Liquid mixing using an automated liquid handling system

Abstract

A method of mixing liquids using an automated liquid handling system includes alternately aspirating liquid volumes of a first liquid and a second liquid from one of a first liquid supply S1 and a second liquid supply S2 into a mixing volume, whereby the aspirated liquid volumes form a liquid stack comprising a succession of alternating interfacial layers of the first liquid and the second liquid in the mixing volume, mixing the interfacial layers of the first liquid and the second liquid with each other by diffusion in the mixing volume to form a mixed liquid, and expelling the mixed liquid from the mixing volume.

Description

Technical Field

[0001] The present technology relates to an automated liquid handling system, and more particularly, to an apparatus and method for mixing liquids using an automated liquid handling system.

Background Art

[0002] Laboratory liquid handling systems are used to transfer and manipulate predetermined amounts of liquid. One or more liquid samples can be supplied into laboratory instrument containers (e.g., microplate or sample tube holders) within the liquid handling system. The liquid handling system can include one or more pipettors used to remove a portion of the sample from the laboratory instrument (e.g., by aspiration) and / or add materials to the sample within the laboratory instrument (e.g., by dispensing). In some cases, it may be desirable or necessary to mix liquids within the system. It may be desirable or necessary to mix the liquids robotically and, in some cases, automatically according to a program. Liquid handling systems, such as robotic laboratory liquid handling systems, typically use turbulent or shear flow induced to mix two liquids having different compositions. These mixing methods rely on high flow rates and may require precise control of the fluid flow. At higher flow rates, a more powerful, usually more expensive and bulky physical infrastructure is required. More precise control typically requires tighter tolerances and thus more difficult and costly manufacturing.

Summary of the Invention

[0003] This specification provides a method and apparatus for mixing liquids in an automated liquid handling system. In one embodiment, a method for mixing liquids using an automated liquid handling system includes: alternately drawing liquid volumes of a first liquid and a second liquid from one of a first liquid supply unit and a second liquid supply unit into a mixing volume, thereby forming a liquid stack in the mixing volume that includes a continuum of alternating interface layers of the first liquid and the second liquid; mixing the interface layers of the first liquid and the second liquid with each other by diffusion in the mixing volume to form a mixed liquid; and discharging the mixed liquid from the mixing volume.

[0004] The second liquid may be different from the first liquid.

[0005] The liquid stack may include at least two layers of a first liquid arranged alternately with at least two layers of a second liquid.

[0006] The liquid stack may include at least two layers of a first liquid arranged alternately with at least two layers of a second liquid.

[0007] Each of the layers of the first and second liquids can have a volume ranging from approximately 3 microliters to 25 microliters.

[0008] In some embodiments, the total volume of the first liquid layer in the liquid stack is greater than the total volume of the second liquid layer in the liquid stack.

[0009] In some embodiments, the total volume of the first liquid layer in the liquid stack is substantially equal to the total volume of the second liquid layer in the liquid stack.

[0010] In some embodiments, mixing the layers forming the interface between the first liquid and the second liquid by diffusion in the mixing volume to form a mixed liquid includes mixing the layers forming the interface between the first liquid and the second liquid by diffusion in the mixing volume for at least 4 seconds.

[0011] The first liquid may have a different composition from the second liquid.

[0012] In some embodiments, the first liquid is a reaction mixture containing DNA fragments, and the second liquid contains a ligase enzyme.

[0013] In some embodiments, the first liquid has a different viscosity from the second liquid.

[0014] According to some embodiments, the first liquid has a different chemical composition from the second liquid.

[0015] According to some embodiments, the mixing volume is elongated and has a longitudinal axis, and the layers of the first liquid and the second liquid in the liquid stack are stacked along the longitudinal axis.

[0016] In some embodiments, each of the layers of the first liquid and the second liquid has a layer diameter ratio of at least 0.016 to the layer height.

[0017] According to several embodiments, each of the layers of the first liquid and the second liquid has a layer liquid volume and an interfacial surface area with an adjacent layer among the layers of the first liquid and the second liquid, and the ratio of the interfacial surface area to the layer liquid volume is at least 0.02 1 / mm.

[0018] According to some embodiments, the operation of alternately drawing the liquid volumes of the first liquid and the second liquid into the mixed volume from either the first liquid supply unit or the second liquid supply unit includes drawing the liquid volumes of the first liquid and the second liquid into a tubular probe through the probe inlet.

[0019] The operation of discharging a mixed liquid from a mixing volume may include discharging the mixed liquid from a tubular probe through the probe inlet.

[0020] According to some embodiments, the tubular probe is provided with a probe passage, and at least a portion of the liquid stack is placed within the probe passage during the operation of mixing the layers forming the interface between the first liquid and the second liquid by diffusion in the mixing volume to form a mixed liquid.

[0021] A flexible conduit defining the conduit passage can be fluid-coupled to a tubular probe, and a pump is fluid-coupled to the probe inlet via the conduit passage, and at least a portion of the liquid stack is located within the conduit passage during the operation of mixing the layers forming the interface between the first liquid and the second liquid by diffusion in the mixing volume to form a mixed liquid.

[0022] The operation of alternately drawing the liquid volumes of the first liquid and the second liquid into the mixed volume from either the first liquid supply unit or the second liquid supply unit may include automatically operating a pump coupled to a tubular probe according to a program to draw the first liquid and the second liquid from the first liquid supply unit and the second liquid supply unit.

[0023] The operation of discharging the mixed liquid from the mixing volume may include automatically operating a pump according to a program to discharge the mixed liquid from the mixing volume so that it exits the probe inlet of a tubular probe.

[0024] The pump can be a syringe pump, bellows pump, peristaltic pump, or screw pump, and can operate by generating pressure or displacing the volume of air or liquid.

[0025] According to several embodiments, the first liquid supply unit comprises a first reservoir for containing a first liquid, and the second liquid supply unit comprises a second reservoir for containing a second liquid, and the operation of alternately drawing the liquid volumes of the first liquid and the second liquid from one of the first and second liquid supply units into a mixed volume involves automatically positioning the probe inlet into the first reservoir according to a program, then automatically drawing a first liquid volume of the first liquid into the tubular probe through the probe inlet according to a program, and then automatically positioning the tubular probe inlet into the second reservoir according to a program. The procedure includes: drawing in a second liquid, then automatically aspirating a second liquid volume of the second liquid into the tubular probe through the probe inlet according to a program, then automatically positioning the tubular probe inlet into the first reservoir according to a program, then automatically aspirating a third liquid volume of the first liquid into the tubular probe through the probe inlet according to a program, then automatically positioning the probe inlet into the second reservoir according to a program, and then automatically aspirating a fourth liquid volume of the second liquid into the tubular probe through the probe inlet according to a program.

[0026] The suction operation may include alternately drawing liquid volumes of the first liquid and the second liquid from one of the first and second liquid supply units through a plurality of probe inlets into each of a plurality of mixed volumes, thereby forming a liquid stack in each of the mixed volumes, each liquid stack comprising a continuum of alternating interface layers of the first liquid and the second liquid in each mixed volume; the mixing operation may include mixing the interface layers of the first liquid and the second liquid with each other by diffusion in each of the mixed volumes to form a mixed liquid; and the discharge operation may include discharging each mixed liquid from each of the mixed volumes.

[0027] Alternately sucking the liquid volumes of the first liquid and the second liquid from one of the first liquid supply section and the second liquid supply section can include automatically operating a single pump actuator according to a program and sucking the liquid volumes of the first liquid and the second liquid through respective ones of the probe inlets.

[0028] In some embodiments, the operation of discharging each mixed liquid from the mixing volume includes discharging a first volume of the mixed liquid into the first reservoir and a second volume of the mixed liquid into the second reservoir, and the method includes alternately sucking the liquid volume of the mixed liquid into the mixing volume from one of the first reservoir and the second reservoir, whereby the sucked liquid volume of the mixed liquid forms a second liquid stack including a continuum of layers alternately interfacing the mixed liquid within the mixing volume, mixing the layers interfacing the mixed liquid with each other by diffusion within the mixing volume to form a second mixed liquid, and discharging the second mixed liquid from the mixing volume.

[0029] In a further aspect, an automated liquid handling system used with a first liquid supply section including a first liquid and a second liquid supply section including a second liquid includes a pressure control mechanism, a mixing volume, and a controller. The controller is configured to operate the pressure control mechanism to alternately suck the liquid volumes of the first liquid and the second liquid into the mixing volume from one of the first liquid supply section and the second liquid supply section, whereby the sucked liquid volume forms a liquid stack including a continuum of layers alternately interfacing the first liquid and the second liquid within the mixing volume, mix the layers interfacing the first liquid and the second liquid with each other by diffusion within the mixing volume to form a mixed liquid, and operate the pressure control mechanism to discharge the mixed liquid from the mixing volume.

[0030] The pressure control mechanism can include a pump and a pump actuator.

[0031] The controller can be configured to automatically operate the pump actuator according to a program to draw in the liquid volumes of the first liquid and the second liquid, to mix the layers forming the interface between the first liquid and the second liquid by diffusion, and to operate the pump actuator to discharge the mixed liquid.

