CN105478754B - The preparation method and cancer cell detection method of Cu nano materials - Google Patents
The preparation method and cancer cell detection method of Cu nano materials Download PDFInfo
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- CN105478754B CN105478754B CN201610004429.7A CN201610004429A CN105478754B CN 105478754 B CN105478754 B CN 105478754B CN 201610004429 A CN201610004429 A CN 201610004429A CN 105478754 B CN105478754 B CN 105478754B
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- C—CHEMISTRY; METALLURGY
- C23—COATING METALLIC MATERIAL; COATING MATERIAL WITH METALLIC MATERIAL; CHEMICAL SURFACE TREATMENT; DIFFUSION TREATMENT OF METALLIC MATERIAL; COATING BY VACUUM EVAPORATION, BY SPUTTERING, BY ION IMPLANTATION OR BY CHEMICAL VAPOUR DEPOSITION, IN GENERAL; INHIBITING CORROSION OF METALLIC MATERIAL OR INCRUSTATION IN GENERAL
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- C23C18/00—Chemical coating by decomposition of either liquid compounds or solutions of the coating forming compounds, without leaving reaction products of surface material in the coating; Contact plating
- C23C18/16—Chemical coating by decomposition of either liquid compounds or solutions of the coating forming compounds, without leaving reaction products of surface material in the coating; Contact plating by reduction or substitution, e.g. electroless plating
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- C23—COATING METALLIC MATERIAL; COATING MATERIAL WITH METALLIC MATERIAL; CHEMICAL SURFACE TREATMENT; DIFFUSION TREATMENT OF METALLIC MATERIAL; COATING BY VACUUM EVAPORATION, BY SPUTTERING, BY ION IMPLANTATION OR BY CHEMICAL VAPOUR DEPOSITION, IN GENERAL; INHIBITING CORROSION OF METALLIC MATERIAL OR INCRUSTATION IN GENERAL
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- C23C18/32—Coating with nickel, cobalt or mixtures thereof with phosphorus or boron
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Abstract
The invention discloses the preparation method of Cu nano materials, comprise the following steps：Bar-shaped ZnO is prepared using Directly depositing, then Ni is plated on ZnO, then the ZnO of nickel plating is coated with Cu, finally the nickel plating ZnO nano material that Cu is coated is modified using hyaluronic acid；Described cancer cell detection method, comprises the following steps：Fluorescein mark is carried out to described Cu nano materials, then co-cultured with cancer cell, then is placed in laser capture microdissection Microscopic observation, the quantity of cancer cell is quantitatively detected according to dyeing quantity.The Cu nano materials prepared by the above method, have prepare simple, good biocompatibility, can and cancer cell specific receptor CD44 the advantage such as combination；The blood that patient need to be only gathered using the method for Cu nano materials detection cancer cell carries out vitro detection, and cancer cell can be just identified in few sample, realizes that ill initial stage being capable of accurate, sensitive, hurtless measure diagnosis cancer.
Description
Technical field
The invention belongs to nano material to prepare and biomedical applications field, more particularly to a kind of hyaluronic acid decorated Cu
The preparation method of the nickel plating ZnO nano material of cladding and the method using the material tests cancer cell.
Background technology
Cancer is to threaten one of human health and the principal disease of life, and the incidence of disease also increasingly rises.At present, largely
Clinical fact proves that the cure rate that carninomatosis becomes initial stage is at a relatively high.Therefore, the survival rate to improving cancer patient is early diagnosed
It is significant.There is an urgent need to find a kind of accurate, sensitive, simple to operate method of early diagnosis for this.
At present, cancer test mode mainly has B ultrasound, CT, MRI, biopsy etc..B ultrasound utilizes section ultrasonogram, can be from angle
Degree, the multi-faceted position for carrying out continuously displaying observation to focus, helping to understand tumour, the relation with surrounding tissue organ are right
The diagnosis and treatment of cancer have valuable help, but its scope of application is smaller.CT, abbreviation electronic computer transverse laminagraphy, can
For checking whole body focus, the scope of carninomatosis stove infringement can be specified, if invade the organ of surrounding, but CT equipment is expensive
Costly, excessive CT, which can exist, induces the danger such as malignant tumour, leukaemia.MRI, abbreviation Magnetic resonance imaging art, it is to brain
Deep tumor, brain stem tumor, tumor of spinal cord, tumour, cardiac tumor etc. can clarify a diagnosis in mediastinum, but be equipped with cardiac pacing
Device, the internal patient for having shell fragment residual, operation to leave metal silver brain clip etc. can not use.Biopsy is divided into Biopsy and aspiration biopsy
Two ways, Biopsy are that biopsy is carried out in surgical procedure, in that case it can be decided that tumor resection scope, help to check the latent of tumour
In diffusion and actual dispersion, cancer staging is determined, so that it is determined that successive treatment scheme；Aspiration biopsy needs drawing in CT, B ultrasound etc.
