WORLD INTELLECTUAL_ PROPERTY ORGANIZATION
`International Bureau
`
`
`
`INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT)
`
`
`
`WO 99/20789
`
`(43) International Publication Date:
`
` (11) International Publication Number:
`
`
`29 April 1999 (29.04.99)
`
`(51) International Patent Classification 6 :
`
`
`
`C12Q 1/00, 1/70, G01N 33/53, 33/536,
`33/532, 31/00, 21/62, C12P 19/34, C07H
`19/00
`
`(21) International Application Number:
`
`PCT/US98/23160
`
`
`
`
`
`
`(81) Designated States: AL, AM, AT, AU, AZ, BA, BB, BG, BR,
`BY, CA, CH, CN, CU, CZ, DE, DK, EE, ES, FI, GE, GE,
`GH, HU, IL, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR,
`
`(22) International Filing Date:
`16 October 1998 (l6.10.98)
`
`LS, LT, LU, LV, MD, MG, MK, MN, MW, MX, NO, NZ,
`PL, PT, RO, RU, SD, SE, SG, SI, SK, TJ, TM, TR, TT,
`UA, UG, US, UZ, VN, YU, ARIPO patent (GH, GM, KE,
`LS, MW, SD, SZ, UG, ZW), Eurasian patent (AM, AZ, BY,
`KG, KZ, MD, RU, TJ, TM), European patent (AT, BE, CH,
`CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL,
`PT, SE), OAPI patent (BF, BJ, CF, CG, CI, CM, GA, GN,
`GW, ML, MR, NE, SN, TD, TG).
`
`
`
`
`
`
`
`(71) Applicant (for all designated States except US): GENICON
`SCIENCES CORPORATION [US/US]; 6450—E106 Lusk
`Boulevard, San Diego, CA 92121 (US).
` Published
`With international search report.
`
`Before the expiration of the time limit for amending the
`claims and to be republished in the event of the receipt of
`
`amendments.
`
`
`
`
`
`
`
`(72) Inventors; and
`(75) Inventors/Applicants (for US only): YGUERABIDE, Juan
`[US/US]; 9505 Poole Street, La Jolla, CA 92037 (US).
`YGUERABIDE, Evangelina, E.
`[US/US];
`9505 Poole
`Street, La Jolla, CA 92037 (US). KOHNE, David, E.
`[US/US]; 364 Nautilus Street, La Jolla, CA 92037 (US).
`JACKSON, Jeffrey, T.
`[US/US]; 12738 Casa Avenida,
`Poway, CA 92064 (US).
`
`(74) Agents: WARBURG, Richard, J. et al.; Lyon & Lyon LLP,
`Suite 4700, 633 West Fifth Street, Los Angeles, CA
`
`90071-2066 (US).
`
`
`
`(57) Abstract
`
`
`
`Method for specific detection of one or more analytes in a sample. The method includes specifically associating any one or more
`analytes in the sample with a scattered-light detectable particle, illuminating any particle associated with the analytes with light under
`conditions which produce scattered light from the particle and in which light scattered from one or more particles can be detected by
`a human eye with less than 500 times magnification and without electronic amplification. The method also includes detecting the light
`scattered by any such particles under those conditions as a measure of the presence of the analytes.
`
`
`
`
`
`
`(54) Title: ANALYTE ASSAY USING PARTICULATE LABELS
`
`Page 1
`
`ILLUMINA, INC. EXHIBIT 1027
`
`(30) Priority Data:
`08/953,713
`
`17 October 1997 (l7.10.97)
`
`US
`
`Page 1
`
`

`
`FOR THE PURPOSES OF INFORMATION ONLY
`
`Codes used to identify States party to the PCT on the front pages of pamphlets publishing international applications under the PCT.
`
`Albania
`Annenia
`Austria
`Australia
`Azerbaijan
`Bosnia and Herzegovina
`Barbados
`Belgium
`Burkina Faso
`Bulgaria
`Benin
`Brazil
`Belarus
`Canada
`Central African Republic
`Congo
`Switzerland
`Cote d’Ivoire
`Cameroon
`China
`Cuba
`Czech Republic
`Germany
`Denmark
`Estonia
`
`AL
`AM
`AT
`AU
`AZ
`BA
`BB
`BE
`BF
`BG
`BJ
`BR
`BY
`CA
`CF
`CG
`CH
`CI
`CM
`CN
`CU
`CZ
`DE
`DK
`EE
`
`
`
`Page 2
`
`
`
`
`ES
`FI
`FR
`GA
`GB
`GE
`GH
`GN
`GR
`HU
`IE
`IL
`IS
`IT
`JP
`KE
`KG
`KP
`
`KR
`KZ
`LC
`LI
`LK
`LR
`
`Spain
`Finland
`France
`Gabon
`United Kingdom
`Georgia
`Ghana
`Guinea
`Greece
`Hungary
`Ireland
`Israel
`Iceland
`Italy
`Japan
`Kenya
`Kyrgyzstan
`Democratic People's
`Republic of Korea
`Republic of Korea
`Kazakstan
`Saint Lucia
`Liechtenstein
`Sri Lanka
`Liberia
`
`LS
`LT
`LU
`LV
`MC
`MD
`MG
`MK
`
`ML
`MN
`MR
`MW
`MX
`NE
`NL
`N0
`NZ
`PL
`PT
`R0
`RU
`SD
`SE
`SG
`
`Lesotho
`Lithuania
`Luxembourg
`Latvia
`Monaco
`Republic of Moldova
`Madagascar
`The fonner Yugoslav
`Republic of Macedonia
`Mali
`Mongolia
`Mauritania
`Malawi
`Mexico
`Niger
`Netherlands
`Norway
`New Zealand
`Poland
`Portugal
`Romania
`Russian Federation
`Sudan
`Sweden
`Singapore
`
`SI
`SK
`SN
`SZ
`TD
`TG
`TJ
`TM
`TR
`TT
`UA
`UG
`US
`UZ
`VN
`YU
`ZW
`
`Slovenia
`Slovakia
`Senegal
`Swaziland
`Chad
`Togo
`Tajikistan
`Turkmenistan
`Turkey
`Trinidad and Tobago
`Ukraine
`Uganda
`United States of America
`Uzbekistan
`Viet Nam
`Yugoslavia
`Zimbabwe
`
`Page 2
`
`

