, under conditions of different content target molecule being present, the assembly and the difference of the content of free golden nanometer particle gone out by Magneto separate, so that the Raman signal of supernatant precipitation changes therewith, the relation changed according to target concentration and Raman signal intensity, can realize the detection of biotin and Streptavidin respectively.The present invention realizes the detection of same system small molecular and protein, and detection sensitivity is high, and actual sample addition recovery result is good, and the foundation for small molecule and the double check system of protein provides good theoretical foundation.
Description
Technical field
The invention belongs to biology sensor detection field, and in particular to one kind is small based on magnetic Nano assembly double check
Molecule and method of protein.
Background technology
It is extremely important in biomedical research and the interaction of clinical practice area research small molecule and protein, such as
Small molecule physiologically and the interaction of the interaction of protein, small-molecule drug and corresponding protein etc., simultaneously
The protein related with signal transduction, physiological metabolism with small molecular phase interaction in most cases by realizing corresponding work(
Can, therefore small molecule and protein are detected and played an important role in Pharmaceutical Analysis and clinical diagnosis field.
Small molecule is different with the physicochemical property of protein, and applicable detection architecture is different, generally requires individually detection body
System could realize the detection respectively of two kinds of materials.At present, the detection method of small molecule mainly includes HPLC MS
(HPLC-MS), gas-chromatography(GC), gas chromatography-mass spectrometry(GC-MS)Deng the detection method of protein mainly includes
EUSA(ELISA), it is protein imprinted(WB), colorimetric method, fluorescence method etc..Therefore, in order to improve detection efficiency,
Testing cost is reduced, detection small molecule and method of protein in same system is developed and is particularly important.
Raman spectrum belongs to molecular vibration spectrum, can reflect the feature structure of molecule, but Raman scattering effect is
Very weak process, with the further development of scientific research, it has been found that raman scattering intensity can be real in coarse metal surface
Now strengthen, cause the extensive research interest of scientific worker.In addition to the structure for analyzing molecules, surface-enhanced Raman
Spectrum also becomes
A kind of detection technique of rapid sensitive, especially using noble metal nanometer material as substrate, Raman signal intensity improves multiple
The order of magnitude, the signal that conventional method can not detect can be realized, or even Single Molecule Detection level can be reached, so that expensive
The research range of metal nanoparticle monomer or assembly in Raman spectrum constantly expands.
The content of the invention
Technical problems to be solved:Existing detection small molecule and method of protein need to enter in independent system
OK, so as to reduce detection efficiency, testing cost is improved.
Technical scheme:The invention discloses a kind of side based on magnetic Nano assembly double check small molecule and protein
Method, comprise the following steps:
(1)The synthesis of magnetic core golden shell composite nanoparticle
The magnetic nanometer powder 10mg newly synthesized is weighed, 100mL ultra-pure water ultrasonic disperse 10min are added, then in stirring
The gold chloride that 5mL mass concentrations are 0.4% is added under state, adding 3mL mass concentrations in the state of ultrasound after stirring is
1% sodium citrate solution, continuation ultrasound is terminated until solution colour by the light yellow black reaction that gradually becomes, in externally-applied magnetic field
In the presence of carry out Magneto separate, supernatant is removed, and cleaned sediment 3 times using Magneto separate with ultra-pure water, so as to obtain golden shell
The magnetic composite of parcel, the thickness of golden shell is 5nm.
(2)The synthesis of golden nanometer particle
The method that golden nanometer particle reduces gold chloride by trisodium citrate synthesizes, and carries out ultraviolet letter by ultra-violet absorption spectrum
Number sign, pass through the sign that transmission electron microscope carries out configuration of surface and particle diameter.
(3)Nanoparticle Modified beacon molecule
By step(2)The golden nanometer particle of synthesis centrifuges 5min under conditions of 8000r/min, removes supernatant with pH7.2's
Golden nanometer particle is resuspended 0.01M PBSs, beacon molecule is added into golden nanometer particle so that its is final concentration of
5 ~ 10 μM, 4h is incubated at room temperature, and 5min is then centrifuged under conditions of 8000r/min and removes supernatant, then will with PBS
It is resuspended.
