CN110104610A - A kind of pollen structure particle and its preparation method and application - Google Patents
A kind of pollen structure particle and its preparation method and application Download PDFInfo
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Abstract
The present invention provides a kind of pollen structure particle and preparation method thereof for capturing circulating tumor cell, which being capable of fast and effeciently enrichment cycles tumour cell.Capture circulating tumor cell technology provided by the present invention is without modified specificity cancer cell identification antibody or aptamer, it can be achieved that the capture of different cancer cell-types, has advantage low in cost, easy to operate.Prepared pollen structure particle can be used for capturing the circulating tumor cell in peripheral blood, be expected to early diagnosis, detection, analysis with cancer patient, the removal of cancer cell and blood purification.
Description
Technical field
The present invention relates to a kind of Biofunctional materials of technical field of analysis and detection and its manufacturing methods, in particular to are used to
The Biofunctional materials and its manufacturing method and purposes of capture circulating tumor cell.In particular it relates to a kind of for catching
Obtain the pollen structure particle and its preparation method and application of circulating tumor cell.
Background technique
Cancer is current one of the disease for threatening human health most serious, and the invasion of tumour and transfer are that cancer patient faces
The main reason for bed is lethal.Circulating tumor cell (Circulating Tumor Cells, CTCs) is the formation or life in tumour
The cell for entering human peripheral circulatory system is detached from growth process from situ tumor lesion.CTCs can be transported to distal end group
It knits, then oozes out, adapt to new microenvironment, ultimately form new transfer stove.Therefore, the quantity and type for monitoring CTCs in blood become
Change, important directive function is suffered from for patient's Index for diagnosis, therapeutic evaluation.Detection and analysis to CTCs in blood, also by
Referred to as " liquid biopsy " technology is considered one of most potential tumour non-invasive diagnosis and real-time curative effect monitoring means, to realization
The individualized treatment of cancer is of great significance.
Since content is extremely low in peripheral blood by CTCs, have very for the sensitivity and specificity of CTCs detection technique
High requirement.It distinguishes for physical properties such as normal cell size, density in tumour cell and blood, and knows for antigen-antibody
Other biochemical property difference, the main separation method of CTCs has filtration method, electrophoresis, magnetic nanoparticle, micro-fluidic core at present
Piece method.Document Small 2015,11,3850, Angew.Chem.Int.Ed.2016,55,1252, Chem.Soc.Rev.,
2017,46,4245 review the methods and techniques of CTCs capture in recent years.Although these methods all have certain CTCs capture
Effect, but also deposit various deficiencies, as material preparation process is complex, and some capture rates are not high or enrichment time is longer etc..
Studies have shown that micro-nanometer structural material has complicated and diversified multilevel structure, pass through itself and cell surface nano junction
The three-dimensional interactions of structure (eg. microvillus, pseudopodium etc.) can significantly affect adherency, growth, differentiation and the albumen table of cell
Up to etc. vital movements.In recent years, the nanostructure (eg. nanoparticle, nano wire, nanofiber etc.) of numerous species, is used for
CTCs capture separation.Although these nano materials can be increased by increasing specific surface area and the touch opportunity of CTCs, to
Further increase the capture rate of CTCs, it is still desirable to epithelial cell adhesion molecule (Epithelial cell adhesion
Molecule, EpCAM) antibody or aptamer be the addition for identifying molecule.Such as document Nano Lett.16,1,766-772
In, Feilong Zhang et al. is caught using indium tin oxide nano wire capture CTCs in the case where being added without EpCAM antibody
The rate of obtaining only has 40%, and after EpCAM antibody is added, capture rate just be can be further improved to 89%.But on occurring due to CTCs
Qualitative change between chrotoplast, and be not that every kind of tumour cell all expresses epithelium mark, therefore uses single EpCAM antibody capture
CTCs often will cause false negative result.In addition the production prices of antibody are expensive, antibody combined using a variety of Specific markers
Use the increase that will lead to cost again；And the stability of aptamer is insufficient, is easily degraded by nuclease.Chinese patent
It is also public respectively in CN201410326400.1, CN201510252919.4, CN201510677697.0, CN201710014061.7
The method or technique of capture CTCs is opened, but there is also above-mentioned similar defects.
Pollen is a kind of natural micro particles, from a wealth of sources, cheap.Pollen has multi-stage micro-nano structure, favorably
It interacts in the pseudopodium of CTCs, can reach nearly 90% or more in the case where not needing modified specificity identification molecule
CTCs capture rate.The method for being used to capture circulating tumor cell using pollen particles is not yet had been reported that at present.
Summary of the invention
The present invention is completed based on existing technical problem is solved, and is followed it is intended that providing one kind for capturing
Pollen structure particle of ring tumour cell and its preparation method and application.
The present invention is achieved by the following technical solutions:
First aspect present invention:
1, a kind of pollen structure particle, the pollen structure particle have core and surface layer, and the surface layer has layer
And upper epidermis, the layer are connect with the upper epidermis by support column, wherein
The upper epidermis has several holes, and pitch of holes range is 50~500nm, and pore diameter range is 50~1000nm,
The porosity ranges of the upper epidermis are 15%~80%,
The altitude range of the support column is 200nm~5000nm.