`
`WO 99/20789
`
`PCT/US98/23160
`
`ANALYTE ASSAY USING PARTICULATE LABELS
`
`w
`
`BACKGROUND OF THE INVENTION
`
`The
`
`following is
`
`an outline of
`
`relevant existing
`
`detection methods. It is also a summary of relevant science
`
`15
`
`to aid the reader
`
`in understanding the details of
`
`the
`
`claimed invention.
`
`It should not be taken as an admission
`
`that any of the cited art is prior art to the claims. The
`
`cited art
`
`is hereby incorporated herein by reference so
`
`that
`
`the general procedures and methods
`
`in that art
`
`that
`
`20
`
`are of use to practice of the present invention need not be
`
`rewritten herein.
`
`In particular,
`
`applicant
`
`incorporates
`
`those sections related to general methods of “binding-pair”
`
`methodology,
`
`and methods
`
`for measurement
`
`of
`
`light
`
`scattering herein.
`
`Sensitive Analyte Assays
`
`Binding—pair
`
`(also known as ligand—receptor, molecular
`
`techniques play an
`like)
`the
`recognition binding and
`important role in many applications of biomedical analysis
`and are gaining importance in the fields of environmental
`
`science, veterinary medicine, pharmaceutical research,
`
`food
`
`and water quality control and the like. For
`
`the detection
`
`25
`
`30
`
`Page 3
`
`Page 3
`
`

`
`WO 99/20789
`
`PCT/US98/23160
`
`of
`
`analytes
`
`at
`
`low concentrations
`
`(less
`
`than about
`
`1
`
`picomole
`
`analyte/sample
`
`volume
`
`analyzed)
`
`the
`
`use
`
`fluorescent,
`
`luminescent,
`
`chemiluminescent,
`
`of
`
`or
`
`electrochemiluminescent
`
`labels and detection methods are
`
`5
`
`often used.
`
`For the detection of low concentrations of analytes in
`
`the field of diagnostics,
`
`the methods of
`
`chemiluminescence
`
`and electrochemiluminescence are gaining wide—spread use.
`
`These
`
`methods
`
`of
`
`chemiluminescence
`
`and
`
`electro-
`
`10
`
`provides
`chemiluminescence
`concentrations of analytes
`
`low
`detect
`to
`means
`a
`by amplifying the number of
`
`luminescent molecules or photon generating events many-
`
`fold,
`
`the resulting “signal amplification" then allowing
`
`for detection of low concentration analytes.
`
`15
`
`In addition,
`
`the method of Polymerase Chain Reaction
`
`(PCR) and other related techniques have gained wide use for
`
`amplifying the number of nucleic acid analytes
`
`in the
`
`sample. By the addition of appropriate enzymes,
`
`reagents,
`
`and temperature cycling methods,
`
`the number of nucleic acid
`
`20
`
`analyte molecules are amplified such that
`be detected by most known detection means.
`
`the analyte can
`The high level
`
`of commercial activity in the development of
`
`new signal
`
`and the development of
`generation and detection systems,
`new types of test kits and instruments utilizing signal and
`analyte molecule amplification attests to the importance
`
`25
`
`and need for sensitive detection methods.
`
`However,
`
`the above mentioned methods of
`
`signal
`
`and
`
`analyte molecule amplification have associated limitations
`which makes
`the detection of analytes by these methods
`
`30
`
`complicated, not easy to use,
`Problems
`of
`interference
`
`time consuming, and
`of
`chemical
`or
`
`costly.
`enzymatic
`
`reactions,
`
`contamination,
`
`complicated
`
`and multi-step
`
`procedures,
`
`limited
`
`adaptability
`
`to
`
`single
`
`step
`
`Page 4
`
`Page 4
`
`

