Electrostatic device and method for immunological detection

• US 6,500,673 B1
• Filed: 08/23/2001
• Issued: 12/31/2002
• Est. Priority Date: 07/28/1998
• Status: Expired due to Term

• Current Assignee: Jacques Toledano
• Original Assignee: Jacques Toledano
• Inventors: Toledano, Jacques
• Primary Examiner(s): Le, Long V.
• Assistant Examiner(s): Counts, Gary W.
• Application Number:
  US09/744,578
• Time in Patent Office: 495 Days
• Field of Search: 422/58, 422/59, 422/68.1, 422/186.26, 422/101, 422/102, 435/7.1, 435/283.2, 436/517, 436/514, 436/518, 204/164, 204/193, 204/415, 204/435, 204/660, 204/665, 204/670, 204/672, 204/674, 204/450, 204/456, 204/403, 204/252, 205/701
• US Class Current: 436/514
• CPC Class Codes: G01N 33/521 Single-layer analytical elements
• CPC Class Codes: G01N 33/54306 Solid-phase reaction mechanisms

Abstract

The invention concerns a device and a method for ultrasensitive detection comprising an electrically inert porous sheet sandwiched between two positively charged fine electrodes. The invention uses the capacity of the electrodes, resulting from their powerful electrostatic properties, for sensing marked antibody complexes related to antigens detected in the sample, and to leave marked antibodies non-complexed with said antigens free. The method consists in depositing a sample droplet on the porous material sheet upstream of the electrodes and in detecting downstream of the electrodes, in a signal-zone, the possible presence of an antigen-antibody complex which has diffused in the porous material. The invention is useful for detecting some millipicogrammes of antigen per milliliter of sample.

Claims

1. A process for the detection of an analyte contained in a fluid or a liquid, by reaction with a specific ligand to form a complex, the analyte or the ligand or both being carriers of neutralized electric charges, or opposite charges when the complex is formed, said process comprising:

causing said fluid or liquid to migrate, after contact with the specific ligand, into a first porous electrically inert material interposed between opposite surfaces of a first pair of electrically charged electrodes of the same polarities, and

detecting in the liquid having migrated into the porous material between the two electrodes, said analyte, ligand or complex, which have not been captured by the electrodes.

2. The process according to claim 1, wherein the polarities of the charge carried by the ligand and those of the electrodes are of opposite sign, and the complexes detected in the liquid having migrated between the two electrodes when the initial liquid contained the analyte.

3. The process according to claim 1, wherein the polarities of the charges carried by the ligand and that of the electrodes are of the same sign and the polarities of the charges carried by the analyte and the complex are of opposite sign, wherein the process further includes the step of detecting an excess of the ligand which do not bind to the analyte and wherein the excess of the ligand is detected when the analyte was initially present.

4. The process according to claim 1, wherein the ligand carries a marker which is detected in a signal zone wherein the liquid migrates through the porous material between the electrodes.

5. The process according to claim 4, wherein the marker is transformable into a colorimetric, fluorescent or chemiluminescent signal, and the signal zone comprises an indicator of the marker.

6. The process according to claim 4, wherein the signal zone is formed from a second porous material, said second porous material in contact with said first porous material which is interposed between the electrodes, so as to permit access in the signal zone to the liquid having first migrated in the first porous material interposed between the electrodes, then into the second porous material comprising the signal zone.

7. The process according to claim 1, wherein the ligand is deposited dry on a predetermined region of the first porous material, in a region upstream of the electrodes, and wherein the fluid or liquid containing the analyte is introduced into said region of said first porous material.

8. The process according to claim 1, wherein:

the first porous material interposed between a preceding pair of electrodes, further comprises an extension beyond said first electrodes,

said liquid or fluid is caused to pass between a second pair of electrodes supplied on opposite zones of said extension of the first porous material, the electrodes or this second pair being charged and of identical polarities to each other, but of a sign opposite to that of the polarities of the electrodes of the first pair, and

the detection is carried out upstream of the second pair of electrodes, after which the ligand has migrated into said first porous material first through the first pair and then through the second pair of electrodes for semiquantitative determinations.

9. The process according to claim 8, wherein the first pair of electrodes is calibrated so as to trap a maximum predetermined quantity of analyte in the form of analyte-ligand complex, and their is detected downstream of the second pair of electrodes an excess of analyte.

10. The process according to claim 7, wherein the analyte carries electrical charges, wherein the fluid or liquid containing the analyte is introduced into the region of the deposited ligand, to form of the complex, thereafter a separate dose of the analyte is introduced into the region, wherein the second dose of the analyte is marked, and the presence of marked analyte is detected in the liquid having migrated between the electrodes.

