CN208104447U - One kind screening circular card box device for tumour cell nucleic acid aptamers - Google Patents
One kind screening circular card box device for tumour cell nucleic acid aptamers Download PDFInfo
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- CN208104447U CN208104447U CN201721897015.8U CN201721897015U CN208104447U CN 208104447 U CN208104447 U CN 208104447U CN 201721897015 U CN201721897015 U CN 201721897015U CN 208104447 U CN208104447 U CN 208104447U
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Abstract
The utility model discloses one kind to screen cartridge device for tumour cell nucleic acid aptamers, including cartridge bodies and positioned at the cartridge reagent storage disk of cartridge bodies bottom；It is provided with turntable, pressing plate and liquid-transfering gun point in cartridge bodies, is provided with air-path interface and air filter film on the shell of cartridge bodies, air-path interface is connect with the side air flue interface of liquid-transfering gun point；It is extended at the top of turntable and forms the first control interface outside cartridge bodies, liquid-transfering gun point is fixed on turntable；Pressing plate is located above turntable, extends at the top of pressing plate and forms the second control interface outside cartridge bodies；Tissue Culture Dish adapter and reagent storage reaction hole location are provided on cartridge reagent storage disk.The closed cartridge device of the utility model, assembly is simple, can be used for automatic operation, entire aptamer screening process all carries out in closed cartridge, the problem of avoiding cross contamination, a wheel or multi-round screening process for achievable aptamer, greatly improves the efficiency of aptamer screening.
Description
Technical field
The utility model belongs to molecular biology field, and in particular to one kind is for tumour cell nucleic acid aptamers screening circle
Shape cartridge device simultaneously can be used for other types suitable for the research experiment screened to tumour cell nucleic acid aptamers
Aptamer screening test, such as albumen, excretion body.
Background technique
Aptamer is a bit of oligonucleotide sequence obtained through in-vitro screening, and high parent can be carried out with corresponding ligand
With the combination of power and strong specificity, its appearance is chemical-biological educational circles and biomedical boundary provide it is a kind of new efficient quick
The research platform of identification, and good application prospect is illustrated in many aspects.
Currently, carrying out the screening of tumour cell nucleic acid aptamers mainly by having been manually done, inefficiency is suitable from screening nucleic acid
Ligand screening starts to being fully completed, and needs to carry out 20 wheel left and right experiments, whole process needs half a year to 1 year, wherein most heavy
The problem of wanting is to will appear cross contamination, is mainly derived from PCR, aptamers transfer etc., once it pollutes, this wheel or even whole
A the selection result just will appear error, it is necessary to re-start screening.Currently, avoiding the method for cross contamination, mainly pass through will be every
A step is separated and is operated in different experiments room, is aerated, is sterilized to laboratory, this mode needs to occupy biggishly
Side arrangement laboratory, and it is more demanding to laboratory technician, even if in this way, the aerial nucleic acid times that is scattered can not so eliminate, in fact
In the experiment on border, cross-contamination issue is always existed.
Utility model content
Purpose of utility model：It is sieved in view of the above-mentioned problems, the utility model proposes one kind for tumour cell nucleic acid aptamers
Circular card box device is selected, which can grasp liquid relief, PCR reaction, aptamers transfer etc. for closed, assembly is simple
Work carries out in a closed cartridge, avoids cross-contamination issue, and can realize automatic operation.
Technical solution：To achieve the goals above, one kind as shown in the utility model is used for tumour cell nucleic acid aptamers
Cartridge device is screened, including cartridge bodies and positioned at the cartridge reagent storage disk of cartridge bodies bottom；It is set in the cartridge bodies
It is equipped with turntable, pressing plate and liquid-transfering gun point, is provided with air-path interface and air filter film, air-path interface and shifting on the shell of cartridge bodies
The side air flue interface of liquid rifle point connects；It is extended at the top of the turntable and forms the first control interface outside cartridge bodies, moved
Liquid rifle point is fixed on turntable；The pressing plate is located above turntable, is extended to outside cartridge bodies at the top of pressing plate and forms the second control
Interface processed；Tissue Culture Dish adapter and reagent storage reaction hole location are provided on the cartridge reagent storage disk.
