Automatic electrophoresis apparatus and method

• US 4,810,348 A
• Filed: 03/16/1987
• Issued: 03/07/1989
• Est. Priority Date: 03/16/1987
• Status: Expired due to Term

• Current Assignee: Helena Laboratories Corporation
• Original Assignee: Helena Laboratories Corporation
• Inventors: Guadagno, Philip A., Sarrine, Robert J., Kelley, Charles D., Garsee, Henry A.
• Primary Examiner(s): Niebling, John F.
• Assistant Examiner(s): STARSIAK, JOHN S
• Application Number:
  US07/026,465
• Time in Patent Office: 722 Days
• Field of Search: 204/182.8, 204/299 R, 436/43, 436/50
• US Class Current: 204/608
• CPC Class Codes: G01N 27/44721 by optical means
• CPC Class Codes: G01N 27/44782 of a plurality of samples

Abstract

An electrophoresis machine is disclosed in which after a microporous support strip and a sample plate are placed in the machine, all electrophoresis processing, scanning and densitometer functions are automatically performed under computer control. The machine includes apparatus for automatic pipetting of liquid samples from the sample plate to the surface of the support strip, apparatus for applying electrophoresis current ot the strip while simultaneously cooling it, apparatus for applying and spreading flourescent staining reagent to the strip, and apparatus for incubating the strip and subsequently drying it. A T.V. camera, mounted to a flourescent scanning box enclosing the application plate, in cooperation with digital processing equipment, electronically scans electrophoresically longitudinally displaced components of the samples for determining relative component densities. An alternative scanning apparatus including a collimator and photomultiplier is also disclosed.

Claims

1. Electrophoresis apparatus comprising

a base,

a support medium including an electrically non-conductive backing having an electrophoresis media thereon and having a reservoir strip of electrically conductive material in communication with said media at each of its longitudinal ends and having at least one sample well on said media between said reservoir strips,

an application plate horizontally disposed on said base and having means for removably securing said support medium to said plate,

a first pair of electrode posts disposed on opposite lateral sides of said support medium and laterally aligned with one of said reservoir strips of said support medium,

a second pair of electrode posts disposed on opposite lateral sides of said microporous support medium and laterally aligned with the other of said reservoir strips of said support medium,

a first electrode bar disposed laterally across said support medium in electrical contact with said first pair of electrode posts and with said one of said reservoir strips,

a second electrode bar disposed laterally across said support medium in electrical contact with said second pair of electrode posts and with said other of said reservoir strips of said support medium,

means for applying a voltage source between said first pair of electrode posts and said second pair of electrode posts, whereby electrical current is passed longitudinally through said support medium substantially uniformly across its lateral extent.

2. Electrophoresis apparatus comprising

a base,

an application plate longitudinally disposed on said base,

an electrophoresis support medium removably disposed on said application plate having longitudinal and lateral dimensions, said support medium including an electrically non-conductive backing having electrophoresis media placed thereon and having first and second electrically conducting reservoir strips disposed laterally at each of the longitudinal ends of the support medium, said electrophoresis media being of substantially uniform thickness forming a planar surface atop said backing and said laterally disposed reservoir strips extending vertically above said planar surface, and

first and second electrodes adapted for connection to opposite polarities of a source of electric potential and adapted for lateral placement across said support medium respectively in electrical contact with said first reservoir strip and with said second reservoir strip.

4. The apparatus of claim 2 wherein each of said first and second electrode means respectively includes a lateral member adapted for alternative placement between an open position thereby allowing manual placement of said support medium on said application plate, and a closed position in electrical contact with said reservoir strip, thereby placing said support medium in circuit with said source of electric potential.

3. Electrophoresis apparatus comprising

a base,

an application plate longitudinally disposed on said base,

an electrophoresis support medium removably disposed on said application plate having longitudinal and lateral dimensions with raised first and second lateral reservoir strips of electrically conductive material at each longitudinal end of said medium,

first and second electrode means adapted for connection to opposite polarities of a source of electric potential and adapted for lateral placement across said support medium respectively in electrical contact with said first reservoir strip and with said second reservoir strip,

wherein each of said first and second electrode means respectively includes,

an electrode pair disposed on opposite lateral sides of said reservoir strips, and

an electrode bar adapted for lateral removable placement across said support medium in electrical contact with said reservoir strip and said electrode pair.

5. The apparatus of claim 3 further comprising,

robotic means for applying a quantity of staining reagent to the surface of said electrophoresis medium, and

robotic means for moving said first electrode bar across the surface of said support medium between said reservoir strips.

6. The apparatus of claim 3 further comprising, robotic means for applying a quantity of staining reagent to the surface of said electrophoresis media, and

robotic means for moving said first electrode bar from said first reservoir strip across the surface of said support medium and for returning said first electrode bar across the surface of said support medium to said first reservoir strip and said first electrode pair.

