Fiber-optic assay apparatus based on phase-shift interferometry

Description of equivalent WO 2005047854 A2

TITLE

0002[0001] Fiber-optic assay apparatus based on phase-shift interferometry.

CROSS REFERENCE TO RELATED APPLICATIONS

0004[0002] This application claims the benefit of U.S. Non-provisional Application No. , filed November 4, 2004, for Hong Tan, et al, entitled "Fiber-Optic Assay

0005Apparatus Based on Phase-Shift Interferometry," Attorney Docket No. 24377-09611US; U.S. Provisional Application No. 60/518,068, filed November 6, 2003; and U.S. Provisional Application No. 60/558,381, filed March 31, 2004 the entire disclosures of which are hereby incorporated by reference in their entirety for all purposes.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

0007[0003] Not applicable.

BACKGROUND OF THE INVENTION

0009Field of the invention [0004] The present invention relates to an apparatus and method for detecting the presence, amount, or rate of binding of one or more analytes in a sample, and in particular,

0010to apparatus and method based on fiber optic interferometry.

0011Description of the Related Art [0005] Diagnostic tests based on a binding event between members of an analyte-anti- analyte binding pair are widely used in medical, veterinary, agricultural and research applications. Typically, such methods are employed to detect the presence or amount or an analyte in a sample, and/or the rate of binding of the analyte to the anti-analyte. Typical analyte-anti-analyte pairs include complementary strands of nucleic acids, antigen-antibody

0012pairs, and receptor-receptor binding agent, where the analyte can be either member of the

0013pair, and the anti-analyte molecule, the opposite member.

0014[0006] Diagnostics methods of this type often employ a solid surface having immobilized anti-analyte molecules to which sample analyte molecules will bind specifically and with high affinity at a defined detection zone. In this type of assay, known as a solid-phase assay, the solid surface is exposed to the sample under conditions that

0015promote analyte binding to immobilized anti-analyte molecules. The binding event can be detected directly, e.g., by a change in the mass, reflectivity, thickness, color or other

0016characteristic indicative of a binding event. Where the analyte is pre-labeled, e.g., with a chromophore, or fluorescent or radiolabel, the binding event is detectable by the presence and/or amount of detectable label at the detection zone. Alternatively, the analyte can be

0017labeled after it is bound at the detection zone, e.g., with a secondary, fluorescent-labeled

0018anti-analyte antibody.

0019[0007] Co-owned U.S. Patent No. 5,804,453, (the '453 patent) which is incorporated herein by reference, discloses a fiber-optic interferometer assay device designed to detect analyte binding to a fiber-optic end surface. Analyte detection is based on a change in the thickness at the end surface of the optical fiber resulting from the binding of analyte molecules to the surface, with greater amount of analyte producing a greater thickness- related change in the interference signal. The change in interference signal is due to a phase shift between light reflected from the end of the fiber and from the binding layer carried on the fiber end, as illustrated particularly in Figs. 7a and 7b of the '453 patent. The device is simple to operate and provides a rapid assay method for analyte detection. [0008] Ideally, an interferometer assay device will yield readily observable changes in spectral peak and valley (extrema) positions within the range of a conventional visible-light

0020spectrometer, that is, in the visible light range between about 450-700 nm, such that relatively small optical thickness changes at the fiber end can be detected as significant changes in the spectral positions of interference wavelength peaks and valleys. One limitation which has been observed with the device described in the '453 patent is the absence of readily identified wavelength spectral extrema over this spectral range.

0021[0009] The present invention is designed to overcome this limitation, preserving the

0022advantages of speed and simplicity of the earlier-disclosed device, but significantly

0023enl ancing sensitivity and accuracy. The present invention also provides a more convenient disposable-head format, as well as a multi-analyte array format, e.g., for gene-chip and protein-chip applications.

SUMMARY OF THE INVENTION

0025[0010] The invention includes, in one aspect, an apparatus for detecting an analyte in a sample, including detecting the presence of analyte, the amount of analyte or the rate of association and/or dissociation of analyte to analyte-binding molecules. The apparatus includes an optical element with a proximal reflecting surface and a distal reflecting surface separated by at least 50 nm. A beam of light from an optical fiber is directed to and

0026reflected from the two reflecting surfaces. The reflected beams are coupled back into the optical fiber and interfere. The optical element also includes a layer of analyte binding molecules that is positioned so that the interference between the reflected beams varies as analyte binds to the layer of analyte binding molecules. [0011] The change in interference can be caused by different physical phenomenon. For example, analyte binding can cause a change in the optical path length or in the physical distance between the two reflecting surfaces. Alternately, analyte binding can cause a change in the index or in the optical absorption of material located between the reflecting

0027surfaces. Analyte binding can also cause the layer of analyte binding molecules to swell, resulting in a change in the interference.

0028[0012] In one particular design, the distal reflecting surface includes the layer of analyte binding molecules. As analyte binds to the layer of analyte binding molecules, the optical path length or the physical distance between the two reflecting surfaces may increase, for

0029example. In another aspect of the invention, a transparent solid material is located between the reflecting surfaces and, optionally, the proximal reflecting surface includes a material with an index greater than that of the transparent solid material. Alternately, an air gap may be located between the reflecting surfaces. In yet another design, the distal reflecting surface is positioned between the proximately reflecting surface and the layer of analyte

0030binding molecules. For example, analyte binding may cause the layer of analyte binding molecules to swell, moving the distal reflecting surface closer to the proximal reflecting surface. In yet another design, the layer of analyte binding molecules is positioned between the two reflecting surfaces. Analyte binding may cause the layer to swell or to change its index, thus changing the interference between the two reflected beams.