Preferably, the turntable bottom is round thin panel, it is solid by cartridge bodies interior channel outstanding
It is fixed, there is aperture on turntable, passes through the fixed liquid-transfering gun point of rifle point spring.
Preferably, the pressing plate bottom is cuboid thin plate, bottom is provided with rag iron spring.
Preferably, being provided with air filtering core in the side air flue interface of the air-path interface and liquid-transfering gun point.It is fixed with
Air filtering core, filter core aperture are less than aptamers, prevent aptamers from spilling into pressure generator, generate pollution.
Further, the filter sizes of the air filter film are less than aptamers.The shell side planar section of cartridge bodies
It is fixed with air filter film, filter sizes are less than aptamers, balance for air pressure in keeping box and external pressure.
Wherein, the Tissue Culture Dish adapter is semiclosed bulge, and bottom end is provided with shaft, can be with cartridge bodies
Part is tightly engaged into.
Preferably, the shaft and bulge are seamlessly connected, the two is to be linked together, and shaft one end projects to cartridge
The shell exterior of main body forms the prominent control point of shaft.The head at the prominent control point of shaft is linear type or other shapes interface,
It stirs by hand or the rotation of motor connecting interface, control pivot swings to control adapter, for controlling culture
Liquid aggregation position in ware.
Further, the reagent storage reaction hole location includes 9 hole locations being successively set on cartridge reagent storage disk,
9 hole locations are respectively library high temperature receiving hole, and library low-temp storage hole, in conjunction with liquid receiving hole, TE buffer receiving hole is given up
Liquid receiving hole, cleaning solution receiving hole, pancreatin digestive juice receiving hole, PCR reacting hole, aptamers shift receiving hole.
Preferably, the reagent storage reaction hole location is circle or rectangular, bottom end is closed with hemispherical or taper ending,
The orifice center point of hole location is distributed on the circumference that the center of circle is overlapped with the round turntable center of circle.
The library high temperature receiving hole and aptamers transfer receiving hole are detachable activity hole location, the opening cork of hole location
Closing, such as rubber stopper, silica gel plug.It can be separated from cartridge, for testing the transfer of aptamers before and after.
Working principle：Carry out the first round aptamer screening start before, by DNA library be put into library high temperature receiving hole,
It is put into conjunction with liquid and is put into PCR reacting hole in conjunction with liquid receiving hole, PCR reaction reagent, TE buffer is put into TE buffer receiving hole, pancreas
Enzymic digestion liquid is put into pancreatin digestive juice receiving hole, cleaning solution is put into cleaning solution receiving hole, and Tissue Culture Dish is put into cell culture
After completion, cartridge bodies and cartridge reagent storage disk are completed for ware adapter.Library high temperature receiving hole is put into 95 DEG C
After metal bath 5min, the first control interface at the top of turntable is rotated, the liquid-transfering gun point being fixed on turntable bottom disc is gone to
Right above the high temperature receiving hole of library, the second control interface at the top of pressing plate is rotated, the cuboid thin plate of pressing plate bottom is gone into shifting
Right above liquid rifle point, the second control interface at the top of pressing plate is firmly oppressed, pressing plate moves down, and liquid-transfering gun point is depressed into the storage of library high temperature
Inside hole, library is sucked by liquid-transfering gun point cavity by the plunger pump connecting with air-path interface, withdraws from and is applied at the top of pressing plate
Power is acted on by rag iron spring elastic force, and pressing plate moves up, and since the elastic force of rifle point spring acts on, moves to library height on liquid-transfering gun point
Above gentle discharge hole, the first control interface at the top of turntable is rotated, the liquid-transfering gun point being fixed on turntable bottom disc is gone to