`
`WO 99/20789
`
`PCT/US98/23160
`
`“homogeneous” (non—separation)
`
`formats, and the requirement
`
`of costly and sophisticated instrumentation are areas that
`
`those in the art are constantly trying to improve.
`
`5
`
`tremendous need for easy to use,
`there is a
`Thus,
`quantitative, multi-analyte, and inexpensive procedures and
`instruments
`for
`the
`detection
`of
`analytes.
`Such
`
`procedures,
`
`test kits,
`
`and instruments would overcome the
`
`disadvantages
`
`and limitations of
`
`the current methods of
`
`signal
`
`and analyte molecule amplification,
`
`and would be
`
`10
`
`useful
`
`in research,
`
`individual point of care situations
`
`(doctor's office, emergency room, out
`
`in the field, etc.),
`
`and in high throughput testing applications.
`
`It is the object of the present invention to provide a
`
`new means
`
`to more easily detect one or more analytes in a
`
`15
`
`sample to low concentrations than was previously possible.
`
`The present
`
`invention can detect
`
`low concentrations of
`
`analytes without
`
`the need for signal or analyte molecule
`
`amplification.
`
`The present
`
`invention provides a signal and detection
`
`20
`
`system for the detection of analytes where the procedures
`
`can be simplified and the amount and types of steps and
`
`reagents reduced.
`
`The present
`
`invention provides for
`
`the
`
`quantitative detection of single or multiple analytes in a
`
`sample. The present invention also provides for substantial
`reductions in the number of different tests and amounts of
`
`25
`
`sample material
`
`that are analyzed.
`
`Such reduction in the
`
`number of individual tests leads to reduced cost and waste
`
`production, especially medically—related waste that must be
`
`disposed of.
`
`30
`
`Light Scattering Detection Methods and Properties of Light
`
`Scattering Particles
`
`Page 5
`
`Page 5
`
`

`
`WO 99/20789
`
`PCT/US98/23160
`
`There is a
`
`large body of
`
`information concerning the
`
`phenomenon of
`
`light scattering by particles,
`
`the use of
`
`particulate labels in diagnostic assays,
`
`and the use of
`
`light scattering methods in diagnostic assays which are now
`
`5
`
`presented in the following discussion of relevant art none
`
`of which is admitted to be prior art to the pending claims.
`
`This art
`
`is provided as a background for understanding of
`
`the novelty and utility of the claimed invention.
`
`The general study of light scattering comprises a very
`
`10
`
`large field. The phenomena of
`
`light scattering has been
`
`studied intensely for about
`
`the last one hundred or
`
`so
`
`years
`
`and
`
`the applications
`
`of
`
`the
`
`knowledge of
`
`light
`
`scattering to different aspects of human endeavor are wide
`and varied.
`
`15
`
`The classical
`
`theory of
`
`light scattering by small,
`
`homogeneous, non light absorbing, spherical particles of a
`
`size of about 1/20 or
`
`less the wavelength of
`
`the incident
`
`radiation was
`
`initially developed by Rayleigh.
`
`Later
`
`a
`
`more general phenomenological theory of light scattering by
`
`20
`
`homogeneous,
`
`spherical
`
`particles
`
`of
`
`any
`
`size
`
`and
`
`composition was developed by Mie. The Mie
`
`theory applies
`
`both to light absorbing and nonabsorbing particles. It has
`
`also been shown from Mie
`
`theory that
`
`the expressions of
`
`Rayleigh can easily be generalized so
`
`as
`
`to apply to
`
`25
`
`particles which absorb
`
`light as long as the particles are
`
`much smaller
`
`than the wavelength of
`
`incident
`
`light.
`
`"these
`
`small
`
`diameter particles, Mie
`
`theory
`
`and
`
`For
`
`the
`
`generalized Rayleigh theory give similar
`
`results. Light
`
`scattering (elastic)
`
`can be viewed from a classical or
`
`30
`
`quantum mechanical point of view. An excellent quantitative
`
`description can be obtained through the classical point of
`view.
`
`A historical background as well as
`
`a description of
`
`Page 6
`
`Page 6
`
`

