Bidirectional transfer of an aliquote of fluid between compartments

Abstract

An apparatus adapted to extract an ingredient from a fluid sample by bidirectional transferring of an aliquot of fluid having magnetic particles therein, said apparatus comprising: an open compartment operable at a constant ambient pressure; a closed compartment having a closed pressure, and an upper zone containing an air pocket adapted to generate internal positive and negative pressure; an intermediate transferring-retaining compartment adapted to temporarily retain a fraction of said aliquot of fluid; a flow barrier member adapted to inhibit a flow of said aliquot of fluid when said closed pressure is substantially equal to said ambient pressure and to allow said flow of fluid when said closed pressure is substantially different from said ambient pressure, the pressure differential controllable by a thermo member; a capturing zone adapted to allow magnetic particles in the fluid to be attracted in response to a magnetic force applied.

1 . An apparatus adapted to extract an ingredient from a fluid sample by bi-directional transferring of an aliquot of fluid having magnetic particles therein, said apparatus comprising:
 an open compartment having a bottom end, an open end, and wherein said open compartment is adapted to apply reagents and remove purified fluid, wherein said open compartment is operable at a substantially constant ambient pressure;   a closed compartment having a closed pressure and having a closed end, a communicating end, said closed compartment having a bottom zone adapted to at least temporarily retain a fraction of said aliquot of fluid, and an upper zone containing an air pocket adapted to generate internal positive and negative pressure;   an intermediate transferring-retaining compartment having an intermediate compartment volume, a first end communicating with said open compartment, and a second end communicating with said closed compartment, said intermediate compartment is adapted to at least temporarily retain a fraction of said aliquot of fluid, wherein said open compartment is reversibly communicating with at least one of said temporary retained fluid fractions;   a flow barrier member, configured between said open and closed compartments and operable with said closed compartment and adapted to inhibit a flow of said aliquot of fluid when said closed pressure is substantially equal to said ambient pressure and to allow said flow of fluid when said closed pressure is substantially different from said ambient pressure, the pressure differential controllable by an external thermo member adapted to alternately change the closed pressure by a cool and a heat cycle, wherein at least one aliquot of fluid is alternately transferrable back and forth into said open compartment in response to changes of the closed pressure;   a capturing zone, having an internal side and an external side definable substantially at either the first or the second end and adapted to allow magnetic particles in the fluid to be attracted to said internal side in response to a magnetic force applied at said external side;   
       wherein said barrier member comprises a restrictive flow zone chosen from the list including: a bore; a capillary tube; and a combination of a bore and a capillary tube. 
     
     
         2 . An apparatus according to  claim 1 , wherein said open compartment is configurable substantially above said closed compartment with said second end positioned within said closed compartment and displaced from said bottom zone wherein said bottom zone is an irreversible waste collection zone, wherein said intermediate compartment volume is adapted to temporarily retain a specified volume of said fluid, and wherein said temporarily retained fluid is adapted to reversibly flow back into said open compartment or irreversibly flow to said waste collection zone, wherein said capturing zone is definable substantially at the first end of said barrier member. 
     
     
         3 . An apparatus according to  claim 1  wherein said closed compartment is configurable substantially above said open compartment and said bottom end is closed, wherein said first end is insertable within said open end and said first end is displaced from said bottom end to allow fluid flow, and wherein said capturing zone is definable at either ends of said barrier member. 
     
     
         4 . An apparatus according to  claim 1 , wherein said open compartment is configurable substantially above said closed compartment, with said second end positioned within said closed compartment, substantially at said closed end, and said capturing zone is definable substantially at either end of said barrier member. 
     
