CN109521202A - A kind of low abundance proteins absolute quantification method based on digital immuno analytical method - Google Patents
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
Abstract
The present invention relates to a kind of low abundance proteins absolute quantification methods based on digital immuno analytical method, this method will capture magnetic bead, target antigen and detection particle are immunoreacted, form immune complex, by the detection particle elution in immune complex, collect concentrate eluant, concentrate is transferred to micro-fluidic grain count chip, detection particle deposition is fixed on micro-fluidic grain count chip after solvent volatilization completely in solution to be concentrated, record by imaging is carried out to all detection particles being fixed on micro-fluidic grain count chip, count the quantity that particle is detected on micro-fluidic grain count chip, obtain the molecular number of target protein.It is an advantage of the invention that can be realized the hypersensitive of disease-associated protein, absolute quantification analysis in clinical sample, standard items are not needed, do not need to draw standard curve, with applied widely, detection sensitivity is high, and analysis result accuracy is high, precision is good, the high advantage of flux.
Description
Technical field
The present invention relates to a kind of low abundance proteins absolute quantification method based on digital immuno analytical method, especially one
Kind belongs to protein hypersensitive analytical technology neck for the hypersensitive absolute quantification analysis method of low-abundance protein in clinical sample
Domain.
Background technique
It is reported that the generation of disease, the unconventionality expression of development and protein or specific protein expression are closely related.It is accurate to survey
Determine the content of disease related protein infectious disease prevention and control, screening for cancer and precisely in terms of be of great significance.Currently, egg
The quantitative method of white matter mainly includes relative quantification and absolute quantitation.The former is the method based on standard curve, is widely used in
Analytical chemistry and clinical examination；The latter does not need protein standard substance, does not need to draw standard curve, is conducive to Simplified analysis step
Suddenly, reduce sample preparation and analysis time, increase detection precision.
Isotopic dilution analysis based on mass spectrum (MS) is considered as the mainstream technology of protein absolute quantitation.Analyze it
Before, the internal standard compound of isotope labelling is added into appropriate amount of sample, by comparing the internal standard compound of isotope labelling and specific natural
The signal difference that peptide fragment generates realizes quantifying for protein.Strictly speaking, belonged to based on mass spectrographic protein absolute quantitation interior
The One point standard of mark method.In addition, the sample pretreatment time of mass spectral analysis is long (such as isotope labelling, trypsin digestion),
Instrument precision is expensive, cannot be quantitative to low abundance proteins, limits it in the application of hypersensitive analysis field.
Numerical analysis technique is a kind of potential biomolecule absolute quantification method.Sample is scattered in multiple short spaces
(such as micropore and microlayer model), single short space contain the quantity of biomolecule less than 6.The signal of short space output is used
" 1 " and " 0 " indicate, respectively represent target molecule with and without.Using Poisson distribution as statistical basis, the concentration of target molecule can be by
The ratio of " 1 ", which calculates, to be obtained.According to above-mentioned theory, digital drop PCR(polymerase chain reaction) technology has been carried out nucleic acid
Absolute copy number analysis.But existing protein numerical analysis technique (Enzyme Linked Immunoadsorbent Assay and digital electrochemical analysis)
It is lost part signal in signal amplification and departure process, still needs to antigen standard and standard curve to realize that protein is fixed
Amount.Therefore, it is necessary to develop new digital immunoassay strategy or method of counting to realize protein absolute quantitation.
Summary of the invention
It is an object of the invention to: in view of the deficiency of the prior art, propose a kind of based on digital immunoassay
The low abundance proteins absolute quantification method of technology, be able to solve existing protein analysis technology can not absolute quantitation the problem of,
Technological gap is filled up.
