Method and apparatus for measurement of binding between a protein and a nucleotide

• US 6,251,688 B1
• Filed: 03/20/1998
• Issued: 06/26/2001
• Est. Priority Date: 03/20/1998
• Status: Expired due to Fees

• Current Assignee: IA, Inc.
• Original Assignee: IA, Inc.
• Inventors: Downward, James G., Erb, Judith L., Ulrich, Otho, Erb-Downward, John R.
• Primary Examiner(s): Chin, Christopher L.
• Application Number:
  US09/045,223
• Time in Patent Office: 1,194 Days
• Field of Search: 385/12, 385/15, 385/25, 385/123, 385/129, 385/130, 422/55, 422/57, 422/82.05, 422/82.08, 422/82.11, 435/6, 435/7, 435/9, 435/287.1, 435/287.2, 435/287.9, 435/288.7, 435/808, 436/510, 436/518, 436/524, 436/527, 436/65, 436/164, 436/172, 436/805, 436/814, 436/817, 436/818
• US Class Current: 436/518
• CPC Class Codes: G01N 2021/6484 Optical fibres
• CPC Class Codes: G01N 2021/7786 Fluorescence
• CPC Class Codes: G01N 21/552 Attenuated total reflection
• CPC Class Codes: G01N 21/6428 Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" in vivo A61B5/00; immunoassay G01N33/53
• CPC Class Codes: G01N 21/648 using evanescent coupling or surface plasmon coupling for the excitation of fluorescence
• CPC Class Codes: G01N 21/7703 using reagent-clad optical fibres or optical waveguides using measurement of total internal reflection or attenuated total reflection G01N21/552; optical fibres or waveguides per se G02B
• CPC Class Codes: Y10S 435/808 Optical sensing apparatus
• CPC Class Codes: Y10S 436/805 Optical property
• CPC Class Codes: Y10S 436/814 Pregnancy
• CPC Class Codes: Y10S 436/817 Steroids or hormones
• CPC Class Codes: Y10S 436/818 Human chorionic gonadotropin
• CPC Class Codes: Y10S 977/924 using nanostructure as support of dna analysis
• CPC Class Codes: Y10S 977/958 Of biomolecule property
• CPC Class Codes: Y10S 977/959 Of disease state

Abstract

A method and apparatus for measuring binding between a plurality of molecules of a first type and a plurality of molecules of a second type is presented. Apparatus utilizes a sensor possessing a waveguide to which have been attached in close proximity to its surface, features resembling molecules of said first type. Light is injected into said waveguide so as to produce an evanescent field at its surface. Molecules of said second type are tagged with a tag belonging to that class of chemicals which alters a characteristic of light, when said light passes through said chemical tag. Apparatus utilizes a rapid means of monitoring the change in optical signal coming from said waveguide as binding proceeds between tagged molecules of type 2 and the feature resembling molecules of type 1 on said waveguide. This allows direct measurement of binding and dissociation rates between the two types of molecules. Methods are provided whereby such data may be used to compute affinity constants, binding activity, complex affinity constants resulting from cooperativity, and kinetic parameters for the molecular pair. Preferred embodiments of the invention illustrate application of the method and apparatus to measuring binding between biological receptors and their nuclear response elements, and the use of this type of measurement for assessment of the activity of hormonal mimics present in a sample, for evaluation of pharmaceuticals intended to treat hormone dependent cancers, and for evaluation of tissue biopsy samples to detect mutant forms of the p53 protein.

Claims

1. An apparatus to monitor the binding affinity between a plurality of a certain molecule of a first type and a plurality of a certain molecule of a second type, said apparatus comprises:

(a) A light source which generates and transmits a certain light signal;

(b) Annularizing means by which a substantially uniformly distributed and cylindrical beam of light containing an initial first amount of light power and which is impingent upon said annularizing means becomes topologically transformed with minimal loss of said initial first amount of light power into an emergent second beam which is substantially uniformly distributed in power within an annular shell or region on the outside of the beam, the inside region being substantially devoid of light power;

(c) An optical fiber waveguide which receives said transmitted light signal in which an evanescent field is generated and having said plurality of a certain molecule of a first type affixed to its lateral surface;

(d) processing means, connected to said light source and to said waveguide for using said evanescent field to determine said binging affinity.

2. The apparatus of claim 1 wherein said molecule of a first type comprises at least a portion of a specific nucleotide, said molecules of a second type comprises at least a portion of a specific protein.