`
`WO 99120789
`
`PCT/US98/23160
`
`the
`
`basic
`
`theories
`
`of
`
`scattered
`
`light
`
`and
`
`other
`
`electromagnetic
`
`radiation is provided in the
`
`following
`
`references; Absorption and Scattering of Light By Small
`
`Particles (1983), C.F. Bohren, D.R. Huffman,
`
`John Wiley and
`
`5
`
`Sons; The Scattering of Light
`
`and Other Electromagnetic
`
`Radiation (1969), M. Kerker, Academic Press.
`
`Further background information of
`
`the phenomenon of
`
`light
`
`scattering
`
`can
`
`be
`
`found
`
`in
`
`the
`
`following
`
`publications.
`
`10
`
`Zsigmondy, Colloids and the Ultramicroscope - A Manual
`
`of Colloid Chemistry and Ultramicroscopy,l914,
`
`John Wiley &
`
`Sons,
`
`Inc.
`
`is described various light scattering properties
`
`of gold particles and other types of particles.
`
`Hunter, Foundation of Colloid Science, Vol,
`
`I,
`
`105,
`
`I5
`
`1991,
`
`describes
`
`use
`
`of
`
`optical
`
`microscopes,
`
`ultramicroscopes,
`
`and electron microscopes
`
`in observation
`
`of particles.
`
`Shaw et al.,
`
`Introduction to Colloid and Surface
`
`Chemistry, 2nd ed., 41, 1970, describe optical properties
`of colloids and the use of electron microscopy,
`and dark
`
`20
`
`field microscopy §.g.,
`
`the ultramicroscope.
`
`Stolz,
`
`SpringerTracts, Vol.
`
`130,
`
`describes
`
`time
`
`resolve light scattering methodologies.
`
`Klein and Metz,
`
`5 Photographic Science and Engineering
`
`25
`
`5-11,
`
`1961,
`
`describes
`
`the
`
`color
`
`of
`
`colloidal
`
`silver
`
`particles in gelatin.
`
`Eversole and Broida,
`
`15 Physical Review 1644-1654,
`
`1977,
`
`describes
`
`the
`
`size
`
`and
`
`shape effects
`
`on
`
`light
`
`scattering from various metal particles such as‘ silver,
`
`30
`
`gold, and copper.
`1970,
`231 Z. Physik 128-143,
`Kreibig and Zacharias,
`describe
`surface
`plasma
`resonances
`in small
`spherical
`
`silver and gold particles.
`
`Page 7
`
`Page 7
`
`

`
`W0 99/20789
`
`PCT/US98/23160
`
`Bloemer et al.,
`
`37 Physical Review 8015-8021,
`
`1988,
`
`describes
`
`the optical properties
`
`of
`
`submicrometer—sized
`
`silver needles and the use of such needles is described in
`
`Bloemer, U.S. Patent 5,l5l,956,where
`
`a
`
`surface plasmon
`
`5
`
`resonance of
`
`small particles of metal
`
`to polarize light
`
`propagating in a wave guide is described.
`
`Wiegel.,
`
`136 Zeitschrift
`
`fur Physik, Bd.,
`
`642-653,
`
`1954, describes the color of colloidal silver and the use
`
`of-electron microscopy.
`
`W
`
`Use of Particles, Light Scattering and Other Methods
`
`for
`
`Detection of Analytes
`
`For about
`
`the last thirty—five years, metal particles
`
`15
`
`including gold and silver have been used as both contrast
`
`enhancement
`
`agents or
`
`light
`
`absorption labels
`
`in many
`
`different types of analytic and/or diagnostic applications.
`The great majority of
`these applications fall under
`the
`category of
`cytoimmunochemistry studies which have used
`gold or silver enhanced gold particles as markers to study
`structural aspects of cellular,
`subcellular,
`or
`tissue
`organization.
`In these studies, metal particles are usually
`detected and localized by electron microscopy,
`including
`
`(backscattered electron
`and BET
`transmission,
`scanning,
`take advantage of
`the electron
`imaging). These methods
`dense nature of metals or the high atomic number of metals
`
`to facilitate the detection of the gold particles by virtue
`
`of
`
`the
`
`large
`
`numbers
`
`of
`
`secondary
`
`and backscattered
`
`(see; Hayat,
`dense metal
`the
`generated by
`electrons
`Immunogold-silver staining reference Page 1 and Chapters 1,
`6, 15; and Hayat, Colloid Gold reference Chapters 1, 5,
`7
`and others).
`
`There have been a few reports of
`
`the use of gold and
`
`20
`
`25
`
`30
`
`Page 8
`
`Page 8
`
`

`
`WO 99/20789
`
`PCT/US98/23160
`
`silver
`
`enhanced
`
`gold particles
`
`in
`
`light microscopic
`
`studies. For example,
`
`in 1978 gold particles were used as
`
`an immunogold stain with detection by light microscopy. A
`
`review of
`
`the use of gold particles in light microscopy
`
`5
`
`(See, Hayat,
`
`Immunogold—Silver Staining Reference Page 3)
`
`published in 1995 discusses this 1978 work and presents the
`
`following analysis:
`
`to use the
`(1978) were the first
`“Geoghehan et al.
`red or pink color of colloidal gold sols for
`light
`microscopical
`immunogold
`staining
`using paraffin
`sections.
`In semithin resin sections red color of
`light scattered from gold particles as small as 14 nm
`was
`seen
`in
`cell
`organelles
`containing
`high
`concentrations
`of
`labeled antigens
`in the
`light
`microscope
`(Lucocq
`and Roth,
`1984).
`Since
`the
`sensitivity
`of
`immunogold
`staining
`in
`light
`is
`microscopy
`inferior
`in comparison with other
`immunocytochemical
`techniques,
`the
`former did not
`gain general acceptance;
`the pinkish color of
`the
`gold deposit is difficult to visualize.”
`
`10
`
`15
`
`20
`
`of
`state
`the
`of
`indication
`an
`is
`paragraph
`This
`the light scattering properties of gold
`understanding of
`and other metal particles
`for diagnostic
`and analytic
`
`25
`
`“In semithin
`studies. The paragraph specifically states
`resin sections
`red color
`of
`light
`scatter
`from gold
`
`in organelles
`seen
`nm was
`14
`as
`small
`as
`particles
`containing high concentrations of
`labeled antigens in the
`
`light microscope.”
`However, with white light
`
`30
`
`illumination,
`
`the scattered
`
`nm gold particles is predominantly green.
`from 14
`light
`Since the particles appear red in the light microscope this
`indicates
`that
`some
`interactions other
`than pure
`light
`scattering are being detected. It is probable that
`the red
`color observed in the light microscope is predominantly
`transmitted light and not scattered light. When the gold
`particles accumulate sufficiently at
`the target site in
`cells,
`tissue sections or some other surface the red color
`
`35
`
`Page 9
`
`Page 9
`
`

