This application is related to pending U.S. application Ser. No. 11/677,674, filed Feb. 22, 2007, pending U.S. application Ser. No. 11/748,023, filed May 14, 2007, pending U.S. application Ser. No. 11/696,369, filed Apr. 4, 2007, and pending U.S. application Ser. No. 11/752,056, filed May 22, 2007, the contents of which are incorporated by reference herein for all purposes.
This invention relates to an apparatus for characterizing molecular binding events for performing binding protein assays and more particularly to such systems employing microarrays.
U.S. Pat. No. 6,594,011 issued Jul. 15, 2003, the entirety of which is incorporated by reference herein for all purposes, discloses an imaging apparatus and method for real time imaging ellipsometry for high throughput sensing of binding events useful in molecular interaction analysis including biotech applications. The apparatus and method disclosed employ the immobilization of an array of binding or capture molecules (“ligands”) on a planar surface of a transparent substrate and the use of a beam of polarized light directed at the underside of the surface in a manner to achieve total internal reflection (TIR) and generate an evanescent field in the plane of the ligands. The ligands are exposed to a biological sample and analytes in the biological sample bind to different patterns of the immobilized ligands in a manner to change the polarization at locations in the array at which binding occurs. An image of the array is compared with a stored image of the initial light polarization shifts to determine the location and magnitude of binding events within the array, thus identifying and quantitating the analytes present in the biological sample.
The apparatus for implementing the foregoing technique typically employs a prism or gratings to achieve the requisite TIR generated evanescent field, the prism being the more practical implementation.
TIR imaging ellipsometry works well for fields of view up to 1-2 cm2, which permits real time imaging of tens of thousands of binding events simultaneously. However, there is a need to be able to image or scan areas which are much larger, such as 128 mm×86 mm (e.g., the area of a 384 well or a 96 multiwell plate) to permit lower costs per test and for multiple tests per patient for large numbers of patients simultaneously which is increasingly a requirement for more clinical diagnostics and personalized medicine. Obviating the need for a single large prism simplifies both the instrument and disposable multiwell plate.
Co-pending U.S. application Ser. No. 11/696,369, filed Apr. 4, 2007, the entirety of which is incorporated by reference herein for all purposes, discloses a multiwell plate in which arrays of ligands are printed on planar side walls of the liquid reservoirs, or wells. The plate is fabricated with transparent material, such as glass or plastic, and a beam of light is directed upwards from the bottom of the plate into the separation between adjacent wells. The direction of the beam is chosen to achieve total internal reflection (TIR) at a well side wall in a manner to generate an evanescent field in the plane of an array of ligands on the interior face of that well. The reflected light from the side wall carries binding information between analytes in a biological sample in the selected well and different patterns of capture molecules in the array immobilized on the addressed side wall.
In accordance with the principles of this invention, a plurality of independent array patterns is immobilized in a stacked format on a single planar wall of a well to correspond to multiple sample chambers stacked along the vertical axis of a well. A corresponding number of separators are formed in the well resulting in a stack of “sample chambers”, with each chamber capable of containing a biological sample, and each sample chamber positioned with respect to a different one of the “vertical” stack of immobilized arrays.
A sample including analytes is introduced to each chamber with (for example) a hypodermic needle which is advanced through the separators in the stack to introduce a sample to a selected chamber. Any opening for the needle in any separator between a selected chamber and the top of a well is small and is the site of an air bubble which permits adjacent chambers in a stack to provide space for a sample without mixing samples.
The scope of the invention is defined by the claims, which are incorporated into this section by reference. A more complete understanding of embodiments of the present invention will be afforded to those skilled in the art, as well as a realization of additional advantages thereof, by a consideration of the following detailed description of one or more embodiments. Reference will be made to the appended sheets of drawings that will first be described briefly.
Embodiments of the present invention and their advantages are best understood by referring to the detailed description that follows. It should be appreciated that like reference numerals are used to identify like elements illustrated in one or more of the figures. It should also be appreciated that the figures may not be necessarily drawn to scale.
The present invention provides an advantageous apparatus and method for performing ligand binding assays using microarrays in a multiwell plate format. Prior to describing embodiments of the present invention in detail, the following definitions are provided for use throughout the present document.
Microwell Plate: A flat plate with multiple “wells” used like small test tubes. The microwell plate has become a standard tool in analytical research and clinical diagnostic testing laboratories with 6, 24, 96, 384 or even 1536 sample wells arranged in a 2:3 rectangular matrix in one example.
