Automatic collecting device for ultracentrifugal strips

Abstract

The utility model relates to the technical field of biochemical extraction and purification, and discloses an automatic collecting device for ultracentrifugal strips, which comprises a pipe frame mechanism, a plurality of centrifugal pipe holes, a plurality of centrifugal pipes, a plurality of centrifugal pipes and a collecting mechanism, the punching mechanism comprises a puncture plate, the puncture plate is provided with puncture needles in one-to-one correspondence with the centrifuge tube holes, the puncture needles are connected with a peristaltic pump through a liquid inlet and outlet pipe, and the other end of the peristaltic pump is connected with a collecting pipe; the movement mechanism is connected with the puncture plate and used for driving the puncture plate to be close to or away from the centrifugal tube and driving the puncture plate to ascend and descend in the height direction; and the peristaltic pump and the movement mechanism are both in signal connection with the control device. According to the automatic ultracentrifugal strip collecting device, full-automatic collection and sampling of target strips are achieved, the sampling efficiency is high, the yield and purity of target samples are higher, and the safety risk that sampling personnel are punctured by a puncture needle is avoided.

Description

Technical Field

[0001] This utility model relates to the field of biochemical extraction and purification technology, and more specifically, to an automatic collection device for ultracentrifuged strips. Background Technology

[0002] Centrifugation, especially ultracentrifugation, is a common method for separating proteins, enzymes, nucleic acids, and cellular subcomponents, and is widely used in the separation, purification, and clarification of biological macromolecules.

[0003] Traditional ultracentrifugation involves manually piercing the bottom of the centrifuge tube with a needle to extract the target band. However, this method is not only prone to injuring the sampling personnel and poses a significant safety hazard, but also prone to sample mixing due to shaking of the centrifuge tube during sampling, reducing the purity of the target band. Furthermore, it is difficult to accurately control the sample volume during sampling, which can easily lead to the problem of collecting too much or too little of the target sample.

[0004] In summary, how to safely, conveniently, and accurately collect and sample the target band after centrifugation is a problem that urgently needs to be solved by those skilled in the art. Utility Model Content

[0005] In view of this, the purpose of this utility model is to provide an automatic collection device for ultracentrifuged strips, which realizes fully automatic collection and sampling of target strips, with high sampling efficiency, higher yield and purity of target samples, and avoids the safety risk of puncture needle injury to sampling personnel.

[0006] To achieve the above objectives, this utility model provides the following technical solution:

[0007] An automatic collection device for ultracentrifuged strips includes:

[0008] The tube rack mechanism has several centrifuge tube holes for placing centrifuge tubes;

[0009] The punching mechanism includes a puncture plate, the puncture plate being provided with puncture needles that correspond one-to-one with the holes of the centrifuge tube, the puncture needles being connected to a peristaltic pump through inlet and outlet pipes, and the other end of the peristaltic pump being connected to a collection pipe;

[0010] The motion mechanism connected to the puncture plate is used to move the puncture plate closer to or away from the centrifuge tube, and to move the puncture plate up and down in the height direction;

[0011] The control device is connected to both the peristaltic pump and the motion mechanism via signal connection.

[0012] Preferably, the tube rack mechanism comprises a tube rack for placing the centrifugal tubes and a tube rack support seat for mounting the ultraviolet lamp, the tube rack support seat is provided with a U-shaped clamping groove facing the punching mechanism, and the tube rack is clamped in the U-shaped clamping groove.

[0013] Preferably, the ultraviolet lamp is mounted at the top end of the tube rack support seat, the length direction of the ultraviolet lamp is parallel to the extension direction of the tube rack, and the length of the ultraviolet lamp is greater than the farthest distance between the two farthest centrifugal tube holes on the tube rack.

[0014] Preferably, the movement mechanism comprises a sliding frame, an X-axis movement mechanism, and a Z-axis movement mechanism, the moving end of the X-axis movement mechanism is connected with the sliding frame, so as to drive the sliding frame to relatively approach or move away from the tube rack mechanism along the X-axis direction.

[0015] The fixed end of the Z-axis movement mechanism is connected with the sliding frame, and the moving end of the Z-axis movement mechanism is connected with the puncture plate, so as to drive the puncture plate to ascend or descend along the height direction.

