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`U5005585242A
`
`[19]
`
`[11} Patent Number:
`
`5,585,242
`
`United States Patent
`Bouma et al.
`[45] Date of Patent: Dec. 17, 1996
`
`
`
`[54] METHOD FOR DETECTION OF NUCLEIC
`ACID USING TOTAL INTERNAL
`REFLECTANCE
`
`[75]
`
`Inventors: Stanley R. Bouma, Grayslake; Omar
`S. Khalil, Libenyville; Edward K.
`Pabich, Chicago, all of 111.
`
`[73] Assignee: Abbott Laboratories, Abbott Park, Ill.
`
`[21] App]. No.: 522,623
`
`[22]
`
`Filed:
`
`Aug. 31, 1995
`
`Related US. Application Data
`
`[63] Continuation of Ser. No. 311,839, Sep. 23, 1994, abandoned,
`which is a continuation of Ser. No. 863,553, Apr. 6, 1992,
`abandoned,
`
`
`. C120 1/68; C12P 19/34
`Int. Cl.‘5 ..
`[51]
`[52] US. Cl.
`................................ 435/6; 435/912; 935/77;
`935/78
`
`........ 435/6, 91.2, 288.
`, 7.21, 7.32; 935/77, 78
`
`[58] Field of Search ............
`435/71, 7.
`
`
`
`[56]
`
`References Cited
`
`U.S. PATENT DOCUMENTS
`
`.. 436/527
`
`5/1984 Hirschfeld .............
`4,447,546
`.. 436/527
`4,558,014 12/1985 Hirschfeld et a].
`
`4,582,809
`4/1986 Block et al.
`.......
`.. 436/527
`
`4,608,344
`8/1986 Carter et al.
`436/34
`
`3/1987 Hirschfeld ......
`250/4581
`4,654,532
`
`7/1987 Mullis et al.
`..
`435/912
`4,683,195
`
`12/1987 Block et al.
`.. 435/514
`4,716,121
`
`4,844,869
`7/1989 Glass ......
`422/68
`3/1990 Block et al.
`. 422/8.“
`4,909,990
`
`...... 435/6
`5.001.051
`3/1991 Miller et al.
`
`6/1992 Urdea .........
`.. 435/6
`5,118,605
`9/0993 Hirschfeld ................................... 435/6
`5,242,797
`
`FOREIGN PATENT DOCUMENTS
`
`0320308A2
`0297379
`0439182A2
`2190189
`2235292
`W090/01157
`
`12/1988 European Pat. 011'.
`1/ 1989 European Pat. 011'.
`1/1991
`European Pat. 011‘.
`3/1987 United Kingdom .
`7/1990 United Kingdom .
`7/1989 WIPO .
`
`.
`.
`.
`
`OTHER PUBLICATIONS
`
`Nicholls et a1. (1989) Journal of Clinical Laboratory Analy-
`sis, vol. 3, pp. 122-135, “Nucleic Acid Analysis by Sand—
`wich Hybridization”.
`Guatelli et al. (1990, Mar.) Proceedings of the National
`Academy of Sciences, vol. 87, pp. 1874—1878, “Isothermal,
`in vitro amplification of nucleic acids by a multienzyrne rxn
`modeled after retroviral amplification”.
`Ostergaard et al. (1991) European Journal of Clinical Micro~
`biology and Infections Diseases, vol. 10(12), 1057—1061,
`see abstract.
`
`Clin Chem. 37/9, 1482—1485 (1991) Polymerase Chain
`Reaction and QHT Replicase Amplification, Cahill.
`Journal of Immunological Methods, 74 (1984) 253—265,
`Immunoassays ata Quartz~Liquid Interface: Theory, Instru-
`mentation, etc, Sutherland.
`
`Analytical Chemistry, vol. 45, No. 4, Apr, 1973, Multiple
`Internal Reflection Fluorescence Spectrometry, Harrick.
`
`Clin Chem. V01. 35, No. 9, 1989, Chemiluminescent Sub-
`
`strates for Alkaline Phosphatase: Application to Ultrasen—
`sitive Enzyme—Linked Immunoassays and DNA Probes,
`Schaap.
`
`Clin Chem. vol. 35, No. 9, 1989, Chemiluminescent Detec»
`[ion of Herpes Simplex Virus I DNA in Blot and ln—Situ
`Hybridization Assays, Bronstein et al.