Ligand: Any molecule that binds to another, in normal usage a soluble molecule such as a hormone or biological molecule that binds to a binding partner or capture molecule. The decision as to which is the ligand and which is the capture molecule is often arbitrary. In the sense of this invention, the ligand refers to that binding element attached to a planar surface and which binds to an analyte molecule in a biological sample.
Total Internal Reflection (TIR): An optical phenomenon that occurs when light strikes a medium boundary at a steep angle. If the refractive index is lower on the other side of the boundary (i.e., the side that does not directly receive the light), no light can pass through and effectively all of the light is reflected. The critical angle is the angle of incidence where total internal reflection begins and continues up to angles of incidence of 90 degrees.
Ellipsometry: A very sensitive optical measurement technique providing capabilities for thin film analysis utilizing the change of polarization of light which is reflected off a sample or transmitted through a sample.
Surface Plasmon Resonance (SPR): The excitation of surface plasmons by light is denoted as a surface plasmon resonance for planar surfaces. Plasmons are collective oscillations of large numbers of electrons in matter, mostly in metals.
Arrays: Ligands affixed to a surface at separate localized regions called spots in an ordered manner thus forming a microscopic pattern where ligand identity is determined by the location (or “address”) of that particular spot.
Binding Protein (Ligand) Assay: A test that uses the binding of proteins (e.g., antibodies) to other ligands (e.g., antigens) to identify and measure the concentration of certain biological substances in blood, urine or other body components. Ligand assays may be used to diagnose disease, drug or vitamin levels, response to therapy or other information of biological relevance. Also, test results can provide information about a disease that may help in planning treatment (for example, when estrogen receptors are measured in breast cancer).
Introducer: The term “introducer” means any instrumentation to introduce a sample into a well. This may include a hypodermic needle, a cannula, a pipette or capillary which may or may not use a preexisting hole in a succession of sample chambers. This may include a multiple “tine” comb-like structure with different length “tines” each with a central channel and where the lengths correspond to the position in the stack of the sample chambers serviced by the sample introducer tine.
Beam: The illuminating beam may or may not be polarized. If polarized, the pattern of the localized changes in the phase of s- and p-polarized light is measured. The system can also be adapted for surface plasma resonance by employing a thin metallic film beneath the ligands as is well understood in the art for producing a surface plasmon interaction.
Insert: An “insert” is defined as an apparatus which provides one or more sample separators to provide multiple sample chambers in a well to allow for 3D stacking of the samples within a single well. The insert need not be transparent nor have stringent surface roughness requirements necessary for good optical surfaces. The insert is made to be placed into wells of a plate in such a manner that sample liquid does not leak between the insert and side walls of the well.
Liner: The liner is a transparent material on which the ligand spots are deposited. The material is typically glass, plastic, or a combination thereof.
Well: A well is any recess in a base plate which is adapted to receive an insert and accept a sample or a multitude of samples.
Side walls: Side walls are the well walls and are preferably planar in order to properly image binding events with the immobilized array pattern. The description is primarily in terms of a four-sided well configuration, but the well can be rectangular, hexagonal, or a variety of geometries as long as each wall is planar. The preferred orientation of a wall is normal to the plane of the base plate on which the walls are formed in order to simplify the positioning of the illumination system to achieve total internal reflection. The necessity of having a planar wall is for current imaging ellipsometry practice. It is contemplated that practical results may also be obtained by “line” scanning a curved wall.
Between Well Spacing: The “between well spacing” is the separation between adjacent wells and has to meet specific dimensions in order to allow total internal reflection to occur and to produce the necessary evanescent field in the plane of the array of ligands. The spacing may be filled by transparent material integral with the base plate or by a liner which is added to complete the individual well structure.
Sample: A sample is any biological material which may include analytes which may bind to different patterns of ligands.
3D: The term “3D” applies to the formation of a stratum of separate arrays of ligands on a single face in a single well. The individual arrays are conveniently elongated in a direction horizontal to the well axis and are accessed by a set of sample chambers inserted into a well where each chamber aligns with a corresponding array of the strata of arrays vertically positioned on a wall of a well. An array stratum may be immobilized on each wall of a well. For an insert of seven chambers for example, a single square-shaped well provides for twenty-eight individual tests. This number can be extended by including different patterns of ligands in each of the arrays.
Interface: The interface is defined as the plane where two optical components come together in the optical path of the illuminating beam. The interface is formed in the presence of an index matching medium which renders the interface optically transparent when the components have the same index of refraction.