[0016] Preferably, at least one vertical guide rod is arranged in the sliding frame along the height direction, the puncture plate is provided with a guide hole for the vertical guide rod to pass through, and the puncture plate is slidably sleeved outside the vertical guide rod.

[0017] Preferably, the movement mechanism further comprises a horizontal guide arranged along the X-axis direction, and the sliding frame is slidably connected with the horizontal guide.

[0018] Preferably, the horizontal guide comprises a horizontal guide rod vertically arranged between the tube rack mechanism and a fixed frame arranged parallel to the tube rack mechanism, the sliding frame is provided with a guide hole for the horizontal guide rod to pass through, and the sliding frame is slidably sleeved outside the horizontal guide rod.

[0019] Preferably, the X-axis movement mechanism and the Z-axis movement mechanism each comprise a lead screw motor, the lead screw of the X-axis lead screw motor is threadedly connected with the sliding frame, and the lead screw of the Z-axis lead screw motor is threadedly connected with the puncture plate.

[0020] Preferably, the centrifugal tube holes are uniformly provided with a plurality of elastic pieces in the circumferential direction, and when the centrifugal tube is mounted in the centrifugal tube hole, the centrifugal tube is subjected to elastic compression force of the elastic pieces directed to the center direction.

[0021] Preferably, the liquid inlet and outlet pipe is connected with the peristaltic pump through a multi-channel collector.

[0022] When sampling, first, the motion mechanism is controlled to adjust the height of the puncture plate, so that the puncture needle is opposite to the target strip of the centrifugal tube, then the motion mechanism is controlled to drive the puncture plate to move towards the centrifugal tube, so that the puncture needle pierces the centrifugal tube and is inserted into the target strip, finally, the peristaltic pump is controlled to make the target sample in the target strip be transmitted to the collecting tube through the puncture needle, the liquid inlet and outlet pipe and the peristaltic pump.

[0023] The automatic collecting device for ultrarapid centrifugal strips provided by the utility model realizes full-automatic collecting and sampling of target strips, significantly improves sampling efficiency, and avoids the safety risk of puncture needle injury to sampling personnel.

[0024] The tube rack mechanism limits the shaking of the centrifugal tube during puncture, avoids sample mixing, guarantees the purity of the target sample, and the peristaltic pump not only avoids cross contamination between samples, but also can accurately control the sampling volume, further guarantees the yield and purity of the target sample. BRIEF DESCRIPTION OF DRAWINGS

[0025] In order to more clearly illustrate the technical scheme in the embodiments of the utility model or the prior art, the following will briefly introduce the drawings needed to be used in the embodiment or the prior art description, obviously, the drawings in the following description are only the embodiments of the utility model, and for the ordinary skilled in the art, other drawings can be obtained according to the provided drawings without paying creative labor.

[0026] Figure 1 It is a structure schematic view of the specific embodiment of the automatic collecting device for ultrarapid centrifugal strips provided by the utility model.

[0027] Figure 2 It is a structure schematic view of the tube rack.

[0028] Figure 3 It is a structure schematic view of the sliding frame and the Z-axis motion mechanism.

[0029] Figures 1-3 In the middle:

[0030] 01-centrifugal tube;02-collecting tube;11-tube rack;111-centrifugal tube hole;12-tube rack support seat;13-ultraviolet lamp;2-puncture plate;3-liquid inlet and outlet pipe;4-multichannel collector;5-peristaltic pump;61-sliding frame;62-X-axis screw motor;63-Z-axis screw motor;64-vertical guide rod;65-fixed frame;66-horizontal guide rod;7-collecting tube placing rack;8-control panel. DETAILED DESCRIPTION

[0031] The technical solutions in the embodiments of the utility model will be clearly and completely described below with reference to the drawings in the embodiments of the utility model. Obviously, the described embodiments are only part of the embodiments of the utility model, rather than all the embodiments. Based on the embodiments of the utility model, all other embodiments obtained by those skilled in the art without creative labor fall within the scope of the utility model.

[0032] The utility model discloses a kind of automatic collection devices of ultra-speed centrifugal strip, realize the full-automatic collection sampling of target strip, sampling efficiency is high, the yield and purity of target sample are higher, and avoid the safety risk of puncture needle to prick sampling personnel.

