CN107144610B - Method based on quartz crystal chip in situ quantitation detection gold nano grain adsorption albumen and the interaction of cell membrane associated receptor - Google Patents
Method based on quartz crystal chip in situ quantitation detection gold nano grain adsorption albumen and the interaction of cell membrane associated receptor Download PDFInfo
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- CN107144610B CN107144610B CN201710208042.8A CN201710208042A CN107144610B CN 107144610 B CN107144610 B CN 107144610B CN 201710208042 A CN201710208042 A CN 201710208042A CN 107144610 B CN107144610 B CN 107144610B
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- 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
Abstract
The present invention relates to the methods of a kind of albumen based on the detection gold nano grain chiral surfaces absorption of quartz crystal chip in situ quantitation and the interaction of cell membrane associated receptor, comprising: (1) is pre-processed using 2 mercaptopropionic acid and 2 mercapto ethanol to quartz crystal chip；(2) processed chip is passed through EDC/NHS in QCM-D instrument, by chip surface activated carboxylic；(3) it is passed through the nanogold particle of surface modification penicillamine small molecule, through amido bond in conjunction with chip；(4) it is passed through ligandin, ligandin is made to be adsorbed on 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 easy to operate, practical, has great significance to the research for the structure and function that the albumen that nano grain surface is formed is preced with.
Description
Technical field
The invention belongs to the innovative development fields of dissipativeness quartz crystal microbalance detection method, relate generally to one kind and are based on
Quartz crystal chip in situ quantitation detects the albumen of gold nano grain chiral surfaces absorption and cell membrane associated receptor interacts
Method.
Background technique
Dissipativeness quartz crystal microbalance (QCM-D) utilizes the piezoelectric property of quartz-crystal resonator, by quartz crystal core
The variation of plate electrode surface quality is converted into the frequency variation of quartz crystal oscillator circuit output electric signal, and then is obtained by computer
High-precision data are obtained, are a kind of novel sensitive quality testing instruments, measurement accuracy is up to nanogram level.
After nano material enters in human body, since specific surface area is larger, the albumen in human body fluid can be adsorbed, in material table
Face forms albumen hat.Biological effect, toxicological effect and the metabolic pathway that albumen is preced with after entering human body to nano material suffer from
Decisive action.Therefore, the interfacial structure important in inhibiting of research nano material and protein effect.
There is several hundred kinds of albumen in human body fluid, and since nano material enters in body fluid, simultaneously adhesion protein forms albumen hat,
The albumen being adsorbed structurally and functionally more or less is changing.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；Albumen be adsorbed after structure and function by how
Change, a series of this problem is increasingly paid close attention to by people.Different materials are entered with the difference of the albumen formed in vivo hat
And rule, also people is worth further to study.
Currently, to the interfacial structure research of nano material and protein, there is many difficulties.Although circular dichroism, red
The technologies such as external spectrum, x-ray crystal diffraction can detect the structure changes at different levels for the albumen being adsorbed, but to the albumen being adsorbed
Physiological function change it is still helpless.
Chirality is a fundamental characteristics of nano material, after different chiral nano materials enters human body, to albumen
Suction type also different from, and to the change of protein function difference.But this species diversity is usually smaller, conventional detection means
It is difficult to accurately detect out result.
QCM-D is as micro-mass sensor, and easy to operate, high sensitivity is, it can be achieved that monitor on-line, in chemistry, physics, life
It is widely used in the fields such as object, medicine and Surface Science.In biological field, QCM-D is commonly used in monitoring biology point
Interaction between son, such as: the affinity interaction between Ag-Ab.Usually a kind of life is modified in quartz crystal chip surface
Object molecule, then it is passed directly into another molecule, thus output electric signal caused by the mass change of detection chip electrode surface
Frequency variation.And for nano material-, structure is complicated for albumen hat, currently, detecting about based on quartz crystal chip in situ quantitation
The albumen of gold nano grain chiral surfaces absorption and the technological invention of cell membrane associated receptor interaction are there is not yet report.