(4)Golden magnetic composite nanoparticle modifies Streptavidin
Take 5mL steps(1)The golden magnetic composite nanoparticle of synthesis adsorbs in the presence of externally-applied magnetic field, removes supernatant pH7.2
0.01M PBSs nano-particle is resuspended, and with 0.1M K2CO3PH is adjusted to 8.6 by solution, is then added
500 μ L concentration are 1mg/mL Streptavidin, and 30min is reacted under conditions of slowly vibrating, it is molten then to add 500 μ L BSA
Liquid causes final concentration of the 1% of BSA, then 40min is reacted under conditions of vibration, removes supernatant by externally-applied magnetic field, is used in combination
Nano-particle is resuspended pH7.2 0.01M PBSs, and the strepto- for obtaining golden magnetic composite nanoparticle modification is affine
Element.
(5)Golden nanometer particle modified biological element
Take 2mL steps(3)The golden nanometer particle of preparation, its final concentration of 20nM is measured, add the DNA of biotin modification thereto
Molecule so that DNA final concentration of 40nM, after it is incubated into 6h at room temperature, 5min is centrifuged under conditions of 8000r/min,
Obtain the biotin of golden nanometer particle modification;
The DNA of biotin modification:5’-SH-AAAAA-Biotin-3’.
(6)The preparation of magnetic Nano assembly and the double check of small protein
By 40 μ L steps(4)The nano-particle of preparation and 50 μ L steps(5)The nano-particle mixing of preparation, it is dense to be separately added into 10 μ L
The biotin for 0nM, 0.1nM, 0.5nM, 1nM, 5nM, 10nM, 20nM, 50nM is spent, another group of detection architecture is separately added into concentration
For 0nM, 0.5nM, 1nM, 5nM, 10nM, 20nM, 50nM, 100nM Streptavidin, 0.5 ~ 2h is incubated at ambient temperature,
Subsequent externally-applied magnetic field removes supernatant, and the magnetic nano-complex particle of absorption is resuspended with 50 μ L ultra-pure waters, determines supernatant respectively
Magnetic nano-complex particle is in 1334 cm-1The SERS intensity at place.
Magnetic of the present invention based on described in magnetic Nano assembly double check small molecule and method of protein
The synthesis step of nano-particle is:Anhydrous sodium acetate and FeCl are added in 50mL ethylene glycol3·6H2O so that its final concentration
Respectively 10mM and 2mM, ultrasonic disperse 5min is carried out, is then transferred in reactor, 8h is reacted under conditions of 200 DEG C, instead
Reactor is taken out after should terminating and is allowed to naturally cool to room temperature, with ultra-pure water and ethylene glycol alternately washing 5 times, washs last
Secondary sediment is disperseed with 50mL toluene, is poured into round-bottomed flask, adds 1mL 3- aminopropyl triethoxysilanes, stirring
Lower 80 DEG C of back flow reaction 6h, room temperature is cooled to, then is alternately washed 5 times with ultra-pure water and ethylene glycol, finally by magnetic nanoparticle
The magnetic nanometer that the particle diameter that sub- sediment is dried in vacuo to obtain solid-state is 10nm.
Gold of the present invention based on described in magnetic Nano assembly double check small molecule and method of protein
The synthesis step of nano-particle is:95mL ultra-pure waters are added in clean conical flask, add 2.5mL mass concentrations as 0.4%
Chlorauric acid solution, be placed in heating magnetic stirring apparatus on be heated to seething with excitement, seethe with excitement 10min after, be rapidly added 2.5mL mass concentrations
For 1% citric acid three sodium solution, after solution from it is colourless be changed into claret after continuous heating 5min, continue to stir after stopping heating
Room temperature is cooled to solution, the maximum absorption band of the golden nanometer particle for scanning to obtain by ultra-violet absorption spectrum passes through in 521nm
The particle diameter of transmission electron microscope observation to golden nanometer particle is 10nm.