`
`WO 99/20789
`
`PCT/US98/23160
`
`due to transmitted light will be seen (see also;
`
`J. Roth
`
`(1983)
`
`Immunocytochemistry 2 p217; and Dewaele et al
`
`(1983)
`
`in Techniques in Immunochemistry Vol
`
`2 pl, Eds. Bullock and
`
`Petrusz, Academic Press).
`
`5
`
`3 As mentioned in the above quote,
`
`it appears that
`
`the
`
`sensitivity of
`
`immunogold staining in light microscopy was
`
`believed to be inferior to that of other methods,
`
`and the
`
`use of gold particles
`
`as markers
`
`for
`
`light microscope
`
`detection did not gain general acceptance.
`
`In the 1995
`
`10
`
`review book in Chapter 12,
`
`p198 by Gao and Gao
`
`is the
`
`following quote on the same subject.
`
`initially used only as a marker
`“Colloidal gold was
`for electron microscopy (EM), because of its electron
`dense nature and secondary electron emission feature
`(Horisberger,
`1979).
`Direct
`visualization
`of
`colloidal gold in light microsc0PY (LM) was
`limited.
`The
`size of colloidal gold is
`too small
`to be
`detected at
`the
`light microscope
`level,
`although
`using highly concentrated immunogold cells may be
`stained red by this reagent
`(Geoghegan et al., 1978;
`Roth, 1982; Holgate et al., 1983.”
`
`As mentioned in both of
`
`the above,
`
`the sensitivity of
`
`detection of colloidal gold with light microscopy was
`
`believed to be low.
`The method of silver enhancement of
`gold particles was developed to overcome
`this perceived
`drawback. The
`following is another quote
`from the 1995
`
`review book.
`
`immunogold staining for
`real breakthrough for
`“The
`light microscopy
`came with
`the
`introduction of
`silver-enhancement of colloidal gold particles (20nm)
`bound to immunoglobin in paraffin sections 5 microns
`(Holgate et al., 1983). This approach significantly
`enhanced the sensitivity, efficiency, and accuracy of
`antigen detectability in the light microscope. Using
`IGSS, gold particles as small as lnm in diameter can
`be visualized in the light microscope. Thin section
`subjected to IGSS can also be viewed with the light
`microscope,
`especially by using phase contrast or
`epi—polarization
`illumination
`(Stierhof
`et
`al.,
`1992).”
`
`15
`
`20
`
`25
`
`30
`
`35
`
`40
`
`Page 10
`
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`
`

`
`W0 99/20789
`
`PCT/US98/23160 .
`
`The method of silver enhancement of gold particles is
`
`widely used.
`
`The enhancement method transforms the marker
`
`gold _particle into a
`
`larger metal particle or
`
`an
`
`even
`
`5
`
`larger structure which is microns or greater in dimensions.
`These structures are composed primarily of silver, and such
`
`enlarged particles can be more readily detected visually in
`
`the bright field optical microscope.
`
`Individual enlarged
`
`particles have been visualized by high resolution laser
`
`10
`
`confocal and epipolarization light microscopy. ;g at 26 and
`203.
`
`However,
`even with the use of
`silver
`enhancement
`techniques,
`those in the art
`indicate that
`this will nod
`achieve the sensitivity and specificity of other methods.
`
`15
`
`For example,
`
`in the publication of Vener, T.
`
`I. et. al.,
`
`the authors
`(1991)
`Analytical Biochemistry 198, p308-311
`discuss a new method of sensitive analyte detection called
`
`Latex Hybridization Assay (LHA).
`
`In the method they use
`
`large polymer particles of 1.8 microns in diameter that are
`filled with many highly fluorescent dye molecules as
`the
`
`20
`
`analytes
`bound
`detecting the
`tracer,
`analyte
`fluorescent
`signal. The
`following excerpt
`is
`
`the
`by
`from this
`
`publication:
`
`25
`
`30
`
`35
`
`LHA we have compared our
`“To assess the merits of
`indirect nonradioactive
`technique with
`two other
`techniques described in the
`literature.
`The most
`appropriate
`technique
`for
`comparison
`is
`the
`streptavidin
`colloid
`gold method with
`silver
`enhancement of a hybridization signal, since this is
`a
`competing
`corpuscular
`technique. However,
`this
`method is not very sensitive even with the additional
`step "of silver enhancement:
`8pg of k—phage ‘DNA is
`detected by this method as compared to 0.6 pg or 2 X
`10‘ molecules of K DNA detected by LHA on the nylon
`‘ membrane."
`
`92 Proc. Natl. Acad. Sci. USA, 6379-
`Stimpson et al.,
`6383, July 1995, a real time detection method for detection
`
`Page 11
`
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`
`