`
`Proc. Natl. Acad. Sci. USA, vol. 87, 45144518, Jun. 1990
`
`Imaging ofDNA Seq. with Chemiluminescence, Tizard et al.
`
`Analytical Biochemistry, 180, 95—98 (1989),.4 Comparison
`of Chem. and Colorimetric Substrates in a Hepatitis B Virus
`DNA Hybridization Assay, Bronstein et al.
`
`Journal of Immunological Methods, 8 (1975) 235—240, A
`New Immunoassay Based on Fluorescence Excitation by
`Internal Reflection Spectroscopy, Kronick et al.
`
`Nucleic Acids Research, vol. 16, No. 11, 1988, A Compari—
`son of Non—Radioisotopic Hybridization Assay Methods
`Using Fluorescent, Chem. and Enzyme Labeled Synthetic
`Oligodeoxyribonucleotide Probes, Urdca, et al.
`
`Nucleic Acids Research, vol. 13, No. 7, 1985, The Synthesis
`of Oligonucleotides Containing an Aliphatic Amino Group
`at the 5 ' Terminus: Synthesis of Fluorescent DNA Primers
`for use in DNA Sequence Analysis, Smith.
`
`Bio/Technology, vol. 10, Apr., 1992, Simultaneous Amplifi-
`cation and Detection ofSpecific DNA Sequences, Higuchi et
`al.
`
`Primary ExamineriLisa B. Arthur
`Attorney, Agent, or Firm~Thomas D. Brainard; Paul D.
`Yasger
`
`[57]
`
`ABSTRACT
`
`An apparatus and method for detecting amplified target
`nucleic acid is provided wherein the presence and concen—
`tration of amplified target is determined by total internal
`reflection over the course of the amplification reaction. A
`method and apparatus for detecting target nucleic acid is also
`provided wherein the presence and concentration of target is
`determined by total internal reflection and coupling of the
`target to the TIR element by scissile linkage. An improved
`immunoassay using total internal reflection and dilferential
`temperature cycling is further provided.
`
`13 Claims, 4 Drawing Sheets
`
`Agilent Exhibit 1229
`
`Page 1 of 22
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`Agilent Exhibit 1229
`Page 1 of 22
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`

`

`US. Patent
`
`Dec. 17, 1996
`
`Sheet 1 of 4
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`5,585,242
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`
`
`DETECTION
`EXCITATION
`18
`MEANS AND
`SOURCE AND
`
`16
`OPTICS
`
`
`
`
`OPTICS
`
`10
`
`12
`
`26
`
`22
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`30
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`—////A '/////—
`
`
`20:II Il38
`
`
`
`FIG.1
`
`Agilent Exhibit 1229
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`Page 2 of 22
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`Agilent Exhibit 1229
`Page 2 of 22
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`

`

`US. Patent
`
`Dec. 17, 1996
`
`Sheet 2 of 4
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`5,585,242
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`DETECTOR
`ELECTRONICS
`54
`
`44 45
`
`DETECTOR
`A
`60 -
`
`-
`55 -
`
`EXCHATION
`
`SOURCE
`
`62
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`22
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`42
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`0—///.///.l'////—
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`14 I]
`
`FIG.2
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`Agilent Exhibit 1229
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`Page 3 of 22
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`Agilent Exhibit 1229
`Page 3 of 22
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`

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`US. Patent
`
`Dec. 17, 1996
`
`Sheet 3 0f 4
`
`5,585,242
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`as
`
`70
`
`f
`
`72
`
`64
`
`FlG.3
`
`7B
`
`76
`
`80
`
`/
`
`FIG.4
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`Agilent Exhibit 1229
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`Page 4 of 22
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`Agilent Exhibit 1229
`Page 4 of 22
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`

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`US. Patent
`
`Dec. 17, 1996
`
`Sheet 4 of 4
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`5,585,242
`
`a:
`L”.
`
`9 L
`
`I—
`
`
`
`Agilent Exhibit 1229
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`Page 5 of 22
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`
`
`
`
`mmoEEZdmfimmoEEZmango
`
`<00:
`
`mo._.<Ez_
`
`.mmS moEEE
`
`“EEO
`
`Agilent Exhibit 1229
`Page 5 of 22
`
`
`

`

`1
`METHOD FOR DETECTION OF NUCLEIC
`ACID USING TOTAL INTERNAL
`REFLECTANCE
`
`This application is a continuation of U.S. patent appli-
`cation Ser. No. 08/311,389, filed Sep. 23, 1994, now aban-
`doned which is a continuation of application Ser. No.