In order to reach the goals above, technical scheme is as follows: a kind of based on the low rich of digital immuno analytical method
Protein absolute quantification method is spent, for the system that this method is related to using optical microscopy as imaging platform, micro-fluidic chip is particle
Counting device, number are immunized as analytical model, and micron particles are count detection probe, can realize clinical sample in single molecules level
Hypersensitive, the absolute quantification analysis of middle low abundance proteins.Method includes the following steps:
The first step, the micron particles that the magnetic bead, target antigen (i.e. target protein) and modification of modification capture antibody are detected to antibody
It is immunoreacted, forms ternary immune complex, contain a target protein molecule in an immune complex；Modification capture
The magnetic bead of antibody referred to as capture magnetic bead, the micron particles of modification detection antibody referred to as detection particle；
Second step is eluted the detection particle in ternary immune complex using acid solution, is collected and is concentrated comprising inspection
Survey the eluent of particle；
The detection particle solution of concentration is transferred to micro-fluidic grain count chip, solvent evaporation method fixed test by third step
, the completely rear detection particle of solvent volatilization, which deposits, in solution to be concentrated is fixed on micro-fluidic grain count chip, then uses optics
Microscope carries out record by imaging to all detection particles being fixed on micro-fluidic grain count chip；
The quantity of particle is detected in 4th step, the micro-fluidic grain count chip of statistics to get the molecular number of target protein is arrived.
Absolute quantitation range of the present invention be 180~60000 protein molecules, i.e., 3 × 10-18 M~1 × 10-15 M
(100 μ L sample).
The technical solution that the present invention advanced optimizes is as follows:
Preferably, in third step, under the substrate surface positioning grid auxiliary of micro-fluidic grain count chip, using optical microphotograph
Mirror successively carries out subregion imaging to the detection particle of all fixations, obtains microphoto.
Preferably, in the 4th step, using the detection amounts of particles in software statistics microphoto, as point of target antigen
Subnumber.
Preferably, to detect particle as the direct exhausted of the target protein molecule in count detection probe realization individual particle level
To quantitative analysis, Monitoring lower-cut is 50 molecules, and dynamic range is 180~60000 molecules.
Preferably, in the first step, previously prepared capture magnetic bead and detection particle are needed, the method for preparation capture magnetic bead is such as
Under:
It takes magnetic bead stoste and magnetic bead stoste is cleaned using sodium hydroxide solution and deionized water, add into the magnetic bead stoste after cleaning
Enter EDC solution, stand 0.2 ~ 1 hour at room temperature, Magneto separate removes supernatant, and it is anti-that capture is added to the lower part precipitating after Magneto separate
Liquid solution, concussion is incubated for 0.5 ~ 2 hour at room temperature, cleans to obtain capture magnetic bead using PBS buffer solution；
The method of preparation detection particle is as follows:
It takes micron particles stoste and uses MES buffer solution for cleaning, EDC solution, room is added into the micron particles stoste after cleaning
The lower mixing of temperature 15 minutes, is centrifuged off supernatant, and after using PBS buffer solution cleaning to the lower part precipitating after centrifugation, detection is added
Antibody, concussion is incubated for 0.5 ~ 3 hour at room temperature, PBS buffer solution cleaning, and hydroxylamine hydrochloride solution is then added and mixes 30 minutes, obtains
To detection particle.
Preferably, in the first step, the specific method is as follows for preparation ternary immune complex:
Capture magnetic bead solution is taken, Magneto separate removes supernatant, and target antigen solution, room temperature is added into the lower part precipitating after Magneto separate
After lower concussion is incubated for 0.5 ~ 3 hour, Magneto separate removes supernatant, then confining liquid, room temperature is added into the lower part precipitating after Magneto separate
Lower closing 0.5 ~ 1.5 hour, Magneto separate removes supernatant, and detection particle solution, room temperature are then added into the precipitating after Magneto separate
Lower concussion is incubated for 0.5 ~ 3 hour, forms ternary immune complex.
Preferably, the shape of the detection particle is spherical shape, star, triangle, stick, annular or irregular shape.
Preferably, the detection particle is by one kind of high molecular material, inorganic material or biomaterial or above-mentioned any two
The composite material of kind is made；The size of the detection particle is between 0.5~10 micron.
Preferably, the high molecular material be at least one of polystyrene, silica gel, polyacrylate, epoxy resin,
The inorganic material is at least one of inorganic silicon materials, inorganic carbon material, semiconductor material, and the biomaterial is poly- third
Acrylamide and its derivative gel, polyethylene glycol gel, peptide-based gel, gelatin, calcium alginate gel, cellulose and its derivates
At least one of gel.
Preferably, the optical microscopy is conventional light field microscope, conventional fluorescent microscope or micromation microscope.