0031[0013] hi another aspect, the apparatus includes an optical assembly having first and second reflecting surfaces separated by a distance "d" greater than 50 nm. The optical assembly is composed of a transparent optical element that can have a thickness defined between proximal and distal faces of the element of at least 50 nm, preferably between 400 - 1,000 nm. The first reflecting surface is carried on the distal face of optical element, and is formed of a layer of analyte-binding molecules. The second reflecting surface is fonned by

0032a coating of transparent material having an index of refraction greater than that of the optical element. This coating can be formed of a Ta O₅ layer having a preferred thickness of

0033between 5 and 50 nm. The optical element can be SiO₂, and has a thickness of between

0034about 100-5,000 nm, preferably 400-1,000 nm.

0035[0014] Also included are a light source for directing a beam of light onto the first and second reflecting surfaces, and a detector unit that operates to detect a change in the optical thickness of the first reflecting layer resulting from binding of analyte to the analyte-bindirxg

0036molecules, when the assembly is placed in the solution of analyte. The optical thickness

0037change at the first reflecting layer is related to a shift in a phase characteristic of the interference wave formed by the two light waves reflected from said first and second surfaces. This phase characteristic can be a shift in the spectral position(s) of one or more peaks and valleys of the interference wave, or by a change in the period of a full cycle of the

0038wave.

0039[0015] The light source can include an optical fiber having a distal end adapted to be placed adjacent the second reflecting surface in the assembly, and the apparatus further includes an optical coupling for directing reflected light waves reflected from the assembly to the detector.

0040[0016] In a first embodiment, the optical assembly is fixedly mounted on the optical

0041fiber, with the distal end of the optical fiber in contact with the second reflecting surface. In a second embodiment, the optical assembly further includes a second transparent optical element having an index of refraction less than that of the second coating and a thickness greater than about 100 nm, where the coating of high index of refraction material is sandwiched between the two transparent optical elements, hi this latter embodiment, the

0042assembly is removably attached to the distal end region of the fiber with a spacing of less

0043than 100 nm or greater than 2 μm between the distal end of the fiber and the confronting

0044face of the second transparent optical element in the assembly.

0045[0017] For detecting multiple analytes, such as multiple nucleic acid species, the layer of analyte-binding molecules can be composed of an array of discrete analyte-binding regions, such as single strands of nucleic acid. The regions are effective to bind different analytes. The optical fiber includes a plurality of individual fibers, each aligned with one of the regions, the detector includes a plurality of detection zones, and the optical coupling functions to couple each of the plurality of fibers with one of the zones.

0046[0018] The analyte-binding molecules in the assembly can be, for example, (i) an anti- species antibody molecules, for use in screening hybridoma libraries for the presence of secreted antibody, (ii) antigen molecules, for use in detecting the presence of antibodies

0047specific against that antigen; (iii) protein molecules, for use in detecting the presence of a

0048binding partner for that protein; (iv) protein molecules, for use in detecting the presence of multiple binding species capable of forming a multi-protein complex with the protein; or (v) single stranded nucleic acid molecules, for detecting the presence of nucleic acid binding molecules.

0049[0019] The detector can be a spectrometer for measuring reflected light intensity over a selected range of wavelengths. Alternatively, or in addition, the light source can include a plurality of light-emitting diodes, each with a characteristic spectral frequency, and the detector functions to record light intensity of reflected light at each of the different LED frequencies. In still another embodiment, the light source includes a white-light source and the detector is designed to record light intensity of reflected light at each of a plurality of

0050different wavelengths.

0051[0020] In another aspect, the invention includes a method for detecting the presence or

0052amount of an analyte in a sample solution. The method involves reacting the sample

0053solution with a first reflecting surface formed by a layer of analyte-binding molecules carried on the distal surface of a transparent optical element having a thickness of at least 5O nm, thereby to increase the thickness of the first reflecting layer by the binding of analyte to the analyte-binding molecules in the layer. The change in thickness of the first reflecting layer is measured by detecting a shift in a phase characteristic of the interference wave formed by the two light waves reflected from the first layer and from a second reflecting layer that is formed on the opposite, proximal surface of the optical element and which has

0054an index of refraction greater than that of the optical element.

0055[0021] The detecting step can include directing light from an optical fiber onto the two reflecting surfaces, and directing reflected light from the two surfaces onto a detector

0056through an optical coupling. The detector can be a spectrometer, where the detecting

0057includes measuring a shift in the spectral position of one or more of the interference extrema produced by the two reflecting lightwaves.

0058[0022] Where the method is used for measuring the rate of association of analyte to the second layer, the reacting step can be carried out until a near-maximum increase in thickness of the first reflecting layer is observed. Where the method is used for measuring the rate of dissociation of analyte to the second layer, the reacting steps can include immersing the second layer in a dissociation buffer for a period of time until a decrease in thickness of the first reflecting layer is observed. Where the method is used for measuring the amount of

0059analyte present in the sample, the detecting is carried out over a period sufficient to measure the thickness of the first reflecting layer at a plurality of different time points.

0060[0023] Where the method is used measuring one or more of a plurality of analytes in a

0061sample, the first reflecting layer is composed of an array of discrete analyte-binding regions,

0062the different regions being effective to bind different analytes, and the detecting is effective to detect a change in the thickness of each of the regions resulting from binding of analyte to the analyte-binding molecules.

