CN109486652A - A kind of PCR micro-fluidic chip and preparation method thereof - Google Patents
A kind of PCR micro-fluidic chip and preparation method thereof Download PDFInfo
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- CN109486652A CN109486652A CN201811340297.0A CN201811340297A CN109486652A CN 109486652 A CN109486652 A CN 109486652A CN 201811340297 A CN201811340297 A CN 201811340297A CN 109486652 A CN109486652 A CN 109486652A
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L7/00—Heating or cooling apparatus; Heat insulating devices
- B01L7/52—Heating or cooling apparatus; Heat insulating devices with provision for submitting samples to a predetermined sequence of different temperatures, e.g. for treating nucleic acid samples
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
- B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
- B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
- B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
- B01L2200/02—Adapting objects or devices to another
- B01L2200/026—Fluid interfacing between devices or objects, e.g. connectors, inlet details
- B01L2200/027—Fluid interfacing between devices or objects, e.g. connectors, inlet details for microfluidic devices
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L2300/00—Additional constructional details
- B01L2300/18—Means for temperature control
- B01L2300/1805—Conductive heating, heat from thermostatted solids is conducted to receptacles, e.g. heating plates, blocks
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- Health & Medical Sciences (AREA)
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Abstract
The invention discloses a kind of PCR micro-fluidic chips and preparation method thereof, micro-fluidic chip field, the PCR micro-fluidic chip includes confuser, heating sheet and constant flow pump, and the method for the mutual copy pattern that can be convenient by hot melt adhesive and colloid piece, quickly prepare flow-type PCR microchip, so that its not only manufacturing process is simple and easy, and each module when something goes wrong, the method by heating hot melt adhesive that can be convenient dismantles lower problem module, it comes into operation again after maintenance or replacement component, therefore greatly reduces cost required for processing and maintenance operation.
Description
Technical field
The present invention relates to micro-fluidic chip field, a kind of PCR micro-fluidic chip and preparation method thereof is particularly related to.
Background technique
PCR microchip is processed on the materials such as silicon wafer, sheet glass, silicon rubber with a variety of methods especially MEMS technology
Microchannel, micro- reaction chamber, micro-valve, micro-heater, micro- inductor etc. realize the rapid PCR amplification on chip, with conventional PCR
Technology is compared, have many advantages, such as quickly, efficiently, reagent consumption less, easy to carry, integration density is high, be life science in recent years
One of the research hotspot in field.According to the difference of thermal cycle mode, micro-fluidic pcr chip is broadly divided into static chamber pcr chip
Two kinds of (Static chamber PCR) and continuous flow pcr chip (Continuous-flow or flow-through PCR)
Mode, material have silicon, glass, quartz, metal and organic polymer etc..
But more complicated system of processing especially MEMS is used in these preparation methods, and the whole series completed the process
Chip system safeguard due to various reasons get up it is relatively difficult, some be only used as it is disposable so that experimental cost is higher.Make
With capillary glass tube especially with commercialization capillary column due to its hardness it is larger, preparing continuous-flow PCR
It is bigger that there is curvature when microchip pipeline, causes confuser production difficult, it is difficult to fix and take up space larger.
