`United States Patent
`5,585,242
`Dec. 17, 1996
`Boumaetal.
`(45) Date of Patent:
`
`(11) Patent Number:
`
`NTENAa
`
`US005585242A
`
`[54] METHOD FOR DETECTION OF NUCLEIC
`ACID USING TOTAL INTERNAL
`REFLECTANCE
`
`[75]
`
`Inventors: Stanley R. Bouma, Grayslake; Omar
`S. Khalil, Libertyville; Edward K.
`Pabich, Chicago,all of I.
`
`[73] Assignee: Abbott Laboratories, Abbott Park,Ill.
`
`[21] Appl. No.: 522,623
`
`[22]
`
`Filed:
`
`Aug. 31, 1995
`
`Related U.S. 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.
`
`
`. C12Q 1/68; C12P 19/34
`Int. CL..
`[51]
`[52] U.S. Ch ieee 435/6; 435/91.2; 935/77;
`935/78
`
`teense 435/6, 91.2, 288,
`» 7.21, 7.32; 935/77, 78
`
`[58] Field of Search............
`435/7.1, 7.
`
`
`
`[56]
`
`References Cited
`
`U.S. PATENT DOCUMENTS
`
`Analytical Chemistry, vol. 45, No. 4, Apr., 1973, Multiple
`Internal Reflection Fluorescence Spectrometry, Harrick.
`
`Clin Chem. vol. 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-
`tion of Herpes Simplex Virus I DNA in Blot and In-Situ
`Hybridization Assays, Bronstein et al.
`
`Proc. Natl. Acad. Sci. USA, vol. 87, 4514-4518, Jun. 1990
`imaging ofDNA Seq. with Chemiluminescence, Tizardet al.
`
`Analytical Biochemistry, 180, 95-98 (1989), A 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, etal.
`
`» 436/527
`
`5/1984 Hirschfeld.............
`4,447,546
`- 436/827
`4,558,014 12/1985 Hirschfeld et al.
`
`4,582,809
`4/1986 Block et al.
`.......
`.. 436/527
`
`wee 436/94
`...
`4,608,344
`8/1986 Carter et al.
`
`3/1987 Hirschfeld......
`250/458. 1
`4,654,532
`
`..
`w- 435/91.2
`4,683,195
`7/1987 Mullis et al.
`
`
`12/1987 Block etal. ...
`» 435/514
`4,716,121
`vee 422/68
`4,844,869
`7/1989 Glass......
`
`3/1990 Block etal.
`. 422/811
`4,909,990
`
`Bio/Technology, vol. 10, Apr., 1992, Simultaneous Amplifi-
`5,001,051 3/1991) Miller et ab.weeeseseeeeeseeees 435/6
`
`
`
`cation and Detection ofSpecific DNA Sequences, Higuchi et
`6/1992 Urdea.........
`.. 435/6
`5,118,605
`al.
`9/0993 Hirschfeld 0... ccs ssscensess 435/6
`5,242,797
`
`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.
`
`FOREIGN PATENT DOCUMENTS
`
`0320308A2
`0297379
`0439182A2
`2190189
`2235292
`WO90/01157
`
`12/1988 European Pat. Off.
`1/1989 European Pat. Off.
`1/1991
`European Pat. Off.
`3/1987 United Kingdom .
`7/1990 United Kingdom .
`7/1989 WIPO.
`
`.
`.
`.
`
`OTHER PUBLICATIONS
`
`Nicholls et al. (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
`Academyof Sciences, vol. 87, pp. 1874-1878, “Isothermal,
`in vitro amplification of nucleic acids by a multienzyme rxn
`modeled after retroviral amplification”.
`Ostergaardet 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 Q"T Replicase Amplification, Cahill.
`Journal of Immunological Methods, 74 (1984) 253-265,
`Immunoassays at a Quartz—Liquid Interface: Theory, Instru-
`mentation, etc., Sutherland.
`
`Primary Examiner—Lisa B. Arthur
`Altorney, 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 differential
`temperature cycling is further provided.
