CN107144610A - The method that gold nano grain adsorption albumen interacts with cell membrane associated receptor is detected based on quartz crystal chip in situ quantitation - Google Patents
The method that gold nano grain adsorption albumen interacts with cell membrane associated receptor is detected based on quartz crystal chip in situ quantitation Download PDFInfo
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
Abstract
The present invention relates to a kind of albumen and the method for cell membrane associated receptor interaction that the absorption of gold nano grain chiral surfaces is detected based on quartz crystal chip in situ quantitation, including:(1) quartz crystal chip is pre-processed using 2 mercaptopropionic acids and 2 mercaptoethanols;(2) treated chip is passed through EDC/NHS, by chip surface activated carboxylic in QCM D instruments;(3) nanogold particle of surface modification penicillamine small molecule is passed through, is combined by amido link with chip;(4) ligandin is passed through, makes ligandin absorption in the chiral surfaces of nanogold;(5) it is passed through the cell-derived liposome that associated receptor is contained on surface.The interaction of ligandin and associated receptor is quantified by the power of frequency signal.This method is simple to operate, practical, and the research to the 26S Proteasome Structure and Function of the albumen hat of nano grain surface formation has great significance.
Description
Technical field
The invention belongs to the innovative development field of dissipativeness QCM detection method, relate generally to one kind and be based on
The albumen of quartz crystal chip in situ quantitation detection gold nano grain chiral surfaces absorption interacts with cell membrane associated receptor
Method.
Background technology
Dissipativeness QCM (QCM-D) utilizes the piezoelectric property of quartz-crystal resonator, by quartz crystal core
The change of plate electrode surface quality is converted into the frequency change of quartz crystal oscillator circuit output electric signal, and then is obtained by computer
High-precision data are obtained, are a kind of new sensitive quality testing instruments, its measurement accuracy is up to nanogram level.
After nano material enters in human body, because specific surface area is larger, the albumen in human body fluid can be adsorbed, in material list
Face forms albumen hat.Albumen hat is suffered to biological effect, toxicological effect and the metabolic pathway that nano material enters after human body
Decisive action.Therefore, the interfacial structure important in inhibiting that research nano material is acted on protein.
Hundreds of kinds of albumen are there are in human body fluid, because nano material enters in body fluid and adhesion protein formation albumen hat,
Adsorbed albumen is structurally and functionally more or less being changed.Nano material and albumen are preced with complicated whole as one
Body, for same material, which kind of albumen is adsorbed;How albumen is adsorbed;26S Proteasome Structure and Function after albumen is adsorbed by how
Change, the problem of this is a series of are increasingly paid close attention to by people.Enter the difference of the albumen formed in vivo hat for different materials
And rule, also it is worth people further to study.
At present, the interfacial structure research to nano material and protein has many difficulties.Although circular dichroism, red
The technologies such as external spectrum, x-ray crystal diffraction can detect the structure changes at different levels of adsorbed albumen, but to adsorbed albumen
Physiological function change still feel simply helpless.
Chirality is a fundamental characteristics of nano material, and different chiral nano materials enter after human body, to albumen
Suction type is also otherwise varied, and to the change of protein function difference.But this species diversity generally smaller, conventional detection means
It is difficult to accurately detect out result.
QCM-D is simple to operate as micro-mass sensor, and sensitivity is high, on-line monitoring can be achieved, in chemistry, physics, life
It is widely used in the fields such as thing, medical science and Surface Science.In biological field, QCM-D is generally used for biological point of monitoring
Interaction between son, such as:Affinity interaction between Ag-Ab.It is generally a kind of raw in the modification of quartz crystal chip surface
Thing molecule, then another molecule is passed directly into, so that output electric signal caused by the mass change of detection chip electrode surface
Frequency changes.And it is complicated for nano material-albumen hat, at present, on based on the detection of quartz crystal chip in situ quantitation
The technological invention that the albumen of gold nano grain chiral surfaces absorption interacts with cell membrane associated receptor is there is not yet report.
The content of the invention
In order to distinguish influence of the different chiral nanomaterials to the function of the albumen of absorption, for QCM-D sensitivity it is high this
One feature, the present invention proposes a kind of egg that the absorption of gold nano grain chiral surfaces is detected based on quartz crystal chip in situ quantitation
The method interacted in vain with cell membrane associated receptor.