Summary of the invention
In order to distinguish influence of the different chiral nanomaterials to the function of the albumen of absorption, for QCM-D high sensitivity this
One feature, the invention proposes a kind of eggs based on the detection gold nano grain chiral surfaces absorption of quartz crystal chip in situ quantitation
The white method with the interaction of cell membrane associated receptor.
The present invention devises on the protein ligands and cell of a kind of in situ quantitation detection gold nano grain chiral surfaces absorption
The method of acceptor interaction.Gold nano grain containing chiral surfaces, ligandin, cell membrane surface receptors are successively in situ
It is incorporated in quartz crystal chip surface, the interaction of the ligandin and receptor that are adsorbed is detected, 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 based on the detection gold nano grain chiral surfaces absorption of quartz crystal chip in situ quantitation
The method of associated receptor interaction, comprising the following steps:
1) quartz crystal chip is pre-processed using 2 mercaptopropionic acid and 2 mercapto ethanol；
2) processed quartz crystal chip is put into QCM-D instrument, is passed through EDC/NHS, chip surface carboxyl is living
Change；
3) it is passed through the nanogold particle of surface modification penicillamine small molecule, through amido bond in conjunction with chip；
4) it is passed through ligandin, ligandin is made to be adsorbed on 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 ligand egg
It is white to be quantified with associated receptor interaction.
Specifically, the above method the following steps are included:
1) quartz crystal chip pre-processes: clean quartz crystal chip 2 mercaptopropionic acid and 2 mercapto ethanol are handled
12-24h rinses out the sulfydryl small molecule being not bonded on chip with water three times, stand-by with being dried with nitrogen；
2) the pretreated quartz crystal chip of step 1) activation of quartz crystal chip surface carboxyl: is put into QCM-D instrument
In device, the flow velocity of first 100-150 μ l/min is passed through (in QCM-D instrument, similarly hereinafter) water three times, waits until that signal curve is steady, then with
The flow velocity of 30-50 μ l/min be passed through EDC (1- ethyl -3- [3- dimethylaminopropyl] carbodiimide hydrochloride) containing 75mM and
NHS (n-hydroxysuccinimide) solution 1-2h of 30mM, until signal curve is steady；
3) fixation of the nanogold particle of chiral surfaces: water three times is passed through with the flow velocity of 100-150 μ l/min, Zhi Daoxin
It is number steady, then it is passed through with 20-40 μ l/min flow velocity 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 Hepes (i.e. 4- hydroxyethyl piperazineethanesulfonic acid) buffer of pH=7.4 with 50-
100 μ l/min flow velocitys are passed through, steady to signal, are passed through the ligandin containing 0.5-2mg/ml with 40-60 μ l/min flow velocity
Hepes buffer, signal curve variation, until steady again；
5) ligand-receptor interaction detection: PBS buffer solution is passed through with 40-60 μ l/min flow velocity, 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 velocity 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) it post-processes: after detection, quartz crystal chip being taken out, is immersed in water three times: concentrated ammonia liquor: 30% peroxidating
Hydrogen volume is cleaned 3 times, then blown with nitrogen than 70 DEG C of water-bath, mixed liquor is outwelled after 15min in the solution for 5:1:1 with water three times
It is dry；Quartz crystal chip can be recycled.
Further, the surface of the quartz crystal chip is golden material.
Further, step 1) using the 2 mercaptopropionic acid containing volume fraction 0.97% and contains volume fraction 0.95%
The mixed liquor of 2 mercapto ethanol pre-processes quartz crystal chip.
Further, step 1) specifically includes: 2 mercaptopropionic acid and stoste volume concentration by stoste volume concentration for 97%
It is 95% 2 mercapto ethanol by diluting 100 times again after the mixing of 1:1 volume ratio, be added in tissue culture plate (such as 12 holes
Plate), clean quartz crystal chip is put into hole, is totally submerged by every hole 1-1.5ml, handles 12-24h, is rinsed with water three times
Fall the sulfydryl small molecule being not bonded on chip, it is stand-by with being dried with nitrogen.
