Biosensor and related method

• US 20010053535A1
• Filed: 03/26/2001
• Published: 12/20/2001
• Est. Priority Date: 04/17/2000
• Status: Active Grant

• Current Assignee: Purdue Research Foundation (The Trustees of Purdue University (d/b/a Purdue University))
• Original Assignee: Purdue Research Foundation (The Trustees of Purdue University (d/b/a Purdue University))
• Inventors: Sarikaya, Ayda, Ladisch, Michael R., Bashir, Rashid, Robinson, J. Paul, Bhunia, Arun K., Gomez, Rafael
• Granted Patent: US 6,716,620 B2
• Time in Patent Office: Days
• US Class Current: 435/34
• CPC Class Codes: G01N 33/5438 Electrodes
• CPC Class Codes: G01N 33/56911 Bacteria

Abstract

A microscale biosensor for use in the detection of target biological substances including molecules and cells is a microfluidic system with integrated electronics, inlet-outlet ports and interface schemes, high sensitivity detection of pathogen specificity, and processing of biological materials at semiconductor interfaces. A fabrication process includes an all top-side processing for the formation of fluidic channels, planar fluidic interface ports, integrated metal electrodes for impedance measurements, and a glass cover sealing the non-planar topography of the chip using spin-on-glass as an intermediate bonding layer. Detection sensitivity is enhanced by small fluid volumes, use of a low-conductivity buffer, and electrical magnitude or phase measurements over a range of frequencies.

Claims

1. A method for detecting a microbiological substance, comprising:

providing a microfabricated biosensor chip including integrated detection elements;

delivering a fluid sample to said biosensor chip;

after the delivering of said fluid sample to said biosensor chip, separating at least some contaminants from said fluid sample to at least partially isolate and retain instances of a predetermined type of microbiological material on said biosensor chip, the separating of said contaminants taking place on said biosensor chip; and

after the separating of contaminants from said fluid sample, operating said detection elements to determine whether the separated fluid sample contains microbiological material of said predetermined type.

2. The method defined in

claim 1

, further comprising carrying out a bioseparations process on said fluid sample prior to the delivering of said fluid sample to said biosensor chip.

3. The method defined in

claim 2

wherein said bioseparations process includes adding to said fluid sample a plurality of microscopic carrier elements each provided with a multiplicity of binding agents for coupling said microbiological material to said carrier elements.

4. The method defined in

claim 3

wherein said carrier elements are beads or microspheres.

5. The method defined in

claim 4

wherein the separating of contaminants from said fluid sample on said biosensor chip includes trapping said carrier elements with the coupled microbiological material in a detection chamber on said biosensor chip while flushing remaining portions of said fluid sample from said chamber.

6. The method defined in

claim 5

wherein the trapping of said carrier elements includes providing a filter barrier at an outlet of said detection chamber.

7. The method defined in

claim 2

wherein said bioseparations process includes subjecting a precursor of said fluid sample to a bioactive surface taken from the group consisting of a cation exchange resin and an anion exchange resin.

8. The method defined in

claim 7

wherein said cation exchange resin includes Amberlyst 35 and said anion exchange resin includes IRA 400.

9. The method defined in

claim 1

wherein said predetermined type of microbiological material is a pathogenic strain of bacteria.

10. The method defined in

claim 9

wherein said strain of bacteria includes Listeria monocytogenes.

11. The method defined in

claim 9

, further comprising extracting said fluid sample from a food product prior to delivering of said fluid sample to said bio sensor chip.

12. A biosensor comprising a substrate microfabricated to include as integrated components:

a detection chamber;

a first channel segment extending to an inlet of said detection chamber;

a second channel segment extending from an outlet of said chamber; and

a retention structure for holding, in said chamber, carrier elements entraining a target microbiological species while permitting passage through said detection chamber of contaminant materials in a fluid stream.

13. The biosensor defined in

claim 12

wherein said retention structure includes filter grid or grating on an upstream side of said outlet.

14. The biosensor defined in

claim 12

wherein said retention structure includes a magnetic field generating element.

15. The biosensor defined in

claim 12

wherein said detection chamber is provided with electrodes including interdigitated finger parts.

16. The biosensor defined in

claim 12

wherein said detection chamber has a volume of less than approximately one microliter.

17. A biosensor comprising a substrate microfabricated to include as integrated components:

a detection chamber; and

a channel extending to an inlet of said detection chamber, further comprising a wicking element connected at one end to said substrate so as to be in communication with said channel, for drawing a fluid sample by capillary action to said channel for delivery to said detection chamber.

18. The biosensor defined in

claim 17

wherein said wicking element is attached at said one end by an adhesive to said substrate.

19. The biosensor defined in

claim 17

wherein said substrate is microfabricated to include an inlet groove or trench substantially coplanar with said channel and said detection chamber, said one end of said wicking element being disposed in said inlet groove or trench, said wicking element being coplanar at said one end with said channel and said detection chamber.

20. An integrated microscale biosensor comprising a substrate microfabricated to include as integrated components:

a detection chamber;

a channel extending to an inlet of said detection chamber; and

an inlet groove or trench substantially coplanar with said channel and said detection chamber, further comprising an elongate fluid delivery member having a downstream end disposed in said inlet groove or trench, said fluid delivery member being connected at said downstream end to inlet groove or trench so that at least said downstream end of said fluid delivery member is coplanar with said channel and said detection chamber.

21. The biosensor defined in

claim 20

wherein said elongate fluid delivery member is a microbore tube.

