Classifications

• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
  • G01N33/54366—Apparatus specially adapted for solid-phase testing
  • G01N33/54373—Apparatus specially adapted for solid-phase testing involving physiochemical end-point determination, e.g. wave-guides, FETS, gratings
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
  • G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
  • G01N21/77—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator
  • G01N21/7703—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator using reagent-clad optical fibres or optical waveguides

Definitions

• Fiber-optic assay apparatus based on phase-shift interferometry.
• 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,
• 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
• receptor-receptor binding agent where the analyte can be either member of the
• 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.
• this type of assay known as a solid-phase assay, the solid surface is exposed to the sample under conditions that
• binding event can be detected directly, e.g., by a change in the mass, reflectivity, thickness, color or other
• the binding event is detectable by the presence and/or amount of detectable label at the detection zone.
• the analyte can be 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.
• the analyte can be pre-labeled, e.g., with a chromophore, or fluorescent or radiolabel.
• an interferometer assay device will yield readily observable changes in spectral peak and valley (extrema) positions within the range of a conventional visible-light
• spectrometer 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.
• the present invention is designed to overcome this limitation, preserving the
• 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.
• 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
• 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.
• 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
• Analyte binding can also cause the layer of analyte binding molecules to swell, resulting in a change in the interference.
• the distal reflecting surface includes the layer of analyte binding molecules.
• the optical path length or the physical distance between the two reflecting surfaces may increase, for
• 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
• analyte binding may cause the layer of analyte binding molecules to swell, moving the distal reflecting surface closer to the proximal reflecting surface.
• 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.
• 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
• This coating can be formed of a Ta O 5 layer having a preferred thickness of
• the optical element can be SiO 2 , and has a thickness of between 5 and 50 nm.
• the optical element can be SiO 2 , and has a thickness of between
• a light source for directing a beam of light onto the first and second reflecting surfaces
• 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
• 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
• 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.
• the optical assembly is fixedly mounted on the optical
• 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
• 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.
• 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
• nucleic acid binding molecules for detecting the presence of nucleic acid binding molecules.
• the detector can be a spectrometer for measuring reflected light intensity over a selected range of wavelengths.
• 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.
• 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
• the invention includes a method for detecting the presence or
• the method involves reacting the sample
• 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
• 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
• the detector can be a spectrometer, where the detecting
• the reacting step can be carried out until a near-maximum increase in thickness of the first reflecting layer is observed.
• 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.
• the method is used for measuring the amount of
• 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.
• the first reflecting layer is composed of an array of discrete analyte-binding regions
• the different regions being effective to bind different analytes
• 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.
• Fig. 1 shows the basic system setup for the bioprobe and its apparatus
• Fig. 2 shows an optical assembly formed accordance to one embodiment of the invention
• 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);
• Fig. 4 shows an optical assembly constructed according to another embodiment of the invention
• Fig. 5 shows a disposable multi-analyte optical assembly having an analyte-
• binding array and constructed according to another embodiment of the invention.
• Fig. 6 shows a sequential binding of three molecules
• Fig. 7 shows on and off curves generated from the association and dissociation of antibodies
• Fig. 8 shows the curves of two antibodies binding to their antigen at different concentrations
• 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
• Fig. 10 shows a small molecule binding to a large molecule, negative controls and the base line measurement.
• in vivo refers to processes that occur in a living organism.
• 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
• 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
• polyclonal antibodies such as those produced by inoculating a mammal such as a
• the term includes antibodies produced using genetic engineering methods such as those employing, e.g., SCID
• 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.
• SCFvs single chain variable fragments
• variable sequences usually retain the ability to bind their cognate antigen and so include variable sequences and
• 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
• SNP single nucleotide polymo ⁇ hism
• PBS phosphate buffered saline (0.01 M phosphate buffer, 0.0027 M potassium chloride and 0.137 M sodium chloride, pH 7.4)
• NHS phosphate buffered saline
• MW molecular weight
• Sulfo-SMCC sulfosuccinimidyl 4-[N- maleimidomethyl]cyclohexane-l-carboxylate.
• FIG. 1 shows, in schematic view, an interferometer apparatus 20 constructed in
• the apparatus includes a light
• optical assembly 26 that functions as a sensing element or detector tip and that
• 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
• 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.