Preferably, the micro-fluidic grain count chip is by having the substrate of positioning grid and microporous layers to be bonded.
Preferably, the material of the substrate is glass, polymethyl methacrylate (PMMA), dimethyl silicone polymer
(PDMS), polycarbonate (PC), any one in polystyrene (PS)；The material of the microporous layers is glass, poly- methyl-prop
Any one of e pioic acid methyl ester (PMMA), dimethyl silicone polymer (PDMS), polycarbonate (PC), polystyrene (PS)；It is described
Positioning grid is lines, figure, any one or a few combination in icon.
It is an advantage of the invention that can be realized the hypersensitive of disease-associated protein, absolute quantification analysis in clinical sample,
Standard items are not needed, do not need to draw standard curve, have applied widely, detection sensitivity is high, analysis result accuracy is high,
Precision is good, the high advantage of flux.
Detailed description of the invention
The invention will be further described with reference to the accompanying drawing.
Fig. 1 is digital immunoassay flow diagram in the present invention.
Fig. 2 is the schematic diagram of micro-fluidic grain count chip in the present invention.
Fig. 3 is the light field figure of detection particle in chip micropore in the present invention.
Fig. 4 is that the absolute quantity of particle and the corresponding relationship of CEA molecular number are detected in the present invention.
Fig. 5 is the canonical plotting that Plays addition method of the present invention detects #5 plasma C EA.
Fig. 6 is the canonical plotting that Plays addition method of the present invention detects #10 plasma C EA.
In figure: 1. capture magnetic beads, 2. detection particles, 3. antigens, 4. magnet, 5. basal layers with positioning grid, 6.
Microporous layers, 7. liquid-transfering guns, 8. detection particle suspension liquids, 9. positioning grid lines, 10. detection particles.
Specific embodiment
Following will be combined with the drawings in the embodiments of the present invention, and technical solution in the embodiment of the present invention carries out clear, complete
Site preparation description, it is clear that described embodiments are only a part of the embodiments of the present invention, instead of all the embodiments.It is based on
Embodiment in the present invention, it is obtained by those of ordinary skill in the art without making creative efforts every other
Embodiment shall fall within the protection scope of the present invention.
A kind of low abundance proteins absolute quantification method based on digital immuno analytical method, the system that this method is related to
Optical microscopy is imaging platform, and micro-fluidic chip is grain count device, and number is immunized as analytical model, and micron particles are meter
Number detection probe, can realize the hypersensitive of low abundance proteins, absolute quantification analysis in clinical sample in single molecules level.The party
Method the following steps are included:
The first step, the micron particles that the magnetic bead, target antigen (i.e. target protein) and modification of modification capture antibody are detected to antibody
It is immunoreacted, forms ternary immune complex, it is immune that a target antigen molecules generate an immune complex i.e. one
Contain a target protein molecule in compound；The magnetic bead of modification capture antibody referred to as capture magnetic bead, modifies the micro- of detection antibody
Rice grain referred to as detection particle.
Previously prepared capture magnetic bead and detection particle are needed, the method for preparation capture magnetic bead is as follows:
It takes magnetic bead stoste and magnetic bead stoste is cleaned using sodium hydroxide solution and deionized water, add into the magnetic bead stoste after cleaning
Enter EDC solution, stand 0.2 ~ 1 hour at room temperature, Magneto separate removes supernatant, and it is anti-that capture is added to the lower part precipitating after Magneto separate
Liquid solution, concussion is incubated for 0.5 ~ 2 hour at room temperature, cleans to obtain capture magnetic bead using PBS buffer solution；
The method of preparation detection particle is as follows:
It takes micron particles stoste and uses MES buffer solution for cleaning, EDC solution, room is added into the micron particles stoste after cleaning
The lower mixing of temperature 15 minutes, is centrifuged off supernatant, and after using PBS buffer solution cleaning to the lower part precipitating after centrifugation, detection is added
Antibody, concussion is incubated for 0.5 ~ 3 hour at room temperature, PBS buffer solution cleaning, and hydroxylamine hydrochloride solution is then added and mixes 30 minutes, obtains
To detection particle.