[0032] According to some embodiments, the automatic liquid handling system comprises a tubular probe having a probe inlet. The automatic liquid handling system is configured to alternately draw liquid volumes of a first liquid and a second liquid into a mixed volume from either the first liquid supply unit or the second liquid supply unit through the probe inlet.

[0033] The summary of the present invention does not encompass the scope of these aspects and embodiments. Therefore, while certain aspects and embodiments are presented and / or outlined, it should be understood that these aspects and embodiments are not limited to those presented in the summary of the present invention. Indeed, other aspects and embodiments that may be similar to and / or different from those presented in the summary of the present invention will become apparent from this specification, the accompanying drawings, and / or claims.

[0034] Any aspects and embodiments described in the summary of the present invention but not shown in the appended claims should also be understood as being reserved for later presentation in this application or one or more continuing patent applications.

[0035] It should also be understood that any aspects and embodiments not described in the summary of the present invention and not shown in the appended claims are also reserved for later presentation in one or more continuing patent applications.

[0036] The accompanying drawings, which constitute part of this specification, illustrate embodiments of the present technology. [Brief explanation of the drawing]

[0037] [Figure 1]This is a flowchart illustrating a method for mixing liquids using an automated liquid handling system according to an embodiment of this technology. [Figure 2] This flowchart illustrates a further method for mixing liquids using an automated liquid handling system according to an embodiment of this technology. [Figure 3] This is a front view of an exemplary automated liquid handling system comprising a liquid mixing system according to an embodiment of the present technology. [Figure 4] Figure 3 is a partial cross-sectional view of an automated liquid handling system, illustrating the process of mixing two liquids. [Figure 5] Figure 3 is a partial cross-sectional view of an automated liquid handling system, illustrating the process of mixing two liquids. [Figure 6] Figure 3 is a partial cross-sectional view of an automated liquid handling system, illustrating the process of mixing two liquids. [Figure 7] Figure 3 is a partial cross-sectional view of an automated liquid handling system, illustrating the process of mixing two liquids. [Figure 8] Figure 3 is a partial cross-sectional view of an automated liquid handling system, illustrating the process of mixing two liquids. [Figure 9] This is a magnified partial cross-sectional view of the probe of the automated liquid handling system shown in Figure 3, which houses a layered liquid stack. [Figure 10] Figure 3 is a partial cross-sectional view of the automated liquid handling system, illustrating further steps in the process of mixing two liquids. [Figure 11] Figure 3 is a partial cross-sectional view of the automated liquid handling system, illustrating further steps in the process of mixing two liquids. [Figure 12] Figure 3 is a partial cross-sectional view of the automated liquid handling system, illustrating further steps in the process of mixing two liquids. [Figure 13] Figure 3 is a partial cross-sectional view of the automated liquid handling system, illustrating further steps in the process of mixing two liquids. [Figure 14] Figure 3 is a partial cross-sectional view of the automated liquid handling system, illustrating further steps in the process of mixing two liquids. [Figure 15] Figure 3 is a partial cross-sectional view of the automated liquid handling system, illustrating further steps in the process of mixing two liquids. [Figure 16] Figure 3 is a partial cross-sectional view of the automated liquid handling system, illustrating further steps in the process of mixing two liquids. [Figure 17] Figure 3 is a partial cross-sectional view of an automated liquid handling system, illustrating the process of mixing two liquids using multiple probes. [Figure 18] Figure 3 is a partial cross-sectional view of an automated liquid handling system, illustrating the process of mixing two liquids using multiple probes. [Figure 19] Figure 3 is a partial cross-sectional view of an automated liquid handling system, illustrating the process of mixing two liquids using multiple probes. [Figure 20] This is a schematic diagram showing a controller that forms part of the automated liquid handling system shown in Figure 3. [Modes for carrying out the invention]

[0038] Liquid handler systems, such as robotic laboratory liquid handling systems, typically utilize turbulent or shear flow induced to mix two liquids with different compositions. These mixing methods may rely on high flow velocities and require precise control of the fluid flow. The inventors have found that such techniques are unsuitable for some liquid handling technologies. For example, some automated liquid handlers, such as high-throughput, low-cost liquid handlers, are not configured to generate the turbulence necessary to effectively mix several liquids, such as liquids with different viscosities.

[0039] The apparatus and method according to the embodiments of this technology can address the shortcomings of known liquid mixing methods. In particular, the apparatus and method according to the embodiments of this technology can effectively and rapidly mix volumes of liquids of different compositions using diffusion mixing without high speed or turbulence. The apparatus and method may be particularly advantageous for mixing relatively small volumes of liquids (e.g., microfluidic volumes in the range of about 10 to 200 microliters). This range of liquid volumes may be referred to as the mesofluidic volume range.

[0040] Figure 1 is a flowchart illustrating a method for mixing a first liquid (from a first liquid supply) and a second liquid (from a second liquid supply) according to some embodiments of the present technology. The liquid volumes of the first liquid and the second liquid are alternately drawn into the mixing volume from either the first or second liquid supply, thereby forming a liquid stack in the mixing volume that includes a continuum of alternating interface layers of the first and second liquids (block 52). The interface layers of the first and second liquids are then mixed with each other by diffusion in the mixing volume to form a mixed liquid (block 54). The mixed liquid is then discharged from the mixing volume (block 56). In some embodiments, the second liquid is different from the first liquid.

[0041] Referring to Figure 2, the method may further include further mixing of the mixed liquid, which is the product of the method of Figure 1. Discharging the mixed liquid from the mixing volume (block 56, Figure 1) may include discharging a first volume of the mixed liquid into a first reservoir and a second volume of the mixed liquid into a second reservoir (block 60, Figure 2). The liquid volumes of the mixed liquid are then alternately drawn into the mixing volume from either the first or second reservoir, thereby forming a second liquid stack in the mixing volume, which includes a continuum of alternating interface layers of the mixed liquid (block 62). The interface layers of the mixed liquid are then mixed with each other by diffusion in the mixing volume to form a second mixed liquid (block 64). The second mixed liquid is then discharged from the mixing volume (block 66).

[0042] By layering the first and second liquids multiple times, the total surface area between the first and second liquids increases, thereby increasing the diffusion rate between them. This improved diffusion rate allows for a sufficiently high mixing rate without the need for turbulence.

[0043] In some embodiments, an automated liquid handler draws or aspirates alternating layers of a first liquid and a second liquid from their respective liquid supply units through a tubular probe (e.g., a pipette) into a mixing volume, and discharges the mixed liquid from the tubular probe. The tubular probe can be conveniently and effectively integrated and operated within the liquid handling system.

[0044] Referring to Figure 3, an illustrative liquid mixing system 101 is shown here according to a particular embodiment of the present technology. The illustrated liquid mixing system 101 constitutes part of an automated liquid handling system 110 according to an illustrated embodiment of the present technology. However, it should be understood that the disclosed methods, systems, and apparatus may form part of or be used in conjunction with other designs of liquid handling systems. The liquid mixing system 101 is configured to mix two or more liquids within the liquid handling system 110. The liquid mixing system 101 and the liquid handling system 110 can be used and configured to perform or implement the methods described above with reference to Figures 1 and 2.

[0045] In some embodiments, the liquid handling system 110 includes a first liquid supply unit S1 and a second liquid supply unit S2. As will be described later, the first liquid supply unit S1 contains a volume VSC1 of the first liquid C1, and the second liquid supply unit S2 contains a volume VSC2 of the second liquid C2 (Figure 4). The liquid mixing system 101 operates to mix certain amounts of the first liquid C1 and the second liquid C2 with each other to form a mixed liquid M of component liquids C1 and C2 (Figure 11). In some embodiments, the second liquid C2 is different from the first liquid C1 (for example, component liquids C1 and C2 have different chemical compositions and / or viscosities).

[0046] Referring to Figure 3, the illustrated automated liquid handling system 110 comprises a platform or deck 112, a frame 114, an analyzer 116, a controller 120, and a liquid handler 130. In some embodiments, the automated liquid handling system 110 is a robotic automated liquid handling system.

[0047] For the purpose of this discussion, and as shown in the diagram, the workspace defines the Z-axis, which corresponds to the vertical, and the X-axis and Y-axis, which define both the vertical and horizontal planes.

[0048] In the illustrated embodiment, and also referring to Figure 4, the first liquid supply unit S1 comprises a container 170 having a receptacle or reservoir 171 that contains a volume VSC1 of the first liquid C1. The second liquid supply unit S2 comprises a container 172 having a receptacle or reservoir 173 that contains a volume VSC2 of the second liquid C2. It will be understood that the liquid supply units S1 and S2 can take any preferred form. For example, the containers 170 and 172 may be separate containers or a single container (e.g., a multiwell plate) containing both reservoirs 171 and 173. As a further example, the containers 170 and 172 may be vessels with open lids or vials with closures (e.g., septums) that allow access to the contents. One or both of the liquid supply units S1 and S2 may comprise two or more reservoirs containing the respective liquids C1 and C2.

[0049] In some embodiments, the liquid handling system 110 further comprises one or more mixture storage containers 174 (Figures 4 and 12), 176 (Figure 12), each having a receptacle or reservoir 175, 177 (Figure 12). As will be described later, the mixed liquid M can be discharged into the reservoir(s) 175, 177.

[0050] In some embodiments, the liquid handling system 110 further comprises one or more remixed storage containers 178, each having a receptacle or reservoir 179 (Figure 16). As will be described later, the remixed liquid can be discharged into the reservoir(s) 179.