3. An apparatus of claim 2 wherein said specific protein is a biological receptor and said specific nucleotide is a response means for said receptor.

4. An apparatus of claim 1 wherein said molecules of a first type resemble a ligand and said molecules of a second type comprise at least a portion of a specific protein having affinity for said ligand.

5. An apparatus of claim 4 wherein said specific protein is a biological receptor.

6. The apparatus of claim 1 wherein said molecules of a second type further comprises a molecular tag which when bound to said optical waveguide, produces an alteration in a certain characteristic of light collected from said waveguide in response to said generated evanescent field.

7. The apparatus of claim 6 wherein said molecular tag is a fluorescent molecule.

8. The apparatus of claim 6 wherein said molecular tag is a luminescent molecule.

9. The apparatus of claim 6 wherein said molecular tag absorbs light from said light source.

10. The apparatus of claim 6 wherein said molecular tag alters the polarization of light from said light source.

11. The apparatus of claim 6 wherein said molecules of a second type further comprises a molecular tag which is an enzyme capable of acting upon a substrate so as to produce a chemical substance which when bound to said optical waveguide, produces an alteration in a certain characteristic of light collected from said waveguide.

12. An apparatus of claim 1 wherein said annularizing means comprises:

(a) a large numerical aperture optical fiber cable;

said optical fiber cable having sufficient length to allow an off axis beam injected by a focusing means to propagate as real modes in a substantially confined manner within said optical fiber cable becoming uniformly azimuthally distributed in higher order modes such that the beam will exit the cable as an annular cone of light, the distal end of said cable being the distal end of said annularizing device;

(b) a large numerical aperture optical focusing means located proximally to said optical fiber cable, such as, but not limited to, a lens or lens assembly, said numerical aperture being at least as large as said optical fiber waveguide, said focusing means having an acceptance aperture significantly larger than the diameter of the beam at a first narrow band of wavelengths impingent upon said annularizing device, said focusing means focusing a beam at the first narrow band of wavelengths impingent both normal to the aperture of said focusing means and substantially offset from the optical axis of said focusing means, onto the surface of said optical fiber cable.

13. An apparatus of claim 12 wherein said annularizing means includes a means for adjusting the cone angle of light emergent from said annularizing device so that the cone angle of light entering said optical fiber waveguide such that substantially all rays of light subsequently propagating through said waveguide impinges upon the side surfaces of said waveguide at the proper angle for maximum evanescent coupling of the light at the first narrow band of wavelengths to molecules bound to or near the surface of said optical fiber waveguide.

14. An apparatus of claim 13 wherein said adjusting means is mounted on a translatable mount positioned so that the axis along which translation translation occurs is perpendicular to the longitudinal axis of said focusing means.

15. An apparatus of claim 12 wherein said annularizing optical fiber is butt coupled to the proximal end of said optical fiber waveguide so as to transfer light between the two fibers.

16. An apparatus of claim 15 wherein said annularizing fiber and the side surface of the proximal end of said optical fiber waveguide are both clad with a material having a refractive index which is less than that of the material comprising said optical waveguide and approximately equal to or less than that of the said solution in contact with said waveguide.

17. An apparatus of claim 15 wherein said means of butt coupling said fibers is a coupling capillary comprising a cylindrical tube of capillary dimensions having internal radius so as to permit entry of said clad fibers into the interior of said capillary while constraining the position of said fibers in all directions outward from the radial center of said fibers.

18. An apparatus of claim 16 wherein said processing means comprises:

(a) collecting means for collecting fluorescence from said optical fiber waveguide wherein fluorescence exits the proximal end of said optical fiber waveguide, enters the distal end of said annularizing means, propagates through the cable, is collected in its entirety by a annularizer focusing lens, the numerical aperture of the lens being as large or larger than the numerical aperture of the cable, and is converted into a nearly parallel beam of light containing the desired broad band fluorescence to be measured as well as light at the first narrow band of wavelengths which was reflected from or scattered by a surface within the optical apparatus or fiber sensor;

(b) removing means for removing substantially all light at the first narrow band of wavelengths from the parallel beam of light, such as, but not limited to, a holographic notch filter;

(c) detecting means for detecting the remaining fluorescence the beam of light such as but not limited to a photodiode or photomultiplier which are connected to a plurality of detection electronics appropriate for maximizing signal detection when using modulated or pulsed light sources.