Further, the preparation method packet of the gold nano grain on the hand-type surface containing penicillamine molecular modification described in step 3)
Include following steps:
(1) the penicillamine chirality point of 10mg/ml is added in the nano Au colloid liquid solution for taking 10ml citric acid reduction method to prepare
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 water three times, removes unmodified mould amine molecule, the nanogold of the chiral surfaces containing penicillamine molecular modification is made
Particle.
Further, it in the method for the invention, is often passed through before a kind of solution of new solute, it is necessary to first be passed through and be free of
The solvent of solute, until signal is steady, in order to avoid between different system signal interference.
Further, ligandin solution is placed under the Hepes buffer system of pH=7.4, in order to avoid caused by pH changes
The potential change of albumen, to change the normal physiological configuration of albumen.
Correspondingly, the present invention provides a kind of preparation method of liposome that associated receptor is contained on surface, comprising the following steps:
(1) half adherent HEK293A cell of culture related plasmids transfection, with flow cytomery associated receptor albumen
Expression quantity；
(2) the HEK293A cell that associated receptor is overexpressed is collected, 4-8 × 10 are taken5The cell suspension 10ml, 1000r/ of/ml
Min centrifugation, abandons supernatant culture medium, is resuspended with the PBS buffer solution containing protease inhibitors；
(3) cell suspension is passed through to the carbon fiber filter membrane in the aperture 800nm, the liposome of associated receptor is contained on obtained surface.
Further, the present invention uses HEK293A cell, is on the one hand conducive to transfection, and on the other hand when collecting cell, half is pasted
Wall-like state easily collecting is avoided using trypsase to destroy surface receptor.
Further, the present invention uses protease inhibitors, when can prevent cell from passing through filter membrane, as caused by cell rupture
Protease leakage in lysosome, destroys surface receptor.
The in situ quantitation detection gold nano grain chiral surfaces absorption based on quartz crystal chip that the invention also includes above-mentioned
Albumen and cell membrane associated receptor interaction method research albumen hat in albumen biological effect in terms of application.
It advantages of the present invention and has the beneficial effect that
(1) quartz crystal chip is successively modified, can intuitively detects the matter on each step chip with QCM-D instrument
Amount changes situation.Successively modification, directly contacts with chip surface to avoid ligandin, so as to cause structure change；
(2) it is directly detected in situ using the cell membrane containing associated receptor, can more effectively simulate human internal environment,
It lays a good foundation for the practicability of this method.
The method of the present invention is easy to operate, practical, to the structure and function for the albumen hat that nano grain surface is formed
Research has great significance.
Detailed description of the invention
Fig. 1 be the present invention is based on quartz crystal chip in situ quantitation detect gold nano grain chiral surfaces absorption albumen with
The schematic illustration of the method for cell membrane associated receptor interaction.
Fig. 2 [a] combines the atomic force microscope of the nanogold particle of chiral surfaces for the quartz chip surface in embodiment 1
Phenogram；
Fig. 2 [b] is the atomic force microscope characterization of the chiral surfaces combination transferrins of the nanogold particle in embodiment 1
Figure；
Fig. 2 [c] is the cell-derived liposome that the transferrins mating surface in embodiment 1 contains TfR
Atomic force microscope 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
The QCM-D signal graph of liposome white, containing TfR.
Specific embodiment
The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention..It is not specified in embodiment specific
Technology or conditions person, described technology or conditions according to the literature in the art, or carried out according to product description.It is used
Production firm person is not specified in reagent or instrument, is the conventional products that can be commercially available by regular distributor.
Used QCM-D instrument model is Q-sense E4 (the limited public affairs of hundred Ou Lin of Sweden science and technology in the following example
Department).
The present invention is based on the albumen and cell of the detection gold nano grain chiral surfaces absorption of quartz crystal chip in situ quantitation
The schematic illustration of the method for film associated receptor interaction is shown in Fig. 1.