[0033] The utility model provides an automatic collection device of ultra-speed centrifugal strip, comprising:

[0034] Tube rack mechanism is equipped with a plurality of centrifugal tube holes 111 for placing centrifugal tube 01;

[0035] Punching mechanism, including puncture plate 2, puncture plate 2 is equipped with puncture needle corresponding with centrifugal tube hole 111, puncture needle is connected with peristaltic pump 5 by liquid inlet and outlet pipe 3, another end of peristaltic pump 5 is connected with collection tube 02;

[0036] Movement mechanism connected with puncture plate 2, for driving puncture plate 2 to approach or away from centrifugal tube 01, and drive puncture plate 2 to ascend along height direction;

[0037] Control device, peristaltic pump 5 and movement mechanism are connected with control device signal.

[0038] Among them, tube rack mechanism is used to place and fix centrifugal tube 01, avoid centrifugal tube 01 to shake in puncture process and then cause the sample in centrifugal tube 01 to appear mixing, so as to guarantee the purity of target sample;

[0039] Tube rack mechanism can be placed only on experiment table, also can be fixed on experiment table by fastener such as fastening bolt, connecting pin, to further enhance the stability and reliability of centrifugal tube 01.

[0040] Similarly, collection tube holder 7 for placing collection tube 02 can also be fixed on experiment table by fastener such as fastening bolt, connecting pin.

[0041] Punching mechanism is used to puncture centrifugal tube 01, one end of puncture needle is inserted into target strip in centrifugal tube 01, and the other end is connected with peristaltic pump 5 by liquid inlet and outlet pipe 3, so that peristaltic pump 5 samples target sample of target strip through puncture needle and liquid inlet and outlet pipe 3.

[0042] The puncture needles on the puncture plate 2 can be respectively connected with different peristaltic pumps 5, or connected with the same peristaltic pump 5 to reduce the equipment cost of the automatic collection device; please refer to Figure 1 When the puncture needles correspond to the same peristaltic pump 5, the inlet and outlet liquid pipes 3 can be connected with the peristaltic pump 5 through the multi-channel collector 4 to simplify the connection relationship between the inlet and outlet liquid pipes 3 and the peristaltic pump 5.

[0043] The structure, shape and size of the puncture needle can be determined according to the actual production needs and the existing technology; the inlet and outlet liquid pipes 3 are usually made of rubber pipes, which can not only ensure the sealing performance of the connection between the inlet and outlet liquid pipes 3 and the puncture needle and the peristaltic pump 5, but also have stable chemical properties and are not easy to corrode and age, so the service life is longer.

[0044] The power mechanism is used to drive the punching mechanism, which not only can drive the puncture plate 2 to approach or move away from the tube rack mechanism, so that the puncture needle can be inserted into or withdrawn from the centrifugal tube 01, but also can adjust the height of the puncture plate 2, so that the height of the puncture needle can be adapted to the target strip of different heights.

[0045] It should be noted that the power mechanism drives the puncture plate 2 to approach or move away from the tube rack mechanism, which is not limited to the power mechanism driving the puncture plate 2 to approach or move away from the tube rack mechanism along the normal direction of the tube rack mechanism, but also includes the power mechanism driving the puncture plate 2 to approach or move away from the tube rack mechanism along the inclined direction, as long as it can ensure that the puncture needle accurately penetrates or withdraws from the target strip of the corresponding centrifugal tube 01.

[0046] In order to shorten the moving distance of the punching mechanism and increase the pressure of the puncture needle on the wall surface of the centrifugal tube 01, the power mechanism is preferably arranged to drive the puncture plate 2 to approach or move away from the tube rack mechanism along the normal direction of the tube rack mechanism.

[0047] The power mechanism can be specifically arranged as an electric push rod, a lead screw motor or the like linear power mechanism, and the specific type, model and power thereof can be determined according to the actual production needs and the existing technology, which will not be described here.

[0048] The control device includes a controller for signal connection with the peristaltic pump 5 and the motion mechanism, and a control board 8 for receiving control instructions, and the control board 8 is signal connected with the controller through a cable or wireless signal; in order to facilitate sampling operation, the controller is preferably arranged below the tabletop of the experiment table, and the control board 8 is arranged on the tabletop of the experiment table.