7. The apparatus of claim 3 further comprising,

robotic means for applying a quantity of staining reagent to the surface of said electrophoresis media, and

robotic means for moving said first and second electrode bars across the surface of said support medium between said reservoir strips.

8. The apparatus of claim 3 wherein said electrophoresis support medium includes an application well on its surface, said apparatus further comprising,

robotic means for applying a liquid sample to said well of said support medium, and

means for connecting said electrical potential source between said first and second electrode means whereby electrophoresis electrical current is passed longitudinally through said support medium substantially uniformly across its lateral extent.

9. The apparatus of claim 3 further comprising,

robotic means for applying a quantity of staining reagent to the surface of said electrophoresis medium, and

robotic means for moving said first electrode bar across the surface of said support medium between said reservoir strips.

10. The apparatus of claim 8 further comprising,

thermo-electric transfer means disposed in thermal contact with said application plate, and

means for simultaneously applying an electric current to said thermo electric transfer means,

whereby at least a portion of the heat resulting from said electrophoresis current in said support medium and said application plate is removed by said transfer means.

11. The apparatus of claim 10 wherein said thermo-electric transfer means comprises,

a Peltier device affixed to said application plate,

means for applying current through said Peltier device in a first direction so as to cool said application plate,

duct means disposed below said application plate defining an air path between an inlet and an outlet in said base,

a thermal heat sink in thermal communication with said Peltier device and disposed in said air path defined by said duct means, and

fan means for forcing air from said inlet to said outlet across said thermal heat sink.

12. The apparatus of claim 11 further comprising,

longitudinal members disposed on lateral sides of said application plate and extending vertically above said first and second electrode bars, said longitudinal members having longitudinal slots above said first and second electrode bars, and

a cover plate disposed in said longitudinal slots of said longitudinal members and being longitudinally movable within said slots between a closed position wherein an incubation and drying enclosure is formed about said support medium and said application plate and an open position wherein said cover plate is longitudinally removed from above said support medium.

13. The apparatus of claim 12 further comprising robotic means for moving said cover plate between said closed position and said open position.

14. The apparatus of claim 13 including incubation heater means disposed in thermal contact with said application plate for heating said application plate and said support medium.

15. The apparatus of claim 14 wherein said incubation heater means includes,

means for applying current through said Peltier device in a direction opposite to said first direction.

16. The apparatus of claim 14 further comprising

means for controlling said heater means to apply heat to said application plate and support medium after:

a liquid sample has been applied to said well of said support medium,

said electrophoresis current has been passed longitudinally through said medium,

said reagent has been applied and spread across said surface of said support medium, and

said cover plate means has been returned to its closed position,

said means for controlling said heater means enabling heat application for a predetermined incubation period.

17. The apparatus of claim 14 further comprising

dryer means for applying drying air to said support medium.

18. The apparatus of claim 17 wherein said dryer means includes,

an inlet slot disposed through a first longitudinal side of said application plate,

an outlet slot disposed through a second longitudinal side of said application plate,

inlet duct means defining an inlet air path in communication with said inlet slot,

outlet duct means defining an outlet air path in communication with said outlet slot, and

fan means for forcing air through said inlet duct and said inlet slot across said support medium and out through said outlet slot and said outlet duct.

19. The apparatus of claim 18 further comprising,

heating element means disposed in said inlet duct means for heating air in said inlet duct.

20. The apparatus of claim 16 further comprising,

drying means for applying drying air to said support medium, and

means for controlling said dryer means to apply drying air to said support medium after said heater means has applied heat to said support member for said incubation period,

said means for controlling said dryer means operably enabling drying air application to said support medium for a predetermined drying period.

24. The apparatus of claim 8 wherein said robotic means for applying a liquid sample to said well of said support means includes,

a sample plate having at least one liquid sample well, said sample plate being longitudinally disposed on said base and separated from said application plate,

track means secured to said base,

robotic frame means supported by said track means for longitudinally moving between said sample plate and said application plate,

pipette assembly means secured to said robotic frame means and disposed above said sample plate and said application plate for withdrawing a liquid sample from said liquid sample well when said frame means is longitudinally positioned at said application plate and for applying said liquid sample to said support means when said frame means is longitudinally positioned at said application plate.

25. the apparatus of claim 5 wherein said robotic means for applying a quantity of staining reagent to the surface of said electrophoresis medium includes,

track means secured to said base,

robotic frame means supported by said track means for longitudinally moving between a position longitudinally separated from said application plate and said application plate,

a bottle support member angularly rotatable about a lateral shaft bearingly supported by said frame means,

at least one reagent bottle removably fixed to said bottle support member, said bottle adapted to carry a quantity of staining reagent, and

means for rotating said bottle support member about said lateral shaft when said bottle support member on said frame means is longitudinally positioned above the surface of said electrophoresis media,

whereby staining reagent carried by said bottle is dumped to the surface of said electrophoresis medium.