2, the pollen structure particle according to above-mentioned 1, the diameter range of the support column are 50~500nm.
3, the pollen structure particle according to above-mentioned 1 or 2, the pollen structure particle be from Nelumbonaceae, Chloranthaceae,
The pollen particles of one of Winteraceae, composite family, Cruciferae, Schisandraceae, Theaceae or various plants.
4, the pollen structure particle according to above-mentioned 1~3, the pollen particles are pollen powder of chrysanthemun flower particle and/or rape
Flower pollen particles.
Second aspect of the present invention:
5, one kind above-mentioned 1~4 described in pollen structure particle preparation method, the preparation method the following steps are included:
(1) pollen particles are added in degreasing agent, carry out degreasing is sufficiently stirred, obtains degreasing pollen particles；
(2) the degreasing pollen particles are added in alkaline solution, are stirred, then gained pollen particles are cleaned, dried
It is dry, obtain alkali cleaning pollen particles；
(3) the alkali cleaning pollen particles are added in acid and carry out pickling, by the pollen particles after pickling spend from
Sub- water cleaning, then cleaned with ethyl alcohol, it dries.
6, the preparation method according to above-mentioned 5, the degreasing agent are selected from methanol or acetone.
7, the preparation method according to above-mentioned 5 or 6, the alkaline solution is selected from sodium hydroxide solution or potassium hydroxide is molten
Liquid.
8, the preparation method according to above-mentioned 5~7, the acid are selected from one of concentrated hydrochloric acid, the concentrated sulfuric acid, concentrated nitric acid.
9, the preparation method according to above-mentioned 8, which is characterized in that the acid is the concentrated sulfuric acid.
Third aspect present invention:
10, purposes of the pollen structure particle described in one kind above-mentioned 1~4 in capture circulating tumor cell.
Beneficial effect
Pollen structure particle of the invention can fast and effeciently capture circulating tumor cell.Capture provided by the present invention
Circulating tumor cell technology is without modified specificity cancer cell identification antibody or aptamer, it can be achieved that different cancer cell-types
Capture, have advantage low in cost, easy to operate.Prepared pollen structure microparticle surfaces can be used for capturing in peripheral blood
Circulating tumor cell, be expected to be used for the early diagnosis, detection, analysis of cancer patient, the removal of cancer cell and blood purification.
Detailed description of the invention
Fig. 1 is the stereoscan photograph of pollen powder of chrysanthemun flower structure particles prepared by the embodiment of the present invention 1, and scale bar is 5 μm.
Fig. 2 is the stereoscan photograph of the surface layer side section of pollen powder of chrysanthemun flower structure particles prepared by the embodiment of the present invention 1, than
Example ruler is 100nm.
Fig. 3 be in the embodiment of the present invention 1 before handling pollen powder of chrysanthemun flower with the comparison diagram after processing, scale bar is
5μm。
Fig. 4 is scanning electricity of the pollen powder of chrysanthemun flower structure particles obtained in the embodiment of the present invention 1 after capturing MCF-7 cancer cell
Mirror photo, scale bar are 5 μm.
Fig. 5 is the stereoscan photograph of pollen powder of chrysanthemun flower particle obtained in comparative example 1 of the present invention, after processing, ratio
Ruler is 2 μm.
Description of symbols:
1: upper epidermis；2: layer；3: support column；4: hole；5: cancer cell；6: pseudopodium
Specific embodiment
In order to make the present invention easier to understand, used term is defined as follows.The term and phrase referred to if any
Meaning inconsistent with common art-recognized meanings, that the present invention of being subject to is stated.
As described herein, term " pollen structure particle " refers to the particle with pollen structure.
Pollen structure particle of the invention has core and surface layer, which has layer and upper epidermis (outermost
Layer), which is connect with the upper epidermis by support column.
Aforementioned upper epidermis has several holes, which can penetrate through upper epidermis, can not also penetrate through upper epidermis.Can own
Hole is the hole for penetrating through upper epidermis, and being also possible to a part is the hole for penetrating through upper epidermis, a part of hole not penetrate through upper epidermis.
As long as the hole helps that capture, enrichment can be played the role of, then it is not limited to whether penetrate through.Preferably all holes are perforation
The hole of upper epidermis.
Aforementioned apertures spacing range is 50~500nm, preferably 70~450nm, more preferably 100~400nm.By making hole
Spacing range control can more effectively play the three-dimensional effect of nanostructure, enhance the adherency of cell and catch in above range
It obtains.
The pore diameter range of aforementioned upper epidermis is 50~1000nm, preferably 60~550nm, more preferably 100~450nm.
The porosity ranges of aforementioned upper epidermis be 15%~80%, preferably 20%~75%, more preferably 35%~
69%.The porosity is greater than 80%, then the hole of microparticle surfaces is excessive, can not form, and is unfavorable for loading；If the porosity is less than
15%, then it is unfavorable for that for example tumour cell pseudopodium is more stretches in hole, is unfavorable for realizing capture effect of the invention.
