Microdroplet-manipulation systems and methods for automated execution of molecular biological protocols

Abstract

Disclosed herein are automated systems for performing various biochemical and molecular biological procedures, including processor-controlled execution of protocols involving multiple steps performed in, on, or with liquid microdroplets. Example protocols are the various Polymerase Chain Reaction (PCR) protocols, but the subject systems are not limited to performing PCR protocols. Formation of a microdroplet of the sample for use in the described systems is achieved by bringing an amount of the sample into contact with a hydrophobic milieu, such as a superhydrophobic surface or hydrophobic liquid.

What is claimed is: 
     
         1 . An apparatus, comprising:
 a movement and placement device having a range of movement in at least two dimensions;   a microdroplet-manipulating device coupled to the movement and placement device and placeable by the movement and placement device within the range of movement;   a hydrophobic milieu located within the range of movement; and   a controller operably connected to the movement and placement device and to the microdroplet-manipulating device, the controller being programmable with a protocol in which the movement and placement device is commanded to place the microdroplet-manipulating device relative to the hydrophobic milieu, and the microdroplet-manipulating device is commanded to perform automatically at least two of   placing an amount of a hydrophilic liquid in contact with the hydrophobic milieu sufficiently to form at least one microdroplet of the hydrophilic liquid on or in the hydrophobic milieu,   manipulating the microdroplet while the microdroplet is in contact with the hydrophobic milieu, and   removing at least a portion of a microdroplet from contact with the hydrophobic milieu.   
     
     
         2 . The apparatus of  claim 1 , wherein the controller is further configured to:
 receive a command to stop execution of the protocol;   accept from the user a change, in the protocol, of steps as programmed in the controller; and   resume execution of the protocol with the change.   
     
     
         3 . The apparatus of  claim 1 , wherein the controller is further configured, as requested by a user:
 to stop execution of a first protocol,   to accept from the user a new protocol, and   to commence the second protocol in place of the first protocol.   
     
     
         4 . The apparatus of  claim 1  wherein the controller is further configured, as requested by a user:
 to except from the user a change in a preset protocol of steps as programmed in the controller; and 
 execute the protocol with the change without the user having to change a component of the apparatus. 
 
     
     
         5 . The apparatus of  claim 1 , wherein:
 the hydrophobic milieu comprises a superhydrophobic surface and a hydrophobic liquid having different respective locations in the range of movement; and   the controller is further programmed to command the microdroplet-manipulating device to place an amount of a first hydrophilic liquid on the superhydrophobic surface to form a first microdroplet on the superhydrophobic surface and to place an amount of a second hydrophilic liquid in the hydrophobic liquid to form a second microdroplet in the hydrophobic liquid.   
     
     
         6 . The apparatus of  claim 5 , wherein the controller is further programmed to command the microdroplet-manipulating device to manipulate the first microdroplet on the superhydrophobic surface and to command the microdroplet-manipulating device to manipulate the second microdroplet in the hydrophobic liquid. 
     
     
         7 . The apparatus of  claim 6 , wherein:
 the command to manipulate the first microdroplet on the superhydrophobic surface is part of a first protocol; and   the command to manipulate the second microdroplet in the hydrophobic liquid is part of a second protocol.   
     
     
         8 . The apparatus of  claim 7 , wherein:
 the first protocol comprises extracting genetic material from a sample; and   the second protocol comprises amplifying a predetermined portion of the extracted genetic material.   
     
     
         9 . The apparatus of  claim 1 , wherein the hydrophobic milieu comprises a superhydrophobic surface. 
     
     
         10 . The apparatus of  claim 9 , wherein manipulating the microdroplet on the superhydrophobic surface comprises at least one of:
 moving the microdroplet,   adding a substance to the microdroplet,   removing a substance from the microdroplet,   mixing contents of the microdroplet,   concentrating the microdroplet,   agitating the microdroplet,   changing a volume of the microdroplet,   changing a shape of the microdroplet,   changing a density of the microdroplet,   changing a composition of the microdroplet,   changing a position of the microdroplet,   holding a substance relative to the microdroplet,   merging the microdroplet with another microdroplet,   splitting the microdroplet, and   rotating the microdroplet.   
     
     
         11 . The apparatus of  claim 9 , wherein manipulating the microdroplet on the superhydrophobic surface comprises rotating the microdroplet relative to the surface sufficiently to centrifuge the microdroplet. 
     
     
         12 . The apparatus of  claim 9 , wherein manipulating the microdroplet on the superhydrophobic surface comprises extracting genetic material from the microdroplet. 
     
