Note: Descriptions are shown in the official language in which they were submitted.
2~3631
PATENT
Attorney Docket No. 2558B-384
DETECTION AND IMAGING IN BIOCHEMICAL ASSAYS
USING PHOSPHOR SCREENS
This invention lies in the general field of
biochemical assays and detection methods, with a focus on
methods of labelling species and detecting the labels. In
particular, the invention relates to macromolecule detection
involving nonisotopic labelling methods.
A procedure essential to all molecular biology
laboratories is the detection and imaging of macromolecules.
This is used in protein assays, DNA sequencing, gene mapping,
and any number of other experiments and determinations. The
most common method used is by tagging or labelling the molecule
or sequence of interest with a radioactive species, then
recording an autoradiographic image of the radioactive emission
on x-ray film.
Disadvantages associated with the use of radioactive
species are that they present a handling hazard to the
laboratory technician, they are difficult to dispose of, and
their radioactive decay requires that they be periodically
replaced with fresh materials. Labels which do not involve
radioactive species are not readily translated into spatial
images on a film or screen, and are generally of lower
sensitivity than their radioactive counterparts.
X-ray films also have their limitations. The dynamic
range of a typical x-ray film is about fiftyfold, which limits
the degree to which one can obtain quantitative information
from the film. Also, long exposures are generally required to
obtain a satisfactory image, due to the limited sensitivity of
the film to the B-particle emissions used in most radioactive
label~. In addition, variability is potentially introduced in
the development of the film, since this requires a number of
steps involving unstable solutions.
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A method has now been developed for using phosphor
screens in combination with chemiluminescence to detect
and image macromolecules, replacing the need for
radioisotopes. The macromolecules may now be selectively
labelled with a species which induces a chemiluminescent
reaction in-a substrate, and the substrate exposed to a
phosphor screen, which is capable of responding to the
chemiluminescent emission and indicating its occurrence in
a readily detectable manner.
More specifically the present invention provides a
method for detecting macromolecules immobilized on a
matrix, said method comprising (a) selectively tagging
said macromolecules with a species capable of inducing a
lS chemiluminescent reaction in a liquid-phase
chemiluminescent substrate; (b) placing said matrix, with
said macromolecules so tagged, in contact with said
liquid-phase chemiluminescent substrate to induce said
chemiluminescent reaction; (c) while said matrix and said
liquid-phase chemiluminescent substrate are thus in
contact, exposing said liquid-phase chemiluminescent
substrate to a phosphor screen to excite phosphors
thereon and thereby form an image corresponding to said
macromolecules on said matrix; and (d) detecting said
image as an indication of the presence of said
macromolecules on said matrix.
The use of the substrate as an intermediate element
between the label and the film or screen permits one to
control the formation of the image and the intensity of
the signal and thus the sensitivity of the experiment,
while avoiding the hazard of radioactive materials.
Furthermore, the substrate is in liquid form, which
provides further advantages in terms of enhancing its
contact with the label, and in offering further parameters
for use in controlling and forming the i~age, such
204363 1
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parameters including concentration, light transmissivity
and the ability to accommodate variations in the physical
arrangement and configuration of the solid elements of the
system,
The use of the phosphor screen provides an image with
a high degree of sensitivity, contrast and reproducibility
with relatively short exposure times. Further advantages
include the fact that phosphor screens do not require
chemical treatment to be readable, and that they can trap
a chemiluminescent emission upon receiving it, and retain
the energy of the emission until stimulated by an external
source such as infrared light whereupon the screen
releases its own corresponding emission.
lS Other features, advantages and preferred embodiments
of the invention will be apparent from the description
which follows with reference to the drawings in which:
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FIG. 1 is a representation of an experiment conducted
in accordance with the present invention, in which a
macromolecular immobilization pattern i~ ~ransfered to a
phosphor screen.
FIG. 2 is a representation of an optical system for
generating and detecting signals ~hich ~ranslate the pattern on
the phosphor screen to a visually readable form.
Basic elements of the invEntion are the label, the
chemiluminescent substrate and the phosphor screen. This
detailed discussion will begin ~ith a description of the label
and chemiluminescent substrate.