The layer of pollen structure particle is connect by aforementioned support column with upper epidermis, objectively plays the entire pollen of support
The effect of the skeleton of structure particles.The altitude range of the support column be 200nm~5000nm, preferably 200nm~3000nm, into
One step is preferably 200nm~800nm.The diameter range of the support column is 50~500nm, preferably 100nm~400nm.
Aforementioned pollen structure particle is from Nelumbonaceae, Chloranthaceae, Winteraceae, composite family, Cruciferae, Schisandraceae, mountain
The pollen particles of one of Theaceae or various plants are then not limited to as long as pollen structure particle of the invention can be obtained
Mentioned kind is preferably selected from pollen powder of chrysanthemun flower particle and/or rape petal pollen particle.
The preparation method of pollen structure particle of the invention the following steps are included:
(1) pollen particles are added in degreasing agent, carry out degreasing is sufficiently stirred, obtains degreasing pollen particles；
(2) the degreasing pollen particles are added in alkaline solution, are stirred, then gained pollen particles are cleaned, dried
It is dry, obtain alkali cleaning pollen particles；
(3) the alkali cleaning pollen particles are added in acid and carry out pickling, by the pollen particles after pickling spend from
Sub- water cleaning, then cleaned with ethyl alcohol, it dries.
Further, in above-mentioned steps (1), aforementioned degreasing agent be selected from acetone, methanol, ethyl alcohol, chloroform, methylene chloride and
One of dichloroethanes is a variety of.
Further, in above-mentioned steps (1), degreasing time is suitably selected according to the type of pollen, as long as can incite somebody to action
The lipid material on pollen surface sufficiently removes, and preferably 6~24 hours.
Further, in above-mentioned steps (1), degreasing can be carried out while stirring.It is common that this field can be used in stirring
Method, used agitating mode can be mechanical stirring or magnetic agitation.For mixing time, preferably stir under normal temperature conditions
It mixes 6~24 hours, more preferable mixing time is 12~24 hours.Optionally, in whipping process, using corresponding stirring rate
It is stirred, stirring rate not immobilizes, for example, stirring rate is carried out according to the distribution in pollen particles degreasing agent
Appropriate adjustment can be 300~2000rpm/min, preferably 1000~2000rpm/min.
Further, in above-mentioned steps (2), aforementioned base solution can be selected from potassium hydroxide, sodium hydroxide, bicarbonate
One of potassium, sodium bicarbonate.Preferably sodium hydroxide.The concentration of alkaline solution be 4~10% (w/v), preferably 4~8%
(w/v)。
Further, in above-mentioned steps (2), alkali cleaning can be carried out while stirring.It is common that this field can be used in stirring
Method, used agitating mode can be mechanical stirring or magnetic agitation.For mixing time, preferably stir under normal temperature conditions
It mixes 6~24 hours, more preferable mixing time is 12~24 hours.Optionally, in whipping process, using corresponding stirring rate
It is stirred, stirring rate not immobilizes, for example, distribution of the stirring rate according to degreasing pollen particles in alkaline solution
State carries out appropriate adjustment, can be 300~2000rpm/min, preferably 1000~2000rpm/min.
Further, it in above-mentioned steps (2), is carried out clearly as the pollen particles after the alkali cleaning by obtained by with such as deionized water
It washes.The number cleaned is 1~5 time, preferably 3~5 times.
Further, it in above-mentioned steps (2), is dried after being cleaned to pollen particles, drying can be used
External heating system or internal heating system, but be not specifically limited, such as drying can be normal pressure drying, can also be with
It is decompression drying, preferred vacuumizing and drying.In addition, fixed electric furnace, fluidisation electric furnace, rotation electric furnace, combustion furnace can be used in drying
Or microwave system carries out.
Further, in above-mentioned steps (3), acid used in pickling can be selected from concentrated hydrochloric acid, the concentrated sulfuric acid, concentrated nitric acid etc.,
Wherein, preferably the concentrated sulfuric acid uses.When carrying out pickling using the concentrated sulfuric acid, the concentration of the concentrated sulfuric acid is 75~99% (v/v), is lower than
75%, then body structure surface can not be etched and is carbonized, to cannot achieve effect of the invention.
Further, in above-mentioned steps (3), stirring can be identical as the agitating mode in above-mentioned steps.When for stirring
Between, it preferably stirs 24~72 hours under normal temperature conditions, more preferable mixing time is 48~72 hours.
Further, it in above-mentioned steps (3), is carried out clearly as the pollen particles after the pickling by obtained by with such as deionized water
It washes, and is dried, to obtain alkali cleaning pollen particles.The number cleaned is 1~5 time, preferably 3~5 times.
Further, in above-mentioned steps (3), preferably after being cleaned with such as deionized water, before drying, alcohol is used
Solution carries out secondary cleaning to pollen particles, which is not particularly limited, preferred alcohol.The number cleaned is 1~3
Time, preferably 3 times.
Further, in above-mentioned steps (3), external heating system or internal heating system is can be used in drying, but not
It can be normal pressure drying either with or without concrete restriction, such as drying, be also possible to decompression drying, preferably vacuumizing and drying.In addition, drying
Dry fixed electric furnace, fluidisation electric furnace, rotation electric furnace, combustion furnace or the microwave system of can be used carries out.