An insert 25 is adapted for introduction into well 20. The insert has defined within it a number of sample chambers 25a (seven are illustrated as an example in
Samples are introduced to the sample chambers of the insert through apertures 27 in top surfaces 28 of the sample chambers 30 as shown in
It is to be understood that the immobilization of horizontal patterns of ligands in a stack on a wall of a well provides for a 3D arrangement of independent tests. Thus for a ninety-six well plate, seven arrays on only a single wall of each well permits 672 individual assays to be carried out. For wells with rectangular geometries, four times as many assays (2688) are permitted.
It is noted that the illumination subassembly directs a beam of light into the underside of the plate into the material of the plate between wells. In order to achieve total internal reflection (TIR) for imaging the pattern of binding events on the side walls of wells as illustrated in
sin(A)*√{square root over (n22−n12 sin2(B))}−cos(A)*n1 sin(B)≧n3
where A is the angle between the transmission surface contacting air and the TIR surface contacting the sample, B is the incidence angle of the light in air, n1 is the index of refraction of air, n2 is the index of refraction of the transparent material of the plate and n3 is the index of refraction of the sample (e.g., water, blood, urine, etc.). This formula simplifies dramatically if the wall of the well is normal to the bottom of the plate since then A=90° and the second term in the above equation vanishes. The resulting equation becomes:
In one embodiment compatible with current multiwell plate technology, individual wells of the plate are spaced according to Ansi/SBS standards (e.g., 4.5 mm center to center for 384 well plates and 9 mm center to center for 96 well plates). The spacing of the wells is true to standard. However, there is a minimum sidewall thickness in at least one dimension to allow total internal reflection to occur at precisely the critical angle from the side wall in such a way that the entire surface may be illuminated. This minimum thickness, labeled “t” in
where t is the sidewall thickness, h is the well depth, n2 is the index of refraction of the plate and n3 is the index of refraction of the (sample) material inside the well during measurement. All variables in the equation are labeled in
A 3D stack of arrays may be formed on the faces of a transparent post in a well as disclosed in co-pending U.S. application Ser. No. 11/696,369, filed Apr. 4, 2007 and assigned to the assignee of the present application. The stack of sample chambers may be inserted into the well to surround the post in a manner to position a sample chamber in alignment with an array of the stack of arrays formed on the post.
Parts such as insert 25, shown in
The arrays may be immobilized illustratively on the planar surfaces of a flexible substrate 60 as illustrated in
It is to be understood also that polarized light need not be used herein. It is intended that any measuring and/or sensing technique using an illumination source in a manner to achieve total internal reflection and an evanescent field in the plane of an array or arrays of ligands on a planar side wall of a well be encompassed by the following claims.
The foregoing description of exemplary and preferred embodiments is presented for purposes of illustration and disclosure in accordance with the requirements of the law. It is not intended to be exhaustive or to limit the invention to the precise form(s) described, but only to enable others skilled in the art to understand how the invention may be suited for a particular use or implementation. The possibility of modifications and variations will be apparent to practitioners skilled in the art. This disclosure has been made with respect to the current state of the art, but also contemplates advancements and that adaptations in the future may take into consideration those advancements, namely in accordance with the then current state of the art. It is intended that the scope of the invention be defined by the claims as written and equivalents as applicable. Reference to a claim element in the singular is not intended to mean “one and only one” unless explicitly so stated. Moreover, no element, component, nor method or process step in this disclosure is intended to be dedicated to the public regardless of whether the element, component or step is explicitly recited in the claims.
