Automated Macromolecule Sample Preparation System

Abstract

An apparatus for preparing a macromolecule sample from a complex liquid mixture includes a hydraulic system adapted for control by an automated controller, comprising a pump and one or more valves. The hydraulic system can be controlled by an automated controller to apply the liquid mixture to one or more filters with a pressure differential across each filter. The filters include a rough filter selected to separate, from a macromolecule in a liquid mixture, at least a portion of one or more rough components in the mixture that are larger than the macromolecule. Further included is a fine filter selected to separate from the macromolecule at least a portion of one or more fine components in the mixture that are smaller than the macromolecule. A method for preparing a macromolecule sample includes automatically acquiring a liquid mixture, the mixture including a macromolecule. At least a portion of the other components in the mixture are automatically separated from the macromolecule by applying the mixture to each of one or more filters, with a pressure differential across each filter.

1 . An apparatus for preparing a macromolecule sample, comprising:
 a hydraulic system adapted for control by an automated controller, comprising a pump and one or more valves;   a rough filter selected to separate from a macromolecule in a liquid mixture, at least a portion of one or more rough components in the mixture that are larger than the macromolecule;   a fine filter selected to separate from the macromolecule, at least a portion of one or more fine components in the mixture that are smaller than the macromolecule; and   the hydraulic system being controlled to apply the liquid mixture to each filter with a pressure differential across each filter.   
   
   
       2 . The apparatus of  claim 1 , wherein the fine components comprise salt components. 
   
   
       3 . The apparatus of  claim 2 , further comprising a reservoir supplying a denaturation agent. 
   
   
       4 . The apparatus of  claim 3 , further comprising a heating element adapted for control by the automated controller. 
   
   
       5 . The apparatus of  claim 4 , further comprising a cooling element adapted for control by the automated controller. 
   
   
       6 . The apparatus of  claim 2 , further comprising at least one ionic concentration sensor adapted to communicate with the automated controller. 
   
   
       7 . The apparatus of  claim 6 , further comprising at least one flow sensor adapted to communicate with the automated controller. 
   
   
       8 . The apparatus of  claim 7 , wherein the hydraulic system comprises a second pump adapted for control by the automated controller. 
   
   
       9 . The apparatus of  claim 8 , wherein at least one ionic concentration sensor is a pH sensor. 
   
   
       10 . The apparatus of  claim 9 , further comprising a reservoir supplying a desalination buffer. 
   
   
       11 . The apparatus of  claim 10 , further comprising a reservoir supplying a pH buffer. 
   
   
       12 . The apparatus of  claim 11 , further comprising the automated controller in communication with the valves, the pumps, and the sensors. 
   
   
       13 . The apparatus of  claim 2 , wherein the molecular weight of the macromolecule is between about 1,000 and about 200,000 AMU. 
   
   
       14 . The apparatus of  claim 13 , wherein the molecular weight of the macromolecule is between about 10,000 and about 160,000 AMU. 
   
   
       15 . The apparatus of  claim 14 , wherein the fine filter is selected to separate components of a molecular weight less than about 90% of the molecular weight of the macromolecule. 
   
   
       16 . The apparatus of  claim 15 , wherein the rough filter is selected to separate components of a molecular weight greater than about 110% of the molecular weight of the macromolecule. 
   
   
       17 . The apparatus of  claim 2 , further comprising a lysis unit. 
   
   
       18 . The apparatus of  claim 17 , wherein the lysis unit comprises a sonic disrupter controlled by the automated controller. 
   
   
       19 . The apparatus of  claim 17 , wherein the lysis unit comprises a pump and a reservoir supplying a lysis buffer. 
   
   
       20 . An apparatus for preparing a macromolecule sample, comprising:
 a hydraulic system comprising two or more pumps, buffer reservoirs, a flow sensor, a pH sensor, a heating element, and valves;   at least three filters selected to separate from a macromolecule in a liquid mixture, at least a portion of other components in the mixture, the filters including:   a first stage rough filter selected to separate rough components;   a second stage rough filter selected to separate at least a portion of rough components that pass through the first stage rough filter;   a fine filter to separate fine components comprising salt components;   an automated controller in communication with the valves, the pumps, the heating element, and the sensors that controls the hydraulic system to:   apply the liquid mixture to each filter with a pressure differential across each filter;   direct a desalination buffer from a reservoir through the fine filter into the mixture;   combine a denaturation agent from a reservoir with the macromolecule;   control the heating element to heat the combination of the macromolecule and the denaturation agent until at least partial denaturation of the macromolecule occurs; and   combine a pH buffer from a reservoir to maintain a pH value at the pH sensor in a range from 6 to 8.   
   
   
       21 . The apparatus of  claim 20 , wherein the liquid mixture comprises cells, further comprising a pump that combines a lysis buffer from a reservoir with the cells. 
   
   
       22 . An apparatus for preparing a macromolecule sample, comprising:
 a hydraulic system adapted for control by an automated controller, comprising a pump and one or more valves;   a filter selected to separate, at least in part, a macromolecule in a liquid mixture from one or more salt components in the mixture; and   an automated controller that controls the pump and the valves.   
   