[0049] For example, please refer to Figure 1 The control board 8 is provided with a control switch for controlling the opening and closing of the ultraviolet lamp 13, a control switch for controlling the liquid suction or discharge of the peristaltic pump 5, and a motion control switch for controlling the working state of the motion mechanism, and of course the control switch can also be replaced by a control key or a control button or the like element with similar functions.

[0050] When sampling, first control the motion mechanism to adjust the height of the puncture plate 2, so that the puncture needle is opposite to the target strip of the centrifugal tube 01, then control the motion mechanism to drive the puncture plate 2 to move towards the centrifugal tube 01, so that the puncture needle pierces the centrifugal tube 01 and inserts into the target strip, and finally control the peristaltic pump 5 to transmit the target sample in the target strip to the collection tube 02 through the puncture needle, the liquid inlet and outlet pipe 3 and the peristaltic pump 5.

[0051] After sampling is completed, the peristaltic pump 5 is used to reverse flush the puncture needle, so as to avoid cross contamination caused by the residual sample on the puncture needle during subsequent sampling.

[0052] In the embodiment, full-automatic collection and sampling of the target strip are realized, the sampling efficiency is significantly improved, and the safety risk of the puncture needle piercing the sampling personnel is avoided.

[0053] The tube rack mechanism limits the shaking of the centrifugal tube 01 during puncture, avoids sample mixing, ensures the purity of the target sample, and the peristaltic pump 5 not only avoids cross contamination between samples, but also can accurately control the sampling volume, further ensuring the yield and purity of the target sample.

[0054] On the basis of the above embodiment, the structure of the tube rack mechanism is limited, the tube rack mechanism includes a tube rack 11 for placing the centrifugal tube 01 and a tube rack support seat 12 for installing the ultraviolet lamp 13, the tube rack support seat 12 is provided with a U-shaped clamping groove facing the punching mechanism, and the tube rack 11 is clamped in the U-shaped clamping groove.

[0055] Please refer to Figure 3 The tube rack 11 is uniformly provided with a plurality of centrifugal tube holes 111 along the extension direction, the number of the centrifugal tube holes 111 is determined according to actual production needs, and the shape and size of the centrifugal tube holes 111 are determined according to the size of the centrifugal tube 01 in actual production.

[0056] Preferably, the centrifugal tube holes 111 are uniformly provided with a plurality of elastic pieces in the circumferential direction, when the centrifugal tube 01 is installed in the centrifugal tube hole 111, the centrifugal tube 01 is subjected to the elastic compression force of the elastic pieces pointing to the center direction, so as to more reliably fix the centrifugal tube 01 in the centrifugal tube hole 111 by using the elastic compression force of the elastic pieces.

[0057] In order to meet the sampling needs of centrifugal tubes 01 of different sizes, the tube rack 11 is clamped in the tube rack support seat 12, so as to replace the tube rack 11, but it should be noted that in order to ensure the accurate alignment of the centrifugal tube holes 111 in the tube rack 11 and the puncture needles on the puncture plate 2, the axis positions of the centrifugal tube holes 111 in the tube racks 11 of different sizes are unchanged.

[0058] The tube rack 11 is externally provided with a tube rack support seat 12. The tube rack support seat 12 can support the tube rack 11, further limit the shaking of the centrifugal tube 01 and the tube rack 11 during puncture, and provide installation space for the ultraviolet lamp 13, so that the sampler can identify and confirm the distribution of the bands in the centrifugal tube 01 under ultraviolet light.

[0059] Preferably, the ultraviolet lamp 13 is installed at the top end of the tube rack support seat 12, the length direction of the ultraviolet lamp 13 is parallel to the extension direction of the tube rack 11, and the length of the ultraviolet lamp 13 is greater than the farthest distance between the two centrifugal tube holes 111 on the tube rack 11.

[0060] Compared with moving the ultraviolet lamp 13 up and down to identify the target band, the above-mentioned ultraviolet lamp 13 can irradiate all the centrifugal tubes 01 from top to bottom, which is convenient for the sampler to identify the distribution of different bands in the centrifugal tube 01.

[0061] In the embodiment, the tube rack 11 is clamped in the U-shaped clamping groove of the tube rack support seat 12. This can effectively support the tube rack 11, further reduce the shaking of the tube rack 11 and the centrifugal tube 01 in it during puncture, avoid mixing of the samples in the centrifugal tube 01, and ensure the purity of the samples. It can also meet the sampling needs of centrifugal tubes 01 of different sizes.