Letter of the present invention based on described in magnetic Nano assembly double check small molecule and method of protein
Mark molecule is 4- nitrobenzenethiols.
Letter of the present invention based on described in magnetic Nano assembly double check small molecule and method of protein
Mark final concentration of 6 μM that molecule is 4- nitrobenzenethiols.
Chain of the present invention based on described in magnetic Nano assembly double check small molecule and method of protein
The incubation time of the magnetic nano-complex particle of mould Avidin modification and the golden nanometer particle of biotin modification is 1h.
Beneficial effect:The present invention has synthesized the golden magnetic composite nanoparticle wrapped up using magnetic particle as core golden shell first, makes
Nano-particle with good biocompatibility, stability and while be easy to modified with Magneto separate characteristic.Utilize life
The interaction of thing element and Streptavidin, Streptavidin and biotin are modified in gold-magnetic nanoparticle and gold nano respectively
The surface of particle, and in golden nanometer particle surface modification beacon molecule, under conditions of target molecule, using biotin and
The affine combination of Streptavidin, golden nanometer particle, which is modified to be formed on the surface of golden magnetic particle, defends star topology, passes through Magneto separate
Effect, golden magnetic particle and golden magnetic particle-golden nanometer particle assembly are separated with golden nanometer particle supernatant, Raman in supernatant
Signal intensity diminishes, and Raman signal shows significant enhancing in precipitation.With the addition of target molecule, the quantitative change of assembly
Few, free golden nanometer particle increases, so that the Raman signal intensity in precipitation diminishes, Raman signal intensity increases in supernatant
Greatly, the relation changed according to biotin or Streptavidin and Raman signal intensity, it is possible to achieve biotin or Streptavidin
Independent detection in same system.
Brief description of the drawings
The TEM figures of Fig. 1 satellite shapes nanostructured assembling body.
The standard curve of Fig. 2 biotins detection.
The standard curve of Fig. 3 Streptavidins detection.
Embodiment
Embodiment 1
One kind is based on magnetic Nano assembly double check small molecule and method of protein, comprises the following steps:
(1)The synthesis of magnetic nanometer
Anhydrous sodium acetate and FeCl are added in 50mL ethylene glycol3·6H2O so that its final concentration is respectively 10mM and 2mM,
Ultrasonic disperse 5min is carried out, is then transferred in reactor, 8h is reacted under conditions of 200 DEG C, is reacted reactor after terminating
Taking-up is allowed to naturally cool to room temperature, is alternately washed 5 times with ultra-pure water and ethylene glycol, washs the sediment 50mL of last time
Toluene disperse, pour into round-bottomed flask, add 1mL 3- aminopropyl triethoxysilanes, stir lower 80 DEG C of back flow reaction 6h,
Room temperature is cooled to, then is alternately washed 5 times with ultra-pure water and ethylene glycol, finally does magnetic nanometer sediment progress vacuum
The dry particle diameter for obtaining solid-state is 10nm magnetic nanometer.
(2)The synthesis of magnetic core golden shell composite nanoparticle
The magnetic nanometer powder 10mg newly synthesized is weighed, 100mL ultra-pure water ultrasonic disperse 10min are added, then in stirring
The gold chloride that 5mL mass concentrations are 0.4% is added under state, adding 3mL mass concentrations in the state of ultrasound after stirring is
1% sodium citrate solution, continuation ultrasound is terminated until solution colour by the light yellow black reaction that gradually becomes, in externally-applied magnetic field
In the presence of carry out Magneto separate, supernatant is removed, and cleaned sediment 3 times using Magneto separate with ultra-pure water, so as to obtain golden shell
The magnetic composite of parcel, the thickness of golden shell is 5nm.