The present invention is devised on a kind of protein ligands and cell of the chiral surfaces absorption of in situ quantitation detection gold nano grain
The method of acceptor interaction.Gold nano grain containing chiral surfaces, ligandin, cell membrane surface receptors are successively in situ
With reference in quartz crystal chip surface, the interaction of the adsorbed ligandin of detection and acceptor, so as to further tell
Difference of the gold nano grain of different chiral surfaces to the albumen of absorption in terms of function change.This method is to nano particle-egg
The further research of white hat compound, there is vital meaning.
The present invention is realized by following technical proposals.
A kind of albumen and cell membrane that the absorption of gold nano grain chiral surfaces is detected based on quartz crystal chip in situ quantitation
The method of associated receptor interaction, comprises the following steps:
1) quartz crystal chip is pre-processed using 2 mercaptopropionic acid and 2 mercapto ethanol;
2) treated quartz crystal chip is put into QCM-D instruments, is passed through EDC/NHS, chip surface carboxyl is lived
Change;
3) nanogold particle of surface modification penicillamine small molecule is passed through, is combined by amido link with chip;
4) ligandin is passed through, makes ligandin absorption in the chiral surfaces of nanogold;
5) it is passed through the cell-derived liposome that associated receptor is contained on surface;By the power of frequency signal by part egg
The interaction with associated receptor is quantified in vain.
Specifically, the above method comprises the following steps:
1) quartz crystal chip is pre-processed:By clean quartz crystal chip 2 mercaptopropionic acid and 2 mercapto ethanol processing
12-24h, the sulfydryl small molecule being not bonded on chip is rinsed out with three water, is dried up with nitrogen stand-by;
2) activation of quartz crystal chip surface carboxyl:By step 1) pretreatment quartz crystal chip be put into QCM-D instrument
In device, first 100-150 μ l/min flow velocity is passed through (in QCM-D instruments, similarly hereinafter) three water, when signal curve is steady, then with
30-50 μ l/min flow velocity be passed through EDC (1- ethyls -3- [3- dimethylaminopropyls] carbodiimide hydrochloride) containing 75mM and
30mM NHS (n-hydroxysuccinimide) solution 1-2h, when signal curve is steady;
3) fixation of the nanogold particle of chiral surfaces:Three water are passed through with 100-150 μ l/min flow velocity, Zhi Daoxin
It is number steady, then it is passed through with 20-40 μ l/min flow velocitys the nanogold particle of the chiral surfaces containing penicillamine molecular modification, signature tune
Line is widely varied, until signal is steady;
4) absorption of ligandin:First with pH=7.4 Hepes (i.e. 4- hydroxyethyl piperazineethanesulfonic acids) buffer solution with 50-
100 μ l/min flow velocitys are passed through, steady to signal, and the ligandin containing 0.5-2mg/ml is passed through with 40-60 μ l/min flow velocitys
Hepes buffer solutions, signal curve change, until steady again;
5) ligand-receptor interaction detection:PBS is passed through with 40-60 μ l/min flow velocitys, it is steady to signal,
The cell-derived liposome solutions that associated receptor is contained on surface are passed through with 20-40 μ l/min flow velocitys again, according to the change of signal
Change value, judges that the interaction of ligand-receptor is strong and weak.
The above method is further comprising the steps of:
6) post-process:After detection terminates, quartz crystal chip is taken out, three water are immersed in:Concentrated ammonia liquor:30% peroxidating
Hydrogen volume ratio is 5:1:In 1 solution, mixed liquor is outwelled in 70 DEG C of water-bath after 15min, cleaned 3 times with three water, then blown with nitrogen
It is dry;Quartz crystal chip can be recycled.
Further, the surface of the quartz crystal chip is golden material.
Further, step 1) use the 2 mercaptopropionic acid containing volume fraction 0.97% and contain volume fraction 0.95%
The mixed liquor of 2 mercapto ethanol is pre-processed to quartz crystal chip.
Further, step 1) specifically include:2 mercaptopropionic acid and stoste volume concentration by stoste volume concentration for 97%
1 is pressed for 95% 2 mercapto ethanol:100 times are diluted again after the mixing of 1 volume ratio, are added in Tissue Culture Plate (such as 12 holes
Plate), per hole 1-1.5ml, clean quartz crystal chip is put into hole, is totally submerged, handle 12-24h, rinsed with three water
Fall the sulfydryl small molecule being not bonded on chip, dried up with nitrogen stand-by.
Further, step 3) the hand-type surface containing penicillamine molecular modification gold nano grain preparation method bag
Include following steps:
(1) the nano Au colloid liquid solution for taking 10ml to be prepared with citric acid reducing process, adds 10mg/ml penicillamine chirality point
500 μ l (excess) of son, react at room temperature 12-24h;
(2) nano-Au solution for modifying mould amine molecule is centrifuged 40-60min, abandons supernatant in 9500-12000r/min,
It is resuspended with three water, removes unmodified mould amine molecule, the nanogold of the chiral surfaces containing penicillamine molecular modification is made
Particle.