`07/863,553, filed Apr. 6, 1992, now abandoned.
`
`FIELD OF THE INVENTION
`
`The present invention relates to methods, apparatus, and
`kits for amplifying and/or detecting target nucleic acid using
`total internal reflection (“TIR”) techniques. The invention
`also relates to an improved TIR device and method for
`specific binding assays, including immunoassays.
`
`BACKGROUND DESCRIPTION
`
`The amplification of nucleic acids is useful in a variety of
`applications. For example, nucleic acid amplification meth—
`ods have been used in the identification of genetic disorders
`such as sickle-cell anemia and cystic fibrosis, in detecting
`the presence of infectious organisms, and in typing and
`quantification of DNA and RNA for cloning and sequencing.
`Methods of amplifying nucleic acid sequences are known
`in the art. One method, known as the polymerase chain
`reaction (“PCR”), utilizes
`a pair of oligonucleotide
`sequences called “primers” and thermal cycling techniques
`wherein one cycle of dcnaturation, annealing, and primer
`extension results in a doubling of the target nucleic acid of
`interest. PCR amplification is described further in U.S. Pat.
`Nos. 4,683,195 and 4,683,202, which are incorporated
`herein by reference.
`Another method of amplifying nucleic acid sequences
`known in the art is the ligase chain reaction (“LCR”). Like
`PCR, LCR utilizes thermal cycling techniques. In LCR,
`however, two primary probes and two secondary probes are
`employed instead of the primer pairs used in PCR. By
`repeated cycles of hybridization and ligation, amplification
`of the target is achieved. The ligated amplification products
`are functionally equivalent to either the target nucleic acid or
`its complement. This technique is described more com-
`pletely in EP—A—320 308 and EP-A-439 182.
`Other methods of amplifying nucleic acids known in the
`art
`involve isothermal
`reactions,
`including the reaction
`referred to as Q-beta (“Q0") amplification [See,
`for
`example, Kramer et al., U.S. Pat. No. 4.786.600. W0
`91/043140, Cahill et al., Clin. Chem, 37:1482—1485 (1991);
`Pritchard et al., Ann. Biol. Clin., 48:492—497 (1990)].
`Another isothermal reaction is described in Walker et al.,
`“Isothermal in vitro amplification of DNA by a restriction
`enzyme/DNA polymerase system", Proc. Natl. Acad. Sci,
`89:392—696 (1992). These amplification reactions do not
`require thermal cycling.
`Amplification of nucleic acids using such methods is
`usually performed in a closed reaction vessel such as a
`snap-top vial. After the amplification, the reaction vessel is
`then opened and the amplified product is transferred to a
`detection apparatus where standard detection methodologies
`are used.
`
`is detected by
`the amplified product
`In some cases,
`denaturing the double-stranded amplification products, and
`treating those products with one or more hybridizing probes
`having a detectable label. The unhybridized probes are
`typically separated from the hybridized probe, requiring an
`
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`35
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`4s
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`60
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`65
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`5,585,242
`
`2
`
`extra separation step. Alternatively, the primer or probes
`may be labeled with a hapten as a reporter group. Following
`amplification, the hapten, which has been incorporated into
`the amplification product, can be used for separation and/or
`detection.
`
`In yet another detection method, the amplification prod-
`ucts may be detected by gels stained with ethidium bromide.
`In sum, 32F tracings, enzyme immunoassay [Keller et al., J.
`Clin. Microbiology, 28: 1411—6 (1990)], fluorescence [Urdea
`et al., Nucleic Acids Research, 16:4937-56 (1988); Smith et
`at, Nucleic Acids Research, 13:2399—412 (1985)], and
`chemiluminescence assays and the like can be performed to
`detect nucleic acids in a heterogeneous manner [Bornstein
`and Voyta, Clin. Chem, 35:1856—57 (1989); Bomstein eta1.,
`Anal. Bloc/mm, 180:95—98 (1989); Tizard et al., Proc. Natl.