0063[0024] These and other objects and features of the present invention will become more

0064fully apparent when the following detailed description of the invention is read in

0065conjunction with the accompanying drawings.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWING

0067[0025] These and other features, aspects, and advantages of the present invention will become better understood with regard to the following description, and accompanying drawings, where:

0068[0026] Fig. 1 shows the basic system setup for the bioprobe and its apparatus;

0069[0027] Fig. 2 shows an optical assembly formed accordance to one embodiment of the invention;

0070[0028] Figs. 3A and 3B show a portion of an interference wave over 7 peak and valley orders (3 A), and over in a visible portion of the spectrum (3B);

0071[0029] Fig. 4 shows an optical assembly constructed according to another embodiment of the invention; [0030] Fig. 5 shows a disposable multi-analyte optical assembly having an analyte-

0072binding array and constructed according to another embodiment of the invention;

0073[0031] Fig. 6 shows a sequential binding of three molecules;

0074[0032] Fig. 7 shows on and off curves generated from the association and dissociation of antibodies;

0075[0033] Fig. 8 shows the curves of two antibodies binding to their antigen at different concentrations;

0076[0034] Fig. 9 shows immobilization of bis amino PEG (MW 3300) specifically through an amide bond formation. The PEG (MW 8000) is used as a negative control to monitor

0077non-specific binding of the PEG polymer; and

0078[0035] Fig. 10 shows a small molecule binding to a large molecule, negative controls and the base line measurement.

DETAILED DESCRIPTION OF THE INVENTION

0080Definitions

0081[0036] Terms used in the claims and specification are to be construed in accordance with their usual meaning as understood by one skilled in the art except and as defined as set forth below. Numeric ranges recited in the claims and specification are to be construed as including the limits bounding the recited ranges.

0082[0037] The term "in vivo" refers to processes that occur in a living organism.

0083[0038] An "analyte-binding" molecule refers to any molecule capable of participating in a specific binding reaction with an analyte molecule. Examples include but are not limited to, e.g., antibody-antigen binding reactions, and nucleic acid hybridization reactions. [0039] A "specific binding reaction" refers to a binding reaction that is saturable, usually

0084reversible, and that can be competed with an excess of one of the reactants. Specific binding reactions are characterized by complementarity of shape, charge, and other binding determinants as between the participants in the specific binding reaction. [0040] An "antibody" refers to an immunoglobulin molecule having two heavy chains and two light chains prepared by any method known in the art or later developed and

0085includes polyclonal antibodies such as those produced by inoculating a mammal such as a

0086goat, mouse, rabbit, etc. with an immunogen, as well as monoclonal antibodies produced

0087using the well-known Kohler Milstein hybridoma fusion technique. The term includes antibodies produced using genetic engineering methods such as those employing, e.g., SCID

0088mice reconstituted with human immunoglobulin genes, as well as antibodies that have been

0089humanized using art-known resurfacing techniques.

0090[0041] An "antibody fragment" refers to a fragment of an antibody molecule produced by chemical cleavage or genetic engineering techniques, as well as to single chain variable fragments (SCFvs) such as those produced using combinatorial genetic libraries and phage display technologies. Antibody fragments used in accordance with the present invention

0091usually retain the ability to bind their cognate antigen and so include variable sequences and

0092antigen combining sites.

0093[0042] A "small molecule" refers to an organic compound having a molecular weight less than about 500 daltons. Small molecules are useful starting materials for screening to identify drug lead compounds that then can be optimized through traditional medicinal chemistry, structure activity relationship studies to create new drugs. Small molecule drug compounds have the benefit of usually being orally bioavailable. Examples of small molecules include compounds listed in the following databases: MDL/ACD

0094(http://www.mdli.com/), MDL/MDDR (http://www.mdli.com/), SPECS

0095(http://www.specs.net/), the China Natural Product Database (CNPD)

0096(http://www.neotrident.com/), and the compound sample database of the National Center for

0097Drug Screening (http://www.screen.org.cn/).

0098[0043] Abbreviations used in this application include the following: "ss" refers to

0099single-stranded; "SNP" refers to single nucleotide polymoφhism; "PBS" refers to phosphate buffered saline (0.01 M phosphate buffer, 0.0027 M potassium chloride and 0.137 M sodium chloride, pH 7.4); "NHS" refers to N-hydroxysuccinimide; "MW" refers to molecular weight; "Sulfo-SMCC" refers to sulfosuccinimidyl 4-[N- maleimidomethyl]cyclohexane-l-carboxylate.

0100[0044] It must be noted that, as used in the specification and the appended claims, the

0101singular forms "a," "an" and "the" include plural referents unless the context clearly dictates otherwise.

0102Advantages and utility

0103[0045] The advantages and utility of the invention are illustrated by reference to the Figures and Examples as described in greater detail below. These include the ability to

0104monitor in real time analyte binding reactions without the use of labels, diminishing cost and potential toxicity. A further advantage includes the ability to practice the method using visible wavelength light sources. Yet other advantages are provided by the fiber optic nature of the detector tip that allows binding reactions to be monitored in very small sample volumes, including in "in vitro" spaces, and to bundle fibers to carry out highly multiplexed analyses of binding reactions. [0046] Fig. 1 shows, in schematic view, an interferometer apparatus 20 constructed in

0105accordance with the invention, hi its most basic elements, the apparatus includes a light

0106source 22, an optical assembly 26 that functions as a sensing element or detector tip and that

0107will be detailed further with respect to Figs. 2, 4 and 5 below, and a detector unit 28 for detecting interference signals produced by interfering light waves reflected from the optical

0108assembly 26.