`
`13 Claims, 4 Drawing Sheets
`
`Agilent Exhibit 1229
`Page 1 of 22
`
`Agilent Exhibit 1229
`Page 1 of 22
`
`
`
`U.S. Patent
`
`Dec. 17, 1996
`
`Sheet 1 of 4
`
`5,585,242
`
`DETECTION
`MEANS AND
`OPTICS
`
`18
`
`EXCITATION
`SOURCE AND
`OPTICS
`
`
`
`FIG. 1
`
`Agilent Exhibit 1229
`Page 2 of 22
`
`Agilent Exhibit 1229
`Page 2 of 22
`
`
`
`U.S. Patent
`
`Dec. 17, 1996
`
`Sheet 2 of 4
`
`5,585,242
`
`DETECTOR
`ELECTRONICS
`
`54
`
`44 46
`
`DETECTOR
`
`60
`
`538
`
`56=—
`
`EXCITATION
`SOURCE
`
`40
`
`62
`
`42
`
`HZ Ze
`
`14 3
`
`FIG.2
`
`Agilent Exhibit 1229
`Page 3 of 22
`
`Agilent Exhibit 1229
`Page 3 of 22
`
`
`
`U.S. Patent
`
`Dec. 17, 1996
`
`Sheet 3 of 4
`
`5,585,242
`
`68
`
`70
`
`fo
`
`FIG.4
`
`Agilent Exhibit 1229
`Page 4 of 22
`
`Agilent Exhibit 1229
`Page 4 of 22
`
`
`
`U.S. Patent
`
`Dec. 17, 1996
`
`Sheet 4 of 4
`
`5,585,242
`
`YOLVUINI
`
`T38V1 YOLVLUNI
`
`JUALdVvo
`
` SUOLVUNI
`
`T38V1SYOLVILIN]JUNALAVO
`
`VGOld
`
`Agilent Exhibit 1229
`Page 5 of 22
`
`Agilent Exhibit 1229
`Page 5 of 22
`
`
`
`5,585,242
`
`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.
`
`BACKGROUNDDESCRIPTION
`
`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 cthidium bromide.
`In sum, 2P tracings, enzyme immunoassay[Kelleretal., 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); Bornsteinct al.,
`Anal. Biochem., 180:95-98 (1989); Tizard et al., Proc. Natl.
`Acad. Sci., 78:4515-18 (1990)] or homogeneous manner
`{Amold 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
`erroneousresults.
`
`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 contacttransfer (carryover), or by aerosol
`generation.
`Furthermore, these previously described detection proce-
`dures are time-consumingand laborintensive. 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.
`
`20
`
`25
`
`45
`
`55
`
`The amplification of nucleic acids is useful in a variety of
`applications. For example, nucleic acid amplification meth-
`ods have been usedin 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 RNAfor cloning and sequencing.
`Methodsof amplifying nucleic acid sequences are known
`in the art. One method, known as the polymerase chain
`reaction (“PCR”), utilizes
`a pair of oligonucleotide
`sequencescalled “primers” and thermal cycling techniques
`wherein one cycle of denaturation, 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
`Thus, a need emerges for detecting amplified nucleic
`its complement. This technique is described more com-
`acids in a closed system in order to eliminate the potential
`pletely in EP-A-320 308 and EP-A-439 182.
`for contamination. Also, a need emerges for a method of
`amplifying and detecting the target nucleic acid in an
`Other methods of amplifying nucleic acids known in the
`operationally simple, yet highly sensitive manner. The abil-
`art
`involve isothermal
`reactions,
`including the reaction
`ity to detect the amplification product in a sealed vessel, or
`referred to as Q-beta (“QB”) amplification [See,
`for
`in a closed system, offers useful advantages over existing
`example, Kramer et al., U.S. Pat. No. 4,786,600, WO
`91/04340, Cahill et al., Clin. Chem., 37:1482-1485 (1991);
`prior art methods,
`including the ability to monitor the
`Pritchard et al., Ann. Biol. Clin., 48:492-497 (1990)].
`amplification of target nucleic acid throughout the course of
`the reaction.