19. An apparatus of claim 12 including means for positioning said fiber optic waveguide with respect to a coupling capillary, said means being adapted to slide said fiber optic sensor cartridge into said coupling capillary.

20. An optical sensing apparatus of claim 19 in which an additional means of narrowing the bandwidth of the first wavelength employs a narrow band filter selected from that category of filters which includes multi-layer interference filters and holographic filters, said addition means being situated such that light propagating from said light source impinges on and passes through said additional means of narrowing.

21. An optical sensing apparatus of claim 19 further including additional means for narrowing the bandwidth of the first wavelength using a narrow band filter selected from the group consisting essentially of multi-layer interference filters and holographic filters, said addition means being situated such that light propagating from said light source impinges on and passes through said additional means of narrowing.

22. An apparatus as in claim 19 in which said directing means is a mirror whose cross sectional area is very much smaller than the area of the proximal face of the annularizing device.

23. An apparatus of claim 12 wherein said light source includes means for generating a first narrow band of wavelengths for stimulating fluorescence in molecules at a second band of wavelengths, said narrow band providing improved discrimination between the first narrow band of wavelengths and the fluorescent signal emitted by the fluorescently tagged molecules, said means comprising:

(a) dispersing means for dispersing said light, said dispersing means situated such that light propagating from said light source impinges upon said dispersing means, said impingent light propagating such that each constituent wavelength component of light is angularly dispersed as a function of wavelength so as to angularly separate unwanted wavelength band(s) from wanted wavelength band(s);

(b) directing means for directing said angularly dispersed light along a path of substantial distance, said distance being substantial when the path length is sufficient to spatially separate unwanted wavelength band(s) from wanted wavelength band(s);

(c) Blocking means for intercepting unwanted wavelength bands, said blocking means situated at said substantial distance to said dispersing means.

24. An optical sensing apparatus of claim 23 in which an additional means of narrowing the bandwidth of the first narrow band of wavelengths employs a narrow band filter such as but not limited to a multi-layer interference filter or a holographic filter, situated such that light propagating from said light source impinges on and passes through said filter.

25. An apparatus as in claim 23 wherein said directing means is a mirror whose cross sectional area is very much smaller than the area of the proximal face of the annularizing device.

26. An apparatus of claim 1 wherein said contact between said waveguide surface and said molecules of a second type is achieved by bringing said molecules of a second type into a sensor cartridge, said sensor cartridge comprising in combination:

(a) An optical fiber waveguide, said waveguide having been at least partially stripped of its cladding in a central portion while possessing cladding along the longitudinal surface of its proximal end, and said central portion having been treated so as to hold in proximity to the longitudinal surface of said optical fiber said plurality of a certain molecule of a first type; and

(b) Fluid ferrules which position said fiber assembly within said cylindrical tube, said cylindrical tube being of capillary dimensions, said end caps possessing holes providing means through which solution may enter and exit said capillary tube, and said end caps also provided with sealing means so as to prevent leaking of said solution at points where said optical fiber and said cylindrical tube contact said fluid ferrules.

27. An apparatus of claim 26 wherein said optical fiber waveguide has a protective sheath surrounding said cladding along the longitudinal surface of its proximal end.

28. An apparatus of claim 26 wherein said optical fiber waveguide has a network of hydrophobic regions on said central portion, said regions being spaced so as to prevent large molecules from contacting the surface of said optical fiber while permitting small molecules to contact and said central surface so as to allow chemical sensitization of said central portion of said optical fiber waveguide.

29. An apparatus of claim 28 wherein said hydrophobic regions are achieved by incomplete dissolution of a hydrophobic cladding material on said optical fiber waveguide.

30. An apparatus of claim 29 wherein said cladding material is a member of a class of chemicals known as amorphous copolymers of perfluoro (22, dimethyl-1,3 dioxole) and tetrafluoroethylene and said dissolution is achieved by means of a solvent belonging to that class of chemicals known as perfluoroalkanes.