[0051] The liquid handler 130 (Figure 3) comprises a pressure control mechanism 131, a probe module 140, and a conduit or tube 160. The liquid handler 130 can be controlled by a controller 120.

[0052] The probe module 140 (Figure 3) comprises a probe module base 142 and a tubular probe 150 attached thereto. The liquid handling system 110 comprises a probe positioning system 144. In an exemplary embodiment, the probe positioning system 144 comprises a module positioning actuator 146 and a probe positioning actuator 148. The module positioning actuator 146 is operable by the controller 120 to move the probe module 140 along the X and Y axes relative to the deck 112 and the liquid supply units S1 and S2. The probe positioning actuator 148 is operable by the controller 120 to move the probe 150 up and down along the Z axis relative to the deck 112 and the liquid supply units S1 and S2. However, those skilled in the art will understand that any suitable positioning system can be used to move the probe 150 relative to the liquid supply units S1 and S2. The probe module positioning actuator 146 and the probe positioning actuator 148 may include, for example, electric motors.

[0053] The pressure control mechanism 131 (Figure 3) is fluidly coupled to the probe 150 by a tube 160. The pressure control mechanism 131 comprises a pump 132 and a pump actuator 134. The pump actuator 134 is operable by the controller 120 to drive the pump 132.

[0054] Pump 132 can be any preferred type of pump. In some embodiments, pump 132 is a syringe pump.

[0055] The pump actuator 134 can be any preferred type of force actuator. In some embodiments, the pump actuator 134 is an electric actuator. In some embodiments, the pump actuator 134 is an electric linear actuator.

[0056] Referring to Figure 4, an exemplary probe 150 has a distal end 150A and a proximal end 150B. The probe 150 comprises an elongated body 152 having a tip 153 at the distal end 150A. The lumen or passage 154 of the probe extends axially through the probe 150 along the probe's longitudinal axis PA from the probe inlet 156 (located at the tip 153) to the probe's proximal port 158 ​​(located at the proximal end 150B). In some embodiments, the probe 150 is a pipette or cannula. For example, the probe 150 can be a one-piece pipette as shown.

[0057] Referring to Figure 4, the tube 160 has a distal end 160A and a proximal end 160B on the opposite side (Figure 3). The lumen or passage 164 of the tube extends axially through the tube 160 along the longitudinal axis TA (which may be curved) from the tube inlet 166 (located at the distal end 160A) to the pump 132 (located at the proximal end 160B). The tube inlet 166 is connected to the probe proximal port 158, thereby the probe passage 154 and the tube passage 164 together form a continuous combined passage 167. The tube 160 can be a flexible tube.

[0058] The mixing system 101 comprises a controller 120, a pressure control mechanism 131, a probe 150, and a mixing volume 151 (Figure 4). In some embodiments, the mixing volume 151 is defined by a probe passage 154 and / or a tube passage 164.

[0059] The following describes exemplary operation of system 110 and liquid mixing system 101 according to the method of this technology, with reference to Figures 4 to 11. It should be understood that the following procedure is exemplary and can be modified as desired by the operator. All or part of these operations can be performed by controller 120.

[0060] The liquid supply units S1 and S2 and the mixture storage containers (there may be more than one) 174 and 176 are, for example, installed on the deck. The liquid supply units S1 and S2 can be positioned manually or automatically according to a program by the controller 120.

[0061] The probe module positioning system 144 moves the probe module 140 so that it is vertically aligned with the reservoir 171 of the first liquid supply unit S1. The probe module positioning system 144 then lowers the tip 153 of the probe 150 into the volume VSC1 of the first liquid C1 in the reservoir 171, as shown in Figure 4. The pump actuator 134 then drives the pump 132 to create a vacuum or negative pressure in the probe passage 154, and operates to draw a desired or specified volume VC1 of the first liquid C1 from volume VSC1 into the probe passage 154 through the probe inlet 156. The drawn volume VC1 forms the first layer L1 in the mixed volume 151, as shown in Figure 4.

[0062] Next, the probe module positioning system 144 raises the tip 153 from the reservoir 171, moving the probe module 140 so that it is vertically aligned with the reservoir 173 of the second liquid supply unit S2. Then, as shown in Figure 5, the probe module positioning system 144 lowers the tip 153 of the probe 150 into the volume VSC2 of the second liquid C2 in the reservoir 173. Next, the pump actuator 134 drives the pump 132 to create a vacuum or negative pressure in the probe passage 154, and operates to draw a desired or specified volume VC2 of the second liquid C2 from volume VSC2 into the probe passage 154 through the probe inlet 156. The drawn volume VC2 forms a second layer L2 in the mixed volume 151, as shown in Figure 5.

[0063] In some embodiments, the probe module positioning system 144 then raises the tip 153 from the reservoir 173, moving the probe module 140 so that it is vertically aligned with the reservoir 171 of the first liquid supply unit S1. The probe module positioning system 144 then lowers the tip 153 of the probe 150 into the volume VSC1 of the first liquid C1 in the reservoir 171, as shown in Figure 5. The pump actuator 134 then drives the pump 132 to create a vacuum or negative pressure in the probe passage 154, and operates to draw a desired or specified volume VC1 of the first liquid C1 from the volume VSC1 into the probe passage 154 through the probe inlet 156. The drawn volume VC1 forms a third layer L3 in the mixed volume 151, as shown in Figure 6.

[0064] In some embodiments, the probe module positioning system 144 then raises the tip 153 from the reservoir 171, moving the probe module 140 so that it is vertically aligned or aligned with the reservoir 173 of the second liquid supply unit S2. The probe module positioning system 144 then lowers the tip 153 of the probe 150 into the volume VSC3 of the second liquid C2 in the reservoir 173, as shown in Figure 7. The pump actuator 134 then drives the pump 132 to create a vacuum or negative pressure in the probe passage 154, and operates to draw a desired or specified volume VC2 of the second liquid C2 from the volume VSC2 into the probe passage 154 through the probe inlet 156. The drawn volume VC2 forms a fourth layer L4 in the mixed volume 151, as shown in Figure 7.

[0065] The above can be repeated to gradually form further alternating layers of volume VC1 of the first liquid C1 from liquid supply unit S1 and volume VC2 of the second liquid C2 from liquid supply unit S2, as desired. That is, using the probe module positioning system 144, the probe tip 153 is repeatedly and alternately positioned in liquid supply units S1 and S2, and volumes VC1 and VC2 are drawn in during each position, adding further layers of liquid C1 and C2 below the previously drawn layers. These operations can be continued until a desired number of layers are formed or a desired total volume is drawn into the mixing volume 151.

[0066] Referring to Figure 8, the aspirated liquid volumes VC1 and VC2 form a liquid stack LS in the mixed volume 151. The liquid stack LS includes a continuum SA of alternating layers L1 to L16, which are interfaces of the first liquid C1 and the second liquid C2. The mixed volume 151 is elongated, and layers L1 to L16 overlap along the longitudinal axis XA of the mixed volume 151 (which may be curved) (i.e., layers L1 to L16 overlap along the longitudinal axis PA of the probe and the longitudinal axis TA of the tube). Layers L1 to L16 may also be referred to as layers L in this specification.

[0067] As shown in Figure 9, each layer L1 to L16 is arranged directly adjacent to the others and contacts the upper and lower layers on the same plane at the layer interface I. The liquid volumes VC1 and VC2 of each layer L1 to L16 have interface surfaces E1 and E2 at both ends (except for the outermost layer which has only one interface surface). The upper interface surface E1 of each layer contacts the lower interface surface E2 of the preceding layer at interface I. That is, there are no intervening layers between adjacent layers L1 to L16 containing liquids C1 and C2. However, the boundary between the aspirated liquid volumes VC1 and VC2 may not be separated. This is because drawing in the liquid volumes VC1 and VC2 through passages 154 and 164 causes some stirring of the liquid volumes VC1 and VC2, which may mix with each other at the interface.

[0068] The liquid volumes VC1 and VC2 of each layer L1 to L16 can diffuse into the adjacent liquid volumes VC1 and VC2 of each layer L1 to L16 at the relevant interface I while layers L1 to L16 are present in the mixing volume 151. This diffusion physically mixes the adjacent liquid volumes VC1 and VC2 with each other, thereby forming a mixed liquid M in the mixing volume 151, as shown in Figure 10.

[0069] The probe module positioning system 144 moves the probe module 140 so that it is vertically aligned with or aligned with the reservoir 175 of the container 174, as shown in Figure 10. The probe module positioning system 144 can also lower the tip 153 of the probe 150 to or adjacent to the reservoir 175. The pump actuator 134 then drives the pump 132 to generate positive pressure in the probe passage 154 and operates to discharge a desired or specified volume of the mixed liquid M from the mixing volume 151 into the reservoir 175, as shown in Figure 11. The mixed liquid M is then used or further processed as desired.

[0070] It will be understood that container 174 is just one example of a container for storing the discharged liquid mixture. For example, the liquid mixture M can be discharged into an injector (e.g., in a gas chromatograph) or into a reservoir containing another liquid.