Embodiment 1
(1) quartz crystal chip pre-processes:
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 the 2 mercapto ethanol containing volume fraction 0.95%, every hole 1ml handle 12h, are rinsed with water three times
Fall the sulfydryl small molecule being not bonded on chip, it is stand-by with being dried with nitrogen；
(2) activation of quartz crystal chip surface carboxyl:
Pretreated quartz crystal chip is put into QCM-D instrument, first the flow velocity of 100 μ l/min is passed through water three times, clearly
10min is washed, waits until that signal curve is steady, then be passed through the NHS solution 1h of EDC and 30mM containing 75mM with the flow velocity of 30 μ l/min,
Until signal curve is steady；
(3) preparation and fixation of the nanogold particle of chiral surfaces:
The nano Au colloid liquid solution for taking 10ml citric acid reduction method to prepare respectively, is separately added into the L-type and D of 10mg/ml
500 μ l (excess) of type penicillamine chiral molecules reacts at room temperature 12h；
The nano-Au solution 9500r/min of mould amine molecule will be modified, be centrifuged 1h, abandon supernatant, be resuspended with water three times, removed
The nanogold particle of the chiral surfaces containing penicillamine molecular modification is made in unmodified mould amine molecule；
Water three times is passed through with the flow velocity of 100 μ l/min, is passed through until signal is steady, then with 20 μ l/min flow velocitys containing blueness
The nanogold particle of the chiral surfaces of mould amine molecule modification, signal curve is widely varied, until signal is steady；
Quartz chip surface combines the atomic force microscope phenogram of the nanogold particle of chiral surfaces to see figure Fig. 2 [a]；
(4) absorption of ligandin:
First it is passed through with the Hepes buffer of pH=7.4 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 of the transferrins containing 1mg/ml, signal curve variation, until steady again；
The atomic force microscope 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 cell that plasmid transfection TfR is overexpressed is cultivated, flow cytomery is used
TfR expressing quantity；
The HEK293A cell that TfR is overexpressed is collected, takes 5 × 105The cell suspension 10ml, 1000r/ of/ml
Min centrifugation, abandons supernatant culture medium, is resuspended with the PBS buffer solution containing protease inhibitors；
Cell suspension is passed through to the carbon fiber filter membrane in the aperture 800nm, the cell that TfR is contained on obtained surface spreads out
Raw liposome；
PBS buffer solution 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 combine the nanogold particles of chiral surfaces, transferrins, containing transferrins by
The QCM-D signal graph of the liposome of body is shown in Fig. 3.Abscissa refers to the time in Fig. 3, and ordinate is frequency signal, and Au is nanogold
Grain, transferrin are transferrins, and liposome is cell-derived liposome.
Further, the above method is further comprising the steps of:
(6) it post-processes: after detection, quartz crystal chip being taken out, is immersed in water three times: concentrated ammonia liquor: 30% peroxide
Change hydrogen volume than 70 DEG C of water-bath, mixed liquor is outwelled after 15min in the solution for 5:1:1, is cleaned 3 times with water three times, 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, fall within the scope of the claimed invention without departing from theon the basis of the spirit of the present invention.
Claims (10)
1. a kind of albumen and cell membrane phase based on the detection gold nano grain chiral surfaces absorption of quartz crystal chip in situ quantitation
The method for closing acceptor interaction, which comprises the following steps:
1) quartz crystal chip is pre-processed using 2 mercaptopropionic acid and 2 mercapto ethanol；
2) processed quartz crystal chip is put into QCM-D instrument, is passed through EDC/NHS, by chip surface activated carboxylic；
3) it is passed through the gold nano grain of surface modification penicillamine small molecule, through amido bond in conjunction with chip；
4) it is passed through ligandin, ligandin is made to be adsorbed on the chiral surfaces of gold nano grain；
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 carries out quantitative detection.