Further, in the method for the invention, often it is passed through before a kind of solution of new solute, it is necessary to be first passed through and be free of
The solvent of solute, when signal is steady, in order to avoid between different systems signal interference.
Further, ligandin solution is placed under pH=7.4 Hepes buffer systems, in order to avoid what pH changes were caused
The potential change of albumen, so as to change the normal physiological configuration of albumen.
Correspondingly, the present invention provides a kind of preparation method for the liposome that associated receptor is contained on surface, comprises the following steps:
(1) half adherent HEK293A cells of culture related plasmids transfection, with flow cytomery associated receptor albumen
Expression quantity;
(2) the HEK293A cells that associated receptor is overexpressed are collected, takes 4-8 × 105/ ml cell suspension 10ml, 1000r/
Min is centrifuged, and abandons supernatant culture medium, is resuspended with the PBS containing protease inhibitors;
(3) by carbon fiber filter membrane of the cell suspension by 800nm apertures, the liposome of associated receptor is contained on obtained surface.
Further, the present invention uses HEK293A cells, on the one hand beneficial to transfection, on the other hand when collecting cell, and half pastes
Wall-like state easily collecting, it is to avoid using trypsase so as to destroy surface receptor.
Further, the present invention uses protease inhibitors, when can prevent cell by filter membrane, caused by cell rupture
Protease leakage in lysosome, destroys surface receptor.
Present invention additionally comprises the above-mentioned in situ quantitation detection gold nano grain chiral surfaces absorption based on quartz crystal chip
Application of the method that interacts of albumen and cell membrane associated receptor in research albumen hat in terms of the biological effect of albumen.
Advantages of the present invention and have the beneficial effect that:
(1) quartz crystal chip is successively modified, the matter on each step chip can be intuitively detected with QCM-D instruments
Measure change situation.Successively modify, directly contacted with avoiding ligandin with chip surface, so as to cause structural change;
(2) directly detected in the original location using the cell membrane containing associated receptor, can more effectively simulate human internal environment,
Laid a good foundation for the practicality of this method.
The inventive method is simple to operate, practical, the 26S Proteasome Structure and Function being preced with to the albumen of nano grain surface formation
Research has great significance.
Brief description of the drawings
Fig. 1 for the present invention based on quartz crystal chip in situ quantitation detect the albumen of gold nano grain chiral surfaces absorption with
The principle schematic of the method for cell membrane associated receptor interaction.
Fig. 2 a combine the AFM table of the nanogold particle of chiral surfaces for the quartz chip surface in embodiment 1
Levy figure;
Fig. 2 b characterize for the AFM of the chiral surfaces combination transferrins of the nanogold particle in embodiment 1
Figure;
Fig. 2 c contain the cell-derived liposome of TfR for the transferrins mating surface in embodiment 1
AFM phenogram.
Fig. 3 is that the quartz chip surface in situ in embodiment 1 successively combines the nanogold particle of chiral surfaces, turns iron egg
In vain, the QCM-D signal graphs of the liposome containing TfR.
Embodiment
Following examples are used to illustrate the present invention, but are not limited to the scope of the present invention.It is unreceipted specific in embodiment
Technology or condition person, are carried out according to the technology or condition described by document in the art, or according to product description.It is used
Reagent or the unreceipted production firm person of instrument, are the conventional products that can be commercially available by regular distributor.
Used QCM-D INSTRUMENT MODELs are Q-sense E4 (the limited public affairs of the Ou Lin of Sweden hundred science and technology in the following example
Department).
The present invention detects the albumen and cell of gold nano grain chiral surfaces absorption based on quartz crystal chip in situ quantitation
The principle schematic of the method for film associated receptor interaction is shown in Fig. 1.
