CN220450207U - Micro-fluid-control chip for cell electrotransfection in micro-fluid drops based on electrode polarization effect - Google Patents
Micro-fluid-control chip for cell electrotransfection in micro-fluid drops based on electrode polarization effect Download PDFInfo
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Abstract
The utility model discloses an electrode polarization effect-based cell electrotransfection micro-fluidic chip in micro-droplets and an electrotransfection method thereof, wherein the electrotransfection micro-fluidic chip comprises an upper layer detection plate, a middle layer droplet shearing plate and a bottom layer electrode plate which are sequentially arranged from top to bottom; the middle layer liquid drop shear plate is provided with a second detection liquid inlet and a liquid drop operation type medium diversion channel connected with the second detection liquid inlet, and two ends of the liquid drop operation type medium diversion channel are connected with a middle layer medium diversion inlet and a second detection liquid outlet; the upper layer detection plate is provided with a first detection liquid inlet, a medium inlet and a first detection liquid outlet; the bottom electrode plate is provided with a bipolar suspension electrode corresponding to the droplet manipulation type medium diversion channel. The utility model provides a bipolar electrode polarization effect-based micro-droplet cell high-efficiency electrotransfection micro-fluidic chip and an electrotransfection method thereof, which can simplify and efficiently carry out the electrotransfection process.
Description
Technical Field
The utility model relates to the technical field of microfluidic chips, in particular to an electrode polarization effect-based micro-droplet inner cell electrotransfection microfluidic chip.
Background
Currently, biomedical fields are receiving increasing attention, especially transformation medicine and gene medicine in the fields, and gene medicine is an important way to break through medical barriers. In particular, in the cell transfection technique, an exogenous normal gene is introduced into a target cell, and the product produced by the exogenous gene is used for treating and correcting medical problems caused by the abnormal gene.
There are many techniques for cell transfection such as viral transfection, hydrodynamic transfection and electrotransfection. Currently, CAR-T cell-oriented cell transfection is mainly viral transfection, and has the following problems: (1) The production of viral vectors is more complex and expensive and presents a high biological safety risk; (2) The CAR gene is randomly inserted into a host genome, so that the expression of a normal gene is influenced, and a certain cancerogenic risk exists; (3) Viral vectors have difficulty delivering large molecular weight nucleic acids, as well as delivering different drugs simultaneously. Hydrodynamic transfection mainly utilizes the stretching deformation of cells when passing through a narrow channel to form perforations on the surfaces of the cells, so that nucleic acid substances around the cells can be delivered into the cells through the surface perforations, and the purpose of cell transfection is achieved. However, the hydrodynamic transfection method has the defects of low cell transfection flux, poor controllability, low nucleic acid introduction amount and the like, so that the application scene is limited.
In recent years, electrotransfection has demonstrated great advantages in primary T cells, stem T cells, NK cells, etc. that are particularly difficult to transfect, however, existing electrotransfection techniques do not allow for both manipulation of flux, cell activity and transfection controllability; there remains a deficiency in the study of electroporation modulation of cell populations; the nucleic acid utilization rate is low and the introduction rate is slow. Therefore, it has been urgent how to improve the handling flux, controllability and transfection efficiency of cell electrotransfection.
Disclosure of Invention
The utility model overcomes the defects of the prior art, and provides a micro-droplet cell high-efficiency electrotransfection micro-fluidic chip based on bipolar electrode polarization effect and an electrotransfection method thereof, which can simplify and efficiently carry out the electrotransfection process; improves the transfection rate and controllability and the survival rate of cell electrotransfection.
In order to achieve the above purpose, the utility model adopts the following technical scheme: the micro-fluidic chip for cell electrotransfection in micro-droplets based on the electrode polarization effect comprises an upper layer detection plate, a middle layer droplet shearing plate and a bottom layer electrode plate which are sequentially arranged from top to bottom; the middle layer liquid drop shear plate is provided with at least one second detection liquid inlet and a liquid drop control type medium diversion channel connected with the second detection liquid inlet, one end of the liquid drop control type medium diversion channel is connected with a plurality of middle layer medium diversion inlets, and the other end of the liquid drop control type medium diversion channel is connected with a second detection liquid outlet; the upper layer detection plate is provided with a first detection liquid inlet which is in butt joint with a second detection liquid inlet, a medium inlet which is communicated with the medium shunt inlet of the middle layer, and a first detection liquid outlet which is communicated with the second detection liquid outlet; the bottom electrode plate is provided with a bipolar suspension electrode corresponding to the droplet operation type medium diversion channel.