After obtaining pollen structure particle through the above steps, also optionally the pollen structure particle is immobilized on
On carrier.
The pollen structure particle of the invention obtained by above-mentioned manufacturing method, to circulating tumor cell such as human breast carcinoma
Cell (MCF-7 cell), people's epidermis cancer cell (A431 cell), human cervical carcinoma cell (Hela cell) and Non-small cell lung carcinoma
The capture rate of cell (A549 cell) is up to 85% or more, preferably 90% or more.
(1)~step (3) through the above steps, gained pollen structure microparticle surfaces have the hole of several special distributions, table
Face forms multi-stage micro-nano structure, so as to efficient capture CTCs etc..It is of the invention using cheap, from a wealth of sources
Natural pollen particle can effectively reduce use cost without immobilized specific cancer cell identification antibody or aptamer.It should
The pollen structure particle of kind structure is, it can be achieved that the capture to different cancer cell-types can be applied to cancer with preferable broad spectrum activity
The early diagnosis of disease patient.
Hereinafter, enumerating embodiment to illustrate a specific embodiment of the invention, but embodiments of the present invention are not by as follows
The limitation of these embodiments can make any selection and change in the range of not influencing technical effect to be achieved of the invention
More.The person that is not specified actual conditions in embodiment and test example, carries out, examination used according to conventional conditions or manufacturer's recommended conditions
Production firm person is not specified in agent or instrument, and being can be with conventional products that are commercially available.
Embodiment 1
The pollen particles that the present embodiment is selected are natural pollen powder of chrysanthemun flower (average grain diameter is 22 μm), thin in the Chinese Academy of Sciences to buy
The human breast cancer cell (MCF-7 cell) in born of the same parents library is circulating tumor cell model to be captured, is made to capture system of the invention
Further elucidated above and verifying, comprising the following steps:
1. preparing the pollen powder of chrysanthemun flower structure particles for capturing circulating tumor cell
The natural pollen powder of chrysanthemun flower particle of 50g is added in 1L acetone, under conditions of room temperature, with 1000 revs/min
Rate stirs 6 hours, degreasing pollen powder of chrysanthemun flower particle is obtained after filtering.
It is 4% (w/v) that degreasing pollen powder of chrysanthemun flower particle, which is added to concentration, and volume is in the NaOH alkaline solution of 1L, normal
Under conditions of temperature, with 1000 revs/min of rate, stir 12 hours.By the pollen powder of chrysanthemun flower micro particle filtering after alkali cleaning, deionization is used
Water cleans 3 times, remaining alkaline solution is removed, then by pollen powder of chrysanthemun flower particle vacuum drying under normal temperature condition.
Alkali cleaning pollen powder of chrysanthemun flower after drying is added in the concentrated sulfuric acid that concentration is 98% (v/v), under conditions of room temperature,
With 1000 revs/min of rate, stir 24 hours.Pollen powder of chrysanthemun flower particle after pickling is cleaned 3 times with deionized water, is removed residual
Remaining sulfuric acid, then cleaned 3 times with ethyl alcohol, by pollen powder of chrysanthemun flower particle vacuum drying under normal temperature condition, the chrysanthemum flowers that obtain that treated
Powder structure particles.
Gained pollen powder of chrysanthemun flower structure particles are immobilized on a length of 1cm with spray coating method, width is to remain spare on the silicon wafer of 1cm.
The structure of observation gained pollen powder of chrysanthemun flower structure particles under the microscope, wherein Fig. 1 shows what embodiment 1 obtained
The stereoscan photograph (scale bar be 5 μm) of pollen powder of chrysanthemun flower structure particles is shown clearly in the particle upper epidermis (table in Fig. 1
Face) several holes are distributed with；The scanning electron microscope of the surface layer side section of the pollen powder of chrysanthemun flower structure particles obtained Fig. 2 shows embodiment 1
Photo (scale bar 100nm) has layer and upper epidermis, layer and upper epidermis on particle surface layer as can be seen from Figure 2
Between connected by several support columns；Fig. 3 is shown in embodiment 1 before handling pollen powder of chrysanthemun flower (a) and (b) after processing
Comparison diagram (scale bar is 5 μm), as can be seen from Figure 3, although before treatment on surface there are a certain number of holes, hole
Quantity is few, is unevenly distributed, and after process processing of the invention, the quantity and density in hole greatly increase, and are distributed
It is relatively uniform.
Then, a small amount of above-mentioned pollen powder of chrysanthemun flower structure particles are chosen, which is amplified 40,000 times under a scanning electron microscope
Take pictures (100 total), randomly selects the photo under 20 different visuals field, is measured, obtained by nanomeasure software
The parameter of the particle is as follows:
Particle diameter: 22 μm
The pore diameter range of upper epidermis: 65~453nm average value: 135nm
The pitch of holes range of upper epidermis: 70~270nm average value: 160nm
Support column diameter range: 100~220nm average value: 180nm
Support column height range: 430~720nm average value: 540nm
It is 56% by the porosity that Image-J software calculates the pollen powder of chrysanthemun flower structure particles.