[0071] The liquid stack LS can be held in the mixing volume 151 for a period (holding time) between the completion of the formation of the liquid stack LS and the discharge of the mixed liquid M from the probe 150, thereby allowing the liquid volumes VC1 and VC2 of layers L1 to L16 to diffuse as described. The holding time can be a predetermined or specified minimum period. In some embodiments, all layers of the liquid stack LS are diffused together with adjacent interface layers for at least 4 seconds. In some embodiments, all layers of the liquid stack LS are diffused in the mixing volume 151 with adjacent interface layers for a residence or diffusion mixing time ranging from about 4 seconds to 100 seconds.

[0072] Figure 8 shows a liquid stack LS containing 16 layers L, but those skilled in the art will understand that a liquid stack according to an embodiment may contain more or fewer layers. In some embodiments, the liquid stack includes at least three layers of a first liquid C1 and at least two layers of a second liquid C2.

[0073] In some embodiments, each layer L has a liquid volume ranging from about 1 microliter to 100 microliters. In some embodiments, each layer L has a microfluidic liquid volume ranging from about 3 microliters to 25 microliters.

[0074] The layer of the first liquid C1 may have a different volume from the layer of the second liquid C2 (i.e., the liquid handler 130 can draw up or aspirate liquids C1 and C2 of different volumes).

[0075] In some embodiments, the total volume of the first liquid in the liquid stack LS is substantially equal to the total volume of the second liquid in the liquid stack LS.

[0076] In some embodiments, the total volume of the first liquid in the liquid stack LS is different from the total volume of the second liquid in the liquid stack LS.

[0077] In some embodiments, each layer L has a ratio of layer diameter D1 (Figure 9) to layer height H1 (Figure 9) of at least 0.016.

[0078] In some embodiments, the ratio of the area of ​​each interface surface E1, E2 (Figure 9) of each layer to the liquid volume of the layer is at least 0.02 1 / mm.

[0079] In some embodiments, the amounts of liquids C1 and C2 in the liquid supply units S1 and S2 are selected to yield a desired volume ratio of liquids C1 and C2 in the mixed liquid M. Furthermore, the layered volumes VC1 and VC2 are alternately drawn or drawn as described until each supply volume VSC1 and VSC2 is completely drawn into the mixed volume 151.

[0080] In some embodiments, and also with reference to Figures 12-16, the mixed liquid M can be remixed after being discharged from the probe 150 to achieve a more complete and uniform physical mixing of liquids C1 and C2. In this case, a portion of the mixed liquid M (volume VSM1) is discharged from the mixing volume 151 into the reservoir 175 using the pump 132 described above, and another portion of the mixed liquid M (volume VSM2) is discharged from the mixing volume 151 into a second reservoir 177 (for example, in container 176 or another container) using the pump 132.

[0081] The probe module positioning system 144 moves the probe module 140 so that it is vertically aligned with the reservoir 175 and lowers the tip 153 of the probe 150 into volume VSM1, as shown in Figure 12. The pump actuator 134 then drives the pump 132 to create a vacuum or negative pressure in the probe passage 154 and sucks a desired or specified volume VM1 from volume VSM1 into the probe passage 154 through the probe inlet 156. The sucked volume VM1 forms a first layer LM1 in the mixed volume 151, as shown in Figure 12.

[0082] Next, the probe module positioning system 144 raises the tip 153 from the reservoir 175, moves the probe module 140 so that it is vertically aligned with the reservoir 177, and lowers the tip 153 of the probe 150 into volume VSM2, as shown in Figure 13. Then, the pump actuator 134 drives the pump 132 to create a vacuum or negative pressure in the probe passage 154, and operates to draw a desired or specified volume VM2 from volume VSM2 into the probe passage 154 through the probe inlet 156. The drawn volume VM2 forms a second layer LM2 in the mixed volume 151, as shown in Figure 13.

[0083] Similar to the description of the formation of the liquid stack LS, the above can be repeated to gradually form further alternating layers of the mixed liquid M volumes VM1 and VM2 from reservoirs 175 and 177, as desired. That is, using the probe module positioning system 144, the probe tip 153 is repeatedly and alternately placed into the mixed liquid volumes VSM1 and VSM2 in reservoirs 175 and 177, with each volume VM1 and VM2 being aspirated during each placement, thereby stacking further layers of the mixed liquid beneath the previously aspirated layers. These operations can be continued until a desired number of layers are formed or a desired total volume is aspirated into the mixed volume 151.

[0084] Referring to Figure 14, the aspirated liquid volumes VM1 and VM2 form a liquid stack LSM in the mixing volume 151. The liquid stack LSM includes a continuous SAM of alternating layers LM1 to LM16 (collectively referred to as layers LM in this specification) of the first liquid C1 and the second liquid C2. Each layer LM1 to LM16 is directly adjacent and contacts the layer above and below it in the same plane at the layer interface I. The liquid volumes VM1 and VM2 of each layer LM1 to LM16 have interface surfaces at both ends (except for the outermost layer which has only one interface surface). The upper interface surface of each layer contacts the lower interface surface of the preceding layer at interface I.

[0085] The liquid volumes VM1 and VM2 of each layer LM1 to LM16 can diffuse into the adjacent liquid volumes VM1 and VM2 of each layer LM1 to LM16 at the relevant interface I while layers LM1 to LM16 are present in the mixing volume 151. This diffusion physically mixes the adjacent liquid volumes VM1 and VM2 with each other, thereby forming a mixed liquid RM in the mixing volume, as shown in Figure 15.

[0086] The probe module positioning system 144 moves the probe module 140 so that it is vertically aligned with or aligned with the housing reservoir 179, as shown in Figure 15. The probe module positioning system 144 can also lower the tip 153 of the probe 150 to or adjacent to the reservoir 179. The pump actuator 134 then drives the pump 132 to generate positive pressure in the probe passage 154 and operates to discharge a desired or specified volume of the remixed liquid RM from the mixing volume 151 into the reservoir 179, as shown in Figure 16. The remixed liquid RM can then be used or further processed as desired.

[0087] It will be understood that container 178 is just one example of a container for storing the discharged remixed liquid RM. For example, the remixed liquid RM may be discharged into an injector (e.g., in a gas chromatograph) or into a reservoir containing another liquid.

[0088] In some embodiments, the liquid stack LSM is held in the mixing volume 151 for a period (holding time) between the completion of the formation of the liquid stack LSM and the discharge of the remixed liquid RM from the probe 150, thereby allowing the liquid volumes VM1, VM2 of layers LM1 to LM16 to diffuse as described. The holding time can be a predetermined or specified minimum period. In some embodiments, all layers of the liquid stack LSM are diffused together with adjacent interface layers for at least 4 seconds. In some embodiments, all layers of the liquid stack LSM are diffused together with adjacent interface layers for a diffusion time ranging from about 4 seconds to 100 seconds.

[0089] Figure 14 shows a liquid stack LSM containing 16 layers LM1 to LM16, but a liquid stack according to an embodiment may contain more or fewer layers. In some embodiments, the liquid stack LSM includes at least three layers of liquid C1 and at least two layers of liquid C2.

[0090] In some embodiments, each layer LM of the liquid stack LSM has a liquid volume in the range of approximately 3 microliters to 25 microliters.

[0091] In some embodiments, each layer LM of the liquid stack LSM has a layer diameter ratio of at least 0.016 to layer height.

[0092] In some embodiments, the ratio of the area of ​​each interface surface E3, E4 of each layer LM in the liquid stack LSM to the liquid volume of the layer LM is at least 0.02 1 / mm.

[0093] The operations described herein can be performed by or through the controller 120. Actuators 134, 146, 148 and other devices of the automatic liquid handling system 110 can be electronically controlled. According to some embodiments, the controller 120 performs some, and in some embodiments all, of the described suction, dispensing, and probe positioning according to a program. The operations of actuators 134, 146, 148 can be performed entirely automatically and according to a program by the controller 120. The controller 120 may be equipped with an HMI 122 for receiving user commands.

[0094] In some embodiments, the suction and probe movement operations described above, which form the liquid stack LS, are automatically performed by the controller 120 according to a program. In some embodiments, the operation described above for discharging the mixed liquid M is automatically performed by the controller 120 according to a program.

[0095] Each of the above-described suction and probe movement operations for forming the remixed liquid stack LSM can be automatically performed by the controller 120 according to a program. In some embodiments, the above-described operation for discharging the remixed liquid RM is also automatically performed by the controller 120 according to a program.

[0096] As shown in Figures 4 and 8, the mixing volume 151 can include both a portion of the probe passage 154 and a portion of the tube passage 164. That is, the probe passage 154 and the tube passage 164 are used together as the mixing volume 151. In other embodiments, the mixing volume 151 can be defined entirely within the probe passage 154 or entirely within the tube passage 164.

[0097] In some embodiments, the probe passage 154 has an inner diameter in the range of about 0.1 mm to 1 mm.

[0098] In some embodiments, the tube passage 164 has an inner diameter in the range of about 0.5 mm to 1.5 mm.

[0099] In some embodiments, the total volume of the mixed volume 151 is in the range of approximately 10 microliters to 100 microliters.

[0100] In some embodiments, the length of the mixing volume 151 is in the range of approximately 20 mm to 200 mm.

[0101] In some embodiments, the ratio of the length of the mixing volume 151 to the inner diameter of the mixing volume 151 is at least 25.