22. The biosensor defined in

claim 20

wherein said elongate fluid delivery member is a wicking element.

23. The biosensor defined in

claim 20

wherein said substrate is top-side processed only.

24. The biosensor defined in

claim 20

, further comprising a cover attached to said substrate over said detection chamber, said channel, said inlet groove, and said downstream end of said fluid delivery member, said cover having an absence of holes or apertures.

25. A method for manufacturing a bio sensor comprising:

providing a substrate;

processing said substrate to generate a detection chamber and a channel extending to said detection chamber;

further processing said substrate to provide at least one pair of electrodes in said detection chamber; and

exposing the processed substrate to BSA and avidin to adsorb said avidin to said electrodes in the presence of said BSA.

26. The method defined in

claim 25

, further comprising subjecting the exposed processed substrate to a fluid containing a biotinylated antibody specific to a preselected antigen, thereby attaching the antibody to said electrodes via a biotin-avidin link.

27. The method defined in

claim 26

wherein said biotinylated antibody is specific to an antigen on a cell membrane of Listeria monocytogenes.

28. The method defined in

claim 27

wherein said antigen is a 66-kDA protein, further comprising:

culturing monoclonal antibody producing clones of C11E9 and EM-7G1 in growth media in a growth chamber;

harvesting antibodies from culture supernatants by salt precipitation; and

obtaining antibodies from the harvest by purification through size exclusion chromatography followed by protein-A affinity chromatography in an FPLC system.

29. The method defined in

claim 25

, further comprising washing the exposed processed substrate after a predetermined time period.

30. A method for manufacturing a biosensor, comprising:

providing a substrate;

processing said substrate to create a shallow detection chamber and a channel extending to said detection chamber;

after the creation of said detection chamber and said channel, further processing said substrate to deposit at least one pair of electrodes in said detection chamber;

after the deposition of said electrodes, further processing said substrate to create at least one deep groove at a periphery of said substrate, for receiving an elongate fluid delivery element, said channel communicating with said deep groove;

inserting a downstream end of said fluid delivery element into said deep groove; and

attaching said downstream end of said fluid delivery element to said deep groove.

31. The method defined in

claim 30

, further comprising attaching a cover to said substrate over said detection chamber, said channel, said deep groove and said downstream end of said fluid delivery element.

32. A method for detecting a microorganism, comprising:

preparing a fluid sample containing at least one microorganism of a preselected type, said fluid sample having a buffer of a low conductivity liquid, said fluid sample also containing a nonionic nutrient;

disposing said fluid sample in a detection chamber having a volume less than approximately 1 microliter;

maintaining said fluid sample at a predetermined temperature in said detection chamber; and

measuring an electrical parameter of an electrical circuit incorporating said detection chamber and the fluid sample therein

33. The method defined in

claim 32

wherein said electrical parameter is an impedance measure taken from the group consisting of magnitude and phase.

34. The method defined in

claim 32

wherein the measuring of said electrical parameter includes utilizing a four point probe to make a sheet resistivity measurement.

35. The method defined in

claim 32

wherein said microorganism is Listeria Monocytogenes.

36. The method defined in

claim 32

wherein said buffer is a Tris-Glycine buffer.

37. The method defined in

claim 32

wherein said detection chamber has a volume of between about 1 picoliter and about 1 microliter.

38. The method defined in

claim 32

wherein said electrical parameter is an impedance parameter, the measuring of said electrical parameter including measuring the impedance parameter at a plurality of frequencies within a range from 100 Hz to 1 MHz.

39. A method for testing a food product for the presence of a predetermined type of pathogenic bacteria, comprising:

extracting a fluid sample from the food product;

feeding the extracted fluid sample to an integrated microscale biosensor;

subjecting the fluid sample to a bioseparations process to remove extraneous particles including proteins and kinds of bacteria other than the predetermined type of pathogenic bacteria;

binding bacteria of said predetermined type in said fluid sample to at least one substrate body; and

after the feeding of the extracted fluid sample to said chamber, the subjecting of the fluid sample to the bioseparations process, and the binding of the predetermined type of bacteria to the at least one substrate body, measuring an electrical parameter of an electrical circuit incorporating said detection chamber and the fluid sample therein to detect the presence in the fluid sample of living instances of said predetermined type of bacteria.

40. The method defined in

claim 39

wherein the binding of said predetermined type of bacteria is to beads or microspheres floating in said fluid sample.

41. The method defined in

claim 39

wherein the binding of said predetermined type of bacteria is to electrodes in said biosensor.

42. The method defined in

claim 39

wherein subjecting of said fluid sample to said bioseparations process takes place at least partially after feeding of the fluid sample to said biosensor.

43. The method defined in

claim 39

wherein the binding of said predetermined type of bacteria is implemented via antibodies specific to said predetermined type of bacteria.

44. The method defined in

claim 39

, further comprising concentrating said predetermined type of bacteria in said fluid sample prior to the measuring of said electrical parameter.

45. The method defined in

claim 39

wherein said electrical parameter is an impedance parameter.

46. The method defined in

claim 39

wherein said electrical parameter is phase.

47. An integrated microscale biosensor comprising a substrate microfabricated to include as integrated components:

a detection chamber;

a channel extending to an inlet of said detection chamber;

means for feeding a fluid sample to said detection chamber; and

at least one sensor attached to said substrate in operative communication with said detection chamber,

said detection chamber having a volume of between about 1 picoliter and 1 microliter.