(3)The synthesis of golden nanometer particle
The method that golden nanometer particle reduces gold chloride by trisodium citrate synthesizes, and synthesis step is:95mL ultra-pure waters are added
In clean conical flask, the chlorauric acid solution that 2.5mL mass concentrations are 0.4% is added, is placed on heating magnetic stirring apparatus and heats
To boiling, after the 10min that seethes with excitement, the citric acid three sodium solution that 2.5mL mass concentrations are 1% is rapidly added, treats that solution is changed into from colourless
Continuous heating 5min after claret, stop continuing to stir to solution after heating being cooled to room temperature, scanned by ultra-violet absorption spectrum
The maximum absorption band of obtained golden nanometer particle passes through the particle diameter of transmission electron microscope observation to golden nanometer particle in 521nm
For 10nm.
(4)Nanoparticle Modified beacon molecule
By step(3)The golden nanometer particle of synthesis centrifuges 5min under conditions of 8000r/min, removes supernatant with pH7.2's
Golden nanometer particle is resuspended 0.01M PBSs, and beacon molecule 4- nitrobenzenethiols are added into golden nanometer particle, are made
Its final concentration of 6 μM are obtained, is incubated 4h at room temperature, then centrifuges 5min under conditions of 8000r/min and removes supernatant, is delayed with PBS
Fliud flushing is resuspended.
(5)Golden magnetic composite nanoparticle modifies Streptavidin
Take 5mL steps(2)The golden magnetic composite nanoparticle of synthesis adsorbs in the presence of externally-applied magnetic field, removes supernatant pH7.2
0.01M PBSs nano-particle is resuspended, and with 0.1M K2CO3PH is adjusted to 8.6 by solution, is then added
500 μ L concentration are 1mg/mL Streptavidin, and 30min is reacted under conditions of slowly vibrating, it is molten then to add 500 μ L BSA
Liquid causes final concentration of the 1% of BSA, then 40min is reacted under conditions of vibration, removes supernatant by externally-applied magnetic field, is used in combination
Nano-particle is resuspended pH7.2 0.01M PBSs, and the strepto- for obtaining golden magnetic composite nanoparticle modification is affine
Element.
(6)Golden nanometer particle modified biological element
Take 2mL steps(4)The golden nanometer particle of preparation, its final concentration of 20nM is measured, add the DNA of biotin modification thereto
Molecule so that DNA final concentration of 40nM, after it is incubated into 6h at room temperature, 5min is centrifuged under conditions of 8000r/min,
Obtain the biotin of golden nanometer particle modification.
The DNA of biotin modification:5’-SH-AAAAA-Biotin-3’.
(7)The preparation of magnetic Nano assembly and the double check of small protein
By 40 μ L steps(4)The nano-particle of preparation and 50 μ L steps(5)The nano-particle mixing of preparation, it is dense to be separately added into 10 μ L
The biotin for 0nM, 0.1nM, 0.5nM, 1nM, 5nM, 10nM, 20nM, 50nM is spent, another group of detection architecture is separately added into concentration
For 0nM, 0.5nM, 1nM, 5nM, 10nM, 20nM, 50nM, 100nM Streptavidin, 1h is incubated at ambient temperature, then
Externally-applied magnetic field removes supernatant, and the magnetic nano-complex particle of absorption is resuspended with 50 μ L ultra-pure waters, determines supernatant magnetic respectively and receives
Rice compound particle is in 1334 cm-1The SERS intensity at place.According to the concentration of detectable substance and SERS signal intensity it
Between relation establish mark curve learn that linear relationship is good in the range of 0.1 ~ 10nM biotin concentration, the inspection of biotin
Survey is limited to 0.043nM, and linear relationship is good in 0.5 ~ 50nM Streptavidin concentration range, the test limit of Streptavidin
For 0.028nM.