Preparing ternary immune complex, the specific method is as follows:
Capture magnetic bead solution is taken, Magneto separate removes supernatant, and target antigen solution, room temperature is added into the lower part precipitating after Magneto separate
After lower concussion is incubated for 0.5 ~ 3 hour, Magneto separate removes supernatant, then confining liquid, room temperature is added into the lower part precipitating after Magneto separate
Lower closing 0.5 ~ 1.5 hour, Magneto separate removes supernatant, and detection particle solution, room temperature are then added into the precipitating after Magneto separate
Lower concussion is incubated for 0.5 ~ 3 hour, forms ternary immune complex.
The shape for detecting particle is spherical shape, star, triangle, stick, annular and irregular shape etc..Particle is detected by height
Molecular material, one kind of inorganic material or biomaterial or above-mentioned any two kinds of composite material are made；Detect the size of particle
Between 0.5~10 micron；The high molecular material is polystyrene, silica gel, polyacrylate, at least one in epoxy resin
Kind, the inorganic material is at least one of inorganic silicon materials, inorganic carbon material, semiconductor material, and the biomaterial is
Polyacrylamide and its derivative gel, polyethylene glycol gel, peptide-based gel, gelatin, calcium alginate gel, cellulose and its spread out
At least one of biogel.
Second step is eluted the detection particle in ternary immune complex using acid solution, collects and packet is concentrated
The eluent of the particle containing detection.
The detection particle solution of concentration is transferred to micro-fluidic grain count chip, the fixed inspection of solvent evaporation method by third step
Particle is surveyed, detection particle deposition is fixed on micro-fluidic grain count chip after solvent volatilization completely in solution to be concentrated, then is used
Optical microscopy carries out record by imaging to all detection particles being fixed on micro-fluidic grain count chip；In micro-fluidic particle
Under the substrate surface positioning grid auxiliary of counting chip, successively the detection particle of all fixations is divided using optical microscopy
Area's imaging, obtains microphoto.
Optical microscopy is conventional light field microscope, conventional fluorescent microscope or micromation microscope.Micro-fluidic particle meter
Number chip is by having the substrate of positioning grid and microporous layers to be bonded.The material of substrate is glass, polymethyl methacrylate
(PMMA), dimethyl silicone polymer (PDMS), polycarbonate (PC), any one in polystyrene (PS)；The material of microporous layers
Matter is glass, polymethyl methacrylate (PMMA), dimethyl silicone polymer (PDMS), polycarbonate (PC), polystyrene
(PS) any one.Positioning grid is lines, figure, any one or a few combination in icon.
The quantity of particle is detected in 4th step, the micro-fluidic grain count chip of statistics to get the molecular number of target protein is arrived；
Using the detection amounts of particles in software statistics microphoto, the as molecular number of target antigen；Inspection is counted to detect particle
Probing needle realizes that the direct absolute quantification analysis of the target protein molecule in individual particle level, Monitoring lower-cut are 50 molecules, moves
State range is 180~60000 molecules.
1 CEA(carcinoembryonic antigen of embodiment, carcinomebryonic antigen) standard items absolute quantification analysis
Capture magnetic bead preparation: 100 μ L magnetic beads (2 × 10 are pipetted9A/mL) stoste is in 1mL centrifuge tube, using equivalent 0.01M
NaOH solution is cleaned twice, and aliquots of deionized water cleans 3 times, and the 50mg/mL EDC of the 200 fresh configurations of μ L cold water is then added
(i.e. 1- (3- dimethylamino-propyl) -3- ethyl-carbodiimide hydrochloride) solution is incubated at room temperature 30 minutes, and Magneto separate removes supernatant
After liquid, 60 μ L capture antibody-solutions (antibody mass is 120 μ g) are added and adds 100 μ L after incubation at room temperature 30 minutes and contains
The PBS(phosphate buffer saline of 0.1% tween20, phosphate buffered saline solution) buffer (pH=7.4) cleaning 4
It is secondary, capture magnetic bead is finally obtained, 4 DEG C of refrigerators save backup.