Wherein, the calculation of above-mentioned porosity is as follows:
Porosity=(x/N) × 100% (formula 1)
X: hole area N: field area
The porosity in each visual field is calculated by equation 1 above, takes the average value of the porosity in all visuals field then to get arriving
The porosity of pollen structure particle.
2. preparing circulating tumor cell sample to be measured:
100 μ L of MCF-7 cell suspending liquid is taken, is counted with cell counter and calculates its concentration.It draws a certain amount of above-mentioned
Cell suspending liquid, with DMEM cell culture medium to 1 × 105A cell/mL.
3. capture experiment:
The silicon wafer for there are pollen powder of chrysanthemun flower structure particles immobilized in above-mentioned steps 1 is put into tissue culture plate, it then will be above-mentioned
The uniformly mixed cell suspending liquid 1mL of step 2 is added drop-wise to pollen powder of chrysanthemun flower structure particles surface, is subsequently placed in cell incubator,
Capture time is 60 minutes.
As control experiment group 1, the immobilized silicon chip surface for having natural pollen powder of chrysanthemun flower particle is also carried out identical to test sample
Capture circulating tumor cell experiment in product.
After capture time, capture is had to the pollen powder of chrysanthemun flower microparticle surfaces phosphate buffer of circulating tumor cell
(PBS) it cleans three times, with cancer cell 30 minutes of the paraformaldehyde aqueous solution fixed trapped that mass concentration is 4%, by capture
Cancer cell is immersed in the DAPI aqueous solution that concentration is 10 μ g/mL 30 minutes, achievees the purpose that dyeing.Finally fallen in Olympus
Take pictures respectively under 10 times of fluorescence microscope (100 total) is set, the fluorescence photo under 20 different visuals field is randomly selected, is utilized
ImageJ software counts the circulating tumor cell of capture, calculates capture rate.Control experiment group 1 also according to above-mentioned steps into
Row, and calculate capture rate.
The calculation method of capture rate is as follows:
The silicon wafer for having pollen powder of chrysanthemun flower particle immobilized in step 1 is put into tissue culture plate (floor space 2cm2)。
Capture rate=[(x/Ax)/(N/A)] × 100% (formula 2)
The capture quantity of circulating tumor cell in the lower fluorescence photo shone of x:10 times of fluorescence microscope；
The lower fluorescence photo visual field size shone of Ax:10 times of fluorescence microscope；
X/Ax: the cell density of actual acquisition；
N: total injected volume of cell (is " 10 in the present embodiment5A cells/ml ")；
A: cell culture version floor space (is " 2cm in the present embodiment2”)
N/A: the density of cell is launched
The capture rate in each visual field is calculated by equation 2 above, takes the average value of the capture rate in all visuals field then to get arriving
Capture rate of the pollen structure particle to circulating tumor cell.
It is above-mentioned the experimental results showed that, the silicon chip surface that MCF-7 cancer cell has pollen powder of chrysanthemun flower structure particles immobilized stretch out compared with
More pseudopodium are sprawled (referring to fig. 4) in good condition, and pollen powder of chrysanthemun flower microparticle surfaces reach 92% to the capture rate of MCF-7 cancer cell, and
In control experiment group 1, the immobilized silicon wafer for having natural pollen powder of chrysanthemun flower particle is only 7% to the capture rate of MCF-7 cancer cell, shows this
The efficient capture of circulating tumor cell may be implemented in particle.
Embodiment 2
The pollen particles that the present embodiment is selected are natural rape pollen (average grain diameter is 20 μm), thin in the Chinese Academy of Sciences to buy
The human breast cancer cell (MCF-7 cell) in born of the same parents library, people's epidermis cancer cell (A431 cell), human cervical carcinoma cell (Hela cell) and
Non-small cell lung carcinoma cell (A549 cell) be circulating tumor cell model to be captured, to capture system of the invention make into
The elaboration and verifying of one step, comprising the following steps:
1. preparing the rape pollen structure particles for capturing circulating tumor cell
The natural rape pollen particle of 75g is added in 1L methanol, under conditions of room temperature, with 1000 revs/min
Rate stirs 12 hours, degreasing rape pollen particle is obtained after filtering.
It is 6% (w/v) that degreasing rape pollen particle, which is added to concentration, and volume is in the NaOH alkaline solution of 1L, normal
Under conditions of temperature, with 1000 revs/min of rate, stir 18 hours.By the rape pollen micro particle filtering after alkali cleaning, deionization is used
Water cleans 4 times, remaining alkaline solution is removed, then by rape pollen particle vacuum drying under normal temperature condition.
Alkali cleaning rape pollen particle after drying is added in the concentrated sulfuric acid that concentration is 70% (v/v).In the item of room temperature
Under part, with 1000 revs/min of rate, stir 48 hours.Rape pollen particle after pickling is cleaned 3 times with deionized water,
Remaining sulfuric acid is removed, then is cleaned 3 times with ethyl alcohol, by rape pollen particle vacuum drying under normal temperature condition, obtains that treated
Rape pollen structure particles.
Gained rape pollen structure particles are immobilized on a length of 1cm with spray coating method, width is to remain spare on the silicon wafer of 1cm.