Number | Name | Date | Kind |
---|---|---|---|
1637141 | Cooper | Jul 1927 | A |
3858616 | Thiery et al. | Jan 1975 | A |
4146364 | McCormick | Mar 1979 | A |
4238565 | Hornby et al. | Dec 1980 | A |
4256834 | Zuk et al. | Mar 1981 | A |
4508832 | Carter et al. | Apr 1985 | A |
5164589 | Sjoedin | Nov 1992 | A |
5225164 | Astle | Jul 1993 | A |
5229833 | Stewart | Jul 1993 | A |
5234769 | Shevlin | Aug 1993 | A |
5255075 | Cush | Oct 1993 | A |
5313264 | Ivarsson et al. | May 1994 | A |
5341215 | Seher | Aug 1994 | A |
5437840 | King et al. | Aug 1995 | A |
5446534 | Goldman | Aug 1995 | A |
5483346 | Butzer | Jan 1996 | A |
5485277 | Foster | Jan 1996 | A |
5491097 | Ribi et al. | Feb 1996 | A |
5491556 | Stewart et al. | Feb 1996 | A |
5573956 | Hanning | Nov 1996 | A |
5593130 | Hansson et al. | Jan 1997 | A |
5633724 | King et al. | May 1997 | A |
5641640 | Hanning | Jun 1997 | A |
RE35716 | Stapleton et al. | Jan 1998 | E |
5738825 | Rudigier et al. | Apr 1998 | A |
5753518 | Karlsson | May 1998 | A |
5796858 | Zhou et al. | Aug 1998 | A |
5813439 | Herrero et al. | Sep 1998 | A |
5856873 | Naya et al. | Jan 1999 | A |
5922594 | Loefas | Jul 1999 | A |
5922604 | Stapleton et al. | Jul 1999 | A |
5955729 | Nelson et al. | Sep 1999 | A |
5965456 | Malmqvist et al. | Oct 1999 | A |
5972612 | Malmqvist et al. | Oct 1999 | A |
6008010 | Greenberger et al. | Dec 1999 | A |
6008893 | Roos et al. | Dec 1999 | A |
6026053 | Satorius | Feb 2000 | A |
6045996 | Cronin et al. | Apr 2000 | A |
6065501 | Feret et al. | May 2000 | A |
6127183 | Ivarsson et al. | Oct 2000 | A |
6140044 | Besemer et al. | Oct 2000 | A |
6143513 | Loefas | Nov 2000 | A |
6143574 | Karlsson et al. | Nov 2000 | A |
6197595 | Anderson et al. | Mar 2001 | B1 |
6200814 | Malmqvist et al. | Mar 2001 | B1 |
6207381 | Larsson et al. | Mar 2001 | B1 |
6253793 | Dupoiron et al. | Jul 2001 | B1 |
6277330 | Liu et al. | Aug 2001 | B1 |
6289286 | Andersson et al. | Sep 2001 | B1 |
6354333 | Dupoiron et al. | Mar 2002 | B1 |
6355429 | Nygren et al. | Mar 2002 | B1 |
6415825 | Dupoiron et al. | Jul 2002 | B1 |
6475809 | Wagner et al. | Nov 2002 | B1 |
6493097 | Ivarsson | Dec 2002 | B1 |
6503760 | Malmqvist et al. | Jan 2003 | B2 |
D472644 | Dawson et al. | Apr 2003 | S |
6549011 | Flatt | Apr 2003 | B2 |
6589798 | Loefas | Jul 2003 | B1 |
6594011 | Kempen | Jul 2003 | B1 |
D480149 | Dawson et al. | Sep 2003 | S |
6698454 | Sjoelander et al. | Mar 2004 | B2 |
6710870 | Marowsky et al. | Mar 2004 | B1 |
6714303 | Ivarsson | Mar 2004 | B2 |
6806051 | Ellson | Oct 2004 | B2 |
6833920 | Rassman et al. | Dec 2004 | B2 |
6840286 | Espinasse et al. | Jan 2005 | B2 |
6859280 | Kempen | Feb 2005 | B2 |
6882420 | Rassman et al. | Apr 2005 | B2 |
6981526 | Glejbol et al. | Jan 2006 | B2 |
7045287 | Smith et al. | May 2006 | B2 |
7193711 | Rassman et al. | Mar 2007 | B2 |
7195872 | Agrawal et al. | Mar 2007 | B2 |
20020019019 | Hamalainen et al. | Feb 2002 | A1 |
20020154311 | Ivarsson | Oct 2002 | A1 |
20020182717 | Karlsson | Dec 2002 | A1 |
20030022388 | Roos et al. | Jan 2003 | A1 |
20030067612 | Ivarsson | Apr 2003 | A1 |
20030112432 | Yguerabide et al. | Jun 2003 | A1 |
20030148401 | Agrawal et al. | Aug 2003 | A1 |
20030205681 | Modlin | Nov 2003 | A1 |
20030232384 | Kocher et al. | Dec 2003 | A1 |
20040002167 | Andersson et al. | Jan 2004 | A1 |
20040012676 | Weiner et al. | Jan 2004 | A1 |
20040023247 | Xu et al. | Feb 2004 | A1 |
20040030504 | Helt et al. | Feb 2004 | A1 |
20040038268 | Pirrung et al. | Feb 2004 | A1 |
20050148063 | Cracauer et al. | Jul 2005 | A1 |
Number | Date | Country |
---|---|---|
742417 | Feb 2000 | AU |
WO 8911057 | Nov 1989 | WO |
WO 9100467 | Jan 1991 | WO |
WO 9608720 | Mar 1996 | WO |
WO 9638729 | Dec 1996 | WO |
WO 9719375 | May 1997 | WO |
WO 9832002 | Jul 1998 | WO |
WO 03056337 | Jul 2003 | WO |
WO 03102580 | Dec 2003 | WO |