Method and apparatus for high-throughput biological-activity screening of cells and/or compounds

• US 7,699,969 B2
• Filed: 05/02/2002
• Issued: 04/20/2010
• Est. Priority Date: 05/02/2001
• Status: Active Grant

• Current Assignee: Menarini Silicon Biosystems S.P.A
• Original Assignee: Silicon Biosystems, S.p.A.
• Inventors: Altomare, Luigi, Tartagni, Marco, Medoro, Gianni, Guerrieri, Roberto, Manaresi, Nicol
• Primary Examiner(s): NOGUEROLA, ALEXANDER STEPHAN
• Application Number:
  US10/476,467
• Publication Number:
  US 20040191789A1
• Time in Patent Office: 2,910 Days
• Field of Search: 204/164, 204/547, 204/554, 204/643, 204/660
• US Class Current: 204/547
• CPC Class Codes: G01N 33/54366 Apparatus specially adapted for solid-phase testing
• CPC Class Codes: G01N 33/561 Immunoelectrophoresis

Abstract

First entities consisting in cells or microorganisms (BIO) and second entities consisting in compounds or compound units, carried typically by microbeads (BEAD), are trapped selectively within closed movable potential cages (S1) by means of dielectrophoretic force generated by mutually opposed electrodes (M1, M2). The cages are set in relative motion so as to bring about the interaction of selected first and second entities, causing the cages containing them to fuse, whereupon results are obtained preferably by reinstating the original cages and/or observing previously empty adjacent cages. The procedure takes place in a device (DE) with two separate chambers (F, FL) connected one to the other by way of a narrow passage (D) and finished with respective selectively controllable inlets and outlets (I1, I2; O1, O2) through which a liquid or semi-liquid buffer (L) can be pumped in or out.

Claims

1. A method of conducting tests and assays of high throughput and high biological value on a sample containing chemical/biological material consisting of unknown entities characterized in that it comprises the steps of:

(a)—

introducing the sample including said unknown entities into a first chamber of a testing device comprising at least one array of first selectively addressable and energizable electrodes and at least one second electrode positioned opposite and facing the first electrodes;

(b)—

introducing chemical/biological material into a first chamber of said testing device, consisting of known entities identifiable internally of said testing device and having a presumed affinity with the unknown entities;

(c)—

selectively creating closed movable dielectrophoretic potential first and second cages within said first chamber generated by said first electrodes and second electrode and trapping at least a part of said unknown entities within the first moving cages and at least a part of said known entities within the second moving cages;

(d)—

moving at least one of said second movable cages containing the known entities toward said first movable cages containing said unknown entities and causing at least one unknown entity to interact with at least one known entity of at least a first type by bringing about the fusion of at least one first movable cage and one second movable cage containing the relative entities;

(e)—

verifying the creation or otherwise of a stable bond between said at least one unknown entity and said at least one known entity of the first type for determining whether an affinity exists between the two and consequently identifying said at least one unknown entity.

2. A method as in claim 1, wherein the entities introduced into the first chamber of the testing device are known entities of a plurality of different types, identifiable internally of the device, and the moving and verification steps (d) and (e) are repeated for each of said unknown entities not bound stably to a known entity of the first type, selectively causing a known entity of a type different to the first type to interact with each unknown entity until all the types of known entity introduced have been eliminated.

3. A method as in claim 1 comprising the further step, performed before the moving step (d), of identifying the moving cages containing said unknown entities.

4. A method as in claim 3 wherein the identification step is performed with the aid of sensors located internally or externally of said testing device or performed directly on said unknown entities before their introduction into said testing device.

5. A method as in claim 1 wherein said known entities are compound units consisting in microbeads or liposomes carrying a compound selected from a group including at least one antibody for a specific antigen and at least one ligand for a specific receptor and at least one DNA or RNA probe and combinations thereof.

6. A method as in claim 5 wherein said known entities are identified internally of the testing device on the basis of at least one physical characteristic detectable by way of a sensor located internally or externally of said device.

7. A method as in claim 6, wherein said at least one physical characteristic is selected from a group including colour, fluorescence, dielectric constant and combinations thereof.

8. A method as in claim 1 wherein said known entities are introduced into the first chamber of the testing device together with said unknown entities.