     
         5 . A method for extracting an ingredient from the fluid sample using magnetic particles as an active solid support, comprising the steps of:
 I. providing a thermo member adapted for heating and cooling an air pocket positionable in said closed compartment;   II. providing a magnetic member adapted to switch magnetic force on and off; and   III. providing an apparatus comprising;
 an open compartment having a bottom end, an open end, wherein said open compartment is adapted to apply reagents and remove purified fluid, and wherein said open compartment is operable at a substantially constant ambient pressure; 
 a closed compartment having a closed pressure and having a closed end, a communicating end, said closed compartment having a bottom zone adapted to at least temporarily retain a fraction of said aliquot of fluid, and an upper zone containing an air pocket adapted to generate internal positive and negative pressure; 
 an intermediate transferring-retaining compartment having an intermediate compartment volume, a first end communicating with said open compartment, and a second end communicating with said closed compartment, said intermediate compartment is adapted to at least temporarily retain a fraction of said aliquot of fluid, wherein said open compartment reversibly communicates with at least one of said temporary retained fluid fractions; 
 a flow barrier member, configured between said open and closed compartments and operable with said closed compartment and adapted to inhibit a flow of said aliquot of fluid when said closed pressure is substantially equal to said ambient pressure and to allow said flow of fluid when said closed pressure is substantially different from said ambient pressure, the pressure differential controllable by an external thermo member adapted to alternately change the closed pressure by a cool and a heat cycle, wherein at least one aliquot of fluid is alternately transferrable back and forth said open compartment in response to changes of the closed pressure; 
 a capturing zone, having an internal side and an external side definable substantially at either the first or the second end and adapted to allow magnetic particles in the fluid to be attracted to said internal side in response to a magnetic force applied at the external side; 
 the method further comprising the steps of:
 a. inserting said air pocket zone into the thermal zone of a thermo member; 
 b. charging reagents having magnetic particles and said aliquot of fluid to the open compartment; 
 c. applying at least one mixing cycle; 
 d. applying at least one magnetic capturing step; 
 e applying at least one separation step; 
 
   whereby said mixing cycle comprises a bi directional transfer of at least part of said mixture containing said magnetic particles between compartments of said apparatus, by alternating dP between said open and closed pressure, either by generating positive dP and then negative dP, or negative dP and then positive dP, wherein said positive dP is generated by heating said air pocket to a preferred temperature whereas the pressure in said closed pressure become higher than the open pressure and an aliquot of fluid, be it air, liquid or suspension is forced out from said closed compartment, whereas said negative dP is generated by cooling said air pocket to a preferred temperature, whereby an aliquot of fluid, be it air, liquid or suspension is moved from the open to the closed compartment, wherein said mixing cycle is executed while magnetic force is off, wherein the volume of the fluid transferred is proportional to applied dT and to the volume of said air pocket arrested in the thermal zone;   whereby said magnetic capturing step comprises attracting said magnetic particles to said internal side in response to activation of said external magnetic force, while maintaining said air pocket at substantially constant temperature;   whereby said separation step comprises applying either a positive or negative dP while magnetic force is on, whereby said magnetic particles are retained in the capturing zone and the supernatant, substantially free of said magnetic particles, is removed from said capturing compartment.   
     
     
         6 . The method according to  claim 5 , wherein said active magnetic particles interact with an ingredient in said fluid, said active magnetic particles having at least one characteristic chosen from the list including: chemically active; linked; and coated with a reactive ingredient chosen from the list including: an antibody; an enzyme; an ion exchanger; an absorption reagent; an oligonucleotides; a receptors; and a lectin. 
     
     
         7 . The method according to  claim 6  for negative extraction, by removing an interfering ingredient from said fluid sample and collecting said purified supernatant in an open compartment, comprising the steps of;
 a. inserting said air pocket zone into the thermal zone of a thermo member; 
 b. charging reagents having said magnetic particles and an aliquot of sample to said open compartment, 
 c. applying an extraction cycle comprising a sequence of;
 a mixing cycle; 
 a magnetic capturing step; and 
 a separation step; and 
 
 d. collecting said purified supernatant in said open compartment. 
 