Detection particle preparation: 100 μ L particle (100mg/mL) stostes are pipetted into 1mL centrifuge tube, 50mM MES(, that is, 2-
(N- morpholine) ethanesulfonic acid) buffer (pH=5) cleaning twice, be diluted to 1mL, add 10mg EDC, room temperature mixes 15 points
Clock is centrifuged off supernatant, after 0.5mL PBS buffer solution (pH=7.4) cleaning twice, 0.236 g is added and detects antibody, room temperature is incubated
Educate 3h, PBS buffer solution is cleaned twice, is resuspended in 0.5 mL 10mg/mL hydroxylamine hydrochloride solution, is mixed 30 minutes, with containing
The PBS buffer solution of 0.1% tween20 is cleaned 2 times, and detection particle is obtained, and 4 DEG C of refrigerators save backup.
Immune response process: 300 μ L capture magnetic bead solution (2 × 10 is pipetted6A/mL) in 1mL centrifuge tube, Magneto separate removes
Remove supernatant；100 μ L carcinomebryonic antigen (CEA) standard solution are added, are incubated at room temperature 1 hour, Magneto separate removes supernatant；Then plus
Enter 300 μ L confining liquids, room temperature is closed 30 minutes, and Magneto separate removes supernatant；It is eventually adding 100 μ L detection particle solution (1mg/
ML), it is incubated at room temperature 1 hour, forms ternary immune complex, Magneto separate removes supernatant.It is slow using the PBS containing 0.1% tween20
Fliud flushing (pH=7.4) is cleaned ternary immune complex 4 times, and to remove unbonded detection particle, 100 μ L citric acids are then added
Buffer (pH=3.1) elutes ternary immune complex 4 times, and the detection particle in ternary immune complex is eluted, magnetic point
From the detection particle collected in supernatant, using 200 μ L deionized water eccentric cleaning collection liquid 2 times, about 10 μ L(are concentrated into as schemed
1).
Detection grain count: 3~4 μ L of detection particle solution of above-mentioned elution is pipetted in the micro- of micro-fluidic microballoon counting chip
Kong Zhong heats concentrate under the conditions of 90 DEG C and volatilizees completely to solvent, and detection particle is deposited on micropore bottom, repeats above-mentioned mistake
Journey, until the detection particle of all elutions is all deposited on micropore bottom (such as Fig. 2).It is added into micro-fluidic microballoon counting chip
3 μ L deionized waters, covered, then in the case where the positioning grid of chip substrate guides, using optical microscopy to fixed
It detects particle and carries out subregion imaging (such as Fig. 3), the detection amounts of particles of all microphotos of software statistics, the as molecule of CEA
Number.When antigen molecule number changes, the number for detecting particle also changes (such as Fig. 4) therewith
The absolute quantification analysis of 2 plasma C EA of embodiment
CEA standard solution is only replaced with dilution 10 with embodiment 1 by the operating procedure of embodiment 25Human plasma sample again.Blood
The molecular number of slurry samples CEA is equal to the quantity of detection particle, and the concentration of CEA is in combination with sample volume and Avgadro constant meter
It calculates and obtains, the results are shown in Table 1.Table 1 be in 10 plasma samples the absolute quantification analysis result, hospital's testing result of CEA and
The contrast table of Standard entertion analysis result.Wherein, a is dilution 105Blood plasma again, b is whole plasm.
The Standard entertion of 3 plasma C EA of embodiment is analyzed
#5 plasma sample is diluted 105Times, 5 parts of diluted blood plasma (every part of 100 μ L) are taken, 0,5,10,15,20 μ L are separately added into
Concentration is the CEA standard solution of 200aM, and the CEA final concentration of addition is respectively 0,10,20,30,40aM, and subsequent process steps are same
Embodiment 1, it is only necessary to which CEA standard solution is replaced with to the plasma sample of above-mentioned addition CEA.With the CEA final concentration added in blood plasma
For abscissa, the quantity for detecting particle is ordinate, is drawn standard curve (such as Fig. 5).In Fig. 5, the reverse extending of standard curve
Line and X-axis are met at a bit, and the abscissa of the intersection point is 37.3, dilution 105#5 plasma sample CEA concentration again is 37.3
aM.#5 blood plasma stoste CEA concentration is 0.57ng/mL.Under the same terms, Standard entertion curve such as Fig. 6 institute of #10 plasma sample
Show, #10 blood plasma stoste CEA concentration is 0.67ng/mL.