In addition, choosing a small amount of above-mentioned rape pollen structure particles, which is amplified 40,000 times under a scanning electron microscope
Take pictures (100 total), randomly selects the photo under 20 different visuals field, is measured, obtained by nanomeasure software
The parameter of the particle is as follows:
Particle diameter: 24 μm
The pore diameter range of upper epidermis: 240~520nm average value: 290nm
The pitch of holes range of upper epidermis: 250~400nm average value: 330nm
Support column diameter range: 240~440nm average value: 330nm
Support column height range: 250~870nm average value: 520nm
It is 68% by the porosity that method same as Example 1 measures the upper epidermis of the rape pollen structure particles.
2. preparing circulating tumor cell sample to be measured:
Each 100 μ L of people's MCF-7, A431, Hela and A549 cell suspending liquid is taken respectively, is counted and is calculated with cell counter
Its respective concentration；Draw a certain amount of above-mentioned cell suspending liquid respectively, with DMEM cell culture medium respectively suspension to 1 ×
105A cell/mL.
3. capture experiment:
The silicon wafer for there are rape pollen structure particles immobilized in above-mentioned steps 1 is put into tissue culture plate, then by even numbers
The uniformly mixed cell suspending liquid 1mL of step 2 is added drop-wise to rape pollen structure particles surface, is subsequently placed in cell incubator,
Capture time is 60 minutes；The immobilized silicon chip surface for having natural rape pollen particle is directed to respectively as control experiment group 2
People MCF-7, A431, Hela and A549 cell also carry out identical 4 groups of capture circulating tumor cells experiment.
After capture time, capture is had to the rape pollen microparticle surfaces surface phosphate-buffered of circulating tumor cell
Liquid (PBS) cleans three times, with cancer cell 30 minutes of the paraformaldehyde aqueous solution fixed trapped that mass concentration is 4%, will capture
Cancer cell be immersed in concentration be 10 μ g/mL DAPI aqueous solution in 30 minutes, achieve the purpose that dyeing.Finally in Olympus
Take pictures (100 total) respectively under 10 times of inverted fluorescence microscope, randomly selects the fluorescence photo under 20 different visuals field, utilizes
ImageJ software counts the circulating tumor cell of capture, calculates capture rate, the calculation method of capture rate is the same as embodiment 1.
Control experiment group 2 is also carried out according to above-mentioned steps, and calculates capture rate.
The experimental results showed that the rape pollen microparticle surfaces of preparation catch MCF-7, A431, Hela and A549 cancer cell
The rate of obtaining respectively reaches 89%, 92%, 87% and 90%, shows that the efficient capture to different cancerous cell lines may be implemented in the particle,
With preferable broad spectrum activity.
Embodiment 3
The pollen particles that the present embodiment is selected are natural pollen powder of chrysanthemun flower (average grain diameter is 22 μm), to peripheral blood mononuclear cells
Middle addition purchase is circulating tumor cell model to be captured in the human breast cancer cell (MCF-7 cell) of Chinese Academy of Sciences's cell bank,
Capture system of the invention is further elaborated and is verified, comprising the following steps:
1. preparing the pollen powder of chrysanthemun flower particle for capturing circulating tumor cell
The natural pollen powder of chrysanthemun flower particle of 100g is added in 1L acetone.Under conditions of room temperature, with 1000 revs/min
Rate stirs 24 hours, degreasing pollen is obtained after filtering.
It is 10% (w/v) that degreasing pollen, which is added to concentration, and volume is in the NaOH alkaline solution of 1L.In the condition of room temperature
Under, it with 1000 revs/min of rate, stirs 12 hours, the pollen filter after alkali cleaning is cleaned 5 times with deionized water, remove residual
Remaining alkaline solution, then by pollen vacuum drying under normal temperature condition.
Alkali cleaning pollen powder of chrysanthemun flower particle after drying is added in the concentrated sulfuric acid that concentration is 70% (v/v), in the item of room temperature
Under part, with 1000 revs/min of rate, stir 72 hours.Pollen after pickling is cleaned 3 times with deionized water, removal is remaining
Sulfuric acid, then cleaned 3 times with ethyl alcohol, by pollen vacuum drying under normal temperature condition, the pollen powder of chrysanthemun flower structure particles that obtain that treated.
Gained pollen powder of chrysanthemun flower structure particles are immobilized on a length of 1cm with spray coating method, width is to remain spare on the silicon wafer of 1cm.
Then, a small amount of above-mentioned pollen powder of chrysanthemun flower structure particles are chosen, which is amplified 40,000 times under a scanning electron microscope
Take pictures (100 total), randomly selects the photo under 20 different visuals field, is measured, obtained by nanomeasure software
The parameter of the particle is as follows:
Particle diameter: 22 μm
The pore diameter range of upper epidermis: 55~436nm average value: 118nm
The pitch of holes range of upper epidermis: 75~290nm average value: 190nm
Support column diameter range: 110~235nm average value: 189am
Support column height range: 425~718nm average value: 545nm
It is 49% by the porosity that method same as Example 1 measures the upper epidermis of the rape pollen structure particles.