26. The apparatus of claim 6 wherein said robotic means for moving said first electrode bar across the surface of said support medium between said reservoir strips includes,

track means secured to said base,

robotic frame means supported by said track means for longitudinally moving between a position longitudinally separated from said application plate,

a solenoid secured to said robotic frame means,

said solenoid having an electrically actuated plunger movable between an upper position and a lower position,

said plunger having a downwardly facing fork structure at its lower end, and

means for moving said robotic frame means until said plunger is above said first electrode bar for actuating said solenoid causing said plunger to move downwardly to its lower position until said fork of said plunger is about said first electrode bar, and while said plunger is in its lower position for moving said robotic frame toward said second electrode bar and returning said first electrode bar to said first electrode means, thereby operably spreading said staining reagent across the surface of said support medium.

27. The apparatus of claim 13 wherein said robotic means for moving said cover plate between said closed position and said open position includes,

at least one hole formed at a longitudinal end of said cover plate,

track means secured to said base,

robotic frame means supported by said track means for longitudinally moving between a position longitudinally separated from said application plate,

at least one solenoid secured to said robotic frame means,

said solenoid having an electrically actuated plunger movable between an upper position and a lower position, said plunger being laterally aligned with said hole of said cover plate, and

means for moving said robotic frame means until said plunger is longitudinally aligned above said hole in the cover plate, for actuating said solenoid operably causing said plunger to move downwardly to its lower position until said plunger engages said hole, and while said plunger is in its lower position for moving said robotic frame means between said closed position and said open position.

21. Electrophoresis apparatus comprising

a base,

an application plate longitudinally disposed on said base,

an electrophoresis support medium removably disposed on said application plate having longitudinal and lateral dimensions with raised first and second lateral reservoir strips of electrically conductive material at each longitudinal end of said medium,

first and second electrode means adapted for connection to opposite polarities of a source of electric potential and adapted for lateral placement across said support medium respectively in electrical contact with said first reservoir strip and with said second reservoir strip,

wherein

said first and second electrode means include respectively a first pair and a second pair of magnetized posts extending vertically above said application plate, and first and second electrode bars having end portions fabricated of ferro-magnetic material,

said first bar being held in electrical contact at its ends with said pair of magnetized posts by the force of magnetism of said second pair of posts with said ferro-magnetic material of said first bar,

said second bar being held in electrical contact at its ends with said second pair of magnetized posts by the force of magnetism of said second pair of posts with said ferro-magnetic material of said second bar.

22. The apparatus of claim 21 wherein

said first and second electrode bars have a middle portion constructed of electrically conducting stainless steel.

23. The apparatus of claim 21 wherein

said first and second electrode bars have a middle portion constructed of electrically conducting graphite.

28. Electrophoresis apparatus comprising,

a base,

an application plate longitudinally disposed on said base and having top and bottom surfaces,

an electrophoresis support medium removably secured to said top surface of said application plate and adapted to accept a liquid sample, said support medium including an application well on its surface, said support medium having longitudinal and lateral dimensions,

robotic means for applying a liquid sample to said well of said support means including,

a sample plate having at least one liquid sample well, said sample plate being longitudinally disposed on said base and separated from said application plate,

track means secured to said base,

robotic frame means supported by said track means for longitudinally moving between said sample plate and said application plate,

pipette assembly means secured to said robotic frame means and disposed above said sample plate and said application plate for withdrawing a liquid sample from said liquid sample well when said frame means is longitudinally positioned at said application plate and for applying said liquid sample to said support means when said frame means is longitudinally positioned at said application plate, and

means for longitudinally applying an electrophoresis current to said support medium substantially uniformly across its lateral extent operably creating an electrophoretically longitudinally displaced pattern of components of said liquid sample.

29. The apparatus of claim 28 further comprising

thermo-electric heat transfer means disposed in thermal contact with the bottom surface of said application plate, and

means for applying an electric current in a first direction to said thermo electric heat transfer means during application of said electrophoresis current, whereby a portion of the heat resulting from said electrophoresis current in said support medium and said application plate is removed by said heat transfer means.

32. The apparatus of claim 29 further comprising

robotic means for applying staining reagent to said support medium in situ after electrophoresis current has been applied to said medium, and

means for incubating said support medium in situ including means for applying electric current in a second direction to said thermo electric heat transfer means whereby heat is applied to said application plate by said thermo electric heat transfer means.