     
         13 . The apparatus of  claim 9 , wherein manipulating the microdroplet on the superhydrophobic surface comprises serially diluting the microdroplet. 
     
     
         14 . The apparatus of  claim 9 , wherein the controller is further configured to:
 receive a program comprising a first series of steps in the protocol in which at least one step involves automatically manipulating a microdroplet on the superhydrophobic surface; and   execute the first series of steps automatically upon command.   
     
     
         15 . The apparatus of  claim 14 , wherein manipulating a microdroplet on the superhydrophobic surface comprises at least one of:
 moving the microdroplet,   adding a substance to the microdroplet,   removing a substance from the microdroplet,   mixing contents of the microdroplet,   concentrating the microdroplet,   agitating the microdroplet,   changing a volume of the microdroplet,   changing a shape of the microdroplet,   changing a density of the microdroplet,   changing a composition of the microdroplet,   changing a position of the microdroplet,   holding a substance relative to the microdroplet,   merging the microdroplet with another microdroplet,   splitting the microdroplet, and   rotating the microdroplet.   
     
     
         16 . The apparatus of  claim 9 , wherein manipulating the microdroplet further comprises:
 removing the microdroplet from the superhydrophobic surface; and   amplifying a nucleotide sequence in the microdroplet.   
     
     
         17 . The apparatus of  claim 1 , wherein the hydrophobic milieu comprises a hydrophobic liquid. 
     
     
         18 . The apparatus of  claim 17 , wherein manipulating a microdroplet in the hydrophobic liquid comprises at least one of:
 moving the microdroplet,   adding heat to the microdroplet,   removing heat from the microdroplet,   placing the microdroplet in a pendant condition relative to the hydrophobic liquid, and   retracting the microdroplet.   
     
     
         19 . The apparatus of  claim 17 , wherein the controller is further configured to:
 receive a program comprising a first series of steps in the protocol in which at least one step involves automatically manipulating a microdroplet in the hydrophobic liquid; and   execute the first series of steps automatically upon command.   
     
     
         20 . The apparatus of  claim 17 , further comprising a vessel comprising at least one temperature-regulated chamber located within the range of movement, the vessel containing the hydrophobic liquid at a preset temperature. 
     
     
         21 . The apparatus of  claim 20 , wherein the hydrophobic liquid comprises a silicone oil. 
     
     
         22 . The apparatus of  claim 20 , wherein the protocol comprises:
 commanding the microdroplet-manipulating device to move a microdroplet to the hydrophobic liquid in the temperature-regulated chamber;   commanding the microdroplet-manipulating device to manipulate the microdroplet while the microdroplet is submerged in the hydrophobic liquid in the temperature-regulated chamber; and   commanding the microdroplet-manipulating device to remove the microdroplet from the temperature-regulated chamber.   
     
     
         23 . The apparatus of  claim 20 , wherein the vessel comprises at least three temperature-regulated chambers each containing a respective volume of the hydrophobic liquid at a respective temperature. 
     
     
         24 . The apparatus of  claim 23 , wherein:
 the vessel further comprises respective channels connecting the chambers together in a cyclical manner;   each channel also contains a respective volume of the hydrophobic liquid; and   the channels open into the respective chambers.   
     
     
         25 . The apparatus of  claim 20 , wherein the vessel comprises:
 a first temperature-regulated chamber in which the hydrophobic liquid is at a nucleic acid-denaturation temperature;   a second temperature-regulated chamber in which the hydrophobic liquid is at a nucleic acid annealing temperature; and   a third temperature-regulated chamber in which the hydrophobic liquid is at a nucleic acid extension temperature.   
     
     
         26 . The apparatus of  claim 25 , wherein:
 the denaturation temperature is in a range of about 94-96° C.;   the annealing temperature is in the range of about 50-65° C.; and   the extension temperature is in a range of about 70-74° C.   
     
     
         27 . The apparatus of  claim 26 , wherein the controller is further configured to command the movement and positioning device to move the microdroplet-manipulating device in a manner such that the microdroplet, while being kept submerged in the hydrophobic liquid, is moved from one chamber of the vessel to the next according to a preset thermal cycle. 
     
     
         28 . The apparatus of  claim 27 , wherein the controller is further configured to command the microdroplet-manipulating device to hold the microdroplet in each chamber for a preset respective length of time. 
     