Embodiment 1
(1) quartz crystal chip is pre-processed:
Clean quartz crystal chip is placed in 12 orifice plates, the 2- containing volume fraction 0.97% is added in 12 orifice plates
The mixed liquor of mercaptopropionic acid and 2 mercapto ethanol containing volume fraction 0.95%, per hole 1ml, handles 12h, is rinsed with three water
Fall the sulfydryl small molecule being not bonded on chip, dried up with nitrogen stand-by;
(2) activation of quartz crystal chip surface carboxyl:
The quartz crystal chip of pretreatment is put into QCM-D instruments, first 100 μ l/min flow velocity is passed through three water, clearly
10min is washed, when signal curve is steady, then the NHS solution 1h of EDC and 30mM containing 75mM are passed through with 30 μ l/min flow velocity,
When signal curve is steady;
(3) preparation and fixation of the nanogold particle of chiral surfaces:
The nano Au colloid liquid solution for taking 10ml to be prepared with citric acid reducing process respectively, is separately added into 10mg/ml L-type and D
The μ l (excess) of type penicillamine chiral molecules 500, react at room temperature 12h;
The nano-Au solution 9500r/min of mould amine molecule will be modified, centrifuge 1h, abandon supernatant, be resuspended, removed with three water
Unmodified mould amine molecule, is made the nanogold particle of the chiral surfaces containing penicillamine molecular modification;
Three water are passed through with 100 μ l/min flow velocity, until signal is passed through containing green grass or young crops steadily, then with 20 μ l/min flow velocitys
The nanogold particle of the chiral surfaces of mould amine molecule modification, signal curve is widely varied, until signal is steady;
The AFM phenogram that quartz chip surface combines the nanogold particle of chiral surfaces is shown in figure Fig. 2 [a];
(4) absorption of ligandin:
First it is passed through with pH=7.4 Hepes buffer solutions with 50 μ l/min flow velocitys, it is steady to signal, with 50 μ l/min flow velocitys
It is passed through the Hepes buffer solutions of the transferrins containing 1mg/ml, signal curve change, until steady again;
The AFM phenogram of the chiral surfaces combination transferrins of nanogold particle is shown in Fig. 2 [b];
(5) ligand-receptor interaction detection:
The half adherent HEK293A cells that plasmid transfection TfR is overexpressed are cultivated, flow cytomery is used
TfR expressing quantity;
The HEK293A cells that TfR is overexpressed are collected, 5 × 10 are taken5/ ml cell suspension 10ml, 1000r/
Min is centrifuged, and abandons supernatant culture medium, is resuspended with the PBS containing protease inhibitors;
By carbon fiber filter membrane of the cell suspension by 800nm apertures, the cell that TfR is contained on obtained surface spreads out
Raw liposome;
PBS is passed through with 40 μ l/min flow velocitys, it is steady to signal, then the thin of TfR is contained on surface
Liposome solutions derived from born of the same parents are passed through with 30 μ l/min flow velocitys, according to the changing value of signal, judge transferrins-transferrins by
The interaction of body is strong and weak.
Quartz chip surface in situ successively with reference to the nanogold particles of chiral surfaces, transferrins, containing transferrins by
The QCM-D signal graphs of the liposome of body are shown in Fig. 3.Abscissa refers to the time in Fig. 3, and ordinate is frequency signal, and Au is nanogold
Grain, transferrin is transferrins, and liposome is cell-derived liposome.
Further, the above method is further comprising the steps of:
(6) post-process:After detection terminates, quartz crystal chip is taken out, three water are immersed in:Concentrated ammonia liquor:30% peroxide
It is 5 to change hydrogen volume ratio:1:In 1 solution, mixed liquor is outwelled in 70 DEG C of water-bath after 15min, cleaned 3 times with three water, then use nitrogen
Drying.Quartz crystal chip can be recycled.
Although above the present invention is described in detail with a general description of the specific embodiments,
On the basis of the present invention, it can be made some modifications or improvements, this will be apparent to those skilled in the art.Cause
This, these modifications or improvements, belong to the scope of protection of present invention without departing from theon the basis of the spirit of the present invention.
Claims (9)
1. a kind of albumen and cell membrane phase that the absorption of gold nano grain chiral surfaces is detected based on quartz crystal chip in situ quantitation
The method for closing acceptor interaction, it is characterised in that comprise the following steps:
1) quartz crystal chip is pre-processed using 2 mercaptopropionic acid and 2 mercapto ethanol;
2) treated quartz crystal chip is put into QCM-D instruments, EDC/NHS is passed through, by chip surface activated carboxylic;
3) nanogold particle of surface modification penicillamine small molecule is passed through, is combined by amido link with chip;
4) ligandin is passed through, makes ligandin absorption in the chiral surfaces of nanogold;
5) it is passed through the cell-derived liposome that associated receptor is contained on surface;By the power of frequency signal by ligandin with
The interaction of associated receptor is quantified.