[0102] Referring to Figures 17 to 19, further embodiments of a liquid handling system 310 and a mixing system 301 are shown. The liquid handling system 310 and the mixing system 301 can be constructed and operated in the same manner as those described for the liquid handling system 110 and the mixing system 101, except as described below. In the description of the liquid handling system 310, similar components and features are referred to using the same reference numerals as those used in the description of the liquid handling system 110.

[0103] The liquid handling system 310 may include a first liquid supply unit S1' and a second liquid supply unit S2' (Figure 17). The first liquid supply unit S1' corresponds to the first liquid supply unit S1, except that it includes a container 370 having a plurality of reservoirs 371 each containing a volume VSC1 of the first liquid C1. The second liquid supply unit S2' corresponds to the second liquid supply unit S2, except that it includes a container 372 having a plurality of reservoirs 373 each containing a volume VSC2 of the second liquid C2.

[0104] The liquid handling system 310 includes a probe module 340 instead of the probe module 140. The probe module 340 includes a number of (but not limited to four, as shown) tubular probes 150 that are mounted on a module base 342 and move together. A probe positioning system (not shown) corresponding to the probe positioning system 144 is operable to move the probe module 340 and the probes 150 relative to the deck 112 and reservoirs 371, 373.

[0105] The liquid handling system 310 also includes a pressure control mechanism 331 comprising a plurality of pumps 132 and pump actuators 334. Each of the pumps 132 is fluidly coupled to each of the probes 150 by its respective tube 160.

[0106] In some embodiments, the pump actuator 334 is connected to the pump 132 as a common pump actuator that drives the pumps 332 together when in operation. That is, when the pump actuator 334 is operated in one direction or mode, it drives all of the pumps 132, generating negative or suction pressure in the passage 154 of the probe 150, and when the pump actuator 334 is operated in a second direction or mode, it drives all of the pumps 132, generating positive or discharge pressure in the passage 154 of the probe 150. The pump actuator 334 can drive the pumps 132 substantially simultaneously or in sync.

[0107] In another embodiment, each pump 132 is provided with its own pump actuator that can operate to independently control the associated pump 132.

[0108] The liquid handling system 310 can be used substantially in the same manner as the liquid handling system 110 according to the methods of the present invention, except that the liquid volumes VC1 and VC2 are drawn into a mixed volume 151 of a plurality of probes 150 as a group (and substantially simultaneously in some embodiments), diffused within the mixed volume 151 of the plurality of probes 150, and discharged from the mixed volume 151 of the plurality of probes 150.

[0109] For example, as shown in Figure 17, the probe module positioning system 344 moves the probe module 340 to position the probe 150 so that it is vertically aligned with each of the reservoirs 371, and lowers the tip 153 of the probe 150 into each volume VSC1. The pump actuator 334 then drives the pump 132 to create a vacuum or negative pressure in each probe passage 154, and operates to draw a desired or specified volume VC1 of the volume VSC1 into the probe passage 154 of the associated probe 150 through the probe inlet 156. The drawn-in volume VM1 forms each first layer L1 in the mixed volume 151 of the probe 150, as shown in Figure 17.

[0110] Next, the probe module positioning system 344 moves the probe module 340 to position the probe 150 so that it is vertically aligned with each of the reservoirs 373, and lowers the tip 153 of the probe 150 into each volume VSC2, as shown in Figure 18. Then, the pump actuator 334 drives the pump 132 to create a vacuum or negative pressure in each probe passage 154, and operates to draw a desired or specified volume VC2 from the volume VSC2 through the probe inlet 156 into the probe passage 154 of the associated probe 150. The drawn volume VC2 forms each second layer L2 in the mixed volume 151 of the probe 150, as shown in Figure 18.

[0111] Similar to the description of the formation of liquid stacks LS with reference to Figures 4 to 11, the above can be repeated to gradually form each liquid stack LS as desired within the mixing volume associated with each probe 150 of the probe module 340. Each layer of liquid stack LS can be diffused to form its respective mixed liquid within each mixing volume 151. Each mixed liquid can be discharged from the probe 150 as described above. Furthermore, the discharged mixed liquid can be remixed in several probes 150 as described above with reference to Figures 12 to 16.

[0112] In some embodiments, the liquid handler 330 draws each layer L into the four probes 150 substantially simultaneously. In some embodiments, the liquid handler 330 discharges the mixed liquid M (or RM) of each layer from the four probes 150 substantially simultaneously.

[0113] In the mixing system 301 and method described, it will be understood that the mixing procedure in probe 150 is performed as a parallel process. Thus, the mixing system 301 can mix a larger volume of liquid within a given time.

[0114] Referring to Figures 17 to 19, each of the above-described suction, discharge, and probe movement operations can be automatically executed according to the program by the controller 120 as described above, referring to Figures 4 to 16.

[0115] Figures 17 to 19 show a probe module equipped with four probes 150 and a liquid supply unit equipped with four reservoirs, respectively. However, using more or fewer probes and reservoirs, aspiration and mixing can be performed in parallel or simultaneously, as described. Furthermore, two or more of the probes 150 can draw from the same reservoir.

[0116] The first liquid C1 may have a different composition from the second liquid C2.

[0117] In some embodiments, the first liquid C1 has a different chemical composition from the second liquid C2.

[0118] In some embodiments, the first liquid C1 has a different viscosity from the second liquid C2. In some embodiments, one of the liquids C1 and C2 has a viscosity at least 15% higher than the viscosity of the other liquid C1 or C2.

[0119] One or both of the first liquid C1 and the second liquid C2 may be a mixture.

[0120] In some embodiments, the first liquid C1 is a first liquid bioreactor, and the second liquid C2 is a second liquid bioreactor.

[0121] In some embodiments, liquids C1 and C2 are mixed as described herein as part of a process for preparing a DNA sequencing library. In some embodiments, liquids C1 and C2 are mixed as described herein as part of a process for preparing a sample for next-generation sequencing (NGS). In some embodiments, the first liquid C1 is a reaction mixture containing DNA fragments, and the second liquid C2 contains a ligase enzyme and a reaction buffer.

[0122] In some embodiments, liquids C1 and C2 are mixed as described herein as part of a biological reaction. In some embodiments, liquids C1 and C2 are mixed as described herein as part of a SPRI bead washing process. In some embodiments, liquids C1 and C2 are mixed as described herein as part of a DNA oligohybridization assay process.

[0123] In some embodiments, the automated liquid handling system 110 is a high-throughput liquid handling system used to mix two liquids having different viscosities. For example, in some embodiments, the automated liquid handling system 110 is a high-throughput liquid handling system in which a first liquid C1 is a first reactant in the process of preparing a DNA sequencing library, and a second liquid C2 is a second reactant in the process of preparing a DNA sequencing library. The first reactant may contain DNA fragments, and the second reactant may contain a ligation buffer, the second reactant having a lower viscosity than the first reactant. In some embodiments, the first reactant is an end repair and adenylation (ERA) reaction buffer, and the second reactant is a ligation reaction buffer. For example, in a sequencing library preparation protocol (e.g., mixing 36 microliters of ERA and 36 microliters of ligation reaction buffer), the liquid handling system 110 first aspirates 6 microliters of ERA, then 6 microliters of ligation reaction buffer, then 6 microliters of ERA, thereby forming a liquid stack LS of alternating 6 microliters of ERA and ligation reaction buffer.

[0124] Depending on the properties of the first and second liquids or other factors, various parameters can be modified or selected. Examples of these parameters include the height of layer L, the retention time of the liquid stack LS in the mixing volume, the minimum diffusion time between layers L, the number of layers L in the liquid stack LS, the total volume of the liquid stack LS, and the dimensions of the mixing volume 151 (e.g., the inner diameter of the probe passage 154 and the tube passage 164).

[0125] The mixing procedure according to some embodiments may include further steps. For example, the tip 153 may be immersed in a cleaning or rinsing solution between suction steps to prevent or reduce cross-contamination of the liquid supply units S1 and S2.

[0126] In some embodiments, the mixed liquid M (or remixed liquid RM) can be transferred through the tube 160 to a reservoir or device (such as an analyzer 116), and the mixed liquid M (or remixed liquid RM) can be discharged from the mixing volume 151.

[0127] The containers and reservoirs exemplified and described herein are merely examples, and any suitable reservoir can be used. For example, a reservoir for holding and housing liquid volumes VSC1, VSC2, VSM1, VSM2, M, and RM may be a well plate or microwell plate having an integral recess or receptacle for directly containing the liquid sample, or a vial or other individual vessel (which can be mounted on a tray, rack, carrier, or platter), or may include such vessels.

[0128] Although the probe 150 is shown in the figure as a one-piece pipette or cannula, tubular probes of other types and configurations can be used. For example, each probe 150 may include a pipette and a pipette tip that is detachably attached to the pipette. In this case, the tip 153 may form part of the pipette tip.

[0129] The system and holder according to the embodiment of this technology can be used, for example, in biochemical and chemical processing, liquid handling, and laboratory sample analysis. The analytical instrument 116 can be any suitable device or instrument.

