Optical and electrical methods and apparatus for molecule detection

• US 5,653,939 A
• Filed: 08/07/1995
• Issued: 08/05/1997
• Est. Priority Date: 11/19/1991
• Status: Expired due to Term

• Current Assignee: Baylor College of Medicine (Baylor College), Houston Advanced Research Center, Massachusetts Institute of Technology
• Original Assignee: Baylor College of Medicine (Baylor College), Houston Advanced Research Center, Massachusetts Institute of Technology
• Inventors: Chen, Chang-Lee, Burke, Barry E., Hogan, Michael E., Rathman, Dennis D., Mathews, Richard H., Hollis, Mark A., Kosicki, Bernard B., Murphy, R. Allen, Ehrlich, Daniel J., Varma, Rajender Singh, Eggers, Mitch D.
• Primary Examiner(s): Jones, W. Gary
• Assistant Examiner(s): REES, DIANE
• Application Number:
  US08/511,649
• Time in Patent Office: 729 Days
• Field of Search: 435/6, 435/5, 435/91.1, 435/7.1-7.9, 435/288, 435/91.2, 536/25.3, 536/26.6, 536/24.3, 536/33, 422/50, 422/68.1, 422/76, 422/82.01, 422/82.02, 422/82.05, 422/186, 422/186.04, 422/197, 204/91, 204/182.8, 436/149, 436/150, 436/806, 436/807, 436/809, 436/518, 436/516, 436/527, 530/300, 530/350, 350/333, 350/334, 350/338
• US Class Current: 506/3
• CPC Class Codes: B01J 19/0046 Sequential or parallel reactions, e.g. for the synthesis of polypeptides or polynucleotides; Apparatus and devices for combinatorial chemistry or for making molecular arrays synthesis methods per se C40B50/00
• CPC Class Codes: B01J 19/0093 Microreactors, e.g. miniaturised or microfabricated reactors laboratory containers with capillary fluid transport in microfabricated channels or chambers B01L3/5027
• CPC Class Codes: B01J 2219/00317 Microwell devices, i.e. having large numbers of wells
• CPC Class Codes: B01J 2219/00511 Walls of reactor vessels
• CPC Class Codes: B01J 2219/00527 Sheets
• CPC Class Codes: B01J 2219/00585 Parallel processes
• CPC Class Codes: B01J 2219/00596 Solid-phase processes
• CPC Class Codes: B01J 2219/00605 the compounds being directly bound or immobilised to solid supports
• CPC Class Codes: B01J 2219/00612 the surface being inorganic
• CPC Class Codes: B01J 2219/00617 by chemical means
• CPC Class Codes: B01J 2219/00621 by physical means, e.g. trenches, raised areas
• CPC Class Codes: B01J 2219/00626 Covalent
• CPC Class Codes: B01J 2219/00637 by coating it with another layer
• CPC Class Codes: B01J 2219/00653 the compounds being bound to electrodes embedded in or on the solid supports
• CPC Class Codes: B01J 2219/00659 Two-dimensional arrays
• CPC Class Codes: B01J 2219/00689 using computers
• CPC Class Codes: B01J 2219/00702 Processes involving means for analysing and characterising the products
• CPC Class Codes: B01J 2219/00711 Light-directed synthesis
• CPC Class Codes: B01J 2219/00713 Electrochemical synthesis
• CPC Class Codes: B01J 2219/00722 Nucleotides
• CPC Class Codes: B01J 2219/00828 : Silicon wafers or plates
• CPC Class Codes: B01J 2219/00853 : Employing electrode arrangements
• CPC Class Codes: C12Q 1/6825 : Nucleic acid detection involving sensors
• CPC Class Codes: C12Q 1/6832 : Enhancement of hybridisation reaction
• CPC Class Codes: C12Q 1/6837 : using probe arrays or probe chips C12Q1/6874 takes precedence
• CPC Class Codes: C12Q 1/6874 : involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 2565/607 : being a sensor, e.g. electrode
• CPC Class Codes: C40B 40/06 : Libraries containing nucleotides or polynucleotides, or derivatives thereof
• CPC Class Codes: C40B 60/14 : for creating libraries
• CPC Class Codes: G01N 21/253 : for batch operation, i.e. multisample apparatus analytical automats G01N35/00
• CPC Class Codes: G01N 21/6454 : using an integrated detector array
• CPC Class Codes: G01N 27/221 : by investigating the dielectric properties using microwaves G01N22/00; measuring loss factors or dielectric constants per se G01R27/26
• CPC Class Codes: G01N 27/226 : Construction of measuring vessels; Electrodes therefor
• CPC Class Codes: G01N 33/54373 : involving physiochemical end-point determination, e.g. wave-guides, FETS, gratings
• CPC Class Codes: G01N 33/5438 : Electrodes
• CPC Class Codes: Y10S 436/806 : Electrical property or magnetic property
• CPC Class Codes: Y10S 436/807 : Apparatus included in process claim, e.g. physical support structures