`
`WO 99/20789
`
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`
`of DNA hybridization is described.
`
`The authors describe
`
`use of a particulate label on a target DNA which acts as a
`
`10
`
`I5
`
`20
`
`25
`
`30
`
`35
`
`40
`
`the
`illuminated by
`“light-scattering source when
`evanescent wave of the wave guide and only the label
`bound to the
`surface generates
`a
`signal.
`The
`evanescent wave created by the wave guide is used to
`scatter light
`from a particulate label adsorbed at
`multiple DNA capture zones placed on the wave guide
`surface.
`Since an evanescent wave only extends a few
`hundred nanometers
`from the wave guide surface,
`the
`unbound/dissociated label does not scatter light and
`a wash step is not required.
`The signal intensity is
`sufficient
`to
`allow measurement
`of
`the
`surface
`binding and desorption of
`the light—scattering label
`can be studied in real
`time;
`i.e., detection is not
`rate limiting.
`The hybridization pattern on the chip
`can
`be
`evaluated
`visually
`or
`acquired
`for
`quantitative analysis by using a standard CCD camera
`with an 8-bit video frame grabber
`in 1/30 of
`a
`second.”
`
`Experiments were performed with 70 nanometer diameter gold
`
`particles and 200 nanometer diameter
`
`selenium particles.
`
`More
`
`intense
`
`signals were
`
`observed with
`
`the
`
`selenium
`
`particles.
`
`The authors indicate
`
`single-base
`for
`sufficient
`signal
`guide
`“A wave
`discrimination has been generated between 4 and 40 nm
`DNA and is,
`therefore, comparable to a
`fluorescence
`signal system."
`
`This
`
`method
`
`uses
`
`waveguides
`
`and
`
`evanescent
`
`type
`
`illumination.
`
`In addition,
`
`the method is about as sensitive
`
`as current fluorescence-based detection systems. Particles
`
`of 70nm diameter and larger are said to be preferred.
`
`Schutt et al., U.S. Patent 5,017,009, describes
`an
`immunoassay
`system for detection of
`ligands
`or‘
`ligand
`
`binding partners in a heterogenous format.
`
`The system is
`
`based upon detection of
`
`evanescent wave
`from an
`scattered light
`“back
`disturbed by the presence of a colloidal gold label
`brought
`to
`the
`interface
`by
`an
`immunological
`
`10
`
`Page 12
`
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`
`

`
`WO 99/20789
`
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`
`reaction.
`
`... Placement of
`
`the detector at
`
`a back
`
`the critical angle insures
`angle above
`signal-to-noise ratio.”
`
`a
`
`superior
`
`5
`
`the immunoassay system described
`The authors explain that
`utilizes
`scattered
`total
`internal
`reflectance,
`i.e.,
`
`propagation of evanescent waves. They indicate that
`
`the
`
`presence of colloidal gold disrupts propagation of
`
`the
`
`evanescent wave resulting in scattered light which may be
`
`10
`
`light
`detected by a photomultiplier or other
`provide
`a
`responsive
`signal.
`They
`indicate
`
`sensor
`that
`
`to
`an
`
`their
`of
`aspect
`important
`location of the detector.
`
`invention
`
`is
`
`the
`
`physical
`
`“The detector is ideally placed at an angle greater
`than the critical angle and in a
`location whereby
`only light scattered backward toward the light source
`is detected.
`This
`location thereby ideally avoids
`the detection of superior scattered light within the
`bulk liquid medium.”
`
`15
`
`20
`
`Total
`
`internal reflection of
`
`the incident beam is used to
`
`create the evanescent wave mode of
`
`illumination and the
`
`detection
`
`is
`
`performed
`
`on
`
`an
`
`optically—transmissive
`
`surface. The use of specialized apparatus is preferred.
`
`25
`
`Leuvering, U.S. Patent 4,313,734, describes a method
`
`for detection of specific binding proteins by use of
`
`a
`
`labeled component obtained by coupling particles “of
`
`an
`
`aqueous dispersion of a metal, metal compound, or polymer
`
`nuclei coated with a metal or metal
`
`compound having a
`
`30
`
`diameter of at
`
`least
`
`5 nm.” The process
`
`is said to be
`
`especially
`
`suited
`
`for
`
`estimation
`
`of
`
`immunochemical
`
`components such as haptens, antigens and antibodies.
`
`The
`
`metal particles are
`
`said to have already been used as
`
`contrast-enhancing labels in electron microscopy but
`
`their
`
`35
`
`use in immunoassays had apparently
`
`“not previously been reported and has surprisingly
`*
`*
`*
`proved to be possible.
`
`11
`
`Page 13
`
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`
`