2. preparing circulating tumor cell sample to be measured:
Take the fresh anticoagulation of 5mL, into blood be added peripheral blood mononuclear cells separating liquid (Beijing Suo Laibao biotechnology,
P8610), using density-gradient centrifugation method separating periphery blood monocytic cell, peripheral blood mononuclear cells suspension, cell concentration are obtained
It is 0.8 × 106A cell/mL；100 μ L of MCF-7 cell suspending liquid is taken, is counted with cell counter and calculates its concentration；Outward
Be separately added into all blood monocyte suspension cancer cell number (unit: a cell/mL) be 130,168,214,241,399,
534,691,786,812 and 1052 MCF-7 cell is uniformly mixed.
3. capture experiment:
The silicon wafer for there are pollen powder of chrysanthemun flower structure particles immobilized in above-mentioned steps 1 is put into tissue culture plate, then by step 2
Uniformly mixed cell suspending liquid 1mL is added drop-wise to pollen powder of chrysanthemun flower structure particles surface, is subsequently placed in cell incubator, when capture
Between be 60 minutes.
After capture time, the pollen powder of chrysanthemun flower microparticle surfaces phosphate buffer of circulating tumor cell will be captured
(PBS) it cleans three times, with cancer cell 30 minutes of the paraformaldehyde aqueous solution fixed trapped that mass concentration is 4%, by capture
Cancer cell is immersed in the DAPI aqueous solution that concentration is 10 μ g/mL 30 minutes, the Cytokeratin-FITC phosphate of 10 μ g/mL
60 minutes in buffer, achieve the purpose that dyeing.It finally takes pictures respectively under 10 times of laser confocal fluorescence microscope (total
100), randomly select the fluorescence photo under 20 different visuals field, using ImageJ software to the circulating tumor cell of capture into
Row counts, and calculates capture rate, the calculation method of capture rate is the same as embodiment 1.
The experimental results showed that in the pollen powder of chrysanthemun flower microparticle surfaces human peripheral blood sample of preparation, MCF-7 cancer cell cancer cell number
When amount (unit: a cell/mL) is respectively 130,168,214,241,399,534,691,786,812 and 1052, MCF-7 cell
Capture rate difference it is as described in Table 1:
Table 1
From above-mentioned table 1 it is found that the capture rate in the case of difference MCF-7 cancer cell cancer cell number nearly reach 90% with
On, this shows that the pollen structure particle in the case where circulating tumor cell of different injected volumes, may be implemented from peripheral blood
In efficiently capture.
Embodiment 4
The pollen particles that the present embodiment is selected be dandelion pollen (average grain diameter be 12 μm), in addition to this, with implementation
Example 1 is operated under the same conditions, obtains dandelion pollen structure particles.
Then, it is equally operated with the step 2 of embodiment 1 and step 3, capture experiment is carried out under similarity condition, as a result,
To the capture rate of the pollen particles of comparative example 3: 89%.
Then, a small amount of above-mentioned pollen powder of chrysanthemun flower structure particles are chosen, which is amplified 40,000 times under a scanning electron microscope
Take pictures (100 total), randomly selects the photo under 20 different visuals field, is measured, obtained by nanomeasure software
The parameter of the particle is as follows:
Particle diameter: 22 μm
The pore diameter range of upper epidermis: 62~224nm average value: 142nm
The pitch of holes range of upper epidermis: 87~180nm average value: 126nm
Support column diameter range: 76~175nm average value: 139nm
Support column height range: 223~307nm average value: 255nm
It is 47% by the porosity that method same as Example 1 measures the upper epidermis of the rape pollen structure particles.
The above result shows that the efficient capture of circulating tumor cell may be implemented in the particle.
Comparative example 1
The natural pollen powder of chrysanthemun flower particle of 50g is added in 1L acetone, under conditions of room temperature, with 1000 revs/min
Rate stirs 6 hours, degreasing pollen powder of chrysanthemun flower particle is obtained after filtering.The particle is dried 12 hours at 70 DEG C, is then existed
It heats under air environment in Muffle furnace 6 hours (10 DEG C/min of heating rate) at 300 DEG C, then, the particle after heating is existed
Heat 2 hours in 700 DEG C of tube furnaces, then in argon atmosphere (250 ml/min of flow velocity) with 10 DEG C/min of heating rate
Heating, the pollen powder of chrysanthemun flower particle that obtains that treated.Then gained pollen powder of chrysanthemun flower particle is immobilized on a length of 1cm, width as the silicon of 1cm
On piece remains spare.
The structure of observation gained pollen powder of chrysanthemun flower particle under the microscope, referring to Fig. 5.It is found that it should treated chrysanthemum from the Fig. 5
Flower pollen particles volume is obviously shunk, and upper surface is barely perceivable hole.
Then, it is equally operated with the step 2 of embodiment 1 and step 3, capture experiment is carried out under similarity condition, as a result,
Capture rate to the pollen particles of comparative example 1 is 33.2%.
Comparative example 2
In addition to not used other than the concentrated sulfuric acid handled (without pickling) in step 1, other conditions and step 1 and real
It is all the same to apply example 1, obtains pollen powder of chrysanthemun flower structure particles.
