Organic semiconductor recognition complex and system

• US 6,303,316 B1
• Filed: 06/30/2000
• Issued: 10/16/2001
• Est. Priority Date: 07/02/1999
• Status: Expired due to Term

• Current Assignee: Conceptual MindWorks Incorporated
• Original Assignee: Conceptual MindWorks Incorporated
• Inventors: Bruno, John G., Parker, Jill E., Batishko, Charles R., Alls, John L., Kiel, Johnathan L., Holwitt, Eric A.
• Primary Examiner(s): Horlick, Kenneth R.
• Application Number:
  US09/608,706
• Time in Patent Office: 473 Days
• Field of Search: 435/6, 435/91.2, 435/7.1, 536/23.1, 436/94
• US Class Current: 435/6.19
• CPC Class Codes: C12Q 1/6825 Nucleic acid detection involving sensors
• CPC Class Codes: C12Q 1/6837 using probe arrays or probe chips C12Q1/6874 takes precedence
• CPC Class Codes: C12Q 2565/607 being a sensor, e.g. electrode
• CPC Class Codes: Y10T 436/143333 Saccharide [e.g., DNA, etc.]

Abstract

In a recognition complex system, nucleic acid ligands comprising random DNA sequences are operatively coupled to an organic semiconductor and distributed so as to form an array of recognition complexes. When an unknown chemical or biological analyte is applied to the array, the electrical and/or photochemical properties of one or more of the recognition complexes are altered upon binding of the nucleic acid ligand to the analyte. The degree to which the electrical and/or photochemical properties change is a function of the affinity of the nucleic acid ligand sequence for the analyte. The electrical and photochemical changes associated with the array, as a whole, can be used as a unique signature to identify the analyte. In certain embodiments, an iterative process of selection and amplification of nucleic acid ligands that bind to the analyte can be used to generate a new array with greater affinity and specificity for a target analyte, or to produce one or more nucleic acid ligands with high binding affinity for an analyte. The present invention also provides methods for preparing nucleic acid ligands that bind with high affinity to an analyte and using such nucleic acid ligands to neutralize the analyte.

Claims

1. A recognition complex comprising a nucleic acid ligand operably coupled to an organic semiconductor.

2. The recognition complex of claim 1, wherein said nucleic acid ligand comprises random DNA sequences.

3. The recognition complex of claim 1, wherein said nucleic acid ligand is between 20 and 100 bases in length.

4. The recognition complex of claim 1, wherein said nucleic acid ligand is between 100 and 300 bases in length.

5. The recognition complex of claim 1, wherein said nucleic acid ligand is operably coupled to said organic semiconductor by electrostatic binding, non-covalent binding or covalent binding.

6. The recognition complex of claim 5, wherein said electrostatic binding is mediated by magnesium ion.

7. The recognition complex of claim 1, wherein said organic semiconductor is diazoluminomelanin (DALM).

8. The recognition complex of claim 1, wherein said organic semiconductor is a polyphenylene.

9. The recognition complex of claim 1, wherein said organic semiconductor produces an electrochemical signal when an analyte is bound to said nucleic acid ligand.

10. The recognition complex of claim 9, wherein said electrochemical signal is a photochemical signal, a fluorescent signal, a luminescent signal, a change of color or a change in electrical conductivity.

11. The recognition complex of claim 1, wherein said nucleic acid ligand is an aptamer.

12. The recognition complex of claim 1, wherein said nucleic acid ligand is selected for its ability to bind to a specific analyte.

13. The recognition complex of claim 1, wherein said nucleic acid ligand is selected for its ability to neutralize a specific analyte.

14. The recognition complex of claim 1, wherein said nucleic acid ligand is produced by amplification of nucleic acids that bind to a specific analyte.

15. A recognition complex system, comprising one or more recognition complexes according to claim 1, operably coupled to a detection unit.

16. The recognition complex system of claim 15, further comprising a data processing unit.

17. The recognition complex system of claim 16, further comprising a sample collector and an isolator.

18. The recognition complex system of claim 15, wherein said detection unit comprises a charge coupled device (CCD), a CCD camera, a photomultiplier tube, a spectrophotometer or a fluorometer.

19. The recognition complex system of claim 15, wherein said recognition complexes are attached to a surface.

20. The recognition complex system of claim 19, wherein said surface comprises a Langmuir-Bodgett film, functionalized glass, germanium, silicon, PTFE, polystyrene, gallium arsenide, gold, silver, membrane, nylon or PVP.

21. The recognition complex system of claim 19, wherein each recognition complex is positioned between a first and a second electrode.

22. The recognition complex system of claim 21, wherein the first electrode is transparent.

23. The recognition complex system of claim 21, further comprising a detection unit that is capable of detecting conductivity changes in each recognition complex.

24. The recognition complex system of claim 16, further comprising system memory for storing the signals produced by each recognition complex for each of a number of known analytes.

25. The recognition complex system of claim 24, wherein the data processing unit is capable of identifying unknown analytes by comparing the signal produced by each recognition complex for the unknown analyte with the stored signals for known analytes.

26. The recognition complex system of claim 16, wherein the data processing unit comprises a neural network or look up tables.

27. The recognition complex system of claim 18, wherein said recognition complexes are attached to magnetic beads.

28. The recognition complex system of claim 27, wherein said organic semiconductor is DALM and said DALM is covalently or non-covalently attached to said magnetic beads.

29. The recognition complex system of claim 28, wherein said nucleic acid ligands are electrostatically attached to said DALM.

30. The recognition complex system of claim 29, wherein said electrostatic attachment is mediated by magnesium ion.

31. The recognition complex system of claim 28, further comprising a data processing unit, a magnetic electrode and a magnetic filter.