Table 1
Claims (9)
1. a kind of low abundance proteins absolute quantification method based on digital immuno analytical method, which is characterized in that including following
Step:
The micron particles of the magnetic bead of modification capture antibody, target antigen and modification detection antibody are immunoreacted by the first step,
Ternary immune complex is formed, an antigen molecule generates an immune complex；The magnetic bead of modification capture antibody referred to as captures
Magnetic bead, the micron particles of modification detection antibody referred to as detection particle；
Second step is eluted the detection particle in ternary immune complex using acid solution, is collected and is concentrated comprising inspection
Survey the eluent of particle；
The detection particle solution of concentration is transferred to micro-fluidic grain count chip by third step, is detected after solvent volatilization completely
Particle deposition is fixed on micro-fluidic grain count chip, then is fixed on micro-fluidic grain count to all using optical microscopy
Detection particle on chip carries out record by imaging；
The quantity of particle is detected in 4th step, the micro-fluidic grain count chip of statistics to get the molecular number of target protein is arrived.
2. a kind of low abundance proteins absolute quantification method based on digital immuno analytical method according to claim 1,
It is characterized in that, in third step, under the substrate surface positioning grid auxiliary of micro-fluidic grain count chip, using optical microscopy
Subregion imaging successively is carried out to the detection particle of all fixations, obtains microphoto；In 4th step, the inspection in microphoto is counted
Survey amounts of particles, the as molecular number of target antigen；To detect particle as the mesh in count detection probe realization individual particle level
The direct absolute quantification analysis of protein molecular is marked, Monitoring lower-cut is 50 molecules, and dynamic range is 180~60000 molecules.
3. a kind of low abundance proteins absolute quantification method based on digital immuno analytical method according to claim 2,
It is characterized in that, in the first step, the specific method is as follows for preparation ternary immune complex:
Capture magnetic bead solution is taken, Magneto separate removes supernatant, and target antigen solution, room temperature is added into the lower part precipitating after Magneto separate
After lower concussion is incubated for 0.5 ~ 3 hour, Magneto separate removes supernatant, then confining liquid, room temperature is added into the lower part precipitating after Magneto separate
Lower closing 0.5 ~ 1.5 hour, Magneto separate removes supernatant, and detection particle solution, room temperature are then added into the precipitating after Magneto separate
Lower concussion is incubated for 0.5 ~ 3 hour, forms ternary immune complex.
4. a kind of low abundance proteins absolute quantification method based on digital immuno analytical method according to claim 3,
It is characterized in that, the shape of the detection particle is spherical shape, star, triangle, stick, annular or irregular shape.
5. a kind of low abundance proteins absolute quantification method based on digital immuno analytical method according to claim 4,
It is characterized in that, the detection particle is answered by one kind of high molecular material, inorganic material or biomaterial or above-mentioned any two kinds
Condensation material is made；The size of the detection particle is between 0.5~10 micron.
6. a kind of low abundance proteins absolute quantification method based on digital immuno analytical method according to claim 5,
Be characterized in that, the high molecular material be at least one of polystyrene, silica gel, polyacrylate, epoxy resin, it is described
Inorganic material is at least one of inorganic silicon materials, inorganic carbon material, semiconductor material, and the biomaterial is polyacrylamide
Amine and its derivative gel, polyethylene glycol gel, peptide-based gel, gelatin, calcium alginate gel, cellulose and its derivates gel
At least one of.
7. a kind of low abundance proteins absolute quantification method based on digital immuno analytical method according to claim 6,
It is characterized in that, the optical microscopy is conventional light field microscope, conventional fluorescent microscope or micromation microscope.
8. a kind of low abundance proteins absolute quantification method based on digital immuno analytical method according to claim 7,
It is characterized in that, the micro-fluidic grain count chip is by having the substrate of positioning grid and microporous layers to be bonded.
9. a kind of low abundance proteins absolute quantification method based on digital immuno analytical method according to claim 8,
It is characterized in that, the material of the substrate is glass, polymethyl methacrylate, dimethyl silicone polymer, polycarbonate, polyphenyl second
Any one in alkene；The material of the microporous layers is glass, polymethyl methacrylate, dimethyl silicone polymer, poly- carbonic acid
Any one of ester, polystyrene；The grid that positions is any one or a few combination in lines, figure, icon.
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