0109[0047] Light from source 22 is directed onto the optical assembly 26, and reflected back

0110to the detector through an optical coupling assembly indicated by dashed lines at 30. hi a

0111preferred embodiment, the coupling assembly includes a first optical waveguide or fiber 32 extending from the light source to the optical assembly, a second optical waveguide or fiber

011234 which carries reflected light from the optical assembly to the detector, and an optical coupler 36 which optically couples fibers 32, 34. Suitable optical fiber and coupling

0113components are detailed in the above-cited '453 patent. One exemplary coupler is

0114commercially available from many vendors including Ocean Optics (Dunedin, Florida).

0115[0048] Alternatively, the coupling assembling can include a lens system constructed to focus a light beam onto the upper surface of the optical assembly and to direct reflected interfering light from the optical assembly to the detector. The latter system would not

0116require optical fibers, but would impose relatively stringent requirements on the positioning of the lens elements used for the optical coupling.

0117[0049] The light source in the apparatus can be a white light source, such as a light

0118emitting diode (LED) hich produces light over a broad spectrum, e.g., 400 nm or less to 700 nm or greater, typically over a spectral range of at least 100 nm. Alternatively, the light

0119source can be a plurality of sources each having a different characteristic wavelength, such as LEDs designed for light emission at different selected wavelengths in the visible light

0120range. The same function can be achieved by a single light source, e.g., white light source,

0121with suitable filters for directing light with different selected wavelengths onto the optical assembly.

0122[0050] The detector is preferably a spectrometer, such as charge-coupled device (CCD), capable of recording the spectrum of the reflected interfering light from the optical assembly. Alternatively, where the light source operates to direct different selected

0123wavelengths onto the optical assembly, the detector can be a simple photodetector for recording light intensity at each of the different irradiating wavelengths. In still another embodiment, the detector can include a plurality of filters which allows detection of light intensity, e.g., from a white-light source, at each of a plurality of selected wavelengths of the

0124interference reflectance wave. Exemplary light source and detector configurations are

0125described in the above-cited '453 patent, particularly with respect to Figs. 8 and 10 of that

0126patent, and it will be understood that these configurations are suitable for use in the present invention.

0127[0051] Fig. 2 shows an optical assembly 26 constructed in accordance with one embodiment of the invention, and an adjoining portion of the distal end region of an optical

0128fiber 32 to which the optical assembly is fixedly attached. As seen, the assembly 26

0129includes a transparent optical element 38 having first and second reflecting surfaces 42, 40 fonned on its lower (distal) and upper (proximal) end faces, respectively. According to an important feature of the invention, the thickness "d" of the optical element between its distal and proximal surfaces, i.e., between the two reflecting surfaces, is at least 50 nm, and

0130preferably at least 100 nm. An exemplary thickness is between about 100-5,000 nm, preferably 400-1,000 nm. The first reflecting surface 42 is formed of a layer of analyte-

0131binding molecules, such as molecules 44, which are effective to bind analyte molecules 46

0132specifically and with high affinity. That is, the analyte and anti-analyte molecules are opposite members of a binding pair of the type described above, which can include, without limitations, antigen-antibody pairs, complementary nucleic acids, and receptor-binding agent pairs.

0133[0052] The index of refraction of the optical element is preferably similar to that of the

0134first reflecting surface, so that reflection from the lower distal end of the end optical assembly occurs predominantly from the layer formed by the analyte-binding molecules, rather than from the interface between the optical element and the analyte-binding molecules. Similarly, as analyte molecules bind to the lower layer of the optical assembly, light reflection form the lower end of the assembly occurs predominantly from the layer

0135formed by the analyte-binding molecules and bound analyte, rather than from the interface region. One exemplary material forming the optical element is SiO₂, e.g., a high-quality quality glass having an index of refraction of about 1.4-1.5. The optical element can also be formed of a transparent polymer, such as polystyrene or polyethylene, having an index of refraction preferably in the 1.3-1.8 range.

0136[0053] The second reflecting surface in the optical assembly formed as a layer of transparent material having an index of refraction that is substantially higher than that of the optical element, such that this layer functions to reflect a portion of the light directed onto the optical assembly. Preferably, the second layer has a refractive index greater than 1.8. One exemplary material for the second layer is Ta₂O₅ with refractive index equal to 2.1.

0137The layer is typically formed on the optical element by a conventional vapor deposition coating or layering process, to a layer thickness of less than 50 nm, typically between 5 and 30 nm.

0138[0054] The thickness of the first (analyte-binding) layer is designed to optimize the overall sensitivity based on specific hardware and optical components. Conventional immobilization chemistries are used in chemically, e.g., covalently, attaching a layer of analyte-binding molecules to the lower surface of the optical element. For example, a variety of bifunctional reagents containing a siloxane group for chemical attachment to

0139SiO₂, and an hydroxyl, amine, carboxyl or other reaction group for attachment of biological

0140molecules, such as proteins (e.g., antigens, antibodies), or nucleic acids. It is also well known to etch or otherwise treat glass a glass surface to increase the density of hydroxyl groups by which analyte-binding molecules can be bound. Where the optical element is formed of a polymer, such as polystyrene, a variety of methods are available for exposing available chemically-active surface groups, such as amine, hydroxyl, and carboxyl groups.

0141[0055] The analyte-binding layer is preferably formed under conditions in which the distal surface of the optical element is densely coated, so that binding of analyte molecules to the layer forces a change in the thickness of the layer, rather than filling in the layer. The analyte-binding layer can be either a monolayer or a multi-layer matrix.