Lead down, puncture needle is penetrated into diseased region, take living tissue to make pathological diagnosis, aspiration biopsy has and causes needle track implantation, cancerous swelling bleeding
Possibility, it is careful to need during Clinical practice.
The content of the invention
In view of this, an object of the present invention is to provide the preparation method of Cu nano materials, can be obtained by this method
The nickel plating ZnO nano material of hyaluronic acid decorated Cu claddings, the nano material have magnetic Nano material and Cu nanometer materials concurrently
The advantage of material, have and prepare that simple, cost is cheap, does not reunite, good dispersion, easily can be connected with CD44 antibody
The advantages of.
The present invention realizes above-mentioned technical purpose by following technological means：
The preparation method of the Cu nano materials of the present invention, comprises the following steps：
Bar-shaped ZnO is prepared using Directly depositing；
Ni is plated on ZnO, obtains the ZnO of nickel plating.
With the ZnO of Cu cladding nickel plating, that is, obtain the nickel plating ZnO nano material that Cu is coated；
The nickel plating ZnO nano that Cu is coated has been wiped using hyaluronic acid and modified, it comprises the following steps：By hyalomitome
Acid is placed in stirred in water bath with buffer solution, hyaluronic acid is added the nickel plating ZnO that Cu is coated after being scattered in buffer solution, persistently stirs
Mix, unnecessary hyaluronic acid is removed using super filter tube, so as to obtain the nickel plating ZnO nano material of hyaluronic acid decorated Cu claddings
Material.
W metal is that one kind has magnetic, prepares the preferable magnetic material of relatively easy and biocompatibility.Cu change
Property stabilization is learned, its nanoscale has the spectral characteristic of bioaffinity and uniqueness, and Cu price is cheap compared with gold and silver,
Forming nano composite material with the magnetic Nano material of Cu claddings, effectively to overcome magnetic Nano material stability poor, easily group
It is poly-, the shortcomings of surface functional group is few, improve the physical and chemical performance of magnetic material.ZnO is combined with Cu nanometers and can improved
The biocompatibility of Cu nanometers, anti-interference, electric transmission speed, and make its surface be easier to be modified.
Hyaluronic acid, also known as hyaluronic acid or sodium hyaluronate, its specific receptor CD44 are over-expressed in a variety of cancer cells, and
The CD44 acceptors on normal cell surface are then in a kind of inactive silence state, therefore can pass through hyaluronic acid and its acceptor
CD44 combination realizes the targeted imaging analysis or quantitative analysis to cancer cell.
Further, described that Ni is plated on ZnO, it comprises the following steps：By ZnO in the solution of stannous chloride and hydrochloric acid
Sensitization, places into palladium chloride and hydrochloric acid solution and activates, the ZnO after activation is cleaned with deionized water, then again puts ZnO
Enter and carry out chemical plating into the chemical plating pond containing nickel sulfate, sodium hypophosphite, ammonium chloride, sodium citrate, succinic acid and lactic acid, most
The ZnO of plated nickel is filtered afterwards and is washed with deionized and dries, that is, obtains the ZnO of nickel plating.
Further, the ZnO that nickel plating is coated with Cu, it comprises the following steps：The nickel plating ZnO of preparation is moved into deionization
In water, polyethylene glycol is added, disperses to obtain nickel plating ZnO suspension under ultrasound condition；By cuprous oxide powder move into go from
Fully dissolved in sub- water, obtain cuprous oxide suspension；Cuprous oxide suspension is mixed with nickel plating ZnO suspensions, process is quiet
Put, filter, washing, drying after alcohol is washed, that is, obtaining the nickel plating ZnO nano material of Cu claddings.
The second object of the present invention is to provide the cancer cell detection method of Cu nano materials, is detected by this method, only needs
The blood of patient is gathered, and cancer cell can be just identified in few sample, realizes that ill initial stage being capable of accurate, sensitive, nothing
The diagnosis cancer of wound.