     
         29 . The apparatus of  claim 28 , wherein the microdroplet-manipulating device is configured to hold the microdroplet in each chamber while the microdroplet is pendant from the microdroplet-manipulating device and the microdroplet-manipulating device moves the pendant droplet relative to the hydrophobic liquid. 
     
     
         30 . The apparatus of  claim 29 , wherein:
 the microdroplet-manipulating device is further configured to retract the microdroplet as the microdroplet-manipulating device is moving the microdroplet through a respective channel from one chamber to the next in the cycle; and   the microdroplet-manipulating device is further configured to de-retract the microdroplet as the microdroplet is being held by the microdroplet-manipulating device in a subsequent chamber of the cycle.   
     
     
         31 . The apparatus of  claim 30 , wherein the cycle is a PCR cycle. 
     
     
         32 . The apparatus of  claim 1 , wherein the microdroplet-manipulating device comprises a syringe fitted with a tip. 
     
     
         33 . An apparatus, comprising:
 a movement and placement device defining a preset motion range in at least two of x-, y-, and z-dimensions;   a microdroplet-manipulating device coupled to and movable by the controlled movement and placement device in the preset range;   a superhydrophobic surface located in the preset range;   a temperature-controlled vessel located in the preset range and containing a hydrophobic liquid, the vessel comprising multiple interconnected chambers each held at a respective temperature; and   a controller operably connected to the movement and placement device and to the microdroplet-manipulating device, the controller being configured to command the movement and positioning device to place the microdroplet-manipulating device relative to the superhydrophobic surface and to command the movement and placement device to perform automatically at least two of placing an aqueous liquid microdroplet on the superhydrophobic surface, manipulating the microdroplet on the superhydrophobic surface, removing at least a portion of the microdroplet from the superhydrophobic surface, placing the microdroplet in a chamber of the temperature-controlled vessel, manipulating the microdroplet in the chamber, and removing at least a portion of the microdroplet from the chamber.   
     
     
         34 . The apparatus of  claim 33 , wherein the microdroplet-manipulating device comprises a syringe fitted with a tip. 
     
     
         35 . The apparatus of  claim 34 , wherein the controller and syringe are further configured, whenever the microdroplet is in a chamber of the vessel and submerged in the hydrophobic liquid, to hold the microdroplet in a pendant manner from the tip and to move the microdroplet relative to the hydrophobic liquid. 
     
     
         36 . The apparatus of  claim 34 , wherein the microdroplet-manipulating device further comprises a vibration-producing device disposed in contact with the syringe so as to, when actuated, vibrate the syringe in an orbital manner sufficiently to impart a corresponding stirring motion of the microdroplet in contact with the tip. 
     
     
         37 . The apparatus of  claim 33 , wherein the vessel comprises a first chamber containing a hydrophobic liquid held at a nucleic acid-denaturation temperature, a second chamber containing the hydrophobic liquid held at a nucleic acid-annealing temperature, and a third chamber containing the hydrophobic liquid held at a nucleic acid-extending temperature. 
     
     
         38 . The apparatus of  claim 37 , wherein the vessel comprises respective channels connecting the chambers together, each channel containing a respective volume of the hydrophobic liquid. 
     
     
         39 . The apparatus of  claim 38 , wherein:
 the microdroplet-manipulating device comprises a syringe fitted with a tip; and   the controller and syringe are further configured to retract the microdroplet into the tip whenever the microdroplet is being moved, by respective motion of the syringe imparted by the movement and placement device, through a channel from one chamber to the next.   
     
     
         40 . The apparatus of  claim 33 , wherein the controller is programmed to execute at least two protocols on a microdroplet, the protocols including a first protocol comprising at least one step performed with the microdroplet on the superhydrophobic surface and a second protocol comprising at least one step performed with the microdroplet in the vessel submerged in the hydrophobic liquid. 
     
     
         41 . The apparatus of  claim 40 , wherein the first protocol is directed to at least one of releasing genetic material from cells in the microdroplet, diluting the microdroplet, centrifuging the microdroplet, lysing the microdroplet, precipitating genetic material in the microdroplet, collecting genetic material from the microdroplet, washing and drying collected genetic material from the microdroplet, and rehydrating collected genetic material from the microdroplet. 
     
     
         42 . The apparatus of  claim 41 , wherein:
 the microdroplet-manipulating device comprises a syringe fitted with a tip; and   collecting genetic material from the microdroplet comprises adhering the genetic material to the tip.   
     
     
         43 . The apparatus of  claim 42 , wherein collecting genetic material further comprises rotating the tip as the genetic material adheres to the tip. 
     