The label and the substrate may be any materials
tending to produce a chemiluminescent emission upon contact,
including the wide variety of species known in the
chemiluminescence art. The label and the substrate may, for
example, be reactants which combine to form an excited state
which spontaneously degenerates to the ground state with the
release of a fluorescent or phosphorescent emission, or
reactants which combine to form an intermediate which
decomposes spontaneously to an excited state which then
undergoes the same conversion and energy release.
Alternatively, the reaction may be entirely contained in the
substrate. The substrate in such a reaction may be a single
species and the reaction either a conversion or decomposition
entailing an emission, or the substrate may be a mixture of
species which enter into a reaction which results in the
emission.
For substrate-contained reactions, the label may be a
catalyst or an enzyme. Catalyst and enzyme labels are
preferred, particularly enzyme labels, due to their successive
and continued interaction with the reactant substrate
molecules. This permits emissions to continue for prolonged
periods, thereby facilitating recordation-and detection, and
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provides signal amplification which can be controlled by the
amount of substrate made available to the label.
A wide variety of catalysts and enzymes capable of
inducing a chemiluminescent reaction are known, and therefore
suitable for use in the practice of the present invention.
Examples of catalysts are various metals and such species as
adenosine triphosphate. Examples of enzymes are lactate
dehydrogenase, luciferase and phosphatases such as alkaline
phosphatase (AP). Enzymes are preferred, with phosphatases
being particularly preferred, and alkaline phosphatase the most
preferred.
Any known chemiluminescent reaction may be utilized
in the present invention. Peroxide decomposition reactions are
a convenient class. Preferred reactions within this class are
1,2-dioxetane decompositions, including decompositions of
1,2-dioxetanones (~-peroxylactones) and 1,2-dioxetanediones
(peroxyoxalates). Examples of other classes are reactions
involving luminol (3-aminophthalhydrazide) and its analogs, and
organometallics such a p-chlorophenylmagnesium bromide.
Particularly preferred reactions are 1,2-dioxetane
decompositions regulated by enzymes. Compounds containing the
1,2-dioxetane group as well as an ester group of an
orthophosphoric acid are an example of one class of compounds
meeting this description. Decomposition of these compounds is
catalyzed by a phosphatase. One particular example is the
substrate 3-(2'-spiroadamantane)-4-methoxy-4-(3"-
phosphoryloxy)phenyl-1,2-dioxetane with the enzyme alkaline
phosphatase. The former is commercially available from Tropix
Corporation, Bedford, Massachusetts, and from Lumigen
Corporation, Detroit, Michigan, and the latter is comercially
available from a wide range of sources.
The substrate will be in the liquid phase.
Accordingly, the most convenient substrates will be water-
soluble substances, and the liquid phase will be an aqueous
solution. It is understood that the amount of substrate which
will undergo the reaction may not be readily determinable,
since the presence and amount of macromolecular species, and
hence attached label, may not be known. Nevertheless, one
2a.~3l
would seek to include an excess of substrate, based on either
the expected amount or upper limit of macromolecular species
being detected, so that all label immobilized by attachment to
the macromolecular species enters into the reaction and the
level of emission can be related to the amount of label. With
catalyst or enzyme labels, a large excess of substrate is
preferred, such that chemiluminescent light emissions will
continue for sufficient time to allow the remaining steps of
the procedure to be carried out while light of a sufficient
intensity continues to be emitted. In particularly preferred
systems, sufficient substrate is included to result in
chemiluminescent light emissions continuing for at least about
one hour, most preferably at least about 24 hours.
Turning next to the phosphor screen, a wide variety
of phosphors may be used. The proper selection in each
particular application will depend on the chemiluminescent
material, the phosphors being selected to receive and respond
to the emission produced by the particular chemiluminescent
material.