`Acad. Sci, 78:4515—18 (1990)] or homogeneous manner
`[Arnold et al., U.S. Pat. No. 4,950,613; Arnold et al., Clin.
`Chem, 35:1588—1589 (1989); Nelson and Kacian, Clinica
`Chimica Acta, 194:73—90 (1990)].
`In each case, however, these detection procedures have
`serious disadvantages. First, when the reaction vessel con-
`taining a relatively high concentration of the amplified
`product is opened, a splash or aerosol is usually formed.
`Such a splash or aerosol can be sources of potential con-
`tamination, and contamination of negative, or not-yet ampli-
`fied, nucleic acids is a serious problem and may lead to
`erroneous results.
`
`Similar problems concerning contamination may involve
`the work areas and equipment used for sample preparation,
`preparation of the reaction reagents, amplification, and
`analysis of the reaction products. Such contamination may
`also occur through contact transfer (carryover), or by aerosol
`generation.
`Furthermore, these previously described detection proce-
`dures are time-consuming and labor intensive. In the case of
`both hybridization probes and hapten detection, the ampli-
`fication reaction vessel must be opened and the contents
`transferred to another vessel, medium or instrument. Such an
`“open” detection system is disadvantageous as it leads to
`further contamination problems. both airborne and carry—
`over.
`
`Thus, a need emerges for detecting amplified nucleic
`acids in a closed system in order to eliminate the potential
`for contamination. Also, a need emerges for a method of
`amplifying and detecting the target nucleic acid in an
`operationally simple, yet highly sensitive manner. The abil-
`ity to detect the amplification product in a sealed vessel, or
`in a closed system, offers useful advantages over existing
`prior art methods,
`including the ability to monitor the
`amplification of target nucleic acid throughout the course of
`the reaction.
`
`The use of total internal reflection fluorescence techniques
`is known in the art with respect to immunoassays [Hanick,
`et al., Anal. Chem. 452687 (1973)]. Devices and methods
`that use total internal reflection fluorescence for immunoas-
`says have been described in the art by Hirschfield, U.S. Pat.
`Nos. 4,447,564, 4,577,109, and 4,654,532; Hirschfield and
`Block, U.S. Pat. Nos. 4,716,121 and 4,582,809, which are all
`incorporated herein by reference. Other descriptions and
`uses are given by Glass, U.S. Pat. No. 4,844,869; Andarde,
`U.S. Pat. No. 4,368,047; Hirschfield, GB 2,190,189A;
`Lackie, W0 90/067,229; Block, GB 2,235,292A, and Carter
`et al., U.S. Pat. No. 4,608,344.
`Use of total internal reflection elements allows perform
`ing a homogeneous assay (Le. free of separation and wash
`steps) for members of specific binding pairs. Several appli-
`
`Agilent Exhibit 1229
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`5,585,242
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`3
`cations of this principle are known in the art [such as
`Kronick, et al. J. Immunol. Methods, 8:235 (1975) and US.
`Pat. No. 3,604,927] for hapten assays and for immunoassay
`of macromolecules [Sutherland et al, J. Immunol. Methods,
`74:253 (1984)].
`In known total internal reflectance methods, however, the
`slow diffusion of members of specific binding pairs from the
`bulk of the solution to the surface of the TIR element creates
`a limitation in using TIR fluorescence techniques. Thus,
`prior art devices have used capillary tubes or flow cells to
`enhance difl‘usion either by limiting the diffusion distances
`or by continuous exposure to fresh reactant stream, or both.
`But these systems, too, have drawbacks that make them less
`than optimal for clinical biological applications. Capillary
`tubes are difficult to manipulate and are not easily auto—
`mated. Flow cells require extensive washing in an eifort to
`reduce carryover contamination before they can be reused.
`Thus, in addition to a need for contamination-free, closed
`amplification systems, there is also a need in the art for better
`TIR assay systems that are more easily automated and even
`disposable if desired.
`
`SUMMARY OF THE INVENTION
`
`Several objectives and advantages of the present inven—
`tion may be stated. First,
`the invention can monitor the
`presence and/or concentration of target molecules in real
`time. This is particularly of interest in nucleic acid ampli—
`fication reactions. In addition, it is an object of the present
`invention to reduce contamination of other samples and
`other unused vessels and reagents with the amplified target
`nucleic acid through the use of a sealed vessel in which both
`amplification and detection occur. A still further object of the
`present invention is to provide relatively simple and sensi-
`tive methods and apparatus for detecting target nucleic acid
`or other molecules of interest in a reaction sample.