11. The process according to claim 9, wherein the analyte is constituted by a predetermined antigen, the ligand by a specific antibody of the antigen, and the electrodes of the first pair of electrodes have a negative polarity.

12. The process according to claim 11, wherein the first pair of electrodes is calibrated to trap a quantity of an analyte ligand complex, the quantity of trapped antigen cannot exceed that corresponding to its normal content in the original liquid, or fluid, wherein the fluid is a biological fluid from a healthy subject, and the detection is only of the excess antigen relative to the normal content of antigen present in the original fluid or liquid.

13. The process according to claim 12, further comprising a step of detecting an excess in blood plasma of D-Dimer.

14. The process according to claim 1, wherein the analyte is an antibody, the ligand an antigen.

15. The process according to claim 14, wherein the polarities of the first pair of electrodes are of opposite sign to those of the antibodies.

16. The process according to claim 1, wherein the analyte is deposited in a region determined by the first porous material upstream of the electrodes, said region not permitting the retention of a dose of analyte exceeding a predetermined threshold value, a volume of solution containing an electrically charged ligand is deposited in said region whose polarity is opposite that of the electrodes, and the analyte ligand complex in the liquid having migrated between the two electrodes is detected such that the quantity of analyte initially deposited on said region will exceed the dose defined by the predetermined threshold value.

17. The process according to claim 16, wherein the analyte is an antigen and the region or predetermined regions of the porous material are coated with quantities of antibodies responsible for said threshold values.

18. A device for the detection of an analyte contained in a fluid or liquid by reaction with a specific ligand of the analyte to form a complex, the analyte or the ligand or the two being carriers of neutralized electric charges or opposite charges when the complex is formed, said device comprising:

at least one pair of electrodes provided with surfaces which face each other and are electrically charged, with identical polarities;

an electrically inert porous material, interposed between the surfaces and permitting diffusion, between said electrodes, of the liquid to be deposited in a free zone of said porous material, upstream of a liquid path of migration in said porous material; and

means associated with said porous material for the detection in the liquid migrated into the porous material, between said electrodes, said analyte, ligand or complexes, which have not been captured by the electrodes.

19. The device according to claim 18, wherein the electrodes are in the form of sheets that are electrostatically charged, and that the porous material is in the form of a first porous sheet sandwiched between the two electrodes, the first porous sheet extending upstream of said electrodes relative to the direction of migration and comprising, in an external portion, said means for the detection.

20. The device according to claim 18, further comprising display means of a marker carried by the ligand.

21. The device according to claim 19, further comprised of a second porous sheet in contact with a first sheet and wherein the liquid migrates through the first and second sheets to come into contact with an indicator contained in the second sheet.

22. The device according to claim 18, further comprising an opening pierced in one of the electrodes and giving access to the porous material in the region upstream of said electrode, this opening being for the introduction of the fluid or liquid containing the analyte or ligand.

23. The device according to claim 22, wherein the ligand is incorporated in a dry state in the porous material in the same region as the opening.

24. Device according to claim 22, wherein the opening which passes through the first sheet also passes through the porous material and the second electrode of the same pair, the porous sheet being connected upstream of the electrodes to a second porous sheet wherein the liquid migrates through the electrodes, the second sheet being flattened on the under surface of the second electrode, opposite the inlet of the opening, by a transparent sheet, which defines a bottom of the opening, the second porous sheet being provided with an indicator in the region located in alignment with the opening, for reading of the reaction and introduction of agents directly through the opening.

25. The device according to claim 18, wherein said device is in combination with a pipette for removal of a measured volume of the fluid or liquid that contains the analyte, said pipettte including an ampule at one end for containing the volume required for the reaction, of a solution of the ligand, said pipette further includes a detachable closure means for dispensing the solution of the ligand, after introduction by the pipette of the volume of liquid to be detected in the porous material.

26. The device according to claim 18, further comprising a housing for enclosing said electrodes, said porous material and detecting means and a reservoir for containing a solution of the ligand, said housing comprised of elastic material or flexible material, to permit freeing and an introduction of the solution from the reservoir into the porous material by a pressure exerted on walls of the housing.

27. The device according to claim 23, wherein the zone in which the analyte is deposited permits the retention of only a quantity of analyte not exceeding a predetermined threshold value.

28. The device according to claim 27, wherein the analyte is an antigen and the zone or predetermined zones of the porous material are coated with quantities of antibodies responding to said threshold values for retention of corresponding antigens.