0142[0056] The measurement of the presence, concentration, and/or binding rate of analyte to the optical assembly is enabled by the interference of reflected light beams from the two reflecting surfaces in the optical assembly. Specifically, as analyte molecules attach to or detach from the surface, the average thickness of the first reflecting layer changes accordingly. Because the thickness of all other layers remains the same, the interference wave fonned by the light waves reflected from the two surfaces is phase shifted in accordance with this thickness change.

0143[0057] Assume that there are two reflected beams: The first beam is reflected from the

0144first surface, which is the distal end interface between analyte-binding molecules and bound analyte and the sunounding medium; and the second beam is reflected from the second surface, which is the proximal interface between the optical element (the first layer) and the high-index of refraction layer (the second layer). The overall wavelength -dependent

0145intensity of the interference wave is: <img file="WO2005047854A2_D0001.tif" />

0146[0058] where I is the intensity, l_\ and I are the intensity of two interference beams, Δ is the optical path difference, and λ is the wavelength.

0147[0059] When (2πΔ/λ) =Nπ, the curve is at its peak or valley if N is an integer 0, 1, 2,....

0148The thickness of the first layer d = Δ/2n. Therefore, λ = 4nd/N at peaks or valleys (extrema).

0149[0060] For the first several values of N, i. e. , 0, 1, 2, ...7, and assuming a d of 770 nm, the equation gives:

0150[0061] N=0: λ = ∞ (peak)

0151[0062] N =l: λ = 4nd= 4,496.80 nm (Valley)

0152[0063] N =2 : λ = 2nd= 2,248.40 nm (Peak)

0153[0064] N =3 : λ = 4nd/3=l ,498.9 nm (Valley)

0154[0065] N =4: λ = nd= 1 , 124.20nm (Peak )

0155[0066] N =5: λ = 4nd/5=899.36 nm (Valley)

0156[0067] N =6: λ = 2nd/3 =749.47 nm (Peak ) [0068] N =7: λ = 4nd/7 = 642 nm (Valley)

0157[0069] N =8: λ = nd/2 = 562 nm (Peak)

0158[0070] N =9: λ = 4nd/9 = 499.64nm (Valley)

0159[0071] N =10: λ = 4nd/10 = 449.6 nm (Peak )

0160[0072] As can be seen, and illustrated further in Figs. 3A and 3B, at least three peaks/valleys (N=7-9) occur in the visible spectral range.

0161[0073] If the 7^th order valley is used to calculate the change in molecular layer thickness,

0162when the molecular layer attached to the first layer increases from 0 nm to 10 mn, the 7^th

0163order valley will shift to 650.74 mn. Therefore, the ratio between the actual the phase shift of the 7^th order valley and thickness change equals (650.74-642.40)/10 = 0.834.

0164[0074] By contrast, if the initial spacing between the two reflecting layers is made up entirely of the analyte-binding molecules on the end of the fiber, assuming a thickness of this layer of 25 nm, then the first order peak will occur at 146 nm, clearly out of the range of the visible spectrum, so that the device will only see a portion of the region between the 0- order valley and the first order peak, but will not see any peaks, making a shift in the spectral characteristics of the interference wave difficult to measure accurately.

0165[0075] Not until the total thickness of the reflecting layer approaches about 100 nm will the first-order peak appear in the visible spectrum. Assuming a total thickness change of up

0166to 50 nm, the thickness of the optical element can then be as small as 50 nm, but is preferably on the order of several hundred nm, so that the phase shift or change in periodicity of the interference wave can be measured readily by a shift in the spectral positions of higher-order peaks or valleys, e.g., where N=3-10. [0076] The ratio between the actual thickness and the measured phase shift is

0167considered as a key factor of measurement sensitivity. It can be appreciated how one can

0168adjust the thickness of the optical element and its refractive index to improve and optimize the sensitivity to accommodate the electronics and optical designs.

0169[0077] Fig. 4 shows an optical assembly 50 that is removably carried on the distal end of

0170an optical fiber 52 in the assay apparatus. The optical element includes a plurality of

0171flexible gripping arms, such as anns 54, that are designed to slide over the end of the fiber

0172and grip the fiber by engagement of an annular rim or detente 56 on the fiber with complementary-shaped recesses formed in the arms, as shown. This attachment serves to

0173position the optical assembly on the fiber to provide an air gap 58 between the distal end of

0174the fiber and the confronting (upper) face of the assembly, of less than 100 nm

0175or greater than 2 μm. With an air gap of greater than about 100 nm, but less that 2 μm,

0176internal reflection from the upper surface of the optical assembly can contribute significantly to undesirable fringes that can adversely impact the detection accuracy.

0177[0078] With continued reference to Fig. 4, the optical assembly includes a first optical

0178element 60 similar to optical element 38 described above, and having first and second

0179reflective layers 62, 64, respectively, corresponding to above-described reflective layers 40,

018042, respectively. The assembly further includes a second optical element 66 whose thiclαiess is preferably greater than 100 nm, typically at least 200 nm, and whose index of

0181refraction is similar to that of first optical element 60. Preferably, the two optical elements are constructed of the same glass or a polymeric material having an index of refraction of

0182between about 1.4 and 1.6. Layer 64, which is formed of a high index of refraction material, and has a thickness preferably less than about 30 nm, is sandwiched between the 2 optical

0183elements as shown.