     
     
         8 . The method according to  claim 6  for positive extraction of an ingredient by first extracting the ingredient to magnetic particles and eluting a purified ingredient into said open compartment, comprising;
 I) an extraction cycle; II) a washing cycle; and III) an elution cycle, whereby each of said cycles comprise a sequence of; 
 a. a mixing cycle; 
 b. a capturing step; and 
 c. a separation step, by moving the supernatant to another compartment, while magnetic force is on and said magnetic particles are retained in said capturing zone; 
 whereby the extraction cycle comprises the steps of providing sample and active magnetic particles in said open compartment; and applying a sequence of mixing cycle, capture step, and separation step followed by disposing the supernatant, 
 whereby the wash cycle comprises the steps of:
 providing wash buffer in open compartment and repeating the steps: 
 mixing; capturing; and negative separation, by disposing the supernatant; 
 
 whereby the elution cycle comprises the steps of:
 providing an eluting buffer in said open compartment and repeating the steps of: mixing and capturing; and 
 applying a positive separation step, whereby said purified eluent is collected in said open compartment. 
 
 
     
     
         9 . The method according to  claim 8  for positive extraction of an ingredient, by first extracting the ingredient to magnetic particles and then eluting purified ingredient, comprising the steps of:
 providing an apparatus, wherein said open compartment is configurable substantially above said closed compartment with said second end positioned within said closed compartment and displaced from said bottom zone wherein said bottom zone is an irreversibly waste collection zone, where said intermediate compartment volume is adapted to temporarily retain a specified volume of said fluid and wherein said temporarily retain fluid is adapted to reversible flow back into said open compartment or irreversibly to waste collection zone, 
 wherein said capturing zone is substantially definable at said first end, the method comprising the steps of: 
 a. inserting said air pocket of said closed compartment into the thermo zone of said thermo member at a temperature T1; 
 b. charging sample, reagents, magnetic particles into said open compartment; 
 c. applying a moderate mixing cycle wherein said fluid alternates between said open and intermediate compartments; 
 d. applying a capture step at substantially the capturing zone, 
 e. applying a negative separation step by cooling to a temperature T2 while magnetic force is active, whereby the supernatant is forced into said waste zone; 
 f. applying a wash cycle by deactivating said magnetic force and adding a wash buffer; and 
 g repeating said moderate mixing cycle, capturing step, and negative separation step, whereby the washing buffer is forced into said waste zone, 
 h. applying an elution cycle by deactivating said magnetic force and adding an elution buffer; 
 i. repeating said moderate mixing cycle and capturing steps; 
 j. removing purified eluant from said open compartment, using a pipette, while said magnetic force is active. 
 
     
     
         10 . The method according to  claim 7  for negative extraction by removing an interfering ingredient from a fluid sample and collecting the purified supernatant in said open compartment, comprising:
 providing an apparatus, wherein said closed compartment is configurable substantially above said open compartment and said bottom end is closed, wherein said first end is insertable within said open end and said first end is displaced from said bottom end to allow fluid flow, wherein said capturing zone is positioned substantially at the second ends of said barrier member, said method comprising the steps of:
 a. applying regent to said open compartment; 
 b. inserting said air pocket into said thermo zone and inserting said first end into said open end; 
 c. applying a mix cycle; 
 d. applying a negative dP to move the mixture to said upper compartment; 
 e. applying a capture step at said upper capture zone; 
 f. applying a positive separation step by applying heating; and 
 g. collecting a purified fluid in said open compartment. 
 
 
     
     
         11 . The method according to  claim 10  for negative extraction by removing an interfering ingredient from said fluid sample and by collecting the purified supernatant in said open compartment, comprising:
 providing an apparatus wherein said closed compartment is configurable substantially above said open compartment and said bottom end is closed, wherein said first end is insertable within said open end and said first end is displaced from said bottom end to allow fluid flow, wherein said capturing zone is positioned substantially at the first end of said barrier member, the method comprise the steps
 a. applying a negative mix cycle; 
 b. applying a capture step substantially at said first end; 
 c. applying a separation step, while magnet force is on; 
 d. switching off the magnetic force and replacing said open compartment containing magnetic particles with a new open compartment; and 
 e. applying positive dP to move purified fluid from said closed to said open compartment. 
 