In a preferred embodiment of the utility model, the middle layer droplet shear plate is also provided with a medium diversion channel II which is communicated with the droplet operation type medium diversion channel T;
and the second detection liquid inlet is communicated with the second medium diversion channel of the middle-layer liquid drop shearing plate positioned at the lower layer through the main channel.
In a preferred embodiment of the present utility model, the upper layer detection plate is further provided with a first medium diversion channel with a strip structure, the first medium diversion channel is communicated with the medium inlet, and the lower part of the first medium diversion channel is communicated with the medium diversion inlet of the middle layer.
In a preferred embodiment of the present utility model, the second detection liquid inlet and the second detection liquid outlet are respectively located at two ends of the droplet manipulation type medium diversion channel, the middle layer medium diversion inlet is directly communicated with the droplet manipulation type medium diversion channel, and the middle layer medium diversion inlet is located between the second detection liquid inlet and the second detection liquid outlet.
In a preferred embodiment of the utility model, an electrode manipulation area is arranged on the bottom electrode plate, and a plurality of bipolar suspension electrodes which are arranged at intervals are arranged in the electrode manipulation area;
the two ends of the electrode manipulation area are respectively provided with a negative electrode wiring area and a positive electrode wiring area which are connected with the electrode manipulation area;
the negative electrode wiring area is electrically connected with a plurality of negative electrode plates extending to the electrode manipulation area, the positive electrode wiring area is electrically connected with a plurality of positive electrode plates extending to the electrode manipulation area, and both sides of the bipolar suspension electrode are respectively provided with the positive electrode plates and the negative electrode plates.
In a preferred embodiment of the utility model, the bipolar suspension electrode has a width of 80 μm; the electric interval gap between the bipolar suspension electrode and the positive electrode plate and the negative electrode plate of the laser electrodes at two sides is 20 mu m; the distance between the intermediate layer medium diversion inlet and the branch connection section of the liquid drop control type medium diversion channel is 2mm.
In a preferred embodiment of the utility model, the width of the drop manipulation type medium diversion channel is 160 μm, the height is 80 μm, the size of the second detection liquid inlet is 1mm, and the interval between the adjacent second detection liquid outlets is 8mm.
In a preferred embodiment of the utility model, the bottom electrode plate comprises a glass substrate having a thickness of 1.5mm, on which an ITO electrode layer is deposited.
In a preferred embodiment of the utility model, the bottom electrode plate is connected with an electric signal source, the high-frequency signal frequency of the electric signal source is 1MHz, the pulse signal frequency of the electric signal source is 1kHz, the high-frequency voltage of the high-frequency signal is 10V, and the pulse voltage of the pulse signal is 1.5V.
The utility model solves the defects existing in the background technology, and has the beneficial effects that:
the utility model provides a micro-droplet cell high-efficiency electrotransfection micro-fluidic chip based on bipolar electrode polarization effect, which can simplify and efficiently perform the electrotransfection process; the transfection rate and controllability are improved.
Drawings
The utility model will be further described with reference to the drawings and examples.
FIG. 1 is a schematic perspective view of the structure of an electrotransfection microfluidic chip in a micro-droplet based on electrode polarization effect in a preferred embodiment of the present utility model;
FIG. 2 is a schematic diagram of an exploded structure of an electrotransfection microfluidic chip for cells within a micro-droplet based on electrode polarization effect in a preferred embodiment of the present utility model;
FIG. 3 is a schematic view of the structure of an upper layer detection plate according to a preferred embodiment of the present utility model;
FIG. 4 is a schematic view of the structure of a middle layer drop shear plate in a preferred embodiment of the present utility model;
FIG. 5 is a schematic view of the structure of a bottom electrode plate in a preferred embodiment of the present utility model;
FIG. 6 is an enlarged schematic view of bipolar floating electrode in bottom electrode plate in accordance with a preferred embodiment of the present utility model;
the device comprises a 1-upper layer detection plate, a 11-detection liquid inlet I, a 12-medium inlet, a 13-detection liquid outlet I and a 14-medium diversion channel I; 2-middle layer liquid drop shearing plate, 21-second detection liquid inlet, 22-middle layer medium diversion inlet, 23-liquid drop operation type medium diversion channel, 24-second detection liquid outlet, 25-second medium diversion channel; 3-bottom electrode plate, 31-negative electrode wiring area, 32-electrode manipulation area, 33-positive electrode wiring area, 331-bipolar suspension electrode, 332-negative electrode sheet, 333-positive electrode sheet.