[0130] Embodiments of the controller 120 logic may take the form of an entirely software-based embodiment, or an embodiment combining software and hardware aspects, all of which are generally referred to as “circuits” or “modules.” In some embodiments, a circuit includes both software and hardware, and the software is configured to operate with specific hardware having known physical attributes and / or configuration. Furthermore, the controller logic may take the form of a computer program product on a computer-readable storage medium having computer-readable program code embodied in the medium. Any suitable computer-readable medium can be used, including hard disks, CD-ROMs, optical storage devices, transmission media, such as transmission media supporting the Internet or an intranet, or other storage devices.

[0131] Figure 20 is a schematic diagram of a circuit or data processing system 402 that can be used in the controller 120. The circuit and / or data processing system can be incorporated into a digital signal processor 410 in any suitable device or multiple devices. The processor 410 communicates with the HMI 122 and memory 412 via an address / data bus 411. The processor 410 can be any commercially available or custom-designed microprocessor. Memory 412 represents the entire system of memory devices, including software and data used to perform the functions of the data processing system. Memory 412 may include, but is not limited to, devices of the type such as cache, ROM, PROM, EPROM, EEPROM, flash memory, SRAM, and DRAM.

[0132] Figure 20 shows that memory 412 can contain several categories of software and data used in a data processing system, such as an operating system 414, application programs 416, input / output (I / O) device drivers 418, and data 420.

[0133] Data 420 may include instrument-specific data. Figure 20 also shows that data 420 may include liquid supply data 422, laboratory equipment data 424, probe data 426, and procedure data 428.

[0134] The liquid supply unit data 422 may include data relating to or representing the characteristics of the liquid supply units S1 and S2.

[0135] The liquid supply unit data 422 may include, for example, a unique identifier (e.g., serial number) and / or name for each container 170, 172, 174, 176, 178, 370, 372, a unique identifier and / or name for each reservoir 171, 173, 175, 371, 373, and / or a description of the liquids C1, C2 contained in the reservoirs. The liquid supply unit data 422 may include the dimensions of the containers and / or reservoirs. The liquid supply unit data 422 may include positional data representing the spatial or geometric layout or location of the reservoirs.

[0136] The experimental equipment data 424 may include data relating to or representing the characteristics of the container intended to receive the mixed liquid M or the remixed liquid RM.

[0137] The probe data 426 may include probe position data representing the spatial or geometric layout or position of the probe 150 relative to the liquid supply units S1, S2, deck 112, and / or other parts of the system 110.

[0138] The probe data 426 may include, for example, a unique identifier (e.g., serial number) and / or name for each probe 150. The probe data 426 may include the dimensions of the probe 150 and / or probe modules 140, 340. The probe data 426 may include position data representing the spatial or geometric layout or location of the probe 150 and probe modules 140, 340.

[0139] The procedure data 428 may include data representing a protocol or sequence of steps for performing the procedures described herein. The sequence of steps may include all or some of the above steps performed by the controller 120. The sequence of steps may include, for example, an analysis sequence.

[0140] Figure 20 also shows that the application program 416 may include a liquid handler control module 436 for controlling the liquid handler 130 and an analyzer control module 438. The liquid handler control module 436 may include a module for controlling the probe positioning system 144 (e.g., including actuators 146 and 148) and a module for controlling the pressure control mechanism (e.g., including a pump actuator 134). The analyzer control module 438 may be configured to control the operation of the analyzer 116.

[0141] As will be understood by those skilled in the art, the operating system 414 can be any operating system suitable for use with the data processing system. The I / O device driver 418 typically includes software routines accessed through the operating system 414 by the application program 416 to communicate with devices such as I / O data ports (which may be more), data storage, and certain memory components. The application program 416 illustrates a program that performs various functions of the data processing system and may include at least one application that supports the operation according to the embodiment of this art. Finally, the data 420 represents static and dynamic data used by the application program 416, the operating system 414, the I / O device driver 418, and other software programs that may reside in memory 412.

[0142] As will be understood by those skilled in the art, other configurations can be utilized while benefiting from the teachings of this technology. For example, one or more modules can be incorporated into an operating system, an I / O device driver, or other such logical division of a data processing system. Therefore, this technology should not be construed as being limited to the configuration of Figure 20, but is intended to encompass any configuration capable of performing the operations described herein. Furthermore, one or more modules can communicate with other components, such as the controller 120, or can be incorporated into the controller 120, either whole or in part.

[0143] The Art has been described herein with reference to the accompanying drawings illustrating exemplary embodiments of the Art. In the drawings, the relative sizes of areas or feature portions may be exaggerated for clarity. However, the Art can be embodied in many different forms and should not be construed as being limited to the embodiments described herein. Rather, these embodiments are provided to make this disclosure detailed and complete and to fully convey the scope of the Art to those skilled in the art.

[0144] In this specification, terms such as “first,” “second,” etc., may be used to describe various elements, components, areas, layers, and / or sections, but it will be understood that these elements, components, areas, layers, and / or sections should not be limited by these terms. These terms are used solely to distinguish one element, component, area, layer, or section from another area, layer, or section. Accordingly, the term “first element, component, area, layer, or section” discussed below may, without deviation from the teachings of this Art, be used to refer to the “second element, component, area, layer, or section.”

[0145] Spatially relative terms such as "beneath," "below," "lower," "above," and "upper" can be used herein for ease of explanation to describe the relationship between one element or feature and another element or feature(s) shown in the figure. It will be understood that spatially relative terms are intended to include different orientations of the device in use or operation, in addition to the orientation shown in the figure. For example, if the device in the figure is inverted, the element described as "below" or "beneath" of another element or feature will be oriented "above" of the other element or feature. Thus, the exemplary term "below" may include both "above" and "below" orientations. The device may be oriented in other ways (by rotating 90 degrees or to other orientations), and the spatially relative descriptors used herein will be interpreted accordingly.

[0146] Where used herein, unless otherwise explicitly stated, the singular forms “a,” “an,” and “the” are intended to include the plural forms as well. Where used herein, the terms “includes, comprises” and / or “including, comprising” identify the presence of the described feature, complete, step, action, element, and / or component, but do not exclude the presence or addition of one or more other features, complete, step, action, element, component, and / or groups thereof. Where a component is said to be “connected” or “linked” to another component, it is understood that it may be directly connected or linked to the other component, or there may be an intervening component. Where used herein, the term “and / or” includes any and all combinations of one or more of the items listed together.

[0147] The term "automatically" means that an action is performed substantially, and sometimes entirely, without human input or manual intervention, and may be directed or performed according to a program.

[0148] The term "programmatically" refers to actions that are electronically directed and / or primarily executed by computer program modules, code, and / or instructions.

[0149] The term "electronically" includes both wireless and wired connections between components.