2. the method according to claim 1, wherein the following steps are included:
1) quartz crystal chip pre-processes: clean quartz crystal chip 2 mercaptopropionic acid and 2 mercapto ethanol are handled 12-
For 24 hours, the sulfydryl small molecule being not bonded on chip is rinsed out with water three times, it is stand-by with being dried with nitrogen；
2) activation of quartz crystal chip surface carboxyl: the pretreated quartz crystal chip of step 1) is put into QCM-D instrument,
The flow velocity of first 100-150 μ l/min is passed through water three times, waits until that signal curve is steady, then be passed through and contain with the flow velocity of 30-50 μ l/min
The NHS solution 1-2h of the EDC and 30mM of 75mM, until signal curve is steady；
3) fixation of the gold nano grain of chiral surfaces: water three times is passed through with the flow velocity of 100-150 μ l/min, until signal is flat
Surely, then with 20-40 μ l/min flow velocity it is passed through the gold nano grain of the chiral surfaces containing penicillamine molecular modification, signal curve is big
Amplitude variation, until signal is steady；
4) absorption of ligandin: being first passed through with the Hepes buffer of pH=7.4 with 50-100 μ l/min flow velocity, flat to signal
Surely, the Hepes buffer of the ligandin containing 0.5-2mg/ml is passed through with 40-60 μ l/min flow velocity, signal curve changes, until
It is steady again；
5) ligand-receptor interaction detection: PBS buffer solution is passed through with 40-60 μ l/min flow velocity, 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 velocity, according to the changing value of signal,
Judge that the interaction of ligand-receptor is strong and weak.
3. the method according to claim 1, wherein further comprising the steps of: 6) it post-processes: after detection,
Quartz crystal chip is taken out, be immersed in water three times: concentrated ammonia liquor: 30% hydrogen peroxide volume ratio is water-bath in the solution of 5:1:1
70 DEG C, mixed liquor is outwelled after 15min, is cleaned 3 times with water three times, then with being dried with nitrogen；Quartz crystal chip can be recycled.
4. method according to claim 1-3, which is characterized in that the surface of the quartz crystal chip is golden material
Matter.
5. method according to claim 1-3, which is characterized in that step 1), which uses, contains volume fraction 0.97%
2 mercaptopropionic acid and the mixed liquor of the 2 mercapto ethanol containing volume fraction 0.95% quartz crystal chip is pre-processed.
6. according to the method described in claim 5, it is characterized in that, step 1) specifically includes: being 97% by stoste volume concentration
2 mercaptopropionic acid and stoste volume concentration be 95% 2 mercapto ethanol by 1:1 volume ratio mixing after dilute 100 times again, add
Into tissue culture plate, clean quartz crystal chip is put into hole, is totally submerged by every hole 1-1.5ml, handles 12-24h,
The sulfydryl small molecule being not bonded on chip is rinsed out with water three times, it is stand-by with being dried with nitrogen.
7. method according to claim 1-3, which is characterized in that hand of the step 3) containing penicillamine molecular modification
The preparation method of the gold nano grain on property surface the following steps are included:
(1) the nano Au colloid liquid solution for taking 10ml citric acid reduction method to prepare, is added the penicillamine chiral molecules of 10mg/ml
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 gold nano grain of the chiral surfaces containing penicillamine molecular modification is made.
8. method according to claim 1-3, which is characterized in that contain associated receptor in the step 5) surface
The preparation method of liposome, comprising the following steps:
(1) half adherent HEK293A cell of culture related plasmids transfection, with flow cytomery associated receptor protein expression
Amount；
(2) the HEK293A cell that associated receptor is overexpressed is collected, 4-8 × 10 are taken5The cell suspension 10ml, 1000r/min of/ml from
The heart abandons supernatant culture medium, is resuspended with the PBS buffer solution containing protease inhibitors；
(3) cell suspension is passed through to the carbon fiber filter membrane in the aperture 800nm, the liposome of associated receptor is contained on obtained surface.
9. method according to claim 1-3, which is characterized in that be often passed through a kind of new solute solution it
Before, it is necessary to it is first passed through the solvent without solute, until signal is steady.
10. application of the described in any item methods of claim 1-9 in research albumen hat in terms of the biological effect of albumen.
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| CN109239331A (en) * | 2018-09-04 | 2019-01-18 | 南京林业大学 | A method of with bovine serum albumin in-situ modification gold chip |
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