`Another isothermal reaction is described in Walker ct al.,
`“Isothermal in vitro amplification of DNA byarestriction
`Theuseoftotal internal reflection fluorescence techniques
`enzyme/DNA polymerase system”, Proc. Natl. Acad. Sci.,
`is known in the art with respect to immunoassays [Harrick,
`89:392-396 (1992). These amplification reactions do not
`et al., Anal. Chem., 45:687 (1973)]. Devices and methods
`that use total internal reflection fluorescence for immunoas-
`require thermal cycling.
`says have been described in the art by Hirschfield, U.S. Pat.
`Amplification of nucleic acids using such methods is
`Nos. 4,447,564, 4,577,109, and 4,654,532; Hirschfield and
`usually performed in a closed reaction vessel such as a
`Block, U.S. Pat. Nos. 4,716,121 and 4,582,809, whichareall
`snap-top vial. After the amplification, the reaction vessel is
`incorporated herein by reference. Other descriptions and
`then opened and the amplified product is transferred to a
`uses are given by Glass, U.S. Pat. No. 4,844,869; Andarde,
`detection apparatus where standard detection methodologies
`are used.
`U.S. Pat. No. 4,368,047; Hirschfield, GB 2,190,189A;
`Lackie, WO 90/067,229; Block, GB 2,235,292, and Carter
`et al., U.S. Pat. No. 4,608,344.
`Useoftotal internal reflection elements allows perform-
`ing a homogeneousassay(i.e. free of separation and wash
`steps) for members of specific binding pairs. Several appli-
`
`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
`
`Agilent Exhibit 1229
`Page 6 of 22
`
`Agilent Exhibit 1229
`Page 6 of 22
`
`
`
`5,585,242
`
`4
`amplification initiators double as either capture means or
`labe] meansor 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
`elementis in contact with said reaction sample and such that
`one end of the clement 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
`conditionssufficient 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
`elementas a function of the presence or amountoftarget 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 aqueoussolu-
`tions a distance of 1.7 mm or moreis 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
`apparatusfor 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
`fluorophore 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:
`
`25
`
`30
`
`45
`
`50
`
`55
`
`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 sampie 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.
`
`3
`cations of this principle are known in the art [such as
`Kronick,et al. J. Immunol. Methods, 8:235 (1975) and U.S.
`Pat. No. 3,604,927] for hapten assays and for immunoassay
`of macromolecules [Sutherland etal., J. Immunol. Methods,
`74:253 (1984)].
`In knowntotal internal reflectance methods, however, the
`slow diffusion of membersofspecific binding pairs from the
`bulk of the solution to the surface of the TIR elementcreates
`a limitation in using TIR fluorescence techniques. Thus,
`prior art devices have used capillary tubes or flow cells to
`enhance diffusion either by limiting the diffusion distances
`or by continuous exposure to fresh reactant stream, or both.
`Butthese 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 effort 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 needin the art for better
`TIR assay systems that are more easily automated and even
`disposable if desired.
`
`SUMMARYOFTHE 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 meansfor bringing
`said fluorophore within the penetration depth of said ele-
`ment, wherein at least one of said label means and said
`capture meansis specific for said target nucleic acid;
`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 sample, the initiator sequences and
`amplification reagents under conditionssufficient 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 amountoftarget nucleic acid,
`and
`
`detecting within the TIR element a change in fluores-
`cence.
`
`The invention contemplates both covalent attachment and
`specific binding member attachment
`ofinitiators 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
`
`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 embodimentof 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
`Page 7 of 22
`
`Agilent Exhibit 1229
`Page 7 of 22
`
`
`
`5
`FIG. 3 illustrates a unitary fused embodiment comprising
`acylindrical total internal reflection element, a lens therefor,
`and a reaction chamberscal
`
`5,585,242
`
`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
`(N,>N,) from the denser medium (i.e. having the higher
`refractive index; here N,) can be madeto totally internally
`reflect within the medium if it strikes the interface at an
`angle, @,, greater than the critical angle, 8,, where the
`critical angle is defined by the equation:
`
`6.=aresin(N,/N,)
`
`Under these conditions, an electromagnetic waveform
`known as an evanescent waveis 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” (d,) is defined as the distance from the interface at
`which the evanescent wave energy has fallen to 0.368 times
`the energy valueat the interface. [See, Sutherland etal., J.