     
         44 . An apparatus for amplifying genetic material, obtained from a sample, by a thermocycling protocol, the apparatus comprising:
 a movement and placement device;   a microdroplet-manipulating device coupled to the movement and placement device and movable by the movement and placement device in a region of space defined by the movement and placement device;   a multi-chamber array comprising at least three chambers hydraulically connected together and containing a hydrophobic liquid, each channel being held at a respective temperature for a respective stage in the thermocycling protocol; and   a controller operably connected to the movement and placement device and to the microdroplet-manipulating device, the controller being configured to command the movement and placement device to place the microdroplet-manipulating device relative to the chambers of the array in a cyclic manner at respective times, each cycle comprising placing the microdroplet-manipulating device relative to a first chamber of the array at which the microdroplet-manipulating device submerges a microdroplet being carried by the microdroplet-manipulating device in the hydrophobic liquid in the first chamber for a first defined length of time, to transport the microdroplet through the respective channel to a second chamber while keeping the droplet submerged in the hydrophobic liquid for a defined second length of time, to transport the microdroplet through the respective channel to a third chamber while keeping the droplet submerged in the hydrophobic liquid for a defined third length of time, and to return the microdroplet through the respective channel to the first chamber while keeping the droplet submerged in the hydrophobic liquid; and repeating the cycle to produce a desired amplification of the genetic material.   
     
     
         45 . The apparatus of  claim 44 , further comprising a superhydrophobic surface situated within the region of space and reachable by the microdroplet-manipulating device being controllably moved by the movement and placement device. 
     
     
         46 . The apparatus of  claim 45 , wherein the controller is further configured to command the movement and placement device before, during, or after performing the thermocyclic protocol, to place the microdroplet-manipulating device relative to the superhydrophobic surface and to command the microdroplet-manipulating device to perform automatically at least two of placing a liquid microdroplet on the superhydrophobic surface, manipulating the microdroplet on the superhydrophobic surface, and removing at least a portion of the microdroplet from the superhydrophobic surface. 
     
     
         47 . The apparatus of  claim 44 , further comprising a real-time cycle-detection device associated with the multi-chamber array. 
     
     
         48 . The apparatus of  claim 47 , wherein the real-time cycle-detection device comprises, in association with the channel between the third and first chamber, a light source directing light across the chamber so as to be incident on liquid passing through said channel, and a light detector situated to detect light from the source that has passed through said channel. 
     
     
         49 . An apparatus, comprising:
 surface means for forming a microdroplet whenever a corresponding volume of a hydrophilic liquid is situated on the surface means;   means for placing the microdroplet on the surface means and removing the microdroplet from the surface means;   means for manipulating the microdroplet including while the microdroplet is situated on the surface means; and   control means for actuating said placing means and said manipulating means at preset moments in at least one protocol including at least one manipulation of the microdroplet on said surface means.   
     
     
         50 . The apparatus of  claim 49 , wherein said manipulating means comprises one or more of:
 means for moving a microdroplet,   means for adding a substance to a microdroplet,   means for removing a substance from a microdroplet,   means for mixing contents of a microdroplet,   means for concentrating a microdroplet,   means for agitating a microdroplet,   means for removing a portion of a microdroplet,   means for changing a volume of a microdroplet,   means for changing a shape of a microdroplet,   means for changing a density of a microdroplet,   means for changing a composition of a microdroplet,   means for changing a position of a microdroplet,   means for holding a substance relative to a microdroplet,   means for merging the microdroplet with another microdroplet,   means for splitting the microdroplet, and   means for rotating the microdroplet.   
     
     
         51 . The apparatus of  claim 49 , further comprising thermocycling means, wherein said control means further actuates said placing means and said manipulating means to place a microdroplet at preset temperatures in said thermocycling means. 
     
     
         52 . The apparatuss of  claim 51 , further comprising real-time cycle-quantification means. 
     
     
         53 . A method for performing a protocol on a hydrophilic liquid sample, comprising:
 placing an amount of the sample in contact with a first hydrophobic milieu to form at least one microdroplet of the sample in or on the first hydrophobic milieu; and   performing at least one step of a first protocol on, in, or with the microdroplet in or on the first hydrophobic milieu.   
     