Abstract

A method and apparatus are disclosed for identifying molecular structures within a sample substance using a monolithic array of test sites formed on a substrate upon which the sample substance is applied. Each test site includes probes formed therein to bond with a predetermined target molecular structure or structures. A signal is applied to the test sites and certain electrical, mechanical and/or optical properties of the test sites are detected to determine which probes have bonded to an associated target molecular structure.

Claims

1. A method for testing a sample substance for the presence of target nucleic acids comprising the steps of:

a) forming an array of test sites in multiple locations, each site having oligonucleotide probes formed therein of known binding characteristics and wherein the probes in each test site differ from the probes in other test sites in a known predetermined manner, such that the test site location of probes and their binding characteristics are also known;

b) applying the sample substance to the test sites; and

c) detecting a change in the electromagnetic properties of the test sites resulting from the binding of target nucleic acid within the sample substance to probes in the test sites by detection circuitry coupled to individual test sites to determine which target nucleic acid have bonded to a test site;

whereby the presence of a multiplicity of different target nucleic acids in the sample can be detected.

2. The method of claim 1 wherein a test signal is applied to the test sites which is an electro-magnetic signal and forming said array comprises the steps of:

a) forming a first layer on a substrate;

b) forming a second layer on the first layer;

c) forming openings in the second layer into the first layer exposing a portion of the first layer; and

d) forming a pair of electrodes, one electrode on the first layer on the exposed portions in the openings and another electrode on the second layer adjacent to the openings, and wherein said test signal is applied to the electrodes.

3. The method of claim 2 wherein forming said pair of electrodes comprises the steps of depositing metallization on the second layer after the openings are formed;

which metallization forms an upper electrode on the surface of the second layer between openings and a lower electrode on the exposed portions of the first layer.

4. The method of claim 3 wherein the substrate is formed of silicon, and the first layer and second layer are formed of a silicon based dielectric.

5. The method of claim 4 wherein the first and second layers are SiO₂ and Si₃ N₄, respectively, and the metallization comprises Al, Ti, Pt, W, Ta and their silicides or Au.

6. The method of claim 1 wherein said step of detecting comprises detecting the dielectric properties of the test site.

7. The method of claim 1 wherein a test signal is applied to the test sites which is a pulsed or varying frequency signal.

10. The method of claim 1 wherein said sample substance is in a solution or a gel.

17. The method of claim 6 wherein the dielectric property is permittivity.

18. The method of claim 1 wherein the sample substance is in a solid state.

8. The method of clam 1 wherein each test site is formed with a resonant structure which is resonant in a frequency range of electrical signals.

9. The method of claim 8 wherein said detecting step comprises detecting changes in the Q or the resonant frequency of the resonant structure.

19. The method of claim 8 wherein the resonant structure is a transmission line and changes in phase or amplitude of a signal propagated on the line are detected.

11. Apparatus for synthesizing molecular probes in situ comprising:

a) an array of test sites each site containing precursor molecules to be reacted;

b) an array of resistors disposed adjacent to the test site array with a respective resistor located in proximity to a respective test site; and

c) coupling means for coupling a respective resistor to an electrical source for heating a respective resistor to induce a thermal reaction for synthesis of molecules in a respective test site.