Detailed Description
The utility model will now be described in further detail with reference to the drawings and examples, which are simplified schematic illustrations of the basic structure of the utility model, which are presented only by way of illustration, and thus show only the structures that are relevant to the utility model.
Example 1
As shown in fig. 1-6, the micro-fluidic chip for cell electrotransfection in micro-droplets based on electrode polarization effect comprises an upper layer detection plate 1, a middle layer droplet shear plate 2 and a bottom layer electrode plate 3 which are sequentially arranged from top to bottom.
Specifically, the middle layer liquid drop shear plate 2 is provided with at least one second detection liquid inlet 21 and a liquid drop operation type medium diversion channel 23 connected with the second detection liquid inlet 21, one end of the liquid drop operation type medium diversion channel 23 is connected with a plurality of middle layer medium diversion inlets 22, and the other end of the liquid drop operation type medium diversion channel 23 is connected with a second detection liquid outlet 24; the medium diversion channel II 25 is also arranged on the middle layer liquid drop shearing plate 2, and the medium diversion channel II 25 is communicated with the liquid drop operation type medium diversion channel 23T; the second detection liquid inlet 21 and the second detection liquid outlet 24 are respectively positioned at two ends of the liquid drop manipulation type medium diversion channel 23, the middle layer medium diversion inlet 22 is directly communicated with the liquid drop manipulation type medium diversion channel 23, and the middle layer medium diversion inlet 22 is positioned between the second detection liquid inlet 21 and the second detection liquid outlet 24. The second detection liquid inlet 21 is communicated with the second medium diversion channel 25 of the middle-layer liquid drop shearing plate 2 positioned at the lower layer through the main channel.
Specifically, the upper layer detection plate 1 is provided with a first detection liquid inlet 11 which is in butt joint with a second detection liquid inlet 21, a medium inlet 12 which is communicated with a medium split inlet 22 of the middle layer, and a first detection liquid outlet 13 which is communicated with a second detection liquid outlet 24; the bottom electrode plate 3 is provided with a bipolar suspension electrode 331 corresponding to the droplet manipulation type medium diversion channel 23.
Specifically, the upper layer detection plate 1 is further provided with a first medium diversion channel 14 with a strip-shaped structure, the first medium diversion channel 14 is communicated with the medium inlet 12, and the lower part of the first medium diversion channel 14 is communicated with the medium diversion inlet 22 of the middle layer.
Specifically, the bottom electrode plate 3 is provided with an electrode manipulation area 32, and a plurality of bipolar suspension electrodes 331 which are arranged at intervals are arranged in the electrode manipulation area 32; both ends of the electrode manipulation region 32 are provided with a negative electrode wiring region 31 and a positive electrode wiring region 33 connected to the electrode manipulation region 32, respectively; the negative electrode terminal area 31 is electrically connected with a plurality of negative electrode sheets 332 extending to the electrode manipulation area 32, the positive electrode terminal area 33 is electrically connected with a plurality of positive electrode sheets 333 extending to the electrode manipulation area 32, and both sides of the bipolar floating electrode 331 are respectively provided with the positive electrode sheets 333 and the negative electrode sheets 332.