`Accordingly, in a first aspect, the invention is a method of
`detecting amplified target nucleic acid using total internal
`reflection, comprising the steps of:
`providing a reaction vessel having disposed therein (a) a
`reaction sample, (b) a total internal reflection (TIR) element.
`(c) a plurality of members of initiator sequence sets and
`reagents for producing amplification of target nucleic acid
`present in the reaction sample, (d) label means which is
`coupled to a fluorophore, and (e) capture means for bringing
`said fluorophore within the penetration depth of said ele-
`ment, wherein at least one of said label means and said
`capture means is specific for said target nucleic acid;
`producing an evanescent electromagnetic wave in the TlR
`element which penetrates into the reaction sample adjacent
`the element and has an associated penetration depth;
`reacting the reaction sample. the initiator sequences and
`amplification reagents under conditions sufficient to amplify
`target nucleic acid present in the reaction sample to produce
`amplification products;
`capturing said label means within the penetration depth as
`a function of the presence or amount of target nucleic acid;
`and
`
`detecting within the TIR element a change in fluores-
`cence.
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`The invention contemplates both covalent attachment and
`specific binding member attachment of initiators to the
`element
`to bring the fluorophore within the penetration
`depth. Both immunoreactive and polynucleotide specific
`binding pairs are contemplated. It
`is preferred that
`the
`
`65
`
`4
`amplification initiators double as either capture means or
`label means or both.
`
`The invention also provides an apparatus for amplification
`and detection of nucleic acid targets comprising:
`a sealed, static-volumetric reaction vessel adapted to
`contain a reaction sample and reagents for amplification
`a total internal reflection (TIR) element disposed in said
`reaction vessel such that substantial surface area of the
`element is in contact with said reaction sample and such that
`one end of the element protrudes from the vessel;
`means for producing an evanescent electromagnetic wave
`in the TIR element which penetrates into the reaction sample
`adjacent
`the element and has an associated penetration
`depth;
`reacting the reaction
`temperature control means for
`sample and amplification reagents under cyclic temperature
`conditions suflicient to amplify target nucleic acid present in
`the reaction sample and to capture a fluorophore capable of
`emitting fluorescence within the penetration depth of the
`element as a function of the presence or amount of target in
`the sample; and
`means for detecting in the TIR element a change in
`fluorescence.
`
`Preferably the reaction vessel and the TIR element are
`separated from one another by a distance that precludes
`capillary migration. For wettable vessels and aqueous solu-
`tions a distance of 1.7 mm or more is sufficient. The reaction
`vessel may be sealed by a sealing member having a through-
`bore for the TIR element, or by an integral cap/TIR element.
`In another aspect, the invention relates to a method and
`apparatus for detecting nucleic acid in a sample by means of
`signal amplification achieved by destroying, as a function of
`the amount of target present, a scissile link that holds
`fiuorophore near the TIR element. Thus, a decrease in the
`totally internally reflected fluorescence will occur in the
`presence of target.
`In a final aspect, the invention relates to an improved
`method and apparatus for conducting specific binding assays
`with fluorophore labels that are detected or monitored by
`total internal reflectance means. This embodiment of the
`invention includes:
`
`a sealed, static-volumetric reaction vessel adapted to
`contain a reaction sample and reagents for a specific binding
`assay; and
`a total internal reflection (TIR) element disposed in said
`reaction vessel such that substantial surface area of the
`element is in contact with said reaction sample and such that
`one end of the element protrudes from the vessel;
`wherein said static-volumetric reaction vessel and TIR
`element are dimensioned such that the space between the
`element surface and the interior wall of the reaction vessel
`is too great to support capillary migration of an aqueous
`fluid.
`
`The invention also provides kits for detecting amplified
`nucleic acids, comprising PCR or LCR amplification
`reagents and a TIR element having a plurality of coupling
`sites that allow attachment of amplified target nucleic acid.
`BRIEF DESCRIPTION OF THE DRAWINGS
`
`FIG. 1 is a schematic illustration of a detection system in
`accordance with one embodiment of the present invention.
`FIG. 2 illustrates the reaction vessel and associated exci-
`tation and detection optics for a detection system as shown
`in FIG. 1.