[0150] Given the interests of this disclosure, many modifications and changes can be made by those skilled in the art without departing from the spirit and scope of the invention. Therefore, it should be understood that the illustrated embodiments are described for illustrative purposes only and should not be considered as limiting the invention as defined by the appended claims. Accordingly, the appended claims should be read to include not only the combination of elements literally described, but also all equivalent elements to perform substantially the same function and obtain substantially the same results in substantially the same manner. Therefore, the claims should be understood to include those specifically illustrated and described above, conceptual equivalents, and those incorporating the fundamental concepts of the invention. Furthermore, the technical concepts that can be understood from the above embodiments are described below. [Aspect 1] The liquid volumes of the first liquid and the second liquid are alternately drawn into the mixing volume from either the first liquid supply unit or the second liquid supply unit, thereby forming a liquid stack in the mixing volume that includes a continuous layer of alternating interfaces between the first liquid and the second liquid. The layers forming the interface between the first liquid and the second liquid are mixed with each other by diffusion within the mixing volume to form a mixed liquid, Discharging the mixed liquid from the mixing volume, A method of mixing liquids using an automated liquid handling system, including [specific example]. [Aspect 2] The method according to embodiment 1, wherein the second liquid is different from the first liquid. [Aspect 3] The method according to embodiment 1, wherein the liquid stack includes at least two layers of the first liquid arranged alternately and continuously with at least two layers of the second liquid. [Aspect 4] The method according to embodiment 1, wherein the liquid stack includes at least three layers of the first liquid arranged alternately and continuously with at least two layers of the second liquid. [Aspect 5] The method according to embodiment 1, wherein each of the layers of the first liquid and the second liquid has a volume in the range of about 3 microliters to 25 microliters. [Aspect 6] The method according to embodiment 1, wherein the total volume of the first liquid layer in the liquid stack is greater than the total volume of the second liquid layer in the liquid stack. [Aspect 7] The method according to embodiment 1, wherein the total volume of the first liquid layer in the liquid stack is substantially equal to the total volume of the second liquid layer in the liquid stack. [Aspect 8] The method according to embodiment 1, wherein mixing the layers forming the interface between the first liquid and the second liquid by diffusion in the mixing volume to form the mixed liquid comprises mixing the layers forming the interface between the first liquid and the second liquid by diffusion in the mixing volume for at least 4 seconds. [Aspect 9] The method according to embodiment 1, wherein the first liquid has a different composition from the second liquid. [Aspect 10] The method according to embodiment 9, wherein the first liquid is a reaction mixture containing DNA fragments, and the second liquid contains a ligase enzyme. [Aspect 11] The method according to embodiment 1, wherein the first liquid has a viscosity different from that of the second liquid. [Aspect 12] The method according to embodiment 1, wherein the first liquid has a different chemical composition from the second liquid. [Aspect 13] The aforementioned mixing volume is elongated and has a longitudinal axis, The method according to embodiment 1, wherein the layers of the first liquid and the second liquid in the liquid stack are stacked along the longitudinal axis. [Aspect 14] The method according to embodiment 1, wherein each of the layers of the first liquid and the second liquid has a layer diameter ratio of at least 0.016 to the layer height. [Aspect 15] Each of the layers of the first liquid and the second liquid has a layer liquid volume and an interface surface area with an adjacent layer of the layers of the first liquid and the second liquid. The ratio of the interface surface area to the volume of the layer liquid is at least 0.02 The method according to embodiment 1, wherein the density is 1 / mm. [Aspect 16] The method according to embodiment 1, wherein the liquid volumes of the first liquid and the second liquid are alternately drawn into the mixed volume from one of the first liquid supply unit and the second liquid supply unit, and the liquid volumes of the first liquid and the second liquid are drawn into a tubular probe through the probe inlet. [Aspect 17] The method according to embodiment 16, wherein discharging the mixed liquid from the mixing volume includes discharging the mixed liquid from the tubular probe through the probe inlet. [Aspect 18] The tubular probe is provided with a probe passage, The method according to embodiment 16, wherein at least a portion of the liquid stack is placed in the probe passage while the layers forming the interface between the first liquid and the second liquid are mixed by diffusion within the mixing volume to form the mixed liquid. [Aspect 19] A flexible conduit defining the conduit passage is fluidly coupled to the tubular probe. The pump is fluidly coupled to the probe inlet via the conduit passage, The method according to embodiment 16, wherein at least a portion of the liquid stack is placed in the conduit passage while the layers forming the interface between the first liquid and the second liquid are mixed by diffusion within the mixing volume to form the mixed liquid. [Aspect 20] The method according to embodiment 16, wherein alternately drawing the liquid volumes of the first liquid and the second liquid into the mixed volume from one of the first liquid supply unit and the second liquid supply unit is performed by automatically operating a pump coupled to the tubular probe according to a program to draw the first liquid and the second liquid from the first liquid supply unit and the second liquid supply unit. [Aspect 21] The method according to embodiment 20, wherein discharging the mixed liquid from the mixing volume includes automatically operating the pump according to a program to discharge the mixed liquid from the mixing volume so that it exits the probe inlet of the tubular probe. [Aspect 22] The method according to embodiment 20, wherein the pump is a syringe pump, a bellows pump, a peristaltic pump, or a screw-type pump. [Aspect 23] The first liquid supply unit comprises a first reservoir for containing the first liquid, The second liquid supply unit comprises a second reservoir for containing the second liquid, The liquid volumes of the first liquid and the second liquid are alternately drawn into the mixed volume from either the first liquid supply unit or the second liquid supply unit, The probe inlet is automatically positioned within the first reservoir according to the program, Subsequently, the first liquid volume of the first liquid is automatically drawn into the tubular probe through the probe inlet according to the program. Subsequently, the probe inlet is automatically positioned within the second reservoir according to the program, Subsequently, the second liquid volume of the second liquid is automatically aspirated into the tubular probe through the probe inlet according to the program. Subsequently, the probe inlet is automatically positioned within the first reservoir according to the program, Subsequently, the third volume of the first liquid is automatically aspirated into the tubular probe through the probe inlet according to the program. Subsequently, the probe inlet is automatically positioned within the second reservoir according to the program, Subsequently, the fourth liquid volume of the second liquid is automatically aspirated into the tubular probe through the probe inlet according to the program. The method according to embodiment 16, including the method described in embodiment 16. [Aspect 24] The suction involves alternately drawing the liquid volumes of the first liquid and the second liquid from one of the first liquid supply unit and the second liquid supply unit through a plurality of probe inlets into each of a plurality of mixed volumes, thereby forming a liquid stack in each of the mixed volumes, each liquid stack comprising a continuum of alternating interface layers of the first liquid and the second liquid in each of the mixed volumes. The mixing process includes mixing the layers forming the interface between the first liquid and the second liquid by diffusion in each of the mixing volumes to form a mixed liquid. The method according to embodiment 1, wherein the discharging includes discharging each of the mixed liquids from each of the mixed volumes. [Aspect 25] The method according to embodiment 24, wherein alternately aspirating the liquid volumes of the first liquid and the second liquid from one of the first liquid supply unit and the second liquid supply unit is performed by automatically operating a single pump actuator according to a program to aspirate the liquid volumes of the first liquid and the second liquid through the respective probe inlets. [Aspect 26] Discharging each of the aforementioned mixed liquids from the mixing volume includes discharging a first volume of the mixed liquid into a first reservoir and a second volume of the mixed liquid into a second reservoir. The aforementioned method, The liquid volume of the mixed liquid is alternately drawn from one of the first reservoir and the second reservoir into the mixed volume, thereby forming a second liquid stack within the mixed volume, which includes a continuous layer of alternating interfaces of the mixed liquid. The layers forming the interface of the aforementioned mixed liquids are mixed with each other by diffusion within the mixing volume to form a second mixed liquid. Discharge the prepared 2 mixed liquid from the mixing volume, The method according to embodiment 1, further comprising: [Aspect 27] An automatic liquid handling system used with a first liquid supply unit containing a first liquid and a second liquid supply unit containing a second liquid, Pressure control mechanism, Mixing volume and, It is a controller, The pressure control mechanism is operated to alternately draw the liquid volumes of the first liquid and the second liquid into the mixing volume from either the first liquid supply unit or the second liquid supply unit, thereby forming a liquid stack in the mixing volume that includes a continuous layer of alternating interfaces between the first liquid and the second liquid. The layers forming the interface between the first liquid and the second liquid are mixed with each other by diffusion within the mixing volume to form a mixed liquid, The pressure control mechanism is operated to discharge the mixed liquid from the mixing volume, A controller configured to perform the following actions: An automated liquid handling system equipped with [feature]. [Aspect 28] The automatic liquid handling system according to embodiment 27, wherein the pressure control mechanism comprises a pump and a pump actuator. [Aspect 29] The automatic liquid handling system according to embodiment 28, wherein the controller is configured to automatically operate the pump actuator according to a program to aspirate the liquid volumes of the first liquid and the second liquid, to mix the layers forming the interface between the first liquid and the second liquid by diffusion, and to operate the pump actuator to discharge the mixed liquid. [Aspect 30] The automatic liquid handling system according to embodiment 27, comprising a tubular probe having a probe inlet, wherein the automatic liquid handling system is configured to alternately draw the liquid volumes of the first liquid and the second liquid into the mixed volume from one of the first liquid supply unit and the second liquid supply unit through the probe inlet.

Claims

1. A method for mixing liquids using an automated liquid handling system, The liquid volumes of the first liquid and the second liquid are alternately drawn into the mixing volume from either the first liquid supply unit or the second liquid supply unit, and the alternately drawn liquid volumes form a liquid stack in the mixing volume that includes a continuous layer in which the first liquid and the second liquid alternately overlap to form an interface. The layers forming the interface between the first liquid and the second liquid are mixed with each other by diffusion within the mixing volume to form a mixed liquid, Discharging the mixed liquid from the mixing volume, Includes, Discharging the mixed liquid from the mixing volume includes discharging a first volume of the mixed liquid into a first reservoir and a second volume of the mixed liquid into a second reservoir. The aforementioned method, The liquid volume of the mixed liquid is alternately drawn from one of the first reservoir and the second reservoir into the mixed volume, and the drawn liquid volume of the mixed liquid forms a second liquid stack within the mixed volume, which includes a continuous layer in which the mixed liquid alternately overlaps to form an interface. The layers forming the interface of the aforementioned mixed liquids are mixed with each other by diffusion within the mixing volume to form a second mixed liquid. Discharging the prepared 2 mixed liquid from the mixing volume, Methods that further include the above.

2. The method according to claim 1, wherein the second liquid is different from the first liquid.

3. The method according to claim 1, wherein the liquid stack includes at least two layers of the first liquid arranged alternately and continuously with at least two layers of the second liquid.

4. The method according to claim 1, wherein the liquid stack comprises at least three layers of the first liquid arranged alternately and continuously with at least two layers of the second liquid.

5. The method according to claim 1, wherein each of the layers of the first liquid and the second liquid has a volume in the range of 3 microliters to 25 microliters.

6. The method according to claim 1, wherein the total volume of the first liquid layer in the liquid stack is greater than the total volume of the second liquid layer in the liquid stack.

7. The method according to claim 1, wherein the total volume of the first liquid layer in the liquid stack is substantially equal to the total volume of the second liquid layer in the liquid stack.

8. The method according to claim 1, wherein mixing the layers forming the interface between the first liquid and the second liquid by diffusion in the mixing volume to form the mixed liquid comprises mixing the layers forming the interface between the first liquid and the second liquid by diffusion in the mixing volume for at least 4 seconds.

9. The method according to claim 1, wherein the first liquid has a different composition from the second liquid.

10. The method according to claim 9, wherein the first liquid is a reaction mixture containing a DNA fragment, and the second liquid contains a ligase enzyme.

11. The method according to claim 1, wherein the first liquid has a viscosity different from that of the second liquid.

12. The method according to claim 1, wherein the first liquid has a different chemical composition from the second liquid.