More specifically, the width of the bipolar suspension electrode 331 is 80 μm; the electrical gap between the bipolar suspension electrode 331 and the positive electrode sheet 333 and the negative electrode sheet 332 of the two-sided excitation electrode is 20 μm; the branching section distance of the intermediate layer medium split inlet 22 from the droplet-manipulating medium split channel 23 was 2mm. The width of the droplet manipulation type medium flow dividing channel 23 was 160 μm, the height was 80 μm, the size of the detection liquid inlet II 21 was 1mm, and the interval between the adjacent detection liquid outlets II 24 was 8mm. The bottom electrode plate 3 comprises a glass substrate having a thickness of 1.5mm, on which an ITO electrode layer is deposited. Five drop-operated media diverting channels 23, and five sets of bipolar floating electrodes 331 are employed in this embodiment. The number of the first detection liquid outlets 13 corresponds to the number of the droplet manipulation type medium diversion channels 23 and is communicated in a one-to-one correspondence.
Example two
On the basis of the first embodiment, as shown in fig. 1 to 6, the method for electrotransfection of cells in micro-droplets into a microfluidic chip based on electrode polarization effect comprises the following steps:
introducing a detection liquid and shearing the detection liquid into micro-droplets, wherein the method comprises the following steps of: introducing the detection liquid through a detection liquid inlet I11, then entering a medium diversion channel I14, and injecting medium fluid into a medium inlet 12; the detection liquid is sheared into micro-droplets by the medium fluid in a T-shaped structure channel formed by a medium diversion inlet 22, a medium diversion channel II 25 and a droplet manipulation type medium diversion channel 23 of the middle layer droplet shear plate 2. Specifically, the method for obtaining the detection liquid comprises the following steps: culturing the cell sample to be electrotransfected to 70% -80% density, collecting cells, centrifuging to remove supernatant, and re-suspending in physiological saline containing nucleic acid sample to thoroughly mix the two. Specifically, before the electrotransfection method is carried out, the cell electrotransfection micro-fluidic chip is sterilized by using 70% ethanol solution, the surface of the cell electrotransfection micro-fluidic chip is washed by using sterile normal saline, then the normal saline solution containing 2% BSA is added to the cell electrotransfection micro-fluidic chip for incubation for 30 minutes, the residual BSA solution is removed, and the cell electrotransfection micro-fluidic chip is washed by using the normal saline.
Capturing and localizing cellular and nucleic acid material in a microdroplet, comprising the following method: the dielectrophoresis and the alternating current electric heating flow coupling effect formed by the bipolar suspension electrode 331 and the matching of the negative electrode plate 332 and the positive electrode plate 333 are utilized in the electrode manipulation area 32 of the bottom electrode plate 3 to collect the cells and the nucleic acid substances in the detection liquid on the same side.
Electrotransfection of cells is achieved, comprising the following methods: changing the signal connected to the bottom electrode plate 3 from a high-frequency signal to a pulse signal, setting the voltage to a designated value, and establishing electric field distribution on the electric transfection micro-fluidic chip; under the electric field distribution, the plasma membrane of the cells in the detection solution can be locally ruptured, and nucleic acid on the plasma membrane is led into the cells to realize the electrotransfection of the cells. Specifically, the bottom electrode plate 3 on the cell electrotransfection microfluidic chip is connected with an electric signal source, the frequency of a high-frequency signal is set to be 1MHz, the frequency of a pulse signal is set to be 1kHz, the high-frequency voltage is set to be 10V, and the pulse voltage is set to be 1.5V.
After the completion of electrotransfection, the chip was washed with physiological saline, and the cells were collected and centrifuged and analyzed.
Working principle:
the utility model provides a micro-droplet inner cell high-efficiency electrotransfection micro-fluidic chip based on bipolar electrode polarization effect, which integrates the functions of fluid diversion, droplet generation, cell-nucleic acid synchronous capture, cell electroporation and nucleic acid introduction on the chip.
The microfluidic chip mainly comprises a three-layer structure, wherein the three-layer structure comprises an upper detection plate 1 at the upper layer, a middle layer liquid drop shearing plate 2 at the middle layer and a bottom electrode plate 3 at the bottom layer; aims to realize high-flux and high-efficiency cell electrotransfection.
The upper detection plate 1 is an oil phase fluid diversion PDMS channel layer (PDMS, namely polydimethylsiloxane); thus splitting the oil phase (which may be silicone oil, hexadecane, etc.) fluid into multiple streams that enter the drop-operated media manifold 23 of the intermediate layer, with this method, the use of peripheral microfluidic pumps may be reduced, and multiple streams of the same flow rate may be produced using only one microfluidic pump.