`
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`
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`
`technique,
`immunochemical
`sol particle,
`The metal
`invention which has been
`according to the instant
`developed can be not only more sensitive than the
`known radio- and enzyme-immunotechniques, but renders
`it
`furthermore
`possible
`to
`demonstrate
`and
`to
`determine more
`than one
`immunological
`component
`in
`the same test medium simultaneously by utilizing sol
`particles
`of different
`chemical
`compositions
`as
`labels.”
`
`Examples
`
`of metals
`
`include platinum,
`
`gold,
`
`silver,
`
`and
`
`copper or their salts.
`
`the physical properties and/or
`“The measurement of
`the metal and/or
`the
`formed
`the concentration of
`metal containing agglomerate in a certain phase of
`the reaction mixture may take place using numerous
`techniques, which
`are
`in
`themselves
`known.
`As
`examples of
`these techniques there may be cited the
`colorimetric determination,
`in which use is made of
`the
`intense
`colour
`of
`some
`dispersions which
`furthermore
`change
`colour with
`physicochemical
`changes;
`the visual method, which is often already
`applicable to qualitative determinations in view of
`the above-noted fact
`that metal sols are coloured;
`the
`use
`of
`flame
`emission
`spectrophotometry
`or
`another
`plasma-emission
`spectrophotometric method
`which
`renders
`simultaneous determination possible,
`and the highly sensitive method of flame-less atomic
`absorption spectrophotometry.”
`
`Two or more analytes in a sample are preferably detected by
`
`using flame emission spectrophotometry or another plasma-
`
`emission spectrophotometric method. The preferred method of
`
`detection for greatest sensitivity is by flame-less atomic
`
`absorption spectrophotometry.
`U.S.
`
`Swope
`
`Qt
`
`al.,
`
`Patent
`
`5,350,697
`
`describes
`
`apparatus to measure scattered light by having the light
`
`source located to direct
`
`light at
`
`less than the critical
`
`angle toward the sample.
`
`The detector is located to detect
`
`scattered light outside the envelope of the critical angle.
`
`‘
`
`Craig et al.,
`
`U.S.
`
`Patent 4,480,042 describes use of
`
`high refractive index particle reagents in light scattering
`The
`
`preferred particles
`
`are
`
`composed
`
`of
`
`immunoassays.
`
`12
`
`10
`
`U
`
`20
`
`25
`
`30
`
`35
`
`40
`
`Page 14
`
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`
`

`
`WO 99/20789
`
`PCT/US98/231 60 .
`
`polymer
`
`materials.
`
`The
`
`concentration
`
`of
`
`compounds
`
`of
`
`biological
`
`interest was determined by measuring the change
`
`in turbidity caused by particle agglutination or inhibition
`
`of agglutination. The preferred particles are of a diameter
`
`less
`
`than approximately 0.1 p
`
`and greater
`
`than 0.03 p.
`
`“Shorter wavelengths,
`
`such as 340 nm, give larger signal
`
`differences than longer wavelengths, such as 400nm."
`
`Cohen et al., U.S. Patent 4,851,329 and Hansen, U.S.
`
`Patent
`
`5,286,452,
`
`describe methods
`
`for
`
`detection
`
`of
`
`agglutinated particles
`
`by optical pulse particle size
`
`analysis or by use of an optical
`
`flow particle analyzer.
`
`10
`
`H
`
`20
`
`25
`
`30
`
`Page 15
`
`for determination of_
`These systems are said to be useful
`concentrations. These methods
`
`antigen or
`
`antibody
`
`use
`
`sophisticated apparatus and specialized signal processing
`
`means.
`
`Preferred particle diameters are of about 0.1 to 1
`
`micron in diameter for the method of Cohen and about 0.5 to
`
`about 7.0 microns in diameter for the method of Hansen.
`
`Okano et al.,
`
`202 Analytical Biochemistry 120,
`
`1992,
`
`describes
`
`a heterogenous
`
`sandwich immunoassay utilizing
`
`microparticles which
`
`can
`
`be
`
`counted with
`
`an
`
`inverted
`
`optical microscope.~
`
`The microparticles
`
`were
`
`of
`
`approximately
`
`0.76 microns
`
`in
`
`diameter,
`
`and
`
`were
`
`carboxylated microparticles made from acrylate.
`
`Other particle detection methods
`
`are described by
`
`Block,
`
`U.S.
`
`Patent
`
`3,975,084,
`
`Kuroda,
`
`U.S.
`
`Patent
`
`5,274,431, Ford, Jr., U.S. Patent 5,305,073, Euruya, U.S.
`
`Patent 5,257,087,
`
`and by Taniguchi et al., U.S. Patent
`
`5,311,275.
`
`Geoghegan et al.,
`
`7 Immunological Communications 1-12,
`
`1978, describes use of colloidal gold to label rabbit anti-
`
`A
`indirect detection of other antibodies.
`IgG for
`goat
`light and electron microscope were used to detect
`labeled
`particles. The gold particles had an average size of 18-20
`
`13
`
`Page 15
`
`