`Immunol. Meth., 74:253-265 (1984)]. Penetration depth is
`calculated as follows:
`
`MN;
`,
`P 2m{sin2Og — (NAN)? }2
`
`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
`FIG, 4 illustrates a unitary fused embodiment comprising
`that tunnels back into the TIR element is within the total
`a planar or flat total internal reflection element, a beveled
`internal reflection angle and is thus trapped within the TIR
`prismatic lens therefor, and a reaction chamber seal
`element. Accordingly, TIR allows detection of a fluoro-
`FIG, 5A illustrates a preferred configuration for PCR
`phore-labeled target of interest as a function of the amount
`amplification, using a capture initiator andalabel initiator.
`of the target
`in the reaction sample that
`is within the
`FIG. 5B illustrates a preferred configuration for LCR
`penetration depth of the TIR element.
`amplification, using capture initiator and label
`initiator
`B. A First Embodiment
`probe pairs.
`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 anda total internal
`reflection (TIR) element 24, Thereaction 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 embodimentdescribed later) this feature is not
`deemed essential to this embodiment. Accordingly, a ther-
`mocycler device 12 is shown. However, the details of the
`method of thermocycling 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 thermocycler 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 thermostable 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 a typical
`embodiment, the reaction vessel 20 is a microcentrifuge
`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
`
`Factors that tend to increase the penetration depth are:
`increasing angle of incidence, 6,; closely matching indices
`of refraction of the two media (i.e. N,/N,—>1); and increas-
`ing wavelength, A. An examplewill illustrate. If a quartz TIR
`element (N,=1.46) is placed in a aqueous medium (N,=
`1.34), the critical angle, 0,, is 66°. If 500 nm light impacts
`the interface at 0,=70° (ic. greater than the critical angle)
`the d, 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 withinit, is established bythe refractive indices of the
`TIR element and the surrounding medium. For radiation
`initially propagating through a medium ofrefractive index
`Ng, such as air, incident upon a TIR elementof refractive
`index N,, otherwise surrounded by a medium of refractive
`index N,, the maximum acceptance angle, B, may be found
`from the equation:
`
`20
`
`25
`
`30
`
`35
`
`45
`
`50
`
`35
`
`N.A=No sin BN2-N22)"?,
`
`where N.A. is the so-called numerical aperture of the TIR
`element.
`Within the penetration depth, the evanescent wave in the
`less dense medium (typically a reaction solution) can excite
`
`65
`
`Agilent Exhibit 1229
`Page 8 of 22
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`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 ate given in Table 1 below:
`
`TABLE1
`
`Element Material
`Refractive Index
`
`
`1.6
`Quartz
`1.59
`Polystyrene
`1.52
`Glass
`1.49
`Polymethylmethacrylate
`
`Pyrex 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 andit 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 dimensionedto 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 rubber 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 coterminous 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 altemative TIR
`elements shown in FIGS. 3 and 4.It is important, however,
`that a minimum amountof the TIR elementis 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 tendto scatter light incident upon its face. The
`end face 32 may be optically polished, or alternatively, a
`fused quartz TIR clement 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 (e.g. polystyrene), and copolymers of sty-
`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.
`
`20
`
`35
`
`4s
`
`30
`
`55
`
`65
`
`8
`In the embodiment of FIG. 1, opposite end face 34 of the
`TIR element 24 is also polishedflat 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 bylight 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 exposedto 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 membersof specific binding pairs that can
`capture amplification reaction products. The amplified prod-
`uct, typically a double-stranded nucleic acid, comprises a
`pendentfluorophore, 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 24so 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. Oneof 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 energy
`at a different wavelength, as is known in the art. Either the
`capture meansorthe label means, or both, must be specifi-
`cally associated with the presence or amountof target. The
`present TIR invention depends on the ability to bring the
`label means within the penetration depth in amounts that
`correspond to the presence or amount of the target.
`A third reagent system i