12. The apparatus of claim 11 wherein the test site array and resistor array are formed as an integrated structure.

13. Apparatus for synthesizing molecular structures in situ comprising:

a) an array of test sites each site containing precursor molecules to be reacted; and

b) each test site containing electrodes coupled to a voltage source for inducing a reaction to synthesize molecules in a respective test site.

14. The apparatus of claim 13 wherein the array and electrodes are formed in integrated structures.

38. The apparatus of claim 13 wherein the voltage source provides a reverse biased potential applied to the electrodes subsequent to molecule synthesis.

15. A method for synthesizing a plurality of different known molecular structures on an array of test sites comprising:

a) providing an array of test sites;

b) providing a source of chemical reactants disposed adjacent the test sites;

c) providing electrodes associated with respective test sites; and

d) applying a potential to a respective electrode to synthesize said different known molecular structures at said different known sites in the array.

16. Apparatus for enhancing hybridization between a synthesized probe and a target molecule comprising:

a) an array of test sites each site containing a plurality of probes, each test site having probes of known binding characteristics, and wherein the probes in each test site differ from the probes in other test sites in a known predetermined manner;

b) an electrode associated with each site;

c) a source of target molecules applied to said sites; and

d) a voltage source for applying a potential to a respective electrode for attracting said target molecules to said probes.

39. The apparatus of claim 16 wherein the voltage source provides a reverse biased potential applied to the electrodes to aid in the removal of unhybridized target molecules.

20. A method for determining the presence of molecular structures within a sample substance comprising:

a) forming an array of test sites having specific known oligonucleotide probes formed therein selected to bind with said structures of known binding characteristics, each site having oligonucleotide probes which bind to a different structure and wherein the location of the sites containing specific known oligonucleotide probes is also known;

b) dispensing a sample substance into a test site;

c) generating radiation through the test sites; and

d) detecting, by detection circuitry coupled to individual test sites of the array, the difference in the radiation which is absorbed by the respective test sites to determine the presence of the structures which are bonded to a probe.

21. The method of claim 20 wherein the difference is detected by an array of detectors formed of charge-coupled devices which are optically coupled to the test sites of the array.

22. The method of claim 20 wherein the difference is detected by an array of detectors formed integral with the array of test sites.

23. The method of claim 20 wherein the difference is detected by an array of detectors formed separate from the array of test sites.

24. The method of claim 23 wherein the array of detectors is aligned with the array of test sites and radiation is projected through the test sites onto the detector array.

25. The method of claim 24 wherein the radiation is in the form of photons, or radioactive particles.

26. The method of claim 20 wherein the radiation is generated within the test sites by a radioactive, chemical, thermal, chemiluminescent, or luminescent reaction.

27. The method of claim 20 wherein the detectors detect thermal energy occurring when a bonding reaction takes place.

28. A method for attaching probes to a test site formed in a substrate comprising the steps of:

a) forming test sites in a substrate;

b) forming an adhesive material in the test site which material enables probes to attach to the test sites;

c) forming a coating over the adhesive material;

d) contacting the coating with a coating-removal material while selectively initiating a reaction at a selected site which removes the coating at the selected site; and

e) contacting the site in which the coating has been removed with probes which adhere to the adhesive material.

29. The method of claim 28 wherein the probes are presynthesized and the test sites consist of wells, the adhesive material is an epoxide, the coating is formed by hydrolyzing the epoxide, the coating-removal material is an acetate alcohol solution and the reaction is initiated at a selected site by heating the site.

30. The method of claim 28 wherein the reaction is initiated by selectively energizing resistors formed adjacent the test sites to heat the selected test sites.

31. The method of claim 30 wherein non-selected test sites are maintained at a temperature above the desired reaction temperature.

32. The method of claim 28 wherein the reaction is initiated by irradiating selected sites with light.

33. The method of claim 32 wherein the light is in the visible or ultraviolet wavelength range and a photochemical reaction occurs.

34. The method of claim 32 wherein the light originates from a laser which is scanned from site to site to initiate the reaction.

35. The method of claim 32 wherein the light induces local heating of the site to initiate a reaction.

36. The method of claim 32 wherein the light is generated by a light valve which projects light onto selected sites.

37. The method of claim 32 wherein the light source is a light valve for projecting light onto selected sites.