Then, it is equally operated with the step 2 of embodiment 1 and step 3, capture experiment is carried out under similarity condition, as a result,
Capture rate to the pollen particles of comparative example 2 is 63%.
Then, a small amount of above-mentioned pollen powder of chrysanthemun flower structure particles are chosen, which is amplified 40,000 times under a scanning electron microscope
Take pictures (100 total), randomly selects the photo under 20 different visuals field, is measured, obtained by nanomeasure software
The parameter of the particle is as follows:
Particle diameter: 22.5 μm
The pore diameter range of upper epidermis: 37~398nm average value: 98nm
The pitch of holes range of upper epidermis: 82~370nm average value: 235nm
Support column diameter range: 112~245nm average value: 197nm
Support column height range: 415~738nm average value: 532nm
It is 32% by the porosity that method same as Example 1 measures the upper epidermis of the rape pollen structure particles.
Comparative example 3
In addition to being handled in step 1 using concentrated phosphoric acid the alkali cleaning pollen powder of chrysanthemun flower after drying, without the use of the concentrated sulfuric acid
It is handled, in addition to this, other conditions and step and embodiment 1 are all the same, obtain pollen powder of chrysanthemun flower structure particles.
Then, it is equally operated with the step 2 of embodiment 1 and step 3, capture experiment is carried out under similarity condition, as a result,
To the capture rate of the pollen particles of comparative example 3: 65%.
Then, a small amount of above-mentioned pollen powder of chrysanthemun flower structure particles are chosen, which is amplified 40,000 times under a scanning electron microscope
Take pictures (100 total), randomly selects the photo under 20 different visuals field, is measured, obtained by nanomeasure software
The parameter of the particle is as follows:
Particle diameter: 22 μm
The pore diameter range of upper epidermis: 52~431nm average value: 110nm
The pitch of holes range of upper epidermis: 89~301nm average value: 210nm
Support column diameter range: 118~243nm average value: 195nm
Support column height range: 421~725nm average value: 543nm
It is 39% by the porosity that method same as Example 1 measures the upper epidermis of the rape pollen structure particles.
Industrial availability
The present invention relates to a kind of Biofunctional materials of technical field of analysis and detection and its manufacturing methods, in particular to are used to
Capture the Biofunctional materials and its manufacturing method of circulating tumor cell.Prepared pollen structure microparticle surfaces can be used for capturing
Circulating tumor cell in peripheral blood, is expected to be used for the early diagnosis, detection, analysis of cancer patient, the removal of cancer cell and
Blood purification.
Claims (10)
1. a kind of pollen structure particle, which is characterized in that the pollen structure particle has core and surface layer, and the surface layer has
Layer and upper epidermis, the layer are connect with the upper epidermis by support column, wherein
The upper epidermis have several holes, pitch of holes range be 50~500nm, pore diameter range be 50~1000nm, it is described on
The porosity ranges on surface layer are 15%~80%,
The altitude range of the support column is 200nm~5000nm.
2. pollen structure particle according to claim 1, which is characterized in that the diameter range of the support column be 50~
500nm。
3. pollen structure particle according to claim 1 or 2, which is characterized in that the pollen structure particle is from lotus
The pollen particles of one of section, Chloranthaceae, Winteraceae, composite family, Cruciferae, Schisandraceae, Theaceae or various plants.
4. pollen structure particle according to claims 1 to 3, which is characterized in that the pollen particles are that pollen powder of chrysanthemun flower is micro-
Grain and/or rape petal pollen particle.
5. the preparation method of pollen structure particle described in a kind of Claims 1 to 4, which is characterized in that the preparation method includes
Following steps:
(1) pollen particles are added in degreasing agent, carry out degreasing is sufficiently stirred, obtains degreasing pollen particles；
(2) the degreasing pollen particles are added in alkaline solution, are stirred, then gained pollen particles are cleaned, drying obtains
To alkali cleaning pollen particles；
(3) the alkali cleaning pollen particles are added in acid and carry out pickling, by the pollen particles deionized water after pickling
Cleaning, then cleaned with ethyl alcohol, it dries.
6. preparation method according to claim 5, which is characterized in that the degreasing agent is selected from methanol or acetone.
7. preparation method according to claim 5 or 6, which is characterized in that the alkaline solution is selected from sodium hydroxide solution
Or potassium hydroxide solution.
8. the preparation method according to claim 5~7, which is characterized in that the acid is selected from concentrated hydrochloric acid, the concentrated sulfuric acid, dense nitre
One of acid.
9. preparation method according to claim 8, which is characterized in that the acid is the concentrated sulfuric acid.
10. a kind of purposes of the pollen structure particle described in Claims 1 to 4 in capture circulating tumor cell.
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| HONG WANG ET AL.: "Bioinspired Spiky Micromotors Based on Sporopollenin Exine Capsules", 《ADV. FUNCT. MATER.》 * |
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| CN115232790A (en) * | 2022-07-29 | 2022-10-25 | 康元医疗科技(大连)有限公司 | Pollen particle, preparation method, biological functional material using same and application |
| CN115232790B (en) * | 2022-07-29 | 2023-09-12 | 康元医疗科技(大连)有限公司 | Pollen particles, preparation method, biological functional material using the same and application thereof |
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