31. An optical apparatus of claim 1 comprising in combination:

(a) A light source which generates and transmits a certain light signal;

(b) A dispersive element situated such that light propagating from said light source impinges upon said dispersive element;

Said impingement light, upon exiting from said dispersive element, thereafter propagates such that each constituent wavelength component of light is angularly dispersed as a function of wavelength;

Said dispersive element functions to angularly separate unwanted wavelength band(s) from wanted wavelength band(s);

(c) A means of directing said angularly dispersed light along a path of substantial distance;

Said distance is substantial when the path length is sufficient to spatially separate unwanted wavelength band(s) from wanted wavelength band(s);

(d) Blocking element(s) situated at said substantial distance to said dispersive element;

said blocking element(s) intercepting only unwanted wavelength band(s);

selected wavelength band(s) are not being intercepted by said blocking element(s), and thus, continuing to propagate;

(e) Means of directing said selected wavelength band(s) into an optical fiber at an angle so as to cause said wavelength bands to propagate as real modes in a substantially confined manner within said optical fiber such that said selected wavelength band(s) emerge from the distal end of said optical fiber in an annular ring having a certain cone angle;

(f) Means of coupling said optical fiber to a second optical fiber, said second fiber being treated so as to attach a plurality of a certain molecule of a first type, in close proximity to at least a portion of the surface of said waveguide, said surface extending in a direction parallel to the direction of transmission of said light through said waveguide, and said second fiber comprising a part of a fiber optic sensor;

(g) Means of introducing test and reagent solution(s) into contact with the surface of said second optical fiber;

(h) Means of collecting light returning from said second optical fiber and directing said light so as to allow light having a specific characteristic to be focused upon a photodetector, while reflected light from the original light source which lacks said specific characteristic is rejected; and

(i) Means for processing a signal generated by said photodetector.

32. An apparatus of claim 31 wherein said light source comprises a laser diode.

33. An apparatus of claim 31 wherein said specific characteristic of light comprises a certain wavelength bundle produced by fluorescent molecules, said fluorescent molecules having become bound to said plurality of molecules held in close proximity to the surface of said second fiber.

34. An apparatus of claim 31 wherein said certain cone angle is such that light entering said second optical fiber generates an evanescent field at the surface of said second optical fiber.

35. An apparatus of claim 31 wherein the surface of said second optical fiber possesses a network of hydrophobic regions, said regions functioning to reduce nonspecific binding of proteins to said surface.

36. The apparatus of claim 1 wherein said certain first type of molecule is a specific nucleotide and said certain second type of molecule is a specific protein.

37. The apparatus of claim 36 wherein said specific protein is at least a portion of a biological receptor and said specific nucleotide is at least a portion of a biological response element for said biological receptor.

38. The apparatus of claim 1 wherein said certain first type of molecule is a specific ligand and said certain second type of molecule is a specific protein.

39. The apparatus of claim 1 wherein said specific protein is at least a portion of a hormone receptor and said specific ligand is a hormone known to have a binding affinity for said hormone receptor.

40. The apparatus of claim 1 wherein said biological receptor is an estrogen receptor and said specific nucleotide is an estrogen response element.

41. The apparatus of claim 1 wherein said biological receptor is an estrogen receptor and said hormone is an estrogen.

42. An apparatus of claim 1 in which said coupling means focuses light from said annularizing means into said optical fiber waveguide by means of a high numerical aperture optical relay device such as, but not limited to, optical relay devices having numerical aperture at least as large as the numerical aperture of the annularizing device.

43. An apparatus of claim 1 wherein said contact between said waveguide surface and said molecules of a second type includes bringing said molecules of a second type into a sensor cartridge, said sensor cartridge comprising in combination:

(a) An optical fiber waveguide, said waveguide having been at least partially stripped of its cladding in a central portion while retaining for purpose of effective butt coupling, the cladding of claim 23 along the longitudinal surface of its proximal end, and said central portion having been treated so as to hold in proximity to the longitudinal surface of said optical fiber said plurality of a certain molecule of a first type; and

(b) Fluid ferrules which position said fiber assembly within said cylindrical tube, said cylindrical tube being of capillary dimensions, said end caps possessing holes providing means through which solution enters and exits said capillary tube, and said end caps further including sealing means for preventing leaking of said solution at points where said optical fiber and said cylindrical tube contact said fluid ferrules.