The present invention realizes above-mentioned technical purpose by following technological means：
A kind of cancer cell detection method of the present invention, the Cu for having used the preparation method of above-mentioned Cu nano materials to prepare receive
Rice material, comprises the following steps：
A) fluorescein marks, and by described Cu nano materials dissolving in deionized water, adds fluorescein isothiocynate and enters
Line flag；
B) culture of cancer cell, 80%~90% cancer cell converged will be reached after passage, is inoculated in orifice plate, hatch 24h
Afterwards, washed with alkaline phosphatase salting liquid, add the hyaluronic acid decorated Cu nanomaterial solutions of marked by fluorescein isothiocyanate,
And after being fixed using paraformaldehyde solution, wash with alkaline phosphatase salting liquid, add 2% potassium ferrocyanide solution of equivalent with
Stood after 2% hydrogen chloride mixed liquor, lucifuge hatches 18~30min, washes away dyeing liquor, and core fast red is redyed, and removes supernatant, uses alkali
Acid phosphate solution washs again；Each 1 time using 50%, 75%, 85%, 95% and 100% Gradient elution using ethanol, dimethylbenzene is saturating
Bright 2 times, 10~15min every time；Neutral gum mounting, it is placed under laser confocal microscope and observes, the cell membrane of tumour cell
And cytoplasm has Positive fluorescence dyeing, the quantity of cancer cell can be quantitatively detected according to dyeing quantity.
Further, the particle size range of the hyaluronic acid decorated Cu nano materials is 40-60nm, and hyaluronic acid is in Cu bags
The carrying capacity on the plating Ni covered ZnO nano material surface is 0.4~0.5mmol/g.
Further, the cancer cell is one kind in lung carcinoma cell, liver cancer cells, breast cancer cell or stomach cancer cell.
Beneficial effects of the present invention：(1) Cu nano materials have the advantage of magnetic Nano material and Cu nano materials concurrently, have
Preparation is simple, cost is cheap, does not reunite, the advantage such as good dispersion；(2) surface is carried out to Cu nano materials with hyaluronic acid
Modification, can not only improve its stability, and make its surface-functionalized, more easily can be connected with CD44 antibody；(3)
The blood that patient need to be only gathered using the method for hyaluronic acid decorated Cu nano materials detection cancer cell carries out vitro detection, and
Cancer cell can be just identified in few sample, realizes that ill initial stage being capable of accurate, sensitive, hurtless measure diagnosis cancer.
Embodiment
Technical scheme will be described in detail by specific embodiment below：
The preparation method of embodiment 1Cu nano materials
The preparation method of the hyaluronic acid decorated Cu nano materials of the present invention, comprises the following steps：
Bar-shaped ZnO is prepared using Directly depositing；
Ni is plated on ZnO, it comprises the following steps：ZnO is sensitized in the solution of stannous chloride and hydrochloric acid, placed into
Activated into palladium chloride and hydrochloric acid solution, the ZnO after activation is cleaned with deionized water, and then ZnO is put into containing sulphur again
Sour nickel, sodium hypophosphite, ammonium chloride, sodium citrate, succinic acid and lactic acid chemical plating pond in carry out chemical plating, finally by plated nickel
ZnO filter and be washed with deionized and dry, that is, obtain the ZnO of nickel plating.
With the ZnO of Cu cladding nickel plating, it comprises the following steps：The nickel plating ZnO of preparation is moved into deionized water, added poly-
Ethylene glycol, disperse to obtain nickel plating ZnO suspension under ultrasound condition；Cuprous oxide powder is moved into fully molten in deionized water
Solution, obtains cuprous oxide suspension；Cuprous oxide suspension is mixed with nickel plating ZnO suspensions, by standing, filtering, washing,
Alcohol is dried after washing, that is, obtains the nickel plating ZnO nano material of Cu claddings.
The nickel plating ZnO nano material that Cu is coated is modified using hyaluronic acid, it comprises the following steps：By hyalomitome
Acid is placed in stirred in water bath with buffer solution, hyaluronic acid is added the nickel plating ZnO that Cu is coated after being scattered in buffer solution, persistently stirs
Mix, unnecessary hyaluronic acid is removed using super filter tube, so as to obtain the nickel plating ZnO nano material of hyaluronic acid decorated Cu claddings
Material.
The cancer cell detection method of embodiment 2
The cancer cell detection method of the present invention, the preparation method of the Cu nano materials described in embodiment 1 has been used to prepare
The Cu nano materials gone out, comprise the following steps：
A) fluorescein marks, and by described Cu nano materials dissolving in deionized water, adds fluorescein isothiocynate and enters
Line flag, the particle size range of the Cu nano materials is 40-60nm, and hyaluronic acid is 0.4 in the carrying capacity of Cu nano-material surfaces
~0.5mmol/g；
B) culture of cancer cell, the cancer cell are in lung carcinoma cell, liver cancer cells, breast cancer cell or stomach cancer cell
One kind, specific way are：80%~90% cancer cell converged will be reached after passage, with 5 × 105Individual/hole is inoculated in orifice plate, and 30
~37 DEG C, 5%CO2Under the conditions of hatch 24h after, washed with alkaline phosphatase salting liquid, add the copper of marked by fluorescein isothiocyanate
Nanomaterial solution, after hatching 24h, fully washed with alkaline phosphatase salting liquid, the paraformaldehyde solutions of 1ml 4% are added per hole and are consolidated
After determining 20min, washed with alkaline phosphatase salting liquid, after adding 2% potassium ferrocyanide solution and 2% hydrogen chloride mixed liquor of equivalent
5min is stood, under the conditions of 37 DEG C, lucifuge hatching 30min, deionized water washes away dyeing liquor, and core fast red is redyed, and removes supernatant,
Washed again with alkaline phosphatase salting liquid.Each 1 time using 50%, 75%, 85%, 95% and 100% Gradient elution using ethanol, diformazan
Transparent 2 times of benzene, each 10min.Neutral gum mounting, it is placed under inverted microscope and observes and take pictures.
