Light scattering optical waveguide method for detecting specific binding events
First Claim
1. A method for detecting the presence or amount of one or more specific binding analytes in a fluid sample, the method comprising:
- (a) providing a waveguide device, the waveguide device comprising (i) a transparent element having a refractive index greater than that of the fluid sample;
(ii) a light receiving edge; and
(iii) a reactive surface comprising a first specific binding member of at least one cognate binding pair immobilized at a plurality of sites on the surface of the element, other non-situs portions of the reactive surface having no specific binding member immobilized thereon;
wherein said first specific binding member, through intermediate cognate binding pairs if desired, specifically binds at least one analyte;
(b) contacting the reactive surface with a sample suspected to contain said one or more analytes and with a light scattering label attached to a specific binding member of a second cognate binding pair which, through intermediate cognate binding pairs if desired, specifically binds said one or more analytes, in the case of a sandwich assay, or the immobilized first specific binding member of said first cognate binding pair, in the case of a competitive assay;
thereby forming light scattering label complexes attached to the plurality of sites in proportion to the amount of analyte in the sample;
(c) illuminating the light receiving edge of the waveguide with light effective to create total internal reflection within the waveguide, thereby simultaneously illuminating the entire reactive surface;
(d) collecting visually detectable light scattered by said light scattering label;
(e) comparing the degree of light scattering at each situs with either (i) the degree of light scattering at a non-situs portion, or (ii) the degree of light scattering at another situs, or both, whereby light scattering at each situs correlates to the presence or amount of the analyte for which the immobilized specific binding member at that situs is specific.
1 Assignment
0 Petitions
Accused Products
Abstract
A waveguide binding assay method involves detecting the scattering of light directed into the waveguide, the scattering being the result of scattering labels specifically bound to the waveguide within the penetration depth of an evanescent wave. The waveguide may be transparent plastic or glass and the binding is typically by oligonucleotide hybridization or immunological capture. Light scattering labels include colloidal metals or non-metals, including gold, selenium and latex. A light absorbing member consisting of dye or concentrated particles may also be employed to enhance signal. Real-time binding and dissociation can be monitored visually or by video imaging, such as with a CCD camera and frame grabber software. Hybridization mismatches of as few as one base can be distinguished by real-time melting curves.
361 Citations
80 Claims
-
1. A method for detecting the presence or amount of one or more specific binding analytes in a fluid sample, the method comprising:
-
(a) providing a waveguide device, the waveguide device comprising (i) a transparent element having a refractive index greater than that of the fluid sample;
(ii) a light receiving edge; and
(iii) a reactive surface comprising a first specific binding member of at least one cognate binding pair immobilized at a plurality of sites on the surface of the element, other non-situs portions of the reactive surface having no specific binding member immobilized thereon;
wherein said first specific binding member, through intermediate cognate binding pairs if desired, specifically binds at least one analyte;(b) contacting the reactive surface with a sample suspected to contain said one or more analytes and with a light scattering label attached to a specific binding member of a second cognate binding pair which, through intermediate cognate binding pairs if desired, specifically binds said one or more analytes, in the case of a sandwich assay, or the immobilized first specific binding member of said first cognate binding pair, in the case of a competitive assay;
thereby forming light scattering label complexes attached to the plurality of sites in proportion to the amount of analyte in the sample;(c) illuminating the light receiving edge of the waveguide with light effective to create total internal reflection within the waveguide, thereby simultaneously illuminating the entire reactive surface; (d) collecting visually detectable light scattered by said light scattering label; (e) comparing the degree of light scattering at each situs with either (i) the degree of light scattering at a non-situs portion, or (ii) the degree of light scattering at another situs, or both, whereby light scattering at each situs correlates to the presence or amount of the analyte for which the immobilized specific binding member at that situs is specific. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22)
-
-
23. A method for visually detecting the presence or approximate amount of at least one specific binding analyte in a fluid sample, the method comprising:
-
(a) providing a waveguide device, the waveguide device comprising (i) a transparent element having a refractive index greater than that of the fluid sample;
(ii) a light receiving edge; and
(iii) a reactive surface comprising a first specific binding member of at least one cognate binding pair immobilized on at least one test situs on the surface of the element, other non-situs portions of the reactive surface having no specific binding member immobilized thereon;
wherein said first specific binding member, through intermediate cognate binding pairs if desired, specifically binds said analyte;(b) contacting the reactive surface with the sample suspected to contain said analyte and with a light scattering label attached to a first specific binding member of a second cognate binding pair which, through intermediate cognate binding pairs if desired, specifically binds said analyte, in the case of a sandwich assay, or the immobilized first specific binding member, in the case of a competitive assay;
thereby forming light scattering label complexes attached to the situs in proportion to the amount of the analyte in the sample;(c) illuminating the light receiving edge of the waveguide with light effective to create total internal reflection within the waveguide, thereby illuminating the reactive surface; (d) visually examining the reactive surface for light scattering and comparing the degree of light scattering at the test situs with either (i) the degree of light scattering at a non-situs portion, or (ii) the degree of light scattering at another situs, or both, whereby scattering at the situs correlates to the presence or amount of said analyte. - View Dependent Claims (24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36)