0184[0079] In operation, the optical assembly is placed over the distal fiber end and snapped into place on the fiber. The lower surface of the assembly is then exposed to a sample of analyte, under conditions that favor binding of sample analyte to the analyte-binding

0185molecules forming reflective layer 62. As analyte molecules bind to this layer, the thickness of the layer increases, increasing the distance "d" between reflective surfaces 62 and 64. This produces a shift in the extrema of the interference wave produced by reflection from the two layers, as described above with reference to Figs. 3A and 3B. This shift in extrema

0186or wavelength, or wavelength period, in turn, is used to determine the change in thickness at the lower (distal-most) reflecting layer. After use, the optical assembly can be removed and discarded, and replaced with fresh element for a new assay, for assaying the same or a different analyte.

0187[0080] Fig. 5 illustrates an optical assembly and fiber bundle in an embodiment of the invention designed for detecting one or more of a plurality of analytes, e.g. , different-

0188sequence nucleic acid analytes, in a sample. A fiber bundle 72 is composed of an array, e.g.,

0189circular array, for individual optical fibers, such as fibers 74. The optical assembly, indicated generally at 70, is composed of the basic optical elements described above with

0190reference to Fig. 4, but in an array format. Specifically, a first optical element 80 in the element provides at its lower distal surface, an array of analyte-reaction regions, such as

0191regions 84, each containing a layer of analyte-binding molecules effective to bind to one of

0192the different analytes in the sample. Each region forms a first reflective layer in the optical assembly. One preferred sensing provides an array of different-sequence nucleic acids, e.g., cDNAs or oligonucleotides, designed to hybridize specifically with different-sequence

0193nucleic acid analyte species in a sample. That is, the array surface forms a "gene chip" for detecting each of a plurality of different gene sequences.

0194[0081] Also included in the optical assembly are a second optical element 78 and a layer

019579 of high index of refraction material sandwiched between the two optical elements, and which provides the second reflecting surface in the optical assembly. The assembly is carried on the fiber bundle 72 by engagement between a pair of flexible support arm, such as arm 76 and an annular rim or detente 86 on the bundle. With the assembly placed on the fiber bundle, the lower distal ends of the fibers are spaced from the confronting surface of

0196optical element 78 by an air gap 85 whose spacing is preferably less than 100 nm or greater than 2 μm. Further, each of the fibers is aligned with a corresponding assay region of the optical assembly, so that each fiber is directing light on, and receiving reflected light from, its aligned detection region. Similarly, the optical coupler in the apparatus, which serves to couple multiple fibers to the detector, preserves the alignment between the array regions and corresponding positions on an optical detector, e.g., two-dimensional CCD. The materials and thickness dimensions of the various optical-assembly components are similar to those

0197described above with respect to Fig. 4.

0198[0082] The apparatus described in this invention can be used more specifically for the following applications:

0199[0083] (i) with an anti-species antibody carried on the tip, for screening hybridoma

0200expression lines for cell lines with high antibody expression;

0201[0084] (ii) with an antigen carried on the tip, to characterize high affinity antibodies

0202against that antigen;

0203[0085] (iii) with a protein carried on the tip, for identifying and characterizing binding partners (DNA, RNA, proteins, carbohydrates, organic molecules) for that protein;

0204[0086] (iv) with a carbohydrate or glycosyl moiety carried on the tip, for identifying and characterizing binding partners (such as, e.g., DNA, RNA, proteins, carbohydrates, organic molecules) for that carbohydrate;

0205[0087] (v) with a protein thought to participate in a multi-protein complex carried on the tip, for characterizing the binding components and/or kinetics of complex formation;

0206[0088] (vi) with a small protein-binding molecule carried on the tip, for identifying and characterizing protein binders for that molecule;

0207[0089] (vii) with an antibody carried on the tip, for constructing a calibration curve for the analyte using a set of analytes standards. Using this calibration curve, one can then determine the concentration of the analyte in unknown solutions (cell culture supematants, biological samples, process mixtures, etc).

0208[0090] (viii) with a single-stranded nucleic acid, e.g., ssDNA or RNA carried on the tip, for identifying and molecules that bind specifically to the nucleic acid.

0209[0091] Using a temperature control block, the apparatus and method can also be used to

0210monitor the binding and characterize the binding of an immobilized ssDNA to an

0211oligonucleotide in solution to perform SNP analysis.

0212[0092] The following examples illustrate various methods and applications of the invention, but are in no way intended to limit its scope.

EXAMPLES

0214[0093] Below are examples of specific embodiments for carrying out the present

0215invention. The examples are offered for illustrative purposes only, and are not intended to limit the scope of the present invention in any way. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperatures, etc.), but some experimental error and deviation should, of course, be allowed for.

0216[0094] The practice of the present invention will employ, unless otherwise indicated,

0217conventional methods of protein chemistry, biochemistry, recombinant DNA techniques and pharmacology, within the skill of the art. Such techniques are explained fully in the literature. See, e.g., T.E. Creighton, Proteins: Structures and Molecular Properties (W.H. Freeman and Company, 1993); A.L. Lehninger, Biochemistry (Worth Publishers, Inc., current addition); Sambrook, et al., Molecular Cloning: A Laboratory Manual (2nd

0218Edition, 1989); Methods In Enzymology (S. Colo wick and N. Kaplan eds., Academic Press, Inc.); Remington's Pharmaceutical Sciences, 18th Edition (Easton, Pennsylvania: Mack Publishing Company, 1990); Carey and Sundberg Advance d Organic Chemistry 3^rd Ed.

0219(Plenum Press) Vols A and B(1992).

0220Example 1: Small molecule-protein binding reaction. [0095] This example demonstrates the capability to detect the binding of protein to small molecule immobilized on a sensor tip and subsequent bindings of multiple antibodies.