9. A method as in claim 8, comprising the steps of creating a mixture of said known entities and said unknown entities externally of said first chamber and thereupon introducing said mixture into the first chamber.

10. A method as in claim 1 wherein said known entities and said unknown entities are introduced into the first chamber separately and sequentially.

11. A method as in claim 10, wherein said known entities are identified internally of the test by moving the movable cages in which they are trapped to predetermined positions corresponding to predetermined said first electrodes of the array and memorizing the selfsame positions.

12. A method as in claim 1 comprising the further step of counting the number of unknown entities identified on the basis of their binding stably to the known entities.

13. A method as in claim 1 comprising the further steps of:

moving the movable cages in such a way as to transfer only unknown entities bound stably to known entities of a single homogeneous type, and therefore identifiable as being of only a single homogeneous type, into a second chamber of the device communicating with the first chamber by way of a narrow passage;

flushing the entities occupying said second chamber out of the device;

repeating the moving and flushing steps for all other homogeneous types of unknown entities identified, in sequence.

14. A method as in claim 13 comprising the further steps, performed internally of said second chamber and prior to the flushing step, of separating the identified unknown entities from said known entities of a single homogeneous type bound stably to them, and returning the separated known entities to the first chamber by means of the said movable cages.

15. A method as in claim 14 wherein said separating step is accomplished through the agency of dielectrophoretic force, varying one of the operating parameters of the testing device, namely the strength of the electric field between selected electrodes, the frequency of the electric field between selected electrodes, and combinations thereof.

16. A method as in claim 1, wherein said verification step (e) is performed by reinstating a pair of movable cages fused previously and establishing by means of sensors located internally or externally of the testing device whether said entities are present in both movable cages of the pair or in one only.

17. A method as in claim 16 wherein the sensors employed are optical or capacitive.

18. A method as in claim 16 comprising the further step of checking the stable binding force between said at least one unknown entity and said at least one known entity accomplished by varying one of the operating parameters of the testing device namely the amplitude of the voltage between selected electrodes, the frequency of the voltage between selected electrodes, and combinations thereof.

19. A method as in claim 1, wherein said unknown entities are selected from a group including cells, viruses, microorganisms, and compound units composed of cells and/or microbeads and/or liposomes bearing target DNA and/or RNA, used in conducting hybridization tests.

20. A method of conducting tests and assays of high throughput and high biological value between a plurality of first entities selected from a group consisting of cells and microorganisms, and a plurality of second entities consisting of compounds or compound units to be tested for their biological activity in respect of said first entities;

characterized in that it comprises the steps of;

(a)—

introducing said first and said second entities into a first chamber of a testing device comprising at least one array of first selectively addressable and energizable electrodes and at least one second electrode positioned opposite and facing the first electrodes;

(b)—

selectively creating closed movable potential cages internally of the first chamber by means of dielectrophoretic force generated by said first electrodes and second electrode and trapping at least a part of the entities within the movable cages;

(c)—

moving at least one of movable cages containing the said first entities toward movable cages containing the said second entities and causing at least one first entity to interact with at least one second entity of at least a first type by bringing about the fusion of at least one pair of movable cages containing the relative entities;

(d)—

verifying the biological activity of the second entity on the first entity by analyzing the resulting interaction utilizing sensors capable of detecting any evidence in the first entity of at least one of a selected group of effects, namely cytostatic, cytotoxic, mitotic, and expression of a marker.

21. A method as in claim 20 wherein said cytostatic, cytotoxic and mitotic effects are detected by verifying the presence and/or the changed presence of said first entities in the movable cages created around them and/or in proximity to a plurality of first electrodes positioned immediately adjacent to said movable cages that contain the first entities and, prior to the execution of the moving step (c), left vacant or occupied by empty movable cages.

22. A method as in claim 20 wherein said sensors are selected from a group including optical sensors, located internally or externally of the testing device, capacitive sensors, and combinations thereof.

23. A method as in claim 20, comprising the further step of recognizing at least one plurality of entities selected from a group consisting of said first entities and said second entities.

24. A method as in claim 23 wherein the recognition step is conducted before or after said step of introducing the entities into the testing device.

25. A method as in claim 24 wherein said recognition step is conducted by means of sensors selected from a group including optical type sensors located internally or externally of the device, and capacitive type sensors.