Finally illustrate, the above embodiments are merely illustrative of the technical solutions of the present invention and it is unrestricted, although with reference to compared with
The present invention is described in detail good embodiment, it will be understood by those within the art that, can be to the skill of the present invention
Art scheme is modified or equivalent substitution, and without departing from the objective and scope of technical solution of the present invention, it all should cover at this
Among the right of invention.
Claims (6)
- The preparation method of 1.Cu nano materials, it is characterised in that comprise the following steps：Bar-shaped ZnO is prepared using Directly depositing；Ni is plated on ZnO, obtains the ZnO of nickel plating；With the ZnO of Cu cladding nickel plating, that is, obtain the nickel plating ZnO nano material that Cu is coated；The nickel plating ZnO nano material that Cu is coated is modified using hyaluronic acid, it comprises the following steps：By hyaluronic acid with Buffer solution is placed in stirred in water bath, hyaluronic acid is added the nickel plating ZnO that Cu is coated after being scattered in buffer solution, lasting to stir, Unnecessary hyaluronic acid is removed using super filter tube, so as to obtain the nickel plating ZnO nano material of hyaluronic acid decorated Cu claddings.
- 2. the preparation method of Cu nano materials according to claim 1, it is characterised in that it is described that Ni is plated on ZnO, its Comprise the following steps：ZnO is sensitized in the solution of stannous chloride and hydrochloric acid, placed into living in palladium chloride and hydrochloric acid solution Change, the ZnO after activation is cleaned with deionized water, then ZnO is put into containing nickel sulfate, sodium hypophosphite, ammonium chloride, lemon again Chemical plating is carried out in the chemical plating pond of sour sodium, succinic acid and lactic acid, finally the ZnO of plated nickel is filtered and is washed with deionized And dry, that is, obtain the ZnO of nickel plating.
- 3. the preparation method of Cu nano materials according to claim 1, it is characterised in that described to coat nickel plating with Cu ZnO, it comprises the following steps：The nickel plating ZnO of preparation is moved into deionized water, adds polyethylene glycol, is divided under ultrasound condition Dissipate and obtain nickel plating ZnO suspension；Cuprous oxide powder is moved into deionized water and fully dissolved, it is suspended to obtain cuprous oxide Liquid；Cuprous oxide suspension is mixed with nickel plating ZnO suspensions, is dried by standing, filter, wash, after alcohol is washed, that is, obtains Cu The nickel plating ZnO nano material of cladding.
- 4. a kind of cancer cell detection method, it is characterised in that used the Cu nanometer materials as described in claim 1-3 any one The Cu nano materials that the preparation method of material is prepared, comprise the following steps：A) fluorescein marks, and by the dissolving of Cu nano materials in deionized water, adds fluorescein isothiocynate and is marked；B) culture of cancer cell, 80%~90% cancer cell converged will be reached after passage, is inoculated in orifice plate, after hatching 24h, used Alkaline phosphatase salting liquid washs, and adds the solution of the Cu nano materials of marked by fluorescein isothiocyanate, and molten using paraformaldehyde After liquid is fixed, washed with alkaline phosphatase salting liquid, after adding 2% potassium ferrocyanide solution and 2% hydrogen chloride mixed liquor of equivalent Stand, lucifuge hatches 18~30min, washes away dyeing liquor, and core fast red is redyed, and removes supernatant, with alkaline phosphatase salting liquid again Washing；Using Gradient elution using ethanol, transparent 2 times of dimethylbenzene, 10~15min every time；Neutral gum mounting, is placed in laser microscope Lower observation, the cell membrane and cytoplasm of tumour cell have Positive fluorescence dyeing, can quantitatively detect cancer cell according to dyeing quantity Quantity.
- 5. cancer cell detection method as claimed in claim 4, it is characterised in that：The particle size range of the Cu nano materials is 40- 60nm, carrying capacity of the hyaluronic acid on the nickel plating ZnO nano material surface that Cu is coated is 0.4-0.5mmol/g.
- 6. cancer cell detection method as claimed in claim 4, it is characterised in that：The cancer cell be lung carcinoma cell, liver cancer cells, One kind in breast cancer cell or stomach cancer cell.
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