0221The two-layer configuration on the tip of an optic fiber is used for this test. The thickness of

0222the first Ta₂O₅ layer is 25 nm and the thickness of the second SiO layer is 770 nm. The fiber was purchased from Ocean Optics (Dunedin, Florida). It was manually cut into segments that are 40 mm long. Both ends of these segments were polished to standard mirror surface quality. The polishing method used here was exactly the same as those for

0223optical lenses and mirrors. One surface of these fiber segments was outsourced to an optical coating house for Ta₂O₅ layer and SiO₂ layer. This vendor employed an ion-beam assisted physical vapor deposition (IAPVD) coater made by Leybold. IAPVD is a commonly used coating technique for anti-reflection and optical filters. The experimental steps included the following (all steps are performed at room temperature unless otherwise noted):

0224[0096] The fiber tip was coated with a polymer monolayer derivatized with biotin. The

0225polymer monolayer was prepared using a biotinylated lipid (custom). This lipid was using to form a lipid monolayer on the surface of water solution. The monolayer was cross linked using UV light for 15minutes. Clean, dry fibers were then brought in contact with the floating thin film and the biotin polymer was adsorbed onto the fiber tip. The fibers were then diyed at 60°C for 1 hour. The fiber were then stored under ambient conditions [0097] The biosensor tip was immersed in 50 μg/ml streptavidin streptavidin (Pierce

0226Biotechnology, Rockford IL, cat # 21122) in PBS (Invitrogen, Carlsbad, CA; cat #

022714190078) for 9 minutes and then rinsed briefly with PBS.

0228[0098] The same tip was dipped into 10 μg/ml rabbit-anti-streptavidin solution (AbCam,

0229Cambridge, MA; cat # ab6676-1000) in PBS for 36 minutes and then washed with PBS

0230briefly.

0231[0099] Finally, the tip was immersed in 50 μg/mL donkey-anti-rabbit antibody solution antibody (Jackson ImmunoResearch, West Grove, PA; cat# 711-005-152) in PBS for 25 minutes. A final 10 minute rinse was performed in PBS solution.

0232[00100] Fig. 6 shows the real-time response curve for this sequential binding test. The

0233vertical axis is the 7 order valley phase shift in nanometers. It clearly shows the binding of

0234streptavidin to the biotin already immobilized on the tip, and subsequent bindings of anti- streptavidin antibody to streptavidin and a second antibody to this first antibody. The dissociation of the streptavidin layer from the tip was visible (a small reduction in the optical thickness) at 900 seconds.

0235Example 2: Biomolecular interaction analysis of kinetics and affinity of biomolecular interactions.

0236[00101] This example illustrates use of the invention to carry out a biomolecular interaction analysis (BIA) measuring kinetics and affinity of biomolecular interactions. The same tip configuration as described in Example 1 was used. The experimental steps included the following (all steps are perfonned at room temperature unless otherwise noted):

0237[00102] Mercaptosilane coated tips were prepared using the following procedure. Clean, dry fibers were incubated in a mixture of Toluene: hexanoic acid: mercaptopropyltrioxysilane (10:2:1 volumetric ratio) at room temperature for 24 hours. The

0238fibers were rinsed 2X with lOmL toluene for 5 minutes each. The fibers were then rinsed

0239IX with lOmL of ethanol and dried under a stream of argon and stored at ambient conditions.

0240[00103] The biosensor tip was first derivatized by immersion in a with lOμg/ml solution of rabbit-IgG (Jackson hnmunoResearch, West Grove, PA; cat# 309-005-003) in PBS for 1 hour.

0241[00104] The coated tip was dipped into lOμg/ml goat-anti-rabbit antibody solution

0242(Jackson hnmunoResearch, West Grove, PA; cat# 111-005-003) in PBS and remained in it for 15minutes.

0243[00105] The tip was removed and washed in PBS. To facilitate the dissociation of the second antibody from the first antibody, the PBS was agitated manually for 20 minutes.

0244[00106] The tip was then dipped into the same goat-anti-rabbit solution again to show the reproducible association of goat-anti-rabbit to rabbit-IgG.

0245[00107] Fig. 7 shows the on and off curves generated from the association and dissociation of rabbit-IgG and goat-anti-rabbit. The vertical axis is again the 7^th order valley phase shift. The phase shift is directly related to the average thickness with a ratio of 0.834.

0246The ability to detect the on and off curves reliably is essential for measuring interaction kinetics and affinity.

0247Example 3: Calculating affinity constants from antibody-antigen binding and release curves.

0248[00108] This experiment demonstrates the calculation of affinity constants from

0249measuring on and off curves for two antibodies and their antigen. The proprietary antibodies

0250were labeled as Ab-1 and Ab-2. The molecular weight of the antigen was about 30 kilodaltons. The same tip configuration as described in Example 1 was used. The same

0251mercaptosilane fiber preparation as described in Example 2 was used. The experimental

0252steps included (all steps are performed at room temperature unless otherwise noted): [00109] The fiber tip was activated for covalent attachment of the antigen.

0253Mercaptosilane coated fibers were activated by immersing the sensor tips in 50μL of a

025450mg/mL solution of sulfo-SMCC (Pierce Biotechnology, Rockford LL; cat # 22322) in DMF (Sigma-Aldrich Chemical Company, St Louis, MO; cat # 494488) at for 2 hours. The

0255sensor tips were rinsed briefly in DMF and dried;

0256[00110] The antigen was covalently bound to the activated fiber tip by immersing the

0257activated tip in a 20 μg/ml solution of antigen in PBS for 20 minutes. The tip was rinsed with PBS for 2 minutes. Following the PBS rinse, the tip was quenched with an aqueous

0258solution of 100 μM ethanolamine pH 8.5 (Sigma-Aldrich Chemical Company, St Louis,

0259MO; cat # E9508) for 5 minutes and then was rinsed again in PBS for 2 minutes.