`
`Agilent Exhibit 1229
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`5,585,242
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`5
`FIG. 3 illustrates a unitary fused embodiment comprising
`a cylindrical total internal reflection element, a lens therefor,
`and a reaction chamber seal
`
`FIG. 4 illustrates a unitary fused embodiment comprising
`a planar or flat total internal reflection element, a beveled
`prismatic lens therefor, and a reaction chamber seal
`FIG. 5A illustrates a preferred configuration for PCR
`amplification, using a capture initiator and a label initiator.
`FIG. SB illustrates a preferred configuration for LCR
`amplification, using capture initiator and label
`initiator
`probe pairs.
`
`DETAILED DESCRIPTION
`
`A. TIR Principles
`Total internal reflection (“TIR”) is known in the art and
`operates upon the principle that light striking the interface
`between two media having different
`refractive indices
`(N1>N2) from the denser medium (i.c. having the higher
`refractive index; here N,) can be made to totally internally
`reflect within the medium if it strikes the interface at an
`angle, 6R, greater than the critical angle, 0C, where the
`critical angle is defined by the equation:
`
`Gc=arcsin(N2/N,)
`
`Under these conditions, an electromagnetic waveform
`known as an evanescent wave is generated in the less dense
`medium, and the electric field associated with the excitation
`light forms a standing sinusoidal wave, normal
`to the
`interface, is established in the denser medium. The evanes-
`cent wave penetrates into the less dense medium, but its
`energy dissipates exponentially as a function of distance
`from the interface. A parameter known as “penetration
`depth” ((1,) is defined as the distance from the interface at
`which the evanescent wave energy has fallen to 0.368 times
`the energy value at the interface. [See, Sutherland et a]., J.
`Immunol. Meth., 74:253—265 (1984)]. Penetration depth is
`calculated as follows:
`
`NM
`d” _ 21t{sin2BR—(N2/N1)2}m
`
`Factors that tend to increase the penetration depth are:
`increasing angle of incidence, 8R; closely matching indices
`of refraction of the two media (i.c. Nlel-al ); and increas-
`ing wavelength, A. An example will illustrate. If a quartz TIR
`element (N,=l.46) is placed in 3 aqueous medium (N2:
`1.34), the critical angle, 0C, is 66“. If 500 nm light impacts
`the interface at 8R=70° (i.c. greater than the critical angle)
`the dp is approximately 150 nm.
`For cylindrical and fiber optic TIR elements, the maxi-
`mum acceptance angle with regard to the TIR element axis,
`B, for the radiation entering the TIR element and so propa-
`gated witlrin it, is established by the refractive indices of the
`TIR element and the surrounding medium. For radiation
`initially propagating through a medium of refractive index
`No, such as air, incident upon a TIR element of refractive
`index N1, otherwise surrounded by a medium of refractive
`index N2, the maximum acceptance angle, B, may be found
`from the equation:
`
`N.A.=N,, sin that],2 Mom,
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`where NA. is the so-called numerical aperture of the TIR
`element.
`
`65
`
`Within the penetration depth, the evanescent wave in the
`less dense medium (typically a reaction solution) can excite
`
`6
`fluorescence in the sample. The fluorescence tunnels back
`into the TIR element propagates within the TIR element
`along the same path as the standing sinusoidal wave (but at
`a different wavelength) and is detected. All of the radiation
`that tunnels back into the TIR element is within the total
`internal reflection angle and is thus trapped within the TIR
`element. Accordingly, TIR allows detection of a fluoro-
`phorc—labeled target of interest as a function of the amount
`of the target
`in the reaction sample that
`is within the
`penetration depth of the TIR element.
`B. A First Embodiment
`In accordance with a first embodiment of the present
`invention, total internal reflection is used to detect amplified
`target nucleic acid in a reaction vessel. The reaction vessel
`preferably is sealed although a flow cell or a capillary tube
`may be used. Both amplification and detection can take
`place within the same closed reaction vessel, thus minimiz-
`ing contamination risks.
`FIG. 1 illustrates a an amplification and detection system
`10 in accordance with one embodiment of the present
`invention. The system includes a thermal cycling device
`generally represented as 12, a reaction unit generally rep-
`resented as 14, fluorescence excitation source and optics 16
`and fluorescence detection optics 18. The unit 14 includes a
`reaction vessel 20. a sealing member 22 and a total internal
`reflection (TIR) element 24. The reaction vessel 20 is placed
`in a thermal cycling device 12 and is supported by tab
`members 26.