     
         54 . The method of  claim 53 , wherein:
 the first hydrophobic milieu comprises a superhydrophobic surface; and   the at least one step is selected from the group consisting of:
 moving the microdroplet, 
 adding a substance to the microdroplet, 
 removing a substance from the microdroplet, 
 mixing contents of the microdroplet, 
 concentrating the microdroplet, 
 agitating the microdroplet, 
 changing a volume of the microdroplet, 
 changing a shape of the microdroplet, 
 changing a density of the microdroplet, 
 changing a composition of the microdroplet, 
 changing a position of the microdroplet, 
 holding a substance relative to the microdroplet, 
 merging the microdroplet with another microdroplet, 
 splitting the microdroplet, and 
 rotating the microdroplet. 
   
     
     
         55 . The method of  claim 53 , further comprising:
 placing an amount of a second sample in contact with a second hydrophobic milieu to form at least one microdroplet of the second sample in or on the second hydrophobic milieu; and   performing at least one step of a second protocol on, in, or with the microdroplet of the second sample in or on the second hydrophobic milieu.   
     
     
         56 . The method of  claim 55 , wherein:
 the second protocol comprises thermocycling the second sample; and   the second hydrophobic milieu is a hydrophobic liquid; and   the at least one step of the second protocol is produced with the microdroplet of the second sample immersed in the hydrophobic liquid.   
     
     
         57 . The method of  claim 56 , wherein the thermocycling is a respective portion of a PCR protocol. 
     
     
         58 . The method of  claim 57 , wherein the PCR protocol comprises quantitative monitoring, in real time, products of the PCR protocol. 
     
     
         59 . The method of  claim 58 , wherein thermocycling the second sample comprises:
 while keeping the microdroplet of the second sample immersed in the hydrophobic liquid, placing the microdroplet in a first chamber containing the hydrophobic liquid heated to a first temperature;   while keeping the microdroplet of the second sample immersed in the hydrophobic liquid, placing the microdroplet in a second chamber containing the hydrophobic liquid heated to a second temperature;   while keeping the microdroplet of the second sample immersed in the hydrophobic liquid, placing the microdroplet in a third chamber containing the hydrophobic liquid heated to a third temperature; and   repeating at least once the placements in the first, second, and third chambers.   
     
     
         60 . The method of  claim 59 , wherein:
 at least one of the first, second, and third placements comprises a respective incubation time of the microdroplet in the heated hydrophobic liquid in the respective chamber; and   the respective incubation is conducted while the microdroplet is in a pendant condition in the hydrophobic liquid in the respective chamber.   
     
     
         61 . The method of  claim 60 , wherein the respective incubation is conducted while the microdroplet is also being moved relative to the hydrophobic liquid to achieve convective heating of the microdroplet in the hydrophobic liquid in the respective chamber. 
     
     
         62 . The method of  claim 55 , wherein the second sample is a product of the first protocol performed on the first sample. 
     
     
         63 . The method of  claim 53 , wherein:
 the first hydrophobic milieu comprises a hydrophobic liquid; and   the at least one step comprises exposing the first microdroplet to a first temperature while the first microdroplet is immersed in the hydrophobic liquid.   
     
     
         64 . The method of  claim 63 , wherein the at least one step further comprises exposing the first microdroplet to a second temperature while the first microdroplet is immersed in the hydrophobic oil. 
     
     
         65 . The method of  claim 64 , wherein the first and second temperatures are respective portions of a PCR thermocycle. 
     
     
         66 . The method of  claim 53 , further comprising:
 placing an amount of the sample in contact with a second hydrophobic milieu to form at least one microdroplet of the sample in or on the second hydrophobic milieu; and   performing at least one step of a second protocol on, in, or with the microdroplet in or on the second hydrophobic milieu, the second protocol comprising performing PCR on a genetic material in the microdroplet.   
     
     
         67 . The method of  claim 66 , wherein:
 the first hydrophobic milieu comprises a superhydrophobic surface;   the microdroplet is formed on the superhydrophobic surface; and   performing PCR comprises removing the microdroplet from the superhydrophobic surface to a PCR cycler comprising a first chamber, a second chamber, and a third chamber, each containing an inert hydrophobic liquid heated to a respective temperature, wherein the microdroplet is held in each chamber for a specified time according to the respective step in PCR; and   moving the microdroplet from chamber to chamber in a cyclic manner to achieve thermocycling of the microdroplet according to PCR.   
     
     
         68 . The method of  claim 67 , wherein:
 the PCR cycler further comprises respective channels connecting the chambers together, each channel also containing the inert hydrophobic liquid; and   movement of the microdroplet from chamber to chamber is conducted through the respective channels while the microdroplet remains submerged in the inert hydrophobic liquid.