Right above the low-temp storage hole of library, the second control interface at the top of pressing plate is rotated, the cuboid thin plate of pressing plate bottom is gone into shifting
Right above liquid rifle point, interface at the top of pressing plate is firmly oppressed, pressing plate moves down, liquid-transfering gun point is depressed into inside the low-temp storage hole of library,
Liquid-transfering gun point cavity Chinese library is put into the low-temp storage hole of library by plunger pump, after 0 DEG C of storage 10min, with liquid-transfering gun point
Library low-temp storage hole Chinese library is drawn to liquid receiving hole is combined, is mixed by the piping and druming of liquid-transfering gun point and combines liquid in liquid receiving hole
Afterwards, cleaning solution in cleaning solution receiving hole is drawn with liquid-transfering gun point to put into Tissue Culture Dish adapter in Tissue Culture Dish, gently
Tissue Culture Dish supernatant is drawn with liquid-transfering gun point after the prominent control point 2min of the shaft of left-right rotation Tissue Culture Dish adapter 28,
It puts to waste liquid receiving hole, after repeated washing 3 times, draws that combine in liquid receiving hole liquid to move to Tissue Culture Dish suitable with liquid-transfering gun point
In orchestration 28 in Tissue Culture Dish, the shaft in 4 DEG C of environment through motor gently left-right rotation Tissue Culture Dish adapter is prominent
Control point 2h out draws Tissue Culture Dish aqueous supernatant with liquid-transfering gun point, puts to waste liquid receiving hole, drawn and cleaned with liquid-transfering gun point
Cleaning solution is put into Tissue Culture Dish adapter in Tissue Culture Dish in liquid receiving hole, and gently left-right rotation Tissue Culture Dish is adapted to
Tissue Culture Dish supernatant is drawn with liquid-transfering gun point after device 2min, is put to waste liquid receiving hole, after repeated washing 3 times, draws TE buffering
TE buffer is into Tissue Culture Dish in liquid receiving hole 32, then draws pancreatin in pancreatin digestive juice receiving hole with liquid-transfering gun point and digest
After liquid to Tissue Culture Dish 10min, liquid in Tissue Culture Dish is moved in PCR reacting hole with liquid-transfering gun point, is carried out after mixing
PCR amplification, after amplification, liquid-transfering gun point is drawn amplified production and is moved to above aptamers transfer receiving hole, fills in across rubber
Entering in hole, and amplified production is discharged into hole, turn aptamers shift receiving hole, it removed from cartridge, so far, the first round
Aptamer screening is completed, and will be disassembled aptamers transfer receiving hole, is directly installed on the library high temperature of another cartridge
Library high temperature receiving hole of the hole site as new cartridge is stored, directly can carry out the second wheel or the n-th wheel experiment by above-mentioned steps,
Until aptamer screening is completed.
Beneficial effect：It is compared by the prior art, the utility model has the following advantages that：The utility model for tumour it is thin
Born of the same parents' aptamer screening circular card box device is that one kind is closed, assembly is simple, can be used for the cartridge device of automatic operation.
Entire aptamer screening process all carries out in closed cartridge, wherein the PCR for being easy to produce cross contamination is reacted in card
Occur in box, the aptamers library transfer between two cartridges is carried out by closed reagent wells, guarantees that amplified production will not
The problem of spilling into air, avoiding cross contamination.Automation can be passed through by carrying out aptamer screening by cartridge simultaneously
Instrument realizes that the wheel or multi-round screening process of achievable aptamer greatly improve the efficiency of aptamer screening.
Detailed description of the invention
Fig. 1 is the utility model cartridge device overall structure diagram；
Fig. 2 is the structural schematic diagram of cartridge bodies；
Fig. 3 is cartridge bodies side sectional view；
Fig. 4 is the reagent storage dish structure schematic diagram of cartridge device；
Fig. 5 is cartridge device entirety side sectional view.
Specific embodiment
Below in conjunction with drawings and examples, the utility model is described in further detail.