`Amplification reactions using thermal cycling are pres-
`ently preferred over isothermal mechanisms. It is believed
`that convection currents resulting from the successive heat-
`ing and cooling cycles during thermal cycling enhances
`diffusion of molecules in the reaction sample, although (in
`contrast to an embodiment described later) this feature is not
`deemed essential to this embodiment. Accordingly, a ther-
`moeyclcr device 12 is shown. However, the details of the
`method of therrnocycling are not critical to the invention.
`For example, the temperature of the amplification reaction
`may be controlled manually, such as by air or water baths,
`or regulated automatically by a therrnocycler device spe-
`cifically designed for nucleic acid amplification. Thermocy-
`cler devices are commercially available from Perkin-Elmer
`Corporation, (Norwalk, Conn.) and Coy Laboratories, (Ann
`Arbor, Mich.).
`The reaction vessel 20 is made of glass or polymeric
`materials such as polystyrene, polyacrylate and the like, and
`is preferably made of a therrnostable material. Preferably,
`the size of the reaction vessel 20 is selected so as to contain
`relatively small quantities of reaction sample. More prefer-
`ably, the reaction vessel 20 is selected to so as to contain
`from about 50 ul to about 200 ul reaction sample. In atypical
`embodiment, the reaction vessel 20 is a rrricrocentrifuge
`tube, although other configurations are possible and within
`the invention. As will be described (and defined) in more
`detail below in connection with another embodiment, it is
`preferred that the reaction vessel be a “static-volumetric”
`vessel, having a composition (with regard to wettability) and
`a distance between the element surface 38 and the walls of
`the reaction vessel 20 that is sufficiently great to prohibit
`capillary action of an aqueous sample therebetween.
`The TIR element 24 may be preferably any of a number
`of optically transparent materials, including but not limited
`to, glass, quartz, and transparent polymers such as polysty—
`rene or polystyrene copolymers and polyacrylic acids or the
`like, chosen to have an index of refraction greater than that
`of the medium in which it is placed. Preferably the medium
`is an aqueous reaction sample comprising amplification
`
`Agilent Exhibit 1229
`
`Page 8 of 22
`
`Agilent Exhibit 1229
`Page 8 of 22
`
`

`

`5,585,242
`
`7
`reaction reagents and target nucleic acids. Such a reaction
`medium typically will have a refractive index ranging from
`about 1.30 to about 1.38, more typically, about 1.34. Thus,
`for a visible light beam having a wavelength ranging frown
`about 480 to 540 nm, the preferred TIR elements according
`to the invention have refractive indices ranging from about
`1.4 to 1.6. Exemplary materials and their approximate
`refractive indices are given in Table 1 below:
`
`TABLE 1
`
`Element Material
`Refractive Index
`
`
`i.6
`Quartz
`159
`Polystyrene
`1.52
`Glass
`1.49
`Polymethylmethacrylate
`
`Pym 1.47
`
`The TIR element 24 is further chosen to be insoluble and
`non-reactive with the reaction sample. An exemplary TIR
`element 24 is a glass rod with a wide surface area and a
`diameter of approximately 1 millimeter. It will be under-
`stood that the dimensions of the TIR element 24 accommo—
`date the reaction being undertaken and the size of the
`reaction vessel 20. Those skilled in the art will appreciate
`that the surface area of the TIR element 24 should be
`considered and it is believed that to obtain maximum surface
`area binding, the reaction vessel 20 and the TIR element 24
`are preferably long and cylindrical.
`As shown in FIG. 1, a sealing member 22 is configured
`and dimensioned to fit on the open end of the reaction vessel
`20. A centrally disposed bore 30 in the sealing member 22
`is adapted to support an upper end portion of the TIR
`element 24 substantially coaxially within the vessel 20.
`Additionally, the sealing member 22 preferably provides a
`sturdy locating surface (e.g. tab members 26) for positioning
`the unit 14 with respect to the excitation source and optics
`16 and detection optics 18, which will be described in more
`detail in connection with FIG. 2. The sealing member 22 is
`preferably a mbber septum or a polymer or polymer lami-
`nate.