 
     
     
         12 . The method according to  claim 10  for positive extraction of an ingredient, comprising the steps of:
 providing an apparatus wherein said closed compartment is configurable substantially above said open compartment and said bottom end is closed, wherein said first end is insertable within said open end and said first end is displaced from said bottom end to allow fluid flow, wherein said capturing zone is positioned at the second ends of said barrier member, 
 the method comprising the steps:
 a. applying a mix cycle; 
 b. applying negative dP to move fluid from said open to said closed compartment; 
 c. applying a capture step substantially at said second end; 
 d. applying a negative separation step by collecting and disposing the supernatant in said open compartment; 
 e. replacing said open compartment with a tube containing wash buffer; 
 f. repeating steps a to d; 
 g. replacing said open compartment with an elution buffer compartment; 
 h. repeating said negative mix cycle and said capture step; and 
 i. applying a positive separation and collecting a purified fluid in said open compartment. 
 
 
     
     
         13 . A method according to  claim 7  for negative extraction of an interfering ingredient from a fluid sample and collecting purified fluid in an open compartment, comprising:
 providing an apparatus wherein said open compartment is configurable substantially above said closed compartment with said second end positioned within said closed compartment, substantially at said closed end, and wherein said air pocket is positionable at the upper part of said closed compartment, wherein said capturing zone is positioned substantially at said closed end, the method comprises:
 a. charging said sample and said magnetic particles into said open compartment; 
 b. applying a mix cycle; 
 c. applying a capture step at said capturing zone; 
 d. applying a separation step; and 
 e. collecting purified fluid in said open compartment. 
 
 
     
     
         14 . The method according to  claim 8  for testing for the presence of an analyte in a fluid sample and obtaining a quantitative or semi-quantitative signal, the method further comprising;
 a a first positive extraction cycle to extract an ingredient to magnetic particles 
 b a wash cycle to remove impurities 
 c charging an ingredient capable of interacting with said first extracted ingredient, and 
 d applying a second extraction cycle and a second wash cycle; and 
 e charging a substrate capable of interacting with said second extracted ingredient, and 
 f applying a mixing cycle and a capture step, and 
 g reading said signal. 
 
     
     
         15 . A method according to  claim 14  for testing for the presence of an analyte in a fluid sample and obtaining a quantitative or semi-quantitative signal, the method further comprising:
 providing an apparatus, wherein said open compartment is configurable substantially above said closed compartment with said second end positioned within said closed compartment and displaced from said bottom zone wherein fluid in said bottom zone is an irreversible waste collection zone, wherein said intermediate compartment volume is adapted to temporarily retain a specified volume of said fluid and wherein said temporarily retain fluid is adapted to reversibly flow back into said open compartment or to said waste collection zone, wherein said capturing zone is substantially applied at said first end, the method comprising the steps of:
 a. inserting said air pocket into said thermo zone of the thermo member at T1; 
 b. charging said sample, reagents, and magnetic particles into said open compartment; 
 c. applying a first extraction step by moderate mixing cycle wherein the fluid alternates between said open and intermediate compartments; 
 d. applying a capture step at substantially said capturing zone; 
 e. applying a negative separation step by cooling to T2 while magnetic force is active, whereby the supernatant is forced to said waste zone; 
 f. applying a wash cycle by deactivating said magnetic force and adding a wash buffer; 
 g. repeating steps of moderate mixing cycle, capture step and negative separation step, whereby said washing buffer is forced to said waste zone; 
 h. charging a secondary reagent adapted to interact with said first extracted ingredient; 
 i. repeating moderate mix cycle, capture step, and negative separation step; 
 j. applying a wash cycle by; deactivating said magnetic force; adding a wash buffer; and applying a wash cycle; 
 k. charging a substrate; and; 
 l. repeating said moderate mixing cycle and capture step; and 
 m. reading said signal either in said open compartment or by aspirating said supernatant into a new compartment and reading.