30. Electrophoresis apparatus comprising,

a base,

an application plate longitudinally disposed on said base,

an electrophoresis support medium removably secured to said application plate and adapted to accept a liquid sample, said support medium including an application well on its surface, said support medium having longitudinal and lateral dimensions,

robotic means for applying a liquid sample to said application well on said support medium in situ,

means for longitudinally applying an electrophoresis current to said support medium substantially uniformly across its lateral extent operably creating an electrophoretically longitudinally displaced pattern of components of said liquid sample, and

robotic means for applying staining reagent to said support medium in situ after electrophoresis current has been applied to said medium.

31. The apparatus of claim 30 further including

means for incubating said support medium in situ after staining reagent has been applied to said medium.

33. The apparatus of claim 30 further comprising

means for drying said support medium in situ after said support medium and its staining reagent has been incubated.

34. The apparatus of claim 33 further including

means for electronic scanning said support medium in situ after said support medium has been dried.

35. Electrophoresis apparatus comprising,

a base,

an application plate longitudinally disposed on said base and having top and bottom surface,

an electrophoresis support medium removably secured to said top surface of said application plate and adapted to accept a liquid sample, said support medium including an application well on its surface, said support medium having longitudinal and lateral dimensions,

robotic means for applying a liquid sample to said application well on said support medium in situ,

means for longitudinally applying an electrophoresis current to said support medium substantially uniformly across its lateral extent operably creating an electrophoretically longitudinally displaced pattern of components of said liquid sample,

means for staining, incubating and drying said support medium in situ,

means for generating an electronic image of the top surface of said support medium in situ, and

processing means for creating an electronic template about said electronic image of said longitudinally displaced pattern of components of said liquid sample.

36. The apparatus of claim 35 further comprising

processing means for determining from said electronic image within said template a signal representative of the density, as a function of longitudinal displacement of components of said liquid sample.

37. Electrophoresis apparatus comprising

a base,

an application plate longitudinally disposed on said base,

an electrophoresis support medium removably disposed on said application plate,

a sample plate having at least one liquid sample well, said sample plate being longitudinally disposed on said base and separated from said application plate,

track means secured to said base and extending longitudinally between said application plate and said sample plate,

a robotic frame longitudinally disposed on said track means for movement along a longitudinal path between said sample plate and said application plate, said frame having a lateral profile,

robotic pipetting means disposed on said robotic frame for applying a liquid sample from said liquid sample well of said sample plate to said electrophoresis support medium,

means for applying an electrophoresis current longitudinally along said support medium, operably longitudinally separating components of said sample,

automatic means for flourescent staining said longitudinally separated components of said sample,

a scanning box supported by said frame and disposed about said application plate, said box having a back wall, a side wall, an entry wall, a top wall and a front opening,

said front opening including closing means having an open position for allowing placement of said support medium on said application plate and having a closed position for substantially blocking external light from said scanning box,

said entry wall having a opening defining an opening profile, said opening allowing translation of said pipetting means between said sample plate and said application plate, said opening profile being slightly larger than said robotic frame lateral frame profile whereby when said frame is in said opening, light from outside said scanning box is substantially blocked from entry into said box,

flourescent light means secured within said scanning box for flourescently lighting said support medium, and

scanning means secured to said scanning box for determining characteristics of the longitudinal separation of said components of said liquid sample.

38. The apparatus of claim 37 wherein said scanning means includes

imaging device means directed toward said longitudinally separated components of said liquid sample for generating a video signal representative of at least a portion of said sample plate including said separated components,

processing means responsive to said video signal for determining the lateral separation and corresponding density of said longitudinal separation of said components.

39. Electrophoresis apparatus comprising

a base,

an application plate longitudinally disposed on said base,

an electrophoresis support medium removably disposed on said application plate,

a sample plate having at least one liquid sample well, said sample plate being longitudinally disposed on said base and separated from said application plate,

track means secured to said base and extending longitudinally between said application plate and said sample plate,

a robotic frame longitudinally disposed on said track means for movement along a longitudinal path between said sample plate and said application plate,

robotic pipetting means disposed on said robotic frame for applying a liquid sample from said liquid sample well of said sample plate to said electrophoresis support medium,

means for applying an electrophoresis current longitudinally along said support medium, operably longitudinally separating components of said sample,

automatic means for flourescent staining said longitudinally separated components of said sample,

scanning means disposed on said robotic frame for generating electrical signals representative of the intensity of longitudinally separated components of said sample, and

processing means responsive to said electrical signals for determining the lateral separation and corresponding density of said longitudinally separated components.

40. The apparatus of claim 39 wherein said scanning means includes,

a cover having a lateral slit provided therein, said cover facing downwardly toward said support medium,

a flourescent tube disposed inside said cover for operably illuminating said support medium during scanning,

a collimator disposed within said lateral slit and having two ends with one end facing said support medium, and

a photomultiplier tube disposed adjacent the other end of said collimator, said tube operably generating electrical signals representative of the intensity of light collimated through said collimator.