Summary of the invention
In view of this, it is an object of the invention to propose a kind of PCR micro-fluidic chip and preparation method thereof.On solving
It states partly or entirely insufficient in the prior art
Based on a kind of above-mentioned purpose preparation method of PCR micro-fluidic chip provided by the invention, include the following steps:
1) colloid monomer and curing agent are poured into model, heating reaction is solidified into colloid piece, after the finishing of colloid piece
As egative film, then takes identical colloid piece to be cut into colloid item, and colloid item is fixed on egative film, make shape between two colloid items
At groove, egative film mold is obtained；
2) after capillary column being carried out pretreatment drying, it is cut into capillary tube segment, capillary tube segment is individually positioned in egative film
In the groove of mold, and egative film mold is stretched out at both ends, then pours hot melt adhesive to after being completely covered egative film mold to egative film mold,
Solidification is shakeout, egative film mold is removed after the curing be complete, and adjacent capillaries section bending junction is connected to hose, obtains PCR
The confuser of micro-fluidic chip；
3) the colloid piece for taking preparation is cut into the identical size of same egative film and then puts thermally conductive sheet as heating sheet bottom plate
It sets on heating sheet bottom plate, by heater, the temperature-sensing probe on temperature controller, bonding compresses on thermally conductive sheet, then uses hot melt adhesive
All device surfaces are cast in, are shakeout, after its solidification, heating sheet bottom plate is removed, obtains the temperature control heating of PCR micro-fluidic chip
Piece；
4) bonding of the temperature control heating piece of the confuser of PCR micro-fluidic chip and PCR micro-fluidic chip is sealed, is protected
Capillary tube segment and heating sheet square crossing are held, and installs constant flow pump in capillary tube segment inlet and is flowed for controlling in confuser
Speed obtains PCR micro-fluidic chip.
In some optional embodiments, the material of the colloid piece be dimethyl silicone polymer, SE901 electron pouring sealant or
F625 silicon rubber.
In some optional embodiments, the model is the rectangle model of organic glass.
In some optional embodiments, the temperature of the heating reaction is 55-70 DEG C, duration 2-5h.
In some optional embodiments, the mass ratio of the colloid monomer and curing agent is 8-15:1.
In some optional embodiments, it is described pretreatment be the NaOH that capillary is successively used to 0.2M, water, dehydrated alcohol,
Water is cleaned.
In some optional embodiments, the hot melt adhesive is flame-resistant insulation High-temp.-resistant hot-melt adhesive.
In some optional embodiments, the hose is emulsion tube.
In some optional embodiments, the thermally conductive sheet is copper sheet, with a thickness of 0.3-0.5mm.
Meanwhile the embodiment of the present invention also provides a kind of PCR micro-fluidic chip, using the system of the PCR micro-fluidic chip
Preparation Method is prepared；Wherein, the PCR micro-fluidic chip includes confuser, heating sheet and constant flow pump, and the confuser includes
First hot melt adhesive bottom plate and the capillary in S distribution being fixed on the first hot melt adhesive bottom plate, first hot melt adhesive bottom
Plate can be bond plies, and the inlet of the capillary is connected to constant flow pump, for controlling flow velocity in confuser, the capillary
The corner of pipe is connected to hose, for reducing corner；The heating sheet includes the second hot melt adhesive bottom plate and is fixed on institute
The thermally conductive sheet on the second hot melt adhesive bottom plate is stated, temperature controller is connected on the thermally conductive sheet, to control the temperature of PCR experiment.
From the above it can be seen that a kind of PCR micro-fluidic chip provided by the invention and preparation method thereof, passes through hot melt
The method for the mutual copy pattern that glue and colloid piece can be convenient, quickly prepares flow-type PCR microchip, so that it not only makes
Process is simple and easy, and each module when something goes wrong, it may be convenient to the method for passing through heating hot melt adhesive dismantles lower problem
It comes into operation again after module, maintenance or replacement component, therefore greatly reduces cost required for processing and maintenance operation.
It is connected simultaneously in the bending part of capillary using hose, eliminates curved stress significantly, considerably increase hair
The quantity that tubule can be placed on heating sheet also increases the cycle-index of PCR micro-fluidic chip, so that it is more practical.
Detailed description of the invention
Fig. 1 is egative film of embodiment of the present invention mold structure diagram；
Fig. 2 is confuser of embodiment of the present invention structural schematic diagram；
Fig. 3 is heating sheet of embodiment of the present invention structural schematic diagram；
Fig. 4 is the structural schematic diagram of PCR of embodiment of the present invention micro-fluidic chip；
Fig. 5 is the structural schematic diagram for the PCR micro-fluidic chip that the embodiment of the present invention simplifies.