(8)The detection of actual sample
In order to verify feasibility that this method detects in clinical sample, the biotin of various concentrations and Streptavidin are added respectively
It is added in serum sample, the addition concentration of biotin is:0.2nM, 0.8nM, 1.5nM, 3.6nM, 5.2nM, Streptavidin
Adding concentration is:1.5nM, 3.5nM, 5.8nM, 8.3nM, 10.6nM, the TIANZHU XINGNAO Capsul that biotin is measured with the method are
95.4 ~ 98.1%, the TIANZHU XINGNAO Capsul of Streptavidin is 96.2 ~ 99.3%, and addition recovery result is within the acceptable range.
Claims (6)
1. one kind is based on magnetic Nano assembly double check small molecule and method of protein, it is characterised in that including following step
Suddenly:
(1)The synthesis of magnetic core golden shell composite nanoparticle
The magnetic nanometer powder 10mg newly synthesized is weighed, 100mL ultra-pure water ultrasonic disperse 10min are added, then in stirring
The gold chloride that 5mL mass concentrations are 0.4% is added under state, adding 3mL mass concentrations in the state of ultrasound after stirring is
1% sodium citrate solution, continuation ultrasound is terminated until solution colour by the light yellow black reaction that gradually becomes, in externally-applied magnetic field
In the presence of carry out Magneto separate, supernatant is removed, and cleaned sediment 3 times using Magneto separate with ultra-pure water, so as to obtain golden shell
The magnetic composite of parcel, the thickness of golden shell is 5nm;
(2)The synthesis of golden nanometer particle
The method that golden nanometer particle reduces gold chloride by trisodium citrate synthesizes, and carries out ultraviolet letter by ultra-violet absorption spectrum
Number sign, pass through the sign that transmission electron microscope carries out configuration of surface and particle diameter;
(3)Golden nanometer particle modifies beacon molecule
By step(2)The golden nanometer particle of synthesis centrifuges 5min under conditions of 8000r/min, removes supernatant with pH7.2's
Golden nanometer particle is resuspended 0.01M PBSs, beacon molecule is added into golden nanometer particle so that its is final concentration of
5 ~ 10 μM, 4h is incubated at room temperature, and 5min is then centrifuged under conditions of 8000r/min and removes supernatant, then will with PBS
It is resuspended;
(4)Golden magnetic composite nanoparticle modifies Streptavidin
Take 5mL steps(1)The golden magnetic composite nanoparticle of synthesis adsorbs in the presence of externally-applied magnetic field, removes supernatant pH7.2
0.01M PBSs nano-particle is resuspended, and with 0.1M K2CO3PH is adjusted to 8.6 by solution, is then added
500 μ L concentration are 1mg/mL Streptavidin, and 30min is reacted under conditions of slowly vibrating, it is molten then to add 500 μ L BSA
Liquid causes final concentration of the 1% of BSA, then 40min is reacted under conditions of vibration, removes supernatant by externally-applied magnetic field, is used in combination
Nano-particle is resuspended pH7.2 0.01M PBSs, and the strepto- for obtaining golden magnetic composite nanoparticle modification is affine
Element;
(5)Golden nanometer particle modified biological element
Take 2mL steps(3)The golden nanometer particle of preparation, its final concentration of 20nM is measured, add the DNA of biotin modification thereto
Molecule so that DNA final concentration of 40nM, after it is incubated into 6h at room temperature, 5min is centrifuged under conditions of 8000r/min,
Obtain the biotin of golden nanometer particle modification;
The DNA of biotin modification:5’-SH-AAAAA-Biotin-3’;
(6)The preparation of magnetic Nano assembly and the double check of small protein
By 40 μ L steps(4)The nano-particle of preparation and 50 μ L steps(5)The nano-particle mixing of preparation, it is dense to be separately added into 10 μ L
The biotin for 0nM, 0.1nM, 0.5nM, 1nM, 5nM, 10nM, 20nM, 50nM is spent, another group of detection architecture is separately added into concentration
For 0nM, 0.5nM, 1nM, 5nM, 10nM, 20nM, 50nM, 100nM Streptavidin, 0.5 ~ 2h is incubated at ambient temperature,
Subsequent externally-applied magnetic field removes supernatant, and the magnetic nano-complex particle of absorption is resuspended with 50 μ L ultra-pure waters, determines supernatant respectively
Magnetic nano-complex particle is in 1334 cm-1The SERS intensity at place.