• 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 light source in the apparatus can be a white light source, such as a light
• LED emitting diode
• source 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
• the same function can be achieved by a single light source, e.g., white light source,
• 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.
• CCD charge-coupled device
• the detector can be a simple photodetector for recording light intensity at each of the different irradiating wavelengths.
• 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
• 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
• 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
• the first reflecting surface 42 is formed of a layer of analyte-
• binding molecules such as molecules 44, which are effective to bind analyte molecules 46
• 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.
• the index of refraction of the optical element is preferably similar to that of the
• optical element formed by the analyte-binding molecules and bound analyte, rather than from the interface region.
• One exemplary material forming the optical element is SiO 2 , 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.
• 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.
• the second layer has a refractive index greater than 1.8.
• One exemplary material for the second layer is Ta 2 O 5 with refractive index equal to 2.1.
• the 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.
• 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.
• optical element 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.
• 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.
• 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.
• 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.
• I is the intensity
• l ⁇ and I are the intensity of two interference beams
• ⁇ is the optical path difference
• ⁇ is the wavelength
• 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.
• Fig. 4 shows an optical assembly 50 that is removably carried on the distal end of
• the optical element includes a plurality of
• the optical assembly includes a first optical
• element 60 similar to optical element 38 described above, and having first and second
• reflective layers 62, 64 respectively, corresponding to above-described reflective layers 40,
• the assembly further includes a second optical element 66 whose thicl ⁇ iess is preferably greater than 100 nm, typically at least 200 nm, and whose index of
• the two optical elements are constructed of the same glass or a polymeric material having an index of refraction of
• 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
• 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
• 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.
• 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-
• a fiber bundle 72 is composed of an array, e.g.,
• optical assembly 70 is composed of the basic optical elements described above with
• a first optical element 80 in the element provides at its lower distal surface, an array of analyte-reaction regions, such as
• regions 84 each containing a layer of analyte-binding molecules effective to bind to one of
• One preferred sensing provides an array of different-sequence nucleic acids, e.g., cDNAs or oligonucleotides, designed to hybridize specifically with different-sequence
• the array surface forms a "gene chip" for detecting each of a plurality of different gene sequences.
• a second optical element 78 Also included in the optical assembly are a second optical element 78 and a layer
• 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
• 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.
• 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
• nucleic acid e.g., ssDNA or RNA carried on the tip, for identifying and molecules that bind specifically to the nucleic acid.
• Example 1 Small molecule-protein binding reaction.
• 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.
• the two-layer configuration on the tip of an optic fiber is used for this test.
• the first Ta 2 O 5 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
• IAPVD ion-beam assisted physical vapor deposition
• the fiber tip was coated with a polymer monolayer derivatized with biotin.
• polymer 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 ⁇ g/ml streptavidin streptavidin (Pierce
• the tip was immersed in 50 ⁇ g/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.
• donkey-anti-rabbit antibody solution antibody Jackson ImmunoResearch, West Grove, PA; cat# 711-005-152
• Fig. 6 shows the real-time response curve for this sequential binding test.
• streptavidin 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.
• Example 2 Biomolecular interaction analysis of kinetics and affinity of biomolecular interactions.
• 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.
• Toluene hexanoic acid: mercaptopropyltrioxysilane (10:2:1 volumetric ratio) at room temperature for 24 hours.
• the biosensor tip was first derivatized by immersion in a with lO ⁇ g/ml solution of rabbit-IgG (Jackson hnmunoResearch, West Grove, PA; cat# 309-005-003) in PBS for 1 hour.
• rabbit-IgG Jackson hnmunoResearch, West Grove, PA; cat# 309-005-003
• the coated tip was dipped into lO ⁇ g/ml goat-anti-rabbit antibody solution
• 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.
• 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.
• 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.
• the ability to detect the on and off curves reliably is essential for measuring interaction kinetics and affinity.
• Example 3 Calculating affinity constants from antibody-antigen binding and release curves.
• the antigen was covalently bound to the activated fiber tip by immersing the
• 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
• Example 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
• coated fibers were activated by immersing the sensor tips in 50 ⁇ L of a 50mg/mL solution
• 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
• 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
• Example 5 Antibody derivatized tips using NHS-ester chemistry.
• 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,
• an anti-biotin antibody was immobilized to 3 fibers. Immobilization of the antibody was
• the first fiber was exposed to a solution of 200 ⁇ g/mL biotin (Pierce).
• 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 ⁇ g/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.

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