Embodiment
As shown in Figs. 1-5, a kind of to screen cartridge device, including cartridge bodies 1 and position for tumour cell nucleic acid aptamers
Dismountable cartridge reagent storage disk 2 in 1 bottom of cartridge bodies；Turntable 21, pressing plate 22 and liquid relief are provided in cartridge bodies 1
Rifle point 25；The top of turntable 21, which extends to outside cartridge bodies 1, forms the first control interface 38, is protruding to the of the outside of shell
The head of one control interface is linear type or other shapes interface, stirs by hand or motor connects 38 turns of the first control interface
It is dynamic, dial movement is controlled, 21 bottom of turntable is round thin panel, fixes, turns by 1 interior of cartridge bodies channel outstanding
There are two aperture on disk 21, identical two liquid-transfering guns point 25 is fixed on turntable by aperture on turntable 21 and rifle point spring 26,
The pollution of reagent in pipetting processes is prevented, the rotational movement liquid-transfering gun point 25 of turntable 21 moves；Pressing plate 22 is located at turntable 21
Top extends to outside cartridge bodies 1 at the top of pressing plate 22 and forms the second control interface 39；22 upper end of pressing plate is socketed on turntable 22
End, bottom is a cuboid or other shapes panel, and panel bottom end is provided with rag iron spring 27, by the second of enclosure
Second control interface 39 of the compressing of 39 applied force of control interface or rotary pressure plate makes lower end board generate horizontal or vertical displacement,
It is bonded when pressing plate bottom end is moved to 25 top of liquid-transfering gun point with 25 top of liquid-transfering gun point, pushes the second control interface 39 and transport downwards
When dynamic, compressing liquid-transfering gun point 25 is moved downward, and after pressure revocation, pressing plate 22 and liquid-transfering gun point 25 pass through rag iron spring 27 respectively
It sets back with the spring force of rifle point spring 26.
Cartridge bodies 1 are cylinder, and the shell side of cartridge bodies 1 is made of arc surface and plane, outside cartridge bodies 1
There are one or more air-path interfaces 23 outstanding in the plane of shell, air-path interface 23 connects plunger in 1 exterior portion of cartridge bodies
The air pumps pressure generators such as pump, are connected to liquid-transfering gun by rubber tube or other materials pipeline in 1 interior section of cartridge bodies
The side air flue interface of point 25, the suction tapping operation of control rifle point, the side air flue interface of air-path interface 23 and liquid-transfering gun point 25
Inside it is provided with air filtering core.Air filter film 24, the filter hole of air filter film 24 are additionally provided in the plane of the shell of cartridge bodies 1
Diameter is less than aptamers.
Tissue Culture Dish adapter and reagent storage reaction hole location, Tissue Culture Dish are provided on cartridge reagent storage disk 2
Adapter is semiclosed bulge, and bottom end is provided with shaft, and shaft and semiclosed bulge are seamlessly connected, one distal process of shaft
Out to the shell exterior of cartridge bodies 1, the prominent control point 40 of shaft is formed, by projecting to the prominent control of shaft outside cartridge
40 control Tissue Culture Dish adapter side-to-side movement of point reaches in Tissue Culture Dish to drive Tissue Culture Dish side-to-side movement
The purpose of portion's liquid accumulation.Reagent storage reaction hole location includes 9 hole locations being successively set on cartridge reagent storage disk 2, and 8
The hole location and 1 waste liquid hole location of reagent are held, 9 hole locations are respectively library high temperature receiving hole 29, library low-temp storage hole 30, knot
Conjunction liquid receiving hole 31, TE buffer receiving hole 32, waste liquid receiving hole 33, cleaning solution receiving hole 34, pancreatin digestive juice receiving hole 35,
PCR reacting hole 36, aptamers shift receiving hole 37.Each reagent storage reaction hole location be it is round or rectangular, bottom end with hemispherical or
Taper ending closure, the orifice center point of hole location are distributed on the circumference that the center of circle is overlapped with 22 center of circle of turntable.Wherein, library high temperature
Receiving hole 29 and aptamers transfer receiving hole 37 are detachable activity hole location, and the opening of hole location is closed with cork, such as rubber stopper, silicon
Rubber plug etc..25 bottoms of liquid-transfering gun point can wear out rubber stopper and inject liquid into the hole, which is isolated from reagent storage disk 2
Come, can store or be directly installed in another cartridge, realize the transfer of aptamers between two cartridges.
The present embodiment combination HepG2 cell carry out one wheel aptamer screening, key step have library processing, DNA with
Cell combination, cell separation and broken, aptamer amplification, aptamer shift.Used reagent has single stranded DNA literary
Library, in conjunction with liquid, cleaning solution, pancreatin digestive juice, TE buffer, asymmetric PCR reaction reagent etc..