2. according to the method described in claim 1, it is characterised in that comprise the following steps:
1) quartz crystal chip is pre-processed:By clean quartz crystal chip 2 mercaptopropionic acid and 2 mercapto ethanol processing 12-
24h, the sulfydryl small molecule being not bonded on chip is rinsed out with three water, is dried up with nitrogen stand-by;
2) activation of quartz crystal chip surface carboxyl:By step 1) pretreatment quartz crystal chip be put into QCM-D instruments,
First 100-150 μ l/min flow velocity is passed through three water, when signal curve is steady, then is passed through and contains with 30-50 μ l/min flow velocity
75mM EDC and 30mM NHS solution 1-2h, when signal curve is steady;
3) fixation of the nanogold particle of chiral surfaces:Three water are passed through with 100-150 μ l/min flow velocity, until signal is flat
Surely, then with 20-40 μ l/min flow velocitys the nanogold particle of the chiral surfaces containing penicillamine molecular modification is passed through, signal curve is big
Changes in amplitude, until signal is steady;
4) absorption of ligandin:First it is passed through with pH=7.4 Hepes buffer solutions with 50-100 μ l/min flow velocitys, it is flat to signal
Surely, the Hepes buffer solutions of the ligandin containing 0.5-2mg/ml are passed through with 40-60 μ l/min flow velocitys, signal curve changes, until
It is steady again;
5) ligand-receptor interaction detection:PBS is passed through with 40-60 μ l/min flow velocitys, it is steady to signal, then will
The cell-derived liposome solutions that associated receptor is contained on surface are passed through with 20-40 μ l/min flow velocitys, according to the changing value of signal,
Judge that the interaction of ligand-receptor is strong and weak.
3. method according to claim 1 or 2, it is characterised in that further comprising the steps of:6) post-process:Detection terminates
Afterwards, quartz crystal chip is taken out, is immersed in three water:Concentrated ammonia liquor:30% hydrogen peroxide volume ratio is 5:1:In 1 solution,
70 DEG C of water-bath, outwells mixed liquor after 15min, cleaned 3 times with three water, then dried up with nitrogen;Quartz crystal chip is recyclable to be made
With.
4. the method according to claim any one of 1-3, it is characterised in that the surface of the quartz crystal chip is golden material
Matter.
5. the method according to claim any one of 1-3, it is characterised in that step 1) use and contain volume fraction 0.97%
2 mercaptopropionic acid and the mixed liquor of 2 mercapto ethanol containing volume fraction 0.95% quartz crystal chip is pre-processed;
Preferably, step 1) specifically include:The 2 mercaptopropionic acid for being 97% by stoste volume concentration and stoste volume concentration are
95% 2 mercapto ethanol presses 1:100 times are diluted again after the mixing of 1 volume ratio, are added in Tissue Culture Plate, per hole 1-1.5ml,
Clean quartz crystal chip is put into hole, is totally submerged, 12-24h is handled, is rinsed out and be not bonded on chip with three water
Sulfydryl small molecule, with nitrogen dry up it is stand-by.
6. the method according to claim any one of 1-3, it is characterised in that step 3) described contain penicillamine molecular modification
The preparation method of gold nano grain on hand-type surface comprise the following steps:
(1) the nano Au colloid liquid solution for taking 10ml to be prepared with citric acid reducing process, adds 10mg/ml penicillamine chiral molecules
500 μ l, react at room temperature 12-24h;
(2) nano-Au solution for modifying mould amine molecule is centrifuged 40-60min, supernatant is abandoned, with three in 9500-12000r/min
Secondary water is resuspended, and removes unmodified mould amine molecule, and the nanogold particle of the chiral surfaces containing penicillamine molecular modification is made.
7. the method according to claim any one of 1-3, it is characterised in that step 5) surface contains associated receptor
The preparation method of liposome, comprises the following steps:
(1) half adherent HEK293A cells of culture related plasmids transfection, with flow cytomery associated receptor protein expression
Amount;
(2) the HEK293A cells that associated receptor is overexpressed are collected, takes 4-8 × 105/ ml cell suspension 10ml, 1000r/min from
The heart, abandons supernatant culture medium, is resuspended with the PBS containing protease inhibitors;
(3) by carbon fiber filter membrane of the cell suspension by 800nm apertures, the liposome of associated receptor is contained on obtained surface.
8. the method according to claim any one of 1-7, it is characterised in that be often passed through a kind of solution of new solute it
Before, it is necessary to the solvent without solute is first passed through, when signal is steady.
9. application of the method in research albumen hat in terms of the biological effect of albumen described in claim any one of 1-8.
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CN114858917A (en) * | 2021-02-04 | 2022-08-05 | 暨南大学 | Analysis method for dynamically monitoring cell transmembrane transport organic small molecules in real time |
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