`
`WO 99/20789
`
`PCT/US98/23160
`
`nanometers and bright field light microscopy was used. For
`
`electron microscopy, Araldite
`
`silver-gold thin sections
`
`were used.
`
`“Similar percentages of surface labeled cells
`
`were .noted by
`
`immunofluorescence and the colloidal gold
`
`5
`
`bright
`
`field method.”
`
`1-5 particles per cell could be
`
`detected by electron microscopy but
`
`the authors state that:
`
`label were not detected by
`small quantities of
`“Such
`fluorescence or by brightfield microscopy and may represent
`either non-specific and Fc
`receptor bound GAD and GAM,
`10 where a
`low level of surface immunoglobulin (S.Ig)
`on the
`GAD and GAM treated cells.”
`
`Hari et al., U.S. Patent 5,079,172, describes use of
`
`gold particles in antibody reactions and detection of those”
`
`I5
`
`particles using an electron microscope.
`
`15 nanometer gold
`
`particles were
`
`exemplified.
`
`In
`
`the preferred method,
`
`electron microscopy is used.
`
`DeMey et al., U.S. Patent 4,420,558, describes the use
`
`of a bright field light microscopic method for enumerating
`
`20
`
`cells labeled with gold-labeled antibodies. The method uses
`
`a
`
`light microscope in the bright
`
`field arrangement
`
`and
`
`magnifications of 500 or greater with immersion oil
`
`lenses
`
`are used to count gold-labeled peroxidase negative cells.
`
`The visualization of
`
`the labeled-surfaces is based on the
`
`25
`
`aggregate properties of
`
`the gold particles, which, under
`
`the indicated circumstances,
`
`undergo extensive patching,
`
`these patches on the cell surface being resolvable with the
`
`method described.
`
`40 nanometer gold was
`
`found to give
`
`optimal results.
`
`30
`
`De Mey et al., U.S. Patent 4,446,238, describes
`
`a
`
`similar bright field light microscopic immunocytochemical
`
`method
`
`for
`
`localization
`
`of
`
`colloidal
`
`gold
`
`labeled
`
`immunoglobulins
`
`as
`
`a
`
`red colored marker
`
`in histological
`
`sections. The method of
`
`Immuno Gold Staining (IGS)
`
`as
`
`35
`
`described by the authors
`
`l4
`
`Page 16
`
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`
`

`
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`
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`
`an
`is
`end-product
`the
`procedures
`both
`“In
`accumulation of
`large numbers of gold granules over
`antigen—containing areas,
`thus yielding the typical
`reddish colour of colloidal gold sols.”
`
`beBrabander et al., U.S. Patent 4,752,567 describes a
`
`method
`
`for detecting individual metal particles
`
`of
`
`a
`
`diameter smaller than 200nm by use of bright field or epi-
`
`polarization microscopy and contrast
`
`enhancement with a
`
`10
`
`video camera is described.
`
`The inventors state:
`
`15
`
`20
`
`25
`
`the
`in the above mentioned procedures,
`“Typically,
`from
`employed metal particles have
`a diameter of
`about
`10
`to about
`l00nm. This
`is well below the
`resolution limit of bright field microsc0PY. which is
`generally accepted to lie around 200
`nm.
`It
`is
`therefore quite logical
`that all previously known
`visual light microscopic methods are limited in their
`applications
`to
`the
`detection
`of
`immobilized
`aggregates of metal particles.
`Individual particles
`could be observed with ultramicroscopic techniques
`only,
`in particular with electron microscopy.
`It has
`now quite surprisingly been found that
`individual metal particles of a diameter smaller than
`200 nm can be made clearly visible by means of bright
`field light microscopy or epi-polarization microscopy
`in the visible spectrum, provided that
`the resulting
`image
`is
`subjected
`to
`electronic
`contrast
`enhancement.”
`
`30
`
`In subsequent sections the authors state:
`
`“Compared with existing diagnostic methods based
`on sol particle immuno assays,
`the present method has
`a much greater sensitivity.
`Indeed, existing methods
`are
`in
`general
`based
`on
`light
`absorption
`or
`scattering by the bulk of absorbed or suspended metal
`particles. Obviously,
`the observation of colour, e.g.
`on
`a blotting medium,
`requires
`the presence
`of
`massive numbers of particles.
`In contrast therewith,
`the present method makes it possible to observe and
`count
`single particles. Hence,
`the present
`‘method
`will largely facilitate the development of diagnostic
`blots for applications where existing, e.g. visual or
`colorimetric,
`techniques are too less sensitive, e.g.
`for the detection of Hepatitis.”
`
`Schafer et al.,
`
`352 Nature 444-448,
`
`1991, describes
`
`35
`
`40
`
`45
`
`15
`
`Page 17
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`
`

`
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`
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`
`use of nanometer
`
`size particles of gold which could be
`
`observed
`
`Using
`
`video enhanced differential
`
`interference
`
`contrast microscopy.
`was used.
`
`A 40 nanometer d