Before progress first round aptamer screening starts, DNA library is put into library high temperature receiving hole 29, is put in conjunction with liquid
Enter in conjunction with liquid receiving hole 31, PCR reaction reagent is put into PCR reacting hole 36, TE buffer is put into TE buffer receiving hole 32, pancreatin
Digestive juice is put into pancreatin digestive juice receiving hole 35, cleaning solution is put into cleaning solution receiving hole 34, and Tissue Culture Dish is put into cell training
Ware adapter 28 is supported, after completion, cartridge bodies 1 and cartridge reagent storage disk 2 are completed.By library high temperature receiving hole 29
After being put into 95 DEG C of metal bath 5min, first control interface 38 at 21 top of rotation turntable will be fixed on 21 bottom disc of turntable
Liquid-transfering gun point 25 go to right above library high temperature receiving hole 29, second control interface 39 at 22 top of rotation pressing plate, by pressing plate
The cuboid thin plate of 22 bottoms is gone to right above liquid-transfering gun point 25, firmly oppresses the second control interface 39 at the top of pressing plate, pressing plate 22
It moves down, liquid-transfering gun point 25 is depressed into inside the high temperature receiving hole of library, is inhaled library by the plunger pump being connect with air-path interface 23
Enter sharp 25 cavitys of liquid-transfering gun, withdraws from the power for being applied to 22 the second control interface 39 of top of pressing plate, made by 27 elastic force of rag iron spring
With, pressing plate 22 moves up, since the elastic force of rifle point spring 26 acts on, high temperature receiving hole 29 top in library is moved on liquid-transfering gun point 25,
The first control interface 38 for rotating 21 top of turntable, it is low to go to library for the liquid-transfering gun point 25 being fixed on 21 bottom disc of turntable
Right above gentle discharge hole 30, second control interface 39 at 22 top of rotation pressing plate goes to the cuboid thin plate of 22 bottom of pressing plate
Right above liquid-transfering gun point 25,22 the second control interface 39 of top of pressing plate is firmly oppressed, pressing plate 22 moves down, liquid-transfering gun point 25 is depressed into
The sharp 25 cavity Chinese libraries of liquid-transfering gun are put into library low-temp storage hole 30,0 by 30 the inside of library low-temp storage hole by plunger pump
DEG C storage 10min after, with liquid-transfering gun point 25 draw 30 Chinese library of library low-temp storage hole to liquid receiving hole 31 is combined, pass through liquid relief
25 piping and druming of rifle point, which mixes, to be combined in liquid receiving hole 31 after liquid, is drawn cleaning solution in cleaning solution receiving hole 34 with liquid-transfering gun point 25 and is put
Into Tissue Culture Dish adapter 28 in Tissue Culture Dish, the gently prominent control of the shaft of left-right rotation Tissue Culture Dish adapter 28
Tissue Culture Dish supernatant is drawn with liquid-transfering gun point 25 after 40,2min of system point, is put to waste liquid receiving hole 33, after repeated washing 3 times, is used
Liquid-transfering gun point 25, which is drawn, combines liquid in liquid receiving hole 31 to move in Tissue Culture Dish adapter 28 in Tissue Culture Dish, at 4 DEG C
By prominent 40 2h of control point of shaft of motor gently left-right rotation Tissue Culture Dish adapter 28 in environment, with liquid-transfering gun point
25 draw Tissue Culture Dish aqueous supernatant, put to waste liquid receiving hole 33, are drawn with liquid-transfering gun point 25 clear in cleaning solution receiving hole 34
Washing lotion is put into Tissue Culture Dish adapter 28 in Tissue Culture Dish, is gently used after left-right rotation Tissue Culture Dish adapter 2min
Liquid-transfering gun point 25 draws Tissue Culture Dish supernatant, puts to waste liquid receiving hole 33, after repeated washing 3 times, draws the storage of TE buffer
TE buffer draws pancreatin digestive juice in pancreatin digestive juice receiving hole 35 into Tissue Culture Dish, then with liquid-transfering gun point 25 in hole 32
To Tissue Culture Dish 10min, liquid in Tissue Culture Dish is moved in PCR reacting hole 36 with liquid-transfering gun point 25, is mixed laggard
Row PCR amplification, after amplification, liquid-transfering gun point 25 draws amplified production and moves to 37 top of aptamers transfer receiving hole, passes through rubber
In rubber plug access aperture, and amplified production being discharged into hole, turn aptamers shift receiving hole 37, it removed from cartridge, until
This, the screening of first round aptamer is completed, and the aptamers transfer receiving hole 37 disassembled in cartridge is directly installed on
Library high temperature receiving hole of the position of the library high temperature receiving hole 29 of another cartridge as new cartridge can directly press above-mentioned step
It is rapid to carry out the second wheel or the n-th wheel experiment, until aptamer screening is completed.