13. The aforementioned mixing volume is elongated and has a longitudinal axis, The method according to claim 1, wherein the layers of the first liquid and the second liquid in the liquid stack are stacked along the longitudinal axis.

14. The method according to claim 1, wherein each of the layers of the first liquid and the second liquid has a layer diameter ratio of at least 0.016 to the layer height.

15. Each of the layers of the first liquid and the second liquid has a layer liquid volume and an interface surface area with an adjacent layer of the layers of the first liquid and the second liquid. The ratio of the interface surface area to the volume of the layer liquid is at least 0.02 mm －１ The method according to claim 1.

16. The method according to claim 1, wherein alternately drawing the liquid volumes of the first liquid and the second liquid into the mixed volume from one of the first liquid supply unit and the second liquid supply unit includes drawing the liquid volumes of the first liquid and the second liquid into a tubular probe through the probe inlet.

17. The method according to claim 16, wherein discharging the mixed liquid from the mixing volume includes discharging the mixed liquid from the tubular probe through the probe inlet.

18. The tubular probe is provided with a probe passage, The method according to claim 16, wherein at least a portion of the liquid stack is placed in the probe passage while the layers forming the interface between the first liquid and the second liquid are mixed by diffusion within the mixing volume to form the mixed liquid.

19. A flexible conduit defining the conduit passage is fluidly coupled to the tubular probe. The pump is fluidly coupled to the probe inlet via the conduit passage, The method according to claim 16, wherein at least a portion of the liquid stack is placed in the conduit passage while the layers forming the interface between the first liquid and the second liquid are mixed by diffusion within the mixing volume to form the mixed liquid.

20. The method according to claim 16, wherein alternately drawing the liquid volumes of the first liquid and the second liquid into the mixed volume from one of the first liquid supply unit and the second liquid supply unit is performed by automatically operating a pump coupled to the tubular probe according to a program to draw the first liquid and the second liquid from the first liquid supply unit and the second liquid supply unit.

21. The method according to claim 20, wherein discharging the mixed liquid from the mixing volume includes automatically operating the pump according to a program to discharge the mixed liquid from the mixing volume so that it exits the probe inlet of the tubular probe.

22. The method according to claim 20, wherein the pump is a syringe pump, a bellows pump, a peristaltic pump, or a screw-type pump.

23. The first liquid supply unit comprises a first reservoir for containing the first liquid, The second liquid supply unit comprises a second reservoir for containing the second liquid, The liquid volumes of the first liquid and the second liquid are alternately drawn into the mixed volume from either the first liquid supply unit or the second liquid supply unit, The probe inlet is automatically positioned within the first reservoir according to the program, Subsequently, the first liquid volume of the first liquid is automatically drawn into the tubular probe through the probe inlet according to the program. Subsequently, the probe inlet is automatically positioned within the second reservoir according to the program, Subsequently, the second liquid volume of the second liquid is automatically drawn into the tubular probe through the probe inlet according to the program. Subsequently, the probe inlet is automatically positioned within the first reservoir according to the program, Subsequently, the third liquid volume of the first liquid is automatically aspirated into the tubular probe through the probe inlet according to the program. Subsequently, the probe inlet is automatically positioned within the second reservoir according to the program, Subsequently, the fourth liquid volume of the second liquid is automatically drawn into the tubular probe through the probe inlet according to the program. The method according to claim 16, including the method described in claim 16.

24. The suction involves alternately drawing the liquid volumes of the first liquid and the second liquid from one of the first liquid supply unit and the second liquid supply unit through a plurality of probe inlets into each of a plurality of mixed volumes, and forming a respective liquid stack in each of the mixed volumes with the alternately drawn liquid volumes, each liquid stack comprising a continuum of layers in which the first liquid and the second liquid in the respective mixed volume alternately overlap to form an interface. The mixing process includes mixing the layers forming the interface between the first liquid and the second liquid by diffusion in each of the mixing volumes to form a mixed liquid. The method according to claim 1, wherein the discharging includes discharging each of the mixed liquids from each of the mixed volumes.

25. The method according to claim 24, wherein alternately aspirating the liquid volumes of the first liquid and the second liquid from one of the first liquid supply unit and the second liquid supply unit is performed by automatically operating a single pump actuator according to a program to aspirate the liquid volumes of the first liquid and the second liquid through the respective probe inlets.

26. An automatic liquid handling system for mixing liquids, The automatic liquid handling system comprises a probe module including a plurality of tubular probes mounted on a module base and moving together with the module base by a probe positioning system; a pressure control mechanism including a plurality of pumps and pump actuators; and a controller for controlling the probe positioning system and the pressure control mechanism. Each tubular probe has a probe inlet and is individually fluid-coupled to a pump by a tube. The aforementioned controller, Using the probe positioning system, the plurality of tubular probes are positioned and inserted into the first liquid supply unit, The pump generates negative pressure in each tubular probe, thereby drawing the liquid volume of the first liquid from the first liquid supply unit into the plurality of tubular probes through the probe inlets. Using the probe positioning system, the plurality of tubular probes are positioned and inserted into the second liquid supply section, The pump generates negative pressure in each tubular probe, thereby drawing the liquid volume of the second liquid from the second liquid supply unit into the plurality of tubular probes through the probe inlets. The pump and the probe positioning system alternately draw the first liquid volume and the second liquid volume from the first liquid supply unit and the second liquid supply unit into the mixed volume in each tubular probe through the probe inlet, and the alternately drawn first liquid volume and second liquid volume form a first liquid stack in each tubular probe, which includes a continuous layer in which the first liquid and the second liquid alternately overlap to form an interface within the mixed volume in each tubular probe. To mix the layers forming the interface between the first liquid and the second liquid by diffusion within the mixing volume in each tubular probe, thereby forming a mixed liquid in each tubular probe, it is determined whether a predetermined holding time has elapsed for the first liquid stack to be held within the mixing volume in each tubular probe, When it is determined that the predetermined holding time has elapsed, the probe positioning system is used to remix the first volume and the second volume of the mixed liquid, and the pump generates positive pressure in each tubular probe, so that the first volume is discharged from each tubular probe through the probe inlet to the first reservoir and the second volume is discharged to the second reservoir. An automated liquid handling system configured to perform the following actions.

27. An automatic liquid handling system for mixing liquids, The automatic liquid handling system comprises a probe module including a first tubular probe and a second tubular probe mounted on a module base and moving together with the module base by a probe positioning system; a pressure control mechanism including a plurality of pumps and pump actuators; and a controller for controlling the probe positioning system and the pressure control mechanism. The aforementioned controller, Using the probe positioning system, the first tubular probe is inserted into the first liquid supply unit, The pump generates negative pressure in the first tubular probe, thereby drawing the liquid volume of the first liquid from the first liquid supply unit into the first tubular probe. Using the probe positioning system, the first tubular probe is inserted into the second liquid supply unit, The pump generates negative pressure in the first tubular probe, thereby drawing a volume of a second liquid, different from the first liquid, into the first tubular probe from the second liquid supply unit. Using the pump and the probe positioning system, the liquid volume of the first liquid and the liquid volume of the second liquid are alternately drawn into the mixed volume in the first tubular probe, and the alternately drawn liquid volumes of the first liquid and the second liquid form a first liquid stack in the first tubular probe, which includes a continuous layer in which the first liquid and the second liquid alternately overlap and form an interface within the mixed volume in the first tubular probe. To determine whether a predetermined first holding time has elapsed for the first liquid stack to be held within the mixing volume in the first tubular probe in order to mix the layers forming the interface between the first liquid and the second liquid by diffusion within the mixing volume in the first tubular probe and form a first mixed liquid in the first tubular probe, When it is determined that the first holding time has elapsed, the probe positioning system is used and positive pressure is generated in the first tubular probe by the pump to discharge a first volume of the first mixed liquid from the mixed volume in the first tubular probe into the first reservoir. The probe positioning system is used, and positive pressure is generated in the first tubular probe by the pump, causing a second volume of the first mixed liquid to be discharged from the mixed volume in the first tubular probe into the second reservoir. Using the probe positioning system, the second tubular probe is inserted into the first reservoir, The pump generates negative pressure in the second tubular probe to draw the liquid volume of the first mixed liquid from the first reservoir, Using the probe positioning system, the second tubular probe is inserted into the second reservoir, The pump generates negative pressure in the second tubular probe to draw the liquid volume of the first mixed liquid from the second reservoir, Using the pump and the probe positioning system, the liquid volume of the first mixed liquid is alternately drawn from the first reservoir and the second reservoir into the mixing volume in the second tubular probe, and the liquid volumes of the first mixed liquid drawn alternately form a second liquid stack in the second tubular probe, which includes a continuous layer in which the first mixed liquid alternately overlaps and forms an interface within the mixing volume in the second tubular probe. To determine whether a predetermined second holding time has elapsed for the second liquid stack to be held within the mixing volume in the second tubular probe in order to mix the layers forming the interface of the first mixed liquid with each other by diffusion within the mixing volume in the second tubular probe, thereby forming a second mixed liquid in the second tubular probe, When it is determined that the second holding time has elapsed, the pump generates positive pressure in the second tubular probe and discharges the second mixed liquid from the mixed volume in the second tubular probe. An automated liquid handling system configured to perform the following actions.

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