[0062] On the basis of the above-mentioned embodiment, the structure of the motion mechanism is limited. The normal direction of the tube rack mechanism is the X-axis direction, the extension direction of the tube rack mechanism is the Y-axis direction, and the height direction is the Z-axis direction. The motion mechanism includes a sliding frame 61, an X-axis motion mechanism, and a Z-axis motion mechanism. The moving end of the X-axis motion mechanism is connected with the sliding frame 61, so as to drive the sliding frame 61 to relatively approach or move away from the tube rack mechanism along the X-axis direction.

[0063] The fixed end of the Z-axis motion mechanism is connected with the sliding frame 61, and the moving end of the Z-axis motion mechanism is connected with the puncture plate 2, so as to drive the puncture plate 2 to ascend or descend along the height direction.

[0064] Among them, the type of Z-axis motion mechanism and the type of X-axis motion mechanism can be the same or different; both can be set as electric push rods, or can be set as lead screw motors and other linear motion mechanisms.

[0065] Considering the stability of the motion and the convenience of maintenance, it is preferred that the X-axis motion mechanism and the Z-axis motion mechanism both include a lead screw motor. The lead screw of the X-axis lead screw motor 62 is threadedly connected with the sliding frame 61, and the lead screw of the Z-axis lead screw motor 63 is threadedly connected with the puncture plate 2.

[0066] The puncture plate 2 is installed on the sliding frame 61 through a Z-axis movement mechanism. When the Z-axis movement mechanism is extended or rotated, the puncture plate 2 is driven to ascend or descend along the height direction, i.e. the Z-axis direction, relative to the sliding frame 61, so as to realize the accurate alignment of the puncture needle and the target strip of the centrifugal tube 01.

[0067] The sliding frame 61 is connected with the moving end of the X-axis movement mechanism. When the X-axis movement mechanism is extended or rotated, the sliding frame 61 is driven to relatively approach or move away from the tube frame mechanism along the X-axis direction, and then the puncture plate 2 installed on the sliding frame 61 is driven to relatively approach or move away from the tube frame mechanism along the X-axis direction.

[0068] In the embodiment, the X-axis movement mechanism is used to drive the puncture plate 2 to relatively approach or move away from the tube frame mechanism, and the Z-axis movement mechanism is used to drive the puncture plate 2 to ascend or descend along the height direction, so the structure is simple and the adjustment is convenient.

[0069] Preferably, in order to avoid the deviation of the puncture plate 2 when ascending or descending, at least one vertical guide rod 64 arranged in the height direction is arranged in the sliding frame 61, the puncture plate 2 is provided with a guide hole for the vertical guide rod 64 to pass through, and the puncture plate 2 is slidingly sleeved on the vertical guide rod 64, so that the vertical guide rod 64 guides and limits the puncture plate 2.

[0070] Similarly, in order to avoid the deviation of the sliding frame 61 when moving horizontally, the movement mechanism can be provided with a horizontal guide arranged along the X-axis direction, and the sliding frame 61 is slidingly connected with the horizontal guide.

[0071] The horizontal guide can be a guide rail or a sliding block, which is slidingly connected with the sliding frame 61 through a sliding pair, or can be a horizontal guide rod 66.

[0072] Please refer to Figure 1 The automatic collecting device includes a fixed frame 65 arranged parallel to the tube frame mechanism, the horizontal guide includes a horizontal guide rod 66 vertically arranged between the tube frame mechanism and the fixed frame 65, in order to facilitate the replacement of the tube frame 11, the horizontal guide rod 66 is usually arranged between the tube frame support seat 12 and the fixed frame 65, the sliding frame 61 is provided with a guide hole for the horizontal guide rod 66 to pass through, and the sliding frame 61 is slidingly sleeved on the horizontal guide rod 66.

[0073] In the description, each embodiment is described in a progressive manner, and each embodiment focuses on the difference from other embodiments. The same or similar parts between the embodiments can be referred to each other.

[0074] The above has carried out the detailed introduction to the automatic collection device of the ultracentrifugal strip provided by the utility model. The principle and implementation mode of the utility model are described by applying specific examples in this paper, and the above embodiment is only used for helping to understand the method and core idea of the utility model. It should be pointed out that for ordinary skilled person in the art, without departing from the principle of the utility model, the utility model can be improved and modified in several ways, and these improvements and modifications also fall within the protection scope of the utility model claim.