26. A method as in claim 25 wherein said recognition step is conducted internally of said device on the basis of the response of the entities of at least one said plurality of entities to the dielectrophoretic force generated internally of the device.

27. A method as in claim 23 comprising the step, conducted prior to the moving and verification steps (c) and (d), of utilizing said movable cages to establish a predetermined spatial distribution within the device of said at least one plurality of entities of at least one identified type.

28. A method as in claim 20, wherein each of the movable cages trapping said plurality of first entities contains at least one single said first entity.

29. A method as in claim 28, wherein at least a part of the first entities is trapped in a movable dielectrophoretic potential cage having associated with it a sensor capable of generating a signal proportional to the number of first entities present in the cage.

30. A method as in claim 20, wherein said marker is a reporter molecule expressed internally of the first entity.

31. A method as in claim 30 wherein said compound units are selected from a group including microbeads, liposomes, and cells carrying a compound designed to activate the expression of said reporter molecule.

32. A method as in claim 31 wherein said compound is introduced into said first entity during the interaction step.

33. A method as in claim 31 wherein said compound activates the expression of said reporter molecule through a ligand-receptor type mechanism of interaction.

34. A multifunctional testing device (DL) composed of a first module (C) comprising an array (M1) of first electrodes (LIJ) singly and selectively addressable and energizable at least in part, arranged on an insulating support (A1);

a second module comprising at least one second electrode (M2) positioned opposite and facing the first electrodes (LIJ) and an upper supporting structure (A2);

also a spacer element (A3) disposed between the first and the second module and delimiting a liquid or semi-liquid environment (L) during operation, characterized in that the spacer element (A3) is embodied in such a manner as to establish at least one first chamber (F) and at least one second chamber (FL) internally of the device, interconnected hydraulically by at least one narrow passage (D) and delimiting the liquid or semi-liquid environment (L), which is thus divided by the at least one narrow passage (D) into at least two partial environments uninfluenced hydraulically one by the another and coinciding with the at least two chambers (F and FL).

35. A device as in claim 34 wherein the first and second chambers (F and FL) are furnished with selectively and controlledly openable orifices functioning as respective inlets (I1,12) and outlets (O1,O2), and said array (M1) of first electrodes (LIJ) functions as the bottom of said chambers (F and FL) and of the at least one said narrow passage (D) allowing communication between the chambers.

36. A device as in claim 34, wherein said array (M1) of first electrodes (LIJ) is designed to operate together with said second electrode in generating a plurality of dielectrophoretic cages (S1) by means of which to manipulate a biological sample (BIO).

37. A device as in claim 34 wherein said first module (C) is furnished with at least one integrated sensor positioned beneath or in close proximity to at least one of the first electrodes.

38. A device as in claim 37 wherein said at least one sensor is positioned to coincide with said at least one narrow passage (D).

39. A device as in claim 37 wherein said at least one sensor is of optical or capacitive type.

40. A method of transporting first entities consisting in compounds or compound units into second entities, comprising the steps of:

(a)—

introducing the first and second entities into a chamber of a testing device comprising at least one array of first selectively addressable and energizable electrodes and at least one second electrode positioned opposite and facing the first electrodes;

(b)—

selectively creating closed movable potential cages internally of said chamber by means of dielectrophoretic force generated by said electrodes and trapping at least a part of said first entities within first movable cages and of said second entities within second movable cages;

(c)—

identifying and selecting the cages containing first entities;

(d)—

moving at least one of said movable cages containing the first entities toward said movable cages containing the second entities;

(e)—

causing at least one movable cage containing a first entity to fuse with a movable cage containing a second entity, characterized in that said first and second entities and said step of fusing at least one movable cage containing a first entity with a movable cage containing a second entity are such that the first entities are caused to penetrate into the second entities when brought into physical contact with said second entities.

41. A method as in claim 40, wherein said second entities are selected from a group including cells, microorganisms, liposomes, microbeads and the like, and trapped in movable cages.

42. A method as in claim 41, wherein said second entities consisting in cells or microorganisms are fixed to respective microbeads and/or carried on the surface or internally of liposomes.

43. A method as in claim 40, wherein said first entities consisting in said compounds or said compound units are carried directly by cells and/or microbeads and/or liposomes, or by vector designed to penetrate within said second entities once brought into physical contact with the selfsame second entities, the vector in turn being transportable by cells and/or microbeads and/or liposomes.