`
`The TIR element 24 passes through and is supported by
`the sealing member 22 so as to expose as much as possible
`of the TIR element 24 to the interior of the reaction vessel
`20, leaving only an end face 32 unobscured and approxi-
`mately coterrninous with the extremity of the bore 30
`external to the vessel 20. The end face 32 of the TIR element
`24, however, does not have to be coterminous with the
`extremity of the bore 30 as can be seen from alternative TIR
`elements shown in FIGS. 3 and 4. It is important, however,
`that a minimum amount of the TIR element is exposed above
`the sealing member 22 to reduce the dissipation of signals
`received and transmitted by the TIR element 24. Preferably
`the end face 32 is highly transparent and free of blemishes
`which would tend to scatter light incident upon its face. The
`end face 32 may be optically polished, or alternatively, a
`fused quartz TIR element 24 may be cleaved to provide an
`adequate optical surface. Other TIR element configurations
`will be described with reference to FIGS. 3 and 4.
`Alternatively,
`the TIR element may be fabricated by
`injection molding of chemically activated transparent poly-
`mers into an appropriate shape and finish. Chemically-
`activated transparent polymers
`include surface treated
`homopolymers (eg. polystyrene), and copolymers of my
`rene such as styrene maleic anhydride (commercially avail-
`able from ARCO Chemical Company). It is very likely that
`other polymers and copolymers are suitable provided they
`are transparent and chemically activatable.
`
`10
`
`IS
`
`20
`
`30
`
`35
`
`45
`
`50
`
`55
`
`65
`
`8
`In the embodiment of FIG. 1, opposite end face 34 of the
`TIR element 24 is also polished flat or cleaved and, prefer-
`ably,
`is further provided with a black coating, a mirror
`coating or a separate mirror disposed substantially normal to
`the TIR element 24 axis. It is important in the operation of
`the invention to avoid fluorescent excitation of the bulk
`reaction solution by light exiting the TIR element 24 through
`the end face 34. Thus, a black coating (to absorb) or a mirror
`coating (to reflect) are preferred. The mirror coating or
`separate mirror has the added advantage of causing radiation
`trapped in the TIR element 24 to double pass the TIR
`element 24. The end face 34 need not be flat or normal to the
`axis of the element, however, as shown from FIG. 3.
`In the interior of the reaction vessel 20, the TIR element
`24 is exposed to the reaction sample 36. The reaction sample
`36 typically includes a buffered solution of sample compo—
`nents, label means and reagents for amplification (described
`further below). Examples of typical reaction samples for
`particular amplification reactions are provided in Examples
`4—11 below.
`The outer surface 38 of the TIR element 24 is modified,
`as described further below, having a plurality of coupling
`sites that allow attachment of the amplification reaction
`products or other members of specific binding pairs that can
`capture amplification reaction products. The amplified prod-
`uct, typically a double-stranded nucleic acid, comprises a
`pendent fluorophore. as described more fully below. During
`the course of or after the amplification reaction, the ampli-
`fied product and associated fluorophore is brought within the
`penetration depth of the TIR element 24 so that a fluorescent
`signal may be detected.
`C. Reagents and Protocols
`The target nucleic acid of the present invention is that
`nucleic acid sequence sought to be detected. It may comprise
`deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), or
`may be natural or synthetic analogs, fragments, and/or
`derivatives thereof. The target
`is preferably a naturally-
`occurring nucleic acid of prokaryotic or eukaryotic origin,
`including but not limited to, human, human immunodefi-
`ciency virus (HIV), human papilloma virus (HPV), herpes
`simplex virus (HSV), Chlamydia, Mycobacterium, Strepto-
`coccus, and Neisseria. One of skill in the art will recognize
`that
`thousands of other target nucleic acid sources are
`possible. When possible, DNA is often preferred due to its
`better stability.
`As mentioned above, the outer surface 38 of the TIR
`element 24 is modified to include a plurality of coupling
`sites for attachment of “capture means” for bringing fluo—
`rophore within the penetration depth. Various capture means
`are described below, and include covalent bonding mid
`specific binding pair attachment. In addition to the “capture
`means", it is necessary to have a “label means" for absorbing
`and re—emitting the fluorescent energy. The label means
`comprises a fluorophore, which is capable of absorbing
`fluorescent energy at one wavelength and re-emitting en