2. one kind according to claim 1 is based on magnetic Nano assembly double check small molecule and method of protein,
It is characterized in that the synthesis step of described magnetic nanometer is:In 50mL ethylene glycol add anhydrous sodium acetate and
FeCl3·6H2O so that its final concentration is respectively 10mM and 2mM, is carried out ultrasonic disperse 5min, is then transferred in reactor,
8h is reacted under conditions of 200 DEG C, reactor is taken out and is allowed to naturally cool to room temperature by reaction after terminating, with ultra-pure water and ethylene glycol
Alternately washing 5 times, the sediment for washing last time is disperseed with 50mL toluene, is poured into round-bottomed flask, adds 1mL 3- ammonia
Propyl-triethoxysilicane, lower 80 DEG C of back flow reaction 6h are stirred, be cooled to room temperature, then alternately washed with ultra-pure water and ethylene glycol
Wash 5 times, the magnetic nanometer that the particle diameter for finally being dried in vacuo to obtain solid-state by magnetic nanometer sediment is 10nm.
3. one kind according to claim 1 is based on magnetic Nano assembly double check small molecule and method of protein,
It is characterized in that the synthesis step of described golden nanometer particle is:95mL ultra-pure waters are added in clean conical flask, added
2.5mL mass concentrations are 0.4% chlorauric acid solution, are placed on heating magnetic stirring apparatus and are heated to seething with excitement, fast after the 10min that seethes with excitement
Speed adds the citric acid three sodium solution that 2.5mL mass concentrations are 1%, after solution from it is colourless be changed into claret after continuous heating 5min,
Stop continuing to stir to solution after heating being cooled to room temperature, the maximum for the golden nanometer particle for scanning to obtain by ultra-violet absorption spectrum
Absworption peak is 10nm in 521nm, the particle diameter by transmission electron microscope observation to golden nanometer particle.
4. one kind according to claim 1 is based on magnetic Nano assembly double check small molecule and method of protein,
It is characterized in that described beacon molecule is 4- nitrobenzenethiols.
5. one kind according to claim 4 is based on magnetic Nano assembly double check small molecule and method of protein,
It is characterized in that described beacon molecule is final concentration of 6 μM of 4- nitrobenzenethiols.
6. one kind according to claim 1 is based on magnetic Nano assembly double check small molecule and method of protein,
It is characterized in that the incubation for the magnetic nano-complex particle and the golden nanometer particle of biotin modification that described Streptavidin is modified
Time is 1h.
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CN109187484A (en) * | 2018-09-13 | 2019-01-11 | 广西师范大学 | A method of biotin is detected with compatible reaction regulation carbon dots catalysis SERS |
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CN109807344A (en) * | 2019-01-22 | 2019-05-28 | 天津大学 | The gold nanoparticle and the preparation method and application thereof of Norvancomycin modification |
CN110412014A (en) * | 2019-08-28 | 2019-11-05 | 济南大学 | Application of the Surface enhanced Raman scattering paper base sensor in tumor-marker analyte detection |
CN112904017A (en) * | 2021-01-19 | 2021-06-04 | 上海交通大学 | Detection system based on covalent connection for interaction between known molecules and proteins and identification or verification method thereof |
CN113189078A (en) * | 2021-03-04 | 2021-07-30 | 吉林大学 | High-throughput screening method for targeted drugs |
CN113189078B (en) * | 2021-03-04 | 2024-04-16 | 吉林大学 | High-throughput screening method of targeted drugs |
CN115976155A (en) * | 2022-12-25 | 2023-04-18 | 福州大学 | Visualized detection reagent and visualized detection method for SARS-CoV-2 protease |
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