0260[00111] The same tip was immersed in antibody for an association test and the real-time binding data were recorded for 9-15 minutes (depending on the antibody identity and concentration). Once those data were recorded, the tip was again immersed in PBS and agitated to measure the off curve (i.e., dissociation between the immobilized antigen and bound antibody) for 9-15 minutes. The binding (on curve) and dissociation (off curve)

0261measurements were repeated using different concentrations of antibody (25 nM, 150 nM, and 430 nM) and with two different antibodies identified as Ab-1 and Ab-2.

0262[00112] Fig. 8 shows the association and dissociation curves at different concentrations. The test of 25nM Ab-2 was not completed because the association was extremely slow at this concentration. These illustrated curves are plots of the raw data. [00113] K_on, K_off, and Kp were derived from these curves by fitting the raw data with a

0263first order exponential function. By averaging two sets of data, kinetic and affinity

0264coefficients were obtained as follows:

0265<img file="WO2005047854A2_D0002.tif" />

0266Example 4: NHS-ester activated tips. [00114] The same tip configuration as described in Example 1 was used. The same mercaptosilane fiber preparation as described in Example 2 was used. Mercaptosilane

0267coated fibers were activated by immersing the sensor tips in 50 μL of a 50mg/mL solution

0268of sulfo-SMCC (Pierce Biotechnology, Rockford LL; cat # 22322) in DMF (Sigma-Aldrich Chemical Company, St Louis, MO; cat # 494488) at for 2 hours. The sensor tips were rinsed briefly in DMF and dried.

0269[00115] Amine containing molecules can be covalently bound to this surface through formation of a stable amide linkage. Molecules that do not contain free amines are not

0270immobilized through the NHS moiety, but these molecules can still bind to the surface through non-specific binding. This non-specific binding can be multi-layered whereas the covalent immobilization through the NHS esters will be in a single layer controlled by the availability and accessibility of the NHS ester.

0271[00116] hi this set of experiments, a bis amino PEG (MW 3300) (Shearwater Polymers, San Carlos, CA) was used as a test compound to covalently bind to the activated surface. A PEG (MW 8000) (Sigma-Aldrich Chemical Company, St Louis, MO; cat # 04162) that contained no free amino groups was used as a negative control. This negative control was

0272used to look for any non-specific or multi-layered binding that might be inherent to PEG

0273polymers on this surface.

0274[00117] Fig. 9 shows the time course of the treatment of the activated mercaptosilane tip with the test molecules. The activated tip showed a distinct increase in optical thickness upon exposure to the O.lmg/mL bis amino PEG (MW 3300) in PBS. This increase is

0275stopped when the bis amino PEG solution is replaced by the PBS buffer. The activated tip exposed to 0.1 mg/mL PEG (MW8000) in PBS, which contains no amines, shows a small initial increase in optical thickness but the trace quickly becomes flat. From this it can be concluded that the PEG polymer does not have intrinsic non-specific binding and that the binding seen for the bis amino PEG is attributed to the specific covalent immobilization of the amine group.

0276Example 5: Antibody derivatized tips using NHS-ester chemistry. [00118] This example illustrates the binding of a low molecular weight molecule binding to an immobilized high molecular weight molecule. Using the same NHS ester terminated surface described in Example 4 and the same tip configuration as described in Example 1,

0277an anti-biotin antibody was immobilized to 3 fibers. Immobilization of the antibody was

0278accomplished by immersing the activated fiber in a 20 μg/mL solution of mouse anti-biotin

0279antibody (Biodesign, Saco MN; cat # H61504M) in PBS for 1 hour at room temperature. The tip was rinsed with PBS for 2 minutes. Following the PBS rinse, the tip was quenched with an aqueous solution of 100 μM ethanolamine pH 8.5 (Sigma-Aldrich Chemical Company, St Louis, MO; cat # E9508) for 5 minutes and then was rinsed again in PBS for 2

0280minutes.

0281[00119] The first fiber was exposed to a solution of 200 μg/mL biotin (Pierce

0282Biotechnology, Rockford IL; cat # 29129) in PBS. Controls using a solution of sucrose

0283(Sigma-Aldrich Chemical Company, St Louis, MO; cat # S8501) (2mg/mL) and PBS were carried out on the second and the third fibers to determine baseline noise. Data from these tests are shown in Fig. 10. Biotin binding is seen as an increase in optical thickness,

0284whereas exposure to sucrose shows no detectable increase over baseline (PBS).

0285[00120] Another negative control was carried out using an irrelevant antibody (anti- Lewis Y antibody from Calbiochem, San Diego CA; cat# 434636) immobilized in an identical fashion to the anti-biotin antibody above. This immobilized antibody was exposed to a solution of 200 μg/mL biotin. The lack of biotin binding to this antibody indicates that the biotin binding to the anti-biotin antibody is a result of specific interactions and not due to non-specific binding.

0286[00121] ^• While the invention has been particularly shown and described with reference to a preferred embodiment and various alternate embodiments, it will be understood by

0287persons skilled in the relevant art that various changes in form and details can be made therein without departing from the spirit and scope of the invention.

0288[00122] All references, issued patents and patent applications cited within the body of the instant specification are hereby incorporated by reference in their entirety, for all puφoses.