-
-
37. A method for detecting the presence or amount of at least one specific binding analyte in a fluid sample, the method comprising:
-
(a) providing a waveguide device, the waveguide device comprising (i) a transparent element having a refractive index greater than that of the fluid sample;
(ii) a light receiving edge; and
(iii) a reactive surface comprising a first specific binding member of at least one cognate binding pair immobilized at a situs on the surface of the element, other non-situs portions of the reactive surface having no specific binding member immobilized thereon;
wherein said first specific binding member, through intermediate cognate binding pairs if desired, specifically binds said analyte;(b) contacting the reactive surface with the sample suspected to contain said analyte and with a light scattering label attached to a first specific binding member of a second cognate binding pair which, through intermediate cognate binding pairs if desired, specifically binds said analyte, in the case of a sandwich assay, or the immobilized first specific binding member, in the case of a competitive assay;
thereby forming light scattering label complexes attached to said situs in proportion to the amount of analyte in the sample;(c) illuminating the light receiving edge of the waveguide with light effective to create total internal reflection within the waveguide, thereby simultaneously illuminating the entire reactive surface; (d) collecting visually detectable light scattered by said light scattering label at a first time, t1, using a photodetector device; (e) repeating steps (c) and (d) at least once to collect scattered light, if any, from said situs and non-situs portions at a second time, t2 ; and (f) comparing the degree of light scattering at said situs at time t1 with the degree of light scattering at said situs at time t2, whereby the light scattering at the situs correlates to the presence or amount of the specific analyte, and the difference over time in scattering of light provides kinetic information indicative of the amount of analyte present at said situs. - View Dependent Claims (38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48)
-
-
49. A method for determining the nucleotide sequence of segment of unknown nucleic acid or for distinguishing two closely related nucleotide sequences, the method comprising:
-
(a) providing a waveguide device, the waveguide device comprising (i) a transparent element having a refractive index greater than that of the fluid sample;
(ii) a light receiving edge; and
(iii) a reactive surface comprising a plurality of sites having oligonucleotide immobilized thereon, said sites defining an array of oligonucleotides having different sequences for hybridizing with the unknown nucleic acid, other non-situs portions of the surface of said element having no oligonucleotides immobilized thereon;(b) contacting the reactive surface under hybridizing conditions with said unknown nucleic acid wherein said unknown nucleic acid, either directly or through intermediate cognate binding pairs if desired, is labeled with a light scattering label;
thereby forming light scattering label complexes attached at those sites of the reactive surface having an oligonucleotide, complementary to the sequence of the unknown nucleic acid, immobilized thereon;(c) illuminating the light receiving edge of the waveguide with light effective to create total internal reflection within the waveguide, thereby simultaneously illuminating the entire reactive surface; (d) collecting visually detectable light scattered by said light scattering label; (e) comparing the degree of light scattering at each situs with either (i) the degree of light scattering at a non-situs portion;
or (ii) the degree of light scattering at another situs; and(f) further comprising incrementally increasing the stringency conditions at the reactive surface of the waveguide device to initiate dissociation of bound nucleic acid from the sites and repeating steps (d) and (e) at each increment; whereby single base pair differences between the oligonucleotides and the unknown nucleic acid can be distinguished from perfect matches by differences in dissociation properties. - View Dependent Claims (50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62)
-
-
63. A method for detecting the presence or mount of a specific binding analyte in a fluid sample, the method comprising:
-
(a) providing a TIR device, the device comprising (i) a transparent TIR element having a refractive index greater than that of the fluid sample;
(ii) a light receiving edge; and
(iii) a reactive surface comprising a first specific binding member of at least one cognate binding pair immobilized on at least one situs on the surface of the element, other non-situs portions of the reactive surface having no specific binding member immobilized thereon;
wherein said first specific binding member, through intermediate cognate binding pairs if desired, specifically binds said analyte;(b) contacting the reactive surface with (i) the sample suspected to contain said analyte;
(ii) a light scattering label attached to a first specific binding member of a second cognate binding pair which, through intermediate cognate binding pairs if desired, specifically binds said analyte, in the case of a sandwich assay, or the immobilized first specific binding member, in the case of a competitive assay, thereby forming light scattering label complexes attached to said situs in proportion to the amount of analyte in the sample; and
(iii) a solution of a light absorbing member sufficient to impart an effective O.D. of at least 15;(c) illuminating the light receiving edge of the TIR element with light effective to create total internal reflection within the element, thereby illuminating the reactive surface; (d) detecting visually detectable light scattered by said light scattering label and comparing the degree of light scattering at the situs with the degree of light scattering at a non-situs portion, whereby background scattering is minimized by absorbance by the light absorbing material. - View Dependent Claims (64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80)
-
Specification