   
       23 . The apparatus of  claim 22 , further comprising a reservoir supplying a desalination buffer. 
   
   
       24 . The apparatus of  claim 23 , further comprising:
 a reservoir supplying a denaturation agent; and   a heating element adapted for control by an automated controller.   
   
   
       25 . The apparatus of  claim 24 , wherein the filter is selected to separate components that have a molecular weight less than about 90% of the molecular weight of the macromolecule. 
   
   
       26 . An apparatus for preparing a macromolecule sample, comprising:
 a hydraulic system adapted for control by an automated controller, comprising a pump and one or more valves;   a lysis unit that is capable of lysing cells in a liquid mixture comprising cells and a macromolecule; and   a filter selected to separate from the macromolecule, at least a portion of components in the mixture that are larger than the macromolecule, the components comprising insoluble lysed cell components; and   an automated controller that controls the pump and the valves.   
   
   
       27 . The apparatus of  claim 26 , wherein the molecular weight of the macromolecule is between 10,000 and 160,000 AMU. 
   
   
       28 . The apparatus of  claim 27 , wherein the lysis unit comprises a sonic disrupter. 
   
   
       29 . The apparatus of  claim 27 , wherein the lysis unit comprises a reservoir supplying a lysis buffer. 
   
   
       30 . The apparatus of  claim 29 , further comprising:
 a reservoir supplying a denaturation agent; and   a heating element adapted for control by an automated controller.   
   
   
       31 . The apparatus of  claim 30 , wherein the filter is selected to remove insoluble components, further comprising a rough filter selected to separate from the macromolecule, at least in part, soluble components that have a molecular weight greater than about 110% of the molecular weight of the macromolecule. 
   
   
       32 . A method for preparing a macromolecule sample, comprising automatically:
 acquiring a liquid mixture, the mixture comprising a macromolecule and one or more cells;   lysing at least a portion of the cells; and   separating from the macromolecule at least a portion of components larger than the macromolecule, the components comprising insoluble lysed cell components, by applying the mixture to a filter with a pressure differential across the filter.   
   
   
       33 . The method of  claim 32 , further comprising lysing at least a portion of the cells by combining a lysis buffer with the cells. 
   
   
       34 . The method of  claim 33  further comprising increasing the macromolecule concentration by at least 100%. 
   
   
       35 . The method of  claim 34 , further comprising increasing the macromolecule concentration by at least 200%. 
   
   
       36 . The method of  claim 35 , further comprising:
 combining the macromolecule with a denaturation agent; and   heating the macromolecule and denaturation agent until at least partial denaturation of the macromolecule occurs.   
   
   
       37 . The method of  claim 36 , wherein the filter separates insoluble components, further comprising applying the mixture, to a rough filter with a pressure differential across the rough filter, the rough filter selected to separate soluble components that have a molecular weight that is greater than about 110% of the molecular weight of the macromolecule. 
   
   
       38 . An apparatus for preparing a macromolecule sample, comprising:
 means for automatically acquiring a liquid mixture, the mixture comprising a macromolecule and one or more cells;   means for automatically lysing at least a portion of the cells; and   means for automatically separating from the macromolecule at least a portion of components larger than the macromolecule, the components comprising insoluble lysed cell components, by applying the mixture to a filter with a pressure differential across the filter.   
   
   
       39 . An apparatus for preparing a macromolecule sample, comprising:
 a hydraulic system comprising:   a plurality of valves;   a rough pump that draws a liquid mixture from a sample site through:   a first stage rough filter selected to separate rough components; and   a second stage rough filter selected to separate rough components that pass through the first stage rough filter;   a fine pump that:   draws a desalination buffer from a reservoir;   operates cooperatively with the rough pump to:   draw a portion of the liquid mixture through a fine filter, the fine filter selected to separate from the macromolecule fine components that have a molecular weight less than about 90% of the molecular weight of a macromolecule;   direct the desalination buffer through the fine filter to combine the desalination buffer with the macromolecule;   a denaturation pump that operates in combination with the rough pump to direct the macromolecule and the desalination buffer to a denaturing vessel, the denaturing vessel comprising a heating element and a cooling element;   a reservoir supplying a denaturation buffer to the denaturation vessel;   a reservoir supplying a pH buffer;   a pH sensor located at the denaturization conduit;   an automated controller in electronic communication with the pumps, the denaturation vessel, and the sensor, that controls the apparatus to acquire a liquid mixture from the sampling site, the mixture comprising a macromolecule, rough components, and fine components;   separate at least a portion of rough and fine components from the macromolecule;   combine a denaturation buffer with the macromolecule;   heat the denaturation buffer and the macromolecule in the denaturation vessel to denature the macromolecule;   control the pH of the mixture to between about 6 and about 8 by adding pH buffer to the mixture; and   direct the denatured macromolecule to the analysis site.