Egative film mold -1, colloid piece -11, colloid item -12, groove -13, confuser -2, the first hot melt adhesive bottom plate -21 are soft
Pipe -22, capillary -23, heating sheet -3, the second hot melt adhesive bottom plate -31, thermally conductive sheet -32, temperature controller -33, constant flow pump -4 are thermally conductive
Double-sided adhesive -5.
Specific embodiment
To make the objectives, technical solutions, and advantages of the present invention clearer, below in conjunction with specific embodiment, and reference
Attached drawing, the present invention is described in more detail.
It should be noted that all statements for using " first " and " second " are for differentiation two in the embodiment of the present invention
The non-equal entity of a same names or non-equal parameter, it is seen that " first " " second " only for the convenience of statement, does not answer
It is interpreted as the restriction to the embodiment of the present invention, subsequent embodiment no longer illustrates this one by one.
In order to solve some or all of deficiency in the prior art, on the one hand the present invention provides a kind of micro-fluidic cores of PCR
The preparation method of piece, includes the following steps:
1) colloid monomer and curing agent are poured into model, heating reaction is solidified into colloid piece, after the finishing of colloid piece
As egative film, then takes identical colloid piece to be cut into colloid item, and colloid item is fixed on egative film, make shape between two colloid items
At groove, egative film mold is obtained；
2) after capillary column being carried out pretreatment drying, it is cut into capillary tube segment, capillary tube segment is individually positioned in egative film
In the groove of mold, and egative film mold is stretched out at both ends, then pours hot melt adhesive to after being completely covered egative film mold to egative film mold,
Solidification is shakeout, egative film mold is removed after the curing be complete, and adjacent capillaries section bending junction is connected to hose, obtains PCR
The confuser of micro-fluidic chip；
3) the colloid piece for taking preparation is cut into the identical size of same egative film and then puts thermally conductive sheet as heating sheet bottom plate
It sets on heating sheet bottom plate, by heater, the temperature-sensing probe on temperature controller, bonding compresses on thermally conductive sheet, then uses hot melt adhesive
All device surfaces are cast in, are shakeout, after its solidification, heating sheet bottom plate is removed, obtains the temperature control heating of PCR micro-fluidic chip
Piece；
4) bonding of the temperature control heating piece of the confuser of PCR micro-fluidic chip and PCR micro-fluidic chip is sealed, is protected
Capillary tube segment and heating sheet square crossing are held, and installs constant flow pump in capillary tube segment inlet and is flowed for controlling in confuser
Speed obtains PCR micro-fluidic chip.
In some optional embodiments, the present invention provides a kind of preparation method of PCR micro-fluidic chip, specific steps are such as
Under:
According to monomer: curing agent mass ratio is the proportional arrangement dimethyl silicone polymer (PDMS) of 10:1, is cast in organic
In the rectangular die of glass (PMMA), 2~5h is heated at 60 DEG C and obtains cured PDMS piece, is cut to 5*14cm's
Rectangle can equally use the preparation such as SE901 electron pouring sealant, F625 silicon rubber as egative film, this operation), then cut several
Item
The PDMS item of 0.5*5cm needs to carry out the selection of cutting rod number according to experiment, smears in its bottom a small amount of uncured
PDMS, then they are equidistantly placed on above-mentioned PDMS egative film, forms several groove, then heated solidification preparation
PDMS egative film mold, while the rectangle PDMS piece for preparing 5*14cm again is spare.
Using the capillary column (Hebei Yongnian optical fiber factory) of commercialization, outer diameter is 360 μm, internal diameter is 50 μm, always
Length is 60cm, using it is preceding cleaned respectively with the NaOH of 0.2M, water, ethyl alcohol, water after naturally dry.
By above-mentioned capillaries flat after processing in the first groove of the PDMS egative film mold of preparation, thermosol gelgun is used
Milky flame-resistant insulation High-temp.-resistant hot-melt adhesive stick (being purchased from Taobao) is heated, is uniformly poured into the first groove with the hot melt adhesive of thawing
It is interior, after its solidification after, by capillary be bent place No. 2 slot in, same method is poured, is fixed, to the last one it is recessed
Slot, respectively there are the ends of about 3cm at capillary both ends.Finally hot melt adhesive is uniformly cast on all PDMS items, is shakeout, to
It removes PDMS egative film mold after being fully cured, and can prepare the confuser of continuous-flow PCR microchip.