The middle layer liquid drop shearing plate 2 is a micro liquid drop generation and movement PDMS channel layer of the middle layer; the droplet generation and movement PDMS channel layer is provided with a droplet manipulation type medium shunt channel 23, and the droplet manipulation type medium shunt channel 23 functions as a droplet generation and movement channel, and in the droplet manipulation type medium shunt channel 23, a detection liquid (the detection liquid includes a cell-nucleic acid mixture) is injected into the first detection liquid inlet 11, and then forms a micro droplet through the fluid shearing action of the T-shaped channel, and then enters the electrode manipulation area 32 (i.e., the area where the bottom electrode is located).
The bottom electrode plate 3 of the bottom layer comprises a glass substrate and an ITO electrode layer arranged on the glass substrate; the upper layer is a fluid diversion channel which is connected with 5 oil phase inlets of the middle layer. The ITO electrode layer includes an excitation electrode including a negative electrode sheet 332 and a positive electrode sheet 333, and a bipolar suspension electrode 331. And 5 bipolar suspension electrodes 331 are respectively disposed at the middle positions of 5 branch channels of the middle layer, and at this time, the excitation electrodes are disposed at two sides of each channel, so that electrode manipulation regions 32 can be respectively formed in each branch microchannel, and a non-uniform alternating current electric field can be formed in the regions. The three layers are assembled together by plasma bonding technology to form the final microfluidic chip. By utilizing the three-layer micro-fluidic chip structural design, not only can the use quantity of peripheral micro-fluidic pumps be reduced, but also the chip manipulation flux can be increased, the electric field manipulation technology and the liquid drop manipulation technology can be combined, and the controllability of cell manipulation can be enhanced.
When the micro liquid drops enter the electrode area, the exciting electrode is applied with high-frequency alternating current signals to generate induced potential by the bipolar suspension electrode 331, and the alternating current electric heating vortex flow phenomenon is generated in the liquid drops, so that cells and nucleic acid are synchronously captured to one side of the channel by fluid vortex; then pulse electric signals are applied to the excitation electrodes to promote the cells to generate electrotransfection. The chip is of a three-layer micro-fluidic chip structure, and utilizes the fluid diversion channel to conduct multi-channel diversion so as to realize high-flux operation; generating droplets by using a T-shaped channel, and simultaneously wrapping the cell-nucleic acid mixture into the droplets to provide the droplets with independent and pollution-free environments; the method comprises the steps of utilizing an alternating-current electric heating flow phenomenon induced by an alternating-current electric field to control a trans-scale cell and a nucleic acid substance, synchronously capturing the trans-scale cell and the nucleic acid substance to the same region, and providing necessary conditions for high-efficiency cell transfection; electroporation of cells using pulsed electrical signals, the nucleic acids enriched around which are introduced into the cells via the perforations; therefore, the efficient and controllable electrotransfection of the cells is realized, and theoretical and technical support is provided for the preparation of the CAR-T cells.
The above-described preferred embodiments according to the present utility model are intended to suggest that, from the above description, various changes and modifications can be made by the person skilled in the art without departing from the scope of the technical idea of the present utility model. The technical scope of the present utility model is not limited to the description, but must be determined according to the scope of claims.
Claims (9)
1. The cell electrotransfection micro-fluidic chip in the micro-droplet based on the electrode polarization effect is characterized in that: comprises an upper layer detection plate, a middle layer liquid drop shearing plate and a bottom layer electrode plate which are sequentially arranged from top to bottom;
the middle layer liquid drop shear plate is provided with at least one second detection liquid inlet and a liquid drop operation type medium diversion channel connected with the second detection liquid inlet, one end of the liquid drop operation type medium diversion channel is connected with a plurality of middle layer medium diversion inlets, and the other end of the liquid drop operation type medium diversion channel is connected with a second detection liquid outlet;
the upper layer detection plate is provided with a first detection liquid inlet which is in butt joint with the second detection liquid inlet, a medium inlet which is communicated with the medium shunt inlet of the middle layer, and a first detection liquid outlet which is communicated with the second detection liquid outlet;
and the bottom electrode plate is provided with a bipolar suspension electrode corresponding to the liquid drop operation type medium diversion channel.