Phosphors used in the practice of the invention may
be selected from the full range of materials known to possess
the capability of phosphorescence. In general, these are
materials which absorb light and enter an excited state as a
result, then undergo relaxation to the ground state while
emitting light, either at a different intensity or frequency or
over a different time scale, or both. Materials meeting this
description include natural minerals, biological compounds and
synthetically prepared materials and blends. Examples are
metal halophosphates such as Ca5(P04)3(F,Cl):Sb(III),Mn(II),
Sr5(P04)3(Cl):Eu(II), Sr5(po4)3(F~cl):sb(III)~Mn(II) and
[SrEu(II)~5(P04)3Cl; other rare-earth-activated phosphors such
as Y203:Eu(III), SrB407:Eu(II), BaMg2All6027:Eu(II),
Y(V04):Eu(III), Y(V04)P04:Eu(III), Sr2P207:Eu(II),
2 7 ( ), Sr3(P04)2:Eu(II), Sr5Si4Cl6010:Eu(II)
Ba2MgSi207:Eu(II), GdOS:Tb(III), LaOS:Tb(III), LaOBr:Tb(III),
LaOBr:Tm(III) and Ba(F,Cl)2:Eu(II): other aluminate-host
phOsphors such as CeO 6sTbo.3sMgA11l~l9
phosphors such as Zn2SiO4:Mn(II); and fluoride-host phosphors
~q3~31
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such as Y() 7gYbo 2oEro.olF3, La0.86Ybo.l2Ero-o2E3~ and
Yo . ~39Tbo . 3sTmo . oo 1 F3 -
Of particular interest are phosphors which remain in
the excited state until released by external stimulation.
These include many of those listed above, plus others.
Preferred examples are alkaline earth metal sulfides and
selenides, doped with samarium and europium or cerium
oxide, sulfide or fluoride, and further containing a
fusable salt such as lithium fluoride, barium sulfate or
both serving as a flux. I.ists and descriptions of such
materials are found in United States Patent Nos. 4,812,660
~March 14, lg89), 4,822,5~0 (April l8, 1989) and 4,83(),~75
(May 16, 1989), to Lindmayer, J. (Quantex Corporatlon).
The stimulation which releases the energy may be in lS the
form of heat or electromagnetic radiation, such as visible
light, x-rays, ultraviolet radiation and infrared
radiation, depending on the type of phosphor.
The substrate and phosphors will be selected to emit
and absorb, respectively, at the same wavelength, thereby
complementing each other in terms of energy emission and
response. The energy of a single emission will generally
be in the form of a wavelength band, the width of which is
not critical in the general sense. In certain
applications, as described in more detail below, narrowly
defined band widths will serve specific functions. As for
the actual wavelengths of the emissions, in most
applications within the contempla-tion of this invention,
emissions with peak wavelengths falling within -the range
of about 350nm to about 700nm, preferably from about 400nm
to about 600nm will be used.
The use of mixed phosphors, together with a
corresponding mixture of label/substrate systems, presents
further opportunities for enhanced use of the invention.
For example, luminol, a common currently available
chemiluminescent substrate, emits light at 4~8nm when
h ~ 4 3 6 3 ~
6a
activated, and various naphthyl dioxetane isomers
currently available emit li~ht at wavelengths varying from
463nm to 560nm. Of the various phosphors available from
Quantex Corporation, the phosphor designated Q-l~ will
respond to wavelengths of 470nm but not
6 3 ~
higher, while the phosphor designated Q-42 will respond to
wavelengths up to about 600nm.
With these phosphors combined on a single screen, or
any other combination which can similarly discriminate, one can
use multiple label/substrate systems of wavelengths
corresponding to those to which the phosphors are receptive.
The labels may be selectively placed on distinct preselected
groups of macromolecules, and the substrates may be combined in
a single substrate mixture.
Since the phosphors will themselves emit light at
distinct wavelengths, discrimination in the read-out process
may be achieved in a variety of ways, depending on the
particular read-out process used. Using infrared detection for
read-out, for example, a photomultiplier tube can discriminate
between the Q-16 light emissions, which are green, and the Q-42
light emissions, which are orange, through the use of filters.
As a further example of combined systems utilizing
the present invention, mixed phosphors may be used with both
chemiluminescent and radioactive emissions in a multi-signal
system. For example, one set of nucleic acid molecules (or
other macromolecules) may be tagged with a label such as
alkaline phosphatase which induces a chemiluminescent reaction
in the substrate, such as one which emits light at 600nm, and
another set tagged with phosphorus-32, or some other
radioisotope, which emits B radioactive emissions. A mixed
phosphor screen such as a Q-16 and Q-42 combination may then be
used. Since the Q-42 phosphor is insensitive to the B
radioactive emissions, those macromolecules tagged with
phosphorus-32 would be imaged only by the Q-16. The Q