3 pieces of 1*12cm copper sheet (with a thickness of 0.5mm) is uniformly placed in the spare PDMS on piece of preparation, 3 groups of copper sheets are distinguished
Heater, the Pt100 temperature-sensing probe being connected on PID intelligent temperature controller, and with argentiferous heat-conducting silicone grease (thermal coefficient
4.15W/Mk) bonding compresses on above-mentioned copper sheet, is equally cast in all device surfaces with hot melt adhesive, all devices are completely covered
Part shakeouts, and removes PDMS piece after its solidification, acquisition can pass through the heating sheet of PID precise temperature control.
Above-mentioned confuser and heating sheet are sticked together by argentiferous heat-conducting silicone grease, with PID intelligent temperature controller control
Making each heating block temperature is respectively 95 DEG C, 77 DEG C and 60 DEG C, while installing constant flow pump for controlling in the entrance of capillary
Flow velocity can carry out PCR amplification experiment in confuser, can also be cleaned after the end of the experiment.When confuser or heating unit
Part, temperature-sensing probe etc. take out after being melted by the thermosol at the heating place of going wrong when going wrong and are tieed up
It repairs.It is practical and convenient.
Actual fabrication and operation in find, using capillary glass tube especially with commercialization capillary column due to it
Hardness is larger, therefore that there is curvature is bigger when preparing continuous-flow PCR microchip pipeline, between each pipeline between
Away from larger, otherwise it is difficult to will lead to greatly very much confuser production for the bending part stress between two adjacent channels, it is difficult to and it is fixed, therefore
It is difficult to place appropriate number of capillary tube segment on limited heating sheet, therefore proposes another PCR under above-described embodiment
The preparation method of micro-fluidic chip by the way that capillary segmentation to be fixed, and is bent two adjacent capillaries with latex hose
Place is connected to, and reduces the bending stress and arrangement the space occupied of capillary, it is suitable to place on limited heating sheet
The capillary of quantity gives full play to the effect of PCR micro-fluidic chip.
It also found under study for action simultaneously, due to needing just to can be used after being fixed using hot melt adhesive, and heat blob of viscose
Easily deforming causes heating sheet and being bonded for confuser not close, these are unfavorable for dismantling, and influence PCR micro-fluidic chip
Convenience when use and the observation for being unfavorable for student in teaching process.For on the basis of above-mentioned two embodiment at this time more into
One step proposes a kind of preparation method of brief PCR micro-fluidic chip, the more convenient disassembly of the PCR micro-fluidic chip prepared and sight
It examines, and replaces the hot melt adhesive of confuser using bond plies, bond plies are fitted to respectively, PID precise temperature control is installed
Three heat conduction copper sheets on, then capillary segmented is fixed in bond plies, passes through PID precision thermostat and control temperature
Temperature in capillary.It is more advantageous to detachable maintaining and observes the flow condition inside capillary.
On the other hand, based on the above embodiment in PCR micro-fluidic chip preparation method, the present invention also provides a kind of PCR
Micro-fluidic chip is prepared using the preparation method of the PCR micro-fluidic chip；Wherein, the PCR micro-fluidic chip packet
Confuser, heating sheet and constant flow pump are included, the confuser includes the first hot melt adhesive bottom plate and is fixed on first hot melt adhesive bottom
The capillary in S distribution on plate, the first hot melt adhesive bottom plate can be bond plies, the inlet of the capillary and perseverance
Stream pump connection, for controlling flow velocity in confuser, the corner of the capillary is connected to hose, for reducing corner；
The heating sheet includes the second hot melt adhesive bottom plate and the thermally conductive sheet that is fixed on the second hot melt adhesive bottom plate, on the thermally conductive sheet
It is connected with temperature controller, to control the temperature of PCR experiment.