2. The electrode polarization effect-based cell electrotransfection microfluidic chip in a micro-droplet of claim 1, wherein: the medium diversion channel II is also arranged on the middle-layer liquid drop shearing plate and communicated with the liquid drop operation type medium diversion channel T;
and the second detection liquid inlet is communicated with the second medium diversion channel of the middle-layer liquid drop shearing plate positioned at the lower layer through the main channel.
3. The electrode polarization effect-based cell electrotransfection microfluidic chip in a micro-droplet of claim 2, wherein: the upper layer detection plate is also provided with a first medium diversion channel with a strip-shaped structure, the first medium diversion channel is communicated with the medium inlet, and the lower part of the first medium diversion channel is communicated with the medium diversion inlet of the middle layer.
4. The electrode polarization effect-based cell electrotransfection microfluidic chip in micro-droplets of claim 3, wherein: the second detection liquid inlet and the second detection liquid outlet are respectively positioned at two ends of the liquid drop operation type medium diversion channel, the middle layer medium diversion inlet is directly communicated with the liquid drop operation type medium diversion channel, and the middle layer medium diversion inlet is positioned between the second detection liquid inlet and the second detection liquid outlet.
5. The electrode polarization effect-based cell electrotransfection microfluidic chip in a micro-droplet of claim 1, wherein: an electrode control area is arranged on the bottom electrode plate, and a plurality of bipolar suspension electrodes which are distributed at intervals are arranged in the electrode control area;
the two ends of the electrode manipulation area are respectively provided with a negative electrode wiring area and a positive electrode wiring area which are connected with the electrode manipulation area;
the bipolar suspension electrode comprises a bipolar suspension electrode and is characterized in that the negative electrode wiring area is electrically connected with a plurality of negative electrode plates extending to the electrode manipulation area, the positive electrode wiring area is electrically connected with a plurality of positive electrode plates extending to the electrode manipulation area, and both sides of the bipolar suspension electrode are respectively provided with the positive electrode plates and the negative electrode plates.
6. The electrode polarization effect-based cell electrotransfection microfluidic chip in a micro-droplet of claim 1, wherein: the width of the bipolar suspension electrode is 80 μm; the electric interval gap between the bipolar suspension electrode and the positive electrode plate and the negative electrode plate of the laser electrodes at two sides is 20 mu m; the distance between the intermediate layer medium diversion inlet and the branch connection section of the liquid drop control type medium diversion channel is 2mm.
7. The electrode polarization effect-based cell electrotransfection microfluidic chip in a micro-droplet of claim 6, wherein: the width of the liquid drop operation type medium diversion channel is 160 mu m, the height of the liquid drop operation type medium diversion channel is 80 mu m, the size of the second detection liquid inlet is 1mm, and the distance between the adjacent second detection liquid outlets is 8mm.
8. The electrode polarization effect-based cell electrotransfection microfluidic chip in a micro-droplet of claim 7, wherein: the bottom electrode plate comprises a glass substrate with the thickness of 1.5mm, and an ITO electrode layer is arranged on the glass substrate.
9. The electrode polarization effect-based cell electrotransfection microfluidic chip in micro-droplets of claim 8, wherein: the bottom electrode plate is connected with an electric signal source, the high-frequency signal frequency of the electric signal source is 1MHz, the pulse signal frequency of the electric signal source is 1kHz, the high-frequency voltage of the high-frequency signal is 10V, and the pulse voltage of the pulse signal is 1.5V.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202321913364.XU CN220450207U (en) | 2023-07-20 | 2023-07-20 | Micro-fluid-control chip for cell electrotransfection in micro-fluid drops based on electrode polarization effect |
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| CN202321913364.XU CN220450207U (en) | 2023-07-20 | 2023-07-20 | Micro-fluid-control chip for cell electrotransfection in micro-fluid drops based on electrode polarization effect |
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| CN220450207U true CN220450207U (en) | 2024-02-06 |
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| CN202321913364.XU Active CN220450207U (en) | 2023-07-20 | 2023-07-20 | Micro-fluid-control chip for cell electrotransfection in micro-fluid drops based on electrode polarization effect |
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