Claims (10)
1. one kind screens cartridge device for tumour cell nucleic acid aptamers, which is characterized in that including cartridge bodies and be located at card
The cartridge reagent storage disk of box main body bottom；Turntable, pressing plate and liquid-transfering gun point are provided in the cartridge bodies, cartridge bodies
Air-path interface and air filter film are provided on shell, air-path interface is connect with the side air flue interface of liquid-transfering gun point；The turntable
Top extend to and form the first control interface outside cartridge bodies, liquid-transfering gun point is fixed on turntable；The pressing plate, which is located at, to be turned
Above disk, is extended at the top of pressing plate and form the second control interface outside cartridge bodies；It is provided on the cartridge reagent storage disk
Tissue Culture Dish adapter and reagent storage react hole location.
2. according to claim 1 screen cartridge device for tumour cell nucleic acid aptamers, which is characterized in that described
Turntable bottom is round thin panel, is fixed by cartridge bodies interior channel outstanding, there is aperture on turntable, passes through rifle point
The fixed liquid-transfering gun point of spring.
3. according to claim 1 screen cartridge device for tumour cell nucleic acid aptamers, which is characterized in that the pressure
Board bottom portion is cuboid thin plate, and bottom is provided with rag iron spring.
4. according to claim 1 screen cartridge device for tumour cell nucleic acid aptamers, which is characterized in that the gas
Air filtering core is provided in the side air flue interface of road interface and liquid-transfering gun point.
5. according to claim 1 screen cartridge device for tumour cell nucleic acid aptamers, which is characterized in that the sky
The filter sizes of air filter film are less than aptamers.
6. according to claim 1 screen cartridge device for tumour cell nucleic acid aptamers, which is characterized in that described thin
Born of the same parents' culture dish adapter is semiclosed bulge, and bottom end is provided with shaft.
7. according to claim 6 screen cartridge device for tumour cell nucleic acid aptamers, which is characterized in that described turn
Axis and bulge are seamlessly connected, and the two is to be linked together, and shaft one end projects to the shell exterior of cartridge bodies, form shaft
Prominent control point.
8. according to claim 1 screen cartridge device for tumour cell nucleic acid aptamers, which is characterized in that the examination
Agent storage reaction hole location includes 9 hole locations being successively set on cartridge reagent storage disk, and 9 hole locations are respectively library height
Gentle discharge hole, library low-temp storage hole, in conjunction with liquid receiving hole, TE buffer receiving hole, waste liquid receiving hole, cleaning solution receiving hole,
Pancreatin digestive juice receiving hole, PCR reacting hole, aptamers shift receiving hole.
9. according to claim 1 screen cartridge device for tumour cell nucleic acid aptamers, which is characterized in that the examination
Agent storage reaction hole location is circle or rectangular, and bottom end is distributed in hemispherical or taper ending closure, the orifice center point of hole location
On the circumference that the center of circle is overlapped with the turntable center of circle.
10. according to claim 8 screen cartridge device for tumour cell nucleic acid aptamers, which is characterized in that described
Library high temperature receiving hole and aptamers transfer receiving hole are detachable activity hole location, and the opening of hole location is closed with cork.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201721897015.8U CN208104447U (en) | 2017-12-29 | 2017-12-29 | One kind screening circular card box device for tumour cell nucleic acid aptamers |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201721897015.8U CN208104447U (en) | 2017-12-29 | 2017-12-29 | One kind screening circular card box device for tumour cell nucleic acid aptamers |
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| CN208104447U true CN208104447U (en) | 2018-11-16 |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110331075A (en) * | 2019-02-02 | 2019-10-15 | 上海思路迪医学检验所有限公司 | Closed sequencing library prepares cartridge |
-
2017
- 2017-12-29 CN CN201721897015.8U patent/CN208104447U/en active Active
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110331075A (en) * | 2019-02-02 | 2019-10-15 | 上海思路迪医学检验所有限公司 | Closed sequencing library prepares cartridge |
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