As described in Figure 4 it is a kind of PCR micro-fluidic chip provided by the invention, is divided into top heating sheet 3 and corresponds
The lower pipeline piece 2 of cooperation, the confuser 2 are prepared by egative film mold 1, and the structure of egative film mold 1 is as shown in Figure 1, packet
The colloid item 12 for including colloid piece 11 and being fixed on colloid piece 11 is equidistantly to arrange between colloid item 12, adjacent colloidal item 12
Between form groove 13, capillary 23 is placed when being used to prepare confuser 2.The structure of the confuser 2 of preparation is as shown in Fig. 2, pipe
It hot melt adhesive bottom plate 21 and the capillary 23 that is fixed on 21 surface of the first hot melt adhesive bottom plate that road piece 2, which includes the first of bottom, and hair
The first hot melt adhesive bottom plate 21 is stretched out at the both ends of tubule 23, and 23 knee of capillary that is connected is connected to using hose 22, reduces direct
Bending stress caused by bending, the capillary 23 after connection subtract in the S-type arrangement in 21 longitudinal direction of the first hot melt adhesive bottom plate
Few occupied space, capillary 23 can also directly bend to S type, but occupied space is big at this time, and complete stress is big, be unfavorable for multi-quantity
Arrangement and device intensity promotion.
Bonding corresponding with confuser 2 is heating sheet 3, and the structure of the heating sheet 3 is as shown in figure 3, heating sheet 3 includes
The thermally conductive sheet 32 that second hot melt adhesive bottom plate 31 and bonding are fixed on the second hot melt adhesive bottom plate 31, thermally conductive sheet 32 can be multiple groups,
Heater and Pt100 temperature-sensing probe (being to show) installation of thermally conductive sheet 32 and temperature controller 33 connect simultaneously, to control in PCR
The required temperature of experiment.
Confuser 2 and heating sheet 3 are subjected to pairing, and install constant flow pump 4 for controlling PCR in 23 inlet of capillary
Flow velocity when experiment inside pipeline prepares PCR micro-fluidic chip as shown in Figure 4.
In order to facilitate observing and overhauling, the present invention also provides a kind of brief PCR micro-fluidic chip, structure such as Fig. 5 institutes
Show, brief PCR micro-fluidic chip includes heating sheet 3, constant flow pump 4 and bond plies 5, with PCR micro-fluidic chip shown in Fig. 4
Difference in place of be, the first hot melt adhesive bottom plate 21 is replaced with bond plies 5, and bond plies 5 are pasted thermally conductive
On piece 32, the fixation of capillary 23 is carried out.
A kind of PCR micro-fluidic chip provided by the invention and preparation method thereof, can be convenient by hot melt adhesive and colloid piece
Mutual copy pattern method, quickly prepare flow-type PCR microchip so that its not only manufacturing process is simple and easy, and
Each module when something goes wrong, it may be convenient to the method for passing through heating hot melt adhesive dismantles lower problem module, maintenance or replacement portion
It comes into operation again after part, therefore greatly reduces cost required for processing and maintenance operation.
It is connected simultaneously in the bending part of capillary 23 using hose 22, eliminates curved stress significantly, greatly increase
The quantity that capillary 23 can be placed on heating sheet 3 also increases the cycle-index of PCR micro-fluidic chip, so that it is more
Add practical.
It should be understood by those ordinary skilled in the art that: the discussion of any of the above embodiment is exemplary only, not
It is intended to imply that the scope of the present disclosure (including claim) is limited to these examples；Under thinking of the invention, above embodiments
Or can also be combined between the technical characteristic in different embodiments, step can be realized with random order, and be existed such as
Many other variations of the upper different aspect of the invention, for simplicity, they are not provided in details.
The embodiment of the present invention be intended to cover fall into all such replacements within the broad range of appended claims,
Modifications and variations.Therefore, all within the spirits and principles of the present invention, any omission, modification, equivalent replacement, the improvement made
Deng should all be included in the protection scope of the present invention.