32. The recognition complex system of claim 28, further comprising a thermal cycler, a reagent reservoir, a new bead source, a collector and magnetic bead mixer, and one or more pumps.

33. The recognition complex system of claim 16, wherein said recognition complexes are embedded in a matrix.

34. The recognition complex system of claim 33, wherein said matrix is agarose, polyacrylamide or cellulose.

55. The recognition complex system of claim 19, wherein said surface is part of a card or badge.

56. The recognition complex system of claim 55, wherein binding of an analyte to one or more recognition complexes is detectable as a change in color.

57. The recognition complex system of claim 55, wherein binding of an analyte to one or more recognition complexes is detectable as an electrical signal.

58. The recognition complex system of claim 57, wherein said electrical signal activates an alarm.

59. The recognition complex system of claim 58, wherein said alarm is transmitted to a remote location.

60. The recognition complex system of claim 55, wherein said nucleic acid ligands are selected to bind specifically to a biowarfare agent or a chemical warfare agent.

61. The recognition complex system of claim 60, wherein said biowarfare agent is an anthrax spore.

35. A method for identifying an analyte comprising the steps of:

a) generating a recognition complex system comprising one or more recognition complexes each recognition complex comprising a nucleic acid ligand operably coupled to an organic semiconductor;

b) contacting each recognition complex with the analyte;

c) quantifying an electrochemical signal produced by one or more recognition complexes following contact with the analyte, wherein the signal produced by a recognition complex is a function of the affinity the analyte has for the nucleic acid ligand; and

d) identifying the analyte based on the signals produced by the one or more recognition complexes.

36. The method of claim 35, further comprising the steps of:

a) transmitting ultraviolet light through each of the recognition complexes after contacting the recognition complexes with the analyte;

b) measuring the electrochemical signals produced by the recognition complexes; and

c) generating a unique signature for the analyte based on the signals produced by the recognition complexes.

37. The method of claim 35, wherein the signal is a photochemical signal, a fluorescent signal, a luminescent signal, a change of color or a change in electrical conductivity.

45. The method of claim 35, wherein the analyte is a pharmaceutical, a toxin, a poison, an explosive, a pesticide, a bacterium, a virus, a mold, a yeast, a spore, an algae, an amobae, a dinoflagellate, a unicellualr organism, an allergen, a chemical warfare agent, a biohazardous agent, a protein, a lipid, a carbohydrate, a prion, a radiosotope, a vitamin, a heterocyclic aromatic compound, a carcinogen, a mutagen, a narcotic, an amphetamine, a barbiturate, a hallucinogen, a waste product or a contaminant.

46. The method of claim 35, further comprising detecting explosives or illegal drugs in an airport detection system.

47. The method of claim 35, further comprising detecting air-borne pathogens in an air conditioner monitoring system.

48. The method of claim 35, further comprising detecting water-borne pathogens, carcinogens, teratogens or toxins in a water quality monitoring system.

49. The method of claim 35, further comprising detecting pathogens in an operating room monitoring system.

50. The method of claim 35, further comprising detecting allergens, pathogens or contaminants in an food production monitoring system.

51. The method of claim 35, further comprising detecting genetically modified organisms.

52. The method of claim 35, further comprising performing high through-put screening for pharmaceutical compounds.

53. The method of claim 52, wherein said recognition complex comprises a nucleic acid ligand that is selected to bind specifically to a known agonist or antagonist of an enzyme, receptor protein, transport protein, cytokine, transcription factor, protein kinase or structural protein.

54. The method of claim 53, further comprising screening a library of small molecule drug candidates, wherein binding of said candidate to said nucleic acid ligand indicates an affinity of said candidate for said enzyme, receptor protein, transport protein, cytokine, transcription factor, protein kinase or structural protein.

62. The method of claim 35, further comprising performing high through-put screening for pharmaceutical compounds.

38. A method for producing one or more nucleic acid ligands that bind with high affinity to an analyte comprising the steps of:

a) generating multiple recognition complexes, each recognition complex containing a nucleic acid ligand operably coupled to DALM, wherein each nucleic acid ligand contains a random DNA sequence;

b) contacting the recognition complexes with the analyte;

c) separating those recognition complexes that bind to the analyte from those recognition complexes that do not bind to the analyte;

d) amplifying the nucleic acid ligands from the recognition complexes that bind to the analyte;

e) using the amplified nucleic acid ligands to generate a new set of recognition complexes; and

f) repeating steps (b) through (e) until one or more nucleic acid ligands that bind with high affinity to the analyte are produced.

39. The method of claim 38, wherein the recognition complexes are attached to magnetic beads.

40. The method of claim 39, wherein the separating step comprises using a magnetic flow cell, wherein beads attached to recognition complexes that bind to the analyte are separated in the flow cell from beads attached to recognition complexes that do not bind to the analyte.

41. A method for neutralizing an analyte comprising the steps of:

a) producing one or more nucleic acid ligands that bind with high affinity to the analyte;

b) amplifying the one or more nucleic acid ligands;

c) using the amplified nucleic acid ligands to prepare one or more recognition complexes, each recognition complex containing a nucleic acid ligand attached to DALM;

d) contacting the analyte with the recognition complexes under conditions effective to bind the nucleic acid ligand to the analyte; and

e) activating the DALM;

wherein activation of the DALM attached to the nucleic acid ligand is effective to neutralize the analyte.

42. The method of claim 41, wherein the activation comprises exposing the DALM to sunlight, heat, laser radiation, ultraviolet radiation, infrared radiation, radiofrequency radiation or microwave radiation.

43. The method of claim 42, wherein said analyte is a biowarfare agent or a chemical warfare agent.

44. The method of claim 43, wherein said analyte is an anthrax spore.