Claims

1. An ultracentrifuge band automatic collection device, characterized by, The application relates to a centrifugal tube punching and sterilizing device. The centrifugal tube punching and sterilizing device comprises a tube rack mechanism provided with a plurality of centrifugal tube holes (111) for placing centrifugal tubes (01); a punching mechanism comprising a puncture plate (2) provided with puncture needles corresponding to the centrifugal tube holes (111); a peristaltic pump (5) connected with the puncture plate (2) through a liquid inlet and outlet pipe (3); a movement mechanism connected with the puncture plate (2) for driving the puncture plate (2) to approach or move away from the centrifugal tube (01) and to drive the puncture plate (2) to ascend or descend along the height direction; and a control device connected with the peristaltic pump (5) and the movement mechanism. The tube rack mechanism comprises a tube rack (11) for placing the centrifugal tubes (01) and a tube rack support seat (12) for mounting an ultraviolet lamp (13), the tube rack support seat (12) is provided with a U-shaped clamping groove facing the punching mechanism, and the tube rack (11) is clamped in the U-shaped clamping groove. The ultraviolet lamp (13) is mounted at the top end of the tube rack support seat (12), the length direction of the ultraviolet lamp (13) is parallel to the extension direction of the tube rack (11), and the length of the ultraviolet lamp (13) is greater than the farthest distance between two centrifugal tube holes (111) farthest apart on the tube rack (11). The movement mechanism comprises a sliding frame (61), an X-axis movement mechanism and a Z-axis movement mechanism, the moving end of the X-axis movement mechanism is connected with the sliding frame (61) so as to drive the sliding frame (61) to relatively approach or move away from the tube rack mechanism along the X-axis direction, the fixed end of the Z-axis movement mechanism is connected with the sliding frame (61), and the moving end of the Z-axis movement mechanism is connected with the puncture plate (2) so as to drive the puncture plate (2) to ascend or descend along the height direction.

2. The ultracentrifuge strip automatic collection apparatus according to claim 1, characterized by, The sliding frame (61) is provided with at least one vertical guide rod (64) arranged along the height direction, the puncture plate (2) is provided with a guide hole for the vertical guide rod (64) to pass through, and the puncture plate (2) is slidably sleeved outside the vertical guide rod (64).

3. The ultracentrifuge strip automatic collection apparatus according to claim 2, wherein, The movement mechanism further comprises a horizontal guide arranged along the X-axis direction, and the sliding frame (61) is slidably connected with the horizontal guide.

4. The ultracentrifuge strip automatic collection apparatus according to claim 1, wherein, The device further comprises a fixed frame (65) arranged parallel to the tube rack mechanism, the horizontal guide comprises a horizontal guide rod (66) vertically arranged between the tube rack mechanism and the fixed frame (65), the sliding frame (61) is provided with a guide hole for the horizontal guide rod (66) to pass through, and the sliding frame (61) is slidably sleeved outside the horizontal guide rod (66). The X-axis movement mechanism and the Z-axis movement mechanism both comprise a lead screw motor, the lead screw of an X-axis lead screw motor (62) is threadedly connected with the sliding frame (61), and the lead screw of a Z-axis lead screw motor (63) is threadedly connected with the puncture plate (2).

5. The ultracentrifuge strip automatic collection apparatus according to claim 4, wherein, ​ 6. The ultracentrifuge strip automatic collection apparatus of claim 4, wherein, ​ 7. The ultracentrifuge strip automatic collection apparatus of claim 6, wherein, ​ 8. The ultracentrifuge strip automatic collection apparatus of claim 6, wherein, ​ 9. The ultracentrifuge strip automatic collection apparatus according to any one of claims 1 to 8, characterized in that, The centrifugal tube hole (111) is uniformly provided with a plurality of elastic sheets in the circumferential direction, and when the centrifugal tube (01) is installed in the centrifugal tube hole (111), the centrifugal tube (01) is subjected to elastic compression force of the elastic sheets in the direction of the center of the circle.

10. The ultracentrifuge strip automatic collection apparatus according to any one of claims 1 to 8, characterized in that, The inlet and outlet pipe (3) is connected with the peristaltic pump (5) through a multi-channel collector (4).

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