Claims (10)
1. a kind of preparation method of PCR micro-fluidic chip, which comprises the steps of:
1) colloid monomer and curing agent are poured into model, heating reaction is solidified into colloid piece, by conduct after the finishing of colloid piece
Then egative film takes identical colloid piece to be cut into colloid item, and colloid item is fixed on egative film, make to be formed between two colloid items recessed
Slot obtains egative film mold；
2) after capillary column being carried out pretreatment drying, it is cut into capillary tube segment, capillary tube segment is individually positioned in egative film mold
Groove in, and both ends stretch out egative film mold, then pour hot melt adhesive to after being completely covered egative film mold to egative film mold, shakeout
Solidification removes egative film mold after the curing be complete, and adjacent capillaries section bending junction is connected to hose, obtains PCR miniflow
Control the confuser of chip；
3) the colloid piece for taking preparation is cut into the identical size of same egative film and is then placed on thermally conductive sheet as heating sheet bottom plate
On heating sheet bottom plate, by heater, the temperature-sensing probe on temperature controller, bonding is compressed on thermally conductive sheet, is then poured with hot melt adhesive
It in all device surfaces, shakeouts, after its solidification, removes heating sheet bottom plate, obtain the temperature control heating piece of PCR micro-fluidic chip；
4) the temperature control heating piece of the confuser of PCR micro-fluidic chip and PCR micro-fluidic chip is sticked together, keeps capillary
Section and heating sheet square crossing, and constant flow pump is installed for controlling flow velocity in confuser in capillary tube segment inlet, it is micro- to obtain PCR
Fluidic chip.
2. the preparation method of PCR micro-fluidic chip according to claim 1, which is characterized in that the material of the colloid piece
For dimethyl silicone polymer, SE901 electron pouring sealant or F625 silicon rubber.
3. the preparation method of PCR micro-fluidic chip according to claim 1, which is characterized in that the model is organic glass
The rectangle model of glass.
4. the preparation method of PCR micro-fluidic chip according to claim 1, which is characterized in that the temperature of the heating reaction
Degree is 55-70 DEG C, duration 2-5h.
5. the preparation method of PCR micro-fluidic chip according to claim 1, which is characterized in that the colloid monomer and solid
The mass ratio of agent is 8-15:1.
6. the preparation method of PCR micro-fluidic chip according to claim 1, which is characterized in that the pretreatment is by hair
Tubule is successively cleaned with the NaOH of 0.2M, water, dehydrated alcohol, water.
7. the preparation method of PCR micro-fluidic chip according to claim 1, which is characterized in that the hot melt adhesive is insulation
Flame-resistant high-temperature-resistant hot melt adhesive.
8. the preparation method of PCR micro-fluidic chip according to claim 1, which is characterized in that the hose is rubber tube.
9. the preparation method of PCR micro-fluidic chip according to claim 1, which is characterized in that the thermally conductive sheet is copper sheet,
With a thickness of 0.3-0.5mm.
10. a kind of PCR micro-fluidic chip, which is characterized in that using any PCR micro-fluidic chip of claim 1-9
Preparation method is prepared, wherein the PCR micro-fluidic chip includes confuser, heating sheet and constant flow pump, the confuser packet
The capillary in S distribution for including the first hot melt adhesive bottom plate and being fixed on the first hot melt adhesive bottom plate, first hot melt adhesive
Bottom plate can be bond plies, and the inlet of the capillary is connected to constant flow pump, for controlling flow velocity in confuser, the hair
The corner of tubule is connected to hose, for reducing corner；The heating sheet includes the second hot melt adhesive bottom plate and is fixed on
Thermally conductive sheet on the second hot melt adhesive bottom plate is connected with temperature controller on the thermally conductive sheet, to control the temperature of PCR experiment.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN111333025A (en) * | 2020-03-26 | 2020-06-26 | 安徽中医药高等专科学校 | Cold and hot double-control temperature paper cutting chip and preparation method thereof |
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Application publication date: 20190319 |