44. An optical sensing apparatus for stimulating and measuring fluorescence from fluorescently tagged molecules bound to or near to the surface of an optical fiber sensor, said optical apparatus comprising:

(a) A light source selected from the group consisting essentially of a pulsed laser, a laser diode and an LED, said light source generating a beam of light at a first narrow band of wavelengths, to be used for stimulating fluorescence at a second band of wavelengths;

(b) narrowing means for narrowing initial bandwidth of said beam of light at the first narrow band of wavelengths to allow subsequent fluorescence detection means to better discriminate between said first narrow band of wavelengths and said second band of wavelengths, said means comprising;

(i) A dispersive element situated such that light propagating from said light source impinges upon said dispersive element, said impingent light, upon exiting from said dispersive element, thereafter propagating such that each constituent wavelength component of light is angularly dispersed as a function of wavelength, said dispersive element also functioning to angularly separate unwanted wavelength band(s) from wanted wavelength band(s); and

(ii) directing means for directing said angularly dispersed light along a path of substantial distance, said distance being sufficient to spatially separate unwanted wavelength band(s) from wanted wavelength band(s); and

(iii) Blocking element(s) situated at said substantial distance to said dispersive element, said blocking element(s) intercepting only unwanted wavelength band(s) while allowing selected wavelength band(s) to continue to propagate as light possessing a narrowed bandwidth

(c) means for directing said beam of narrowed bandwidth into an annularizing optical device which transforms the approximately cylindric beam of light impingent on the proximal end of said annularizing optical device into an annular cone of light exiting the distal end of the annularizing optical device;

(d) annularizing means by which a substantially uniformly distributed and cylindrical beam of light containing an initial first amount of light power and is impinged upon, said annularizing means becoming topologically transformed with minimal loss of said initial first amount of light power into an emergent second beam which may be collimated, converging or diverging and is substantially uniformly distributed in power within an annular shell or region on the outside of the beam, the inside region being substantially devoid of light power;

said annularizing device consisting of;

(i) A large numerical aperture optical focusing means, said numerical aperture being at least as large as said optical fiber sensor, said focusing means having an acceptance aperture significantly larger than the diameter of the beam containing said first narrow band of wavelengths impingent upon said annularizing device, said focusing means located proximally to and focusing said beam onto the surface of a large numerical aperture optical fiber cable, the numerical aperture of said lens being as large or larger than the numerical aperture of said optical fiber cable, said focusing means including in the focusing, elements of said beam impingent normal to the aperture of said focusing means as well as elements substantially offset from the optical axis of said focusing means;

(ii) Said optical fiber cable having sufficient length to allow the off axis beam injected by the focusing means to propagate as real modes in a substantially confined manner within said optical fiber cable and become uniformly distributed in higher order modes such that the beam exits the cable as an annular cone of light, said annular cone when projected from the distal face of the fiber upon a flat surface shows essentially all emergent light being contained in a thin ring, the thickness of such ring being far smaller than the outer ring diameter;

said cable having the identical numerical aperture and diameter as said optical fiber sensor, the distal end of said cable being the distal end of said annularizing device;

(e) coupling means for coupling the annular cone of light emergent from said optical annularizing device into said optical fiber sensor in which the proximal face of said optical fiber sensor is butt coupled (placed directly in contact with) to the distal end of said annularizing means,

(f) adjusting means for adjusting cone angle of light emergent from said annularizing device such that the cone angle of light entering said optical fiber sensor is adjusted so that substantially all rays of light subsequently propagating through said optical fiber impinge upon the side surfaces of said optical fiber sensor at the proper angle for maximum evanescent coupling of the light at the first narrow band of wavelengths to fluorescent molecules bound to or near the surface of said fiber sensor;

(g) collecting means for collecting fluorescence from said optical fiber sensor wherein fluorescence exits from a proximal end of said optical fiber sensor and enters a distal end of said annularizing means, propagates through a cable, is collected in its entirety by said focusing means, and converted into a nearly parallel beam of light containing the desired broad band fluorescence to be measured as well as light at the first narrow band of wavelengths which was reflected from or scattered by a surface within the optical apparatus or fiber sensor;

(h) blocking means for blocking substantially all light at said first narrow band of wavelengths from said parallel beam of light, said blocking means selected from the group consisting essentially of a holographic notch filter and like filters; and

(i) detecting means for detecting fluorescence, said detecting means selected from the group consisting of a photodiode and a photomultiplier connected to a plurality of detection electronics appropriate for maximizing signal detection when using modulated or pulsed light sources selected from the group consisting essentially of analog to digital converters, synchronous detectors, lock-in amplifiers, and photon counters.

which may be electronically modulated to increase detection sensitivity.

45. An apparatus of claim 44 wherein said butt coupling is replaced by or coupled by means of an high numerical aperture optical relay devices, said numerical aperture of said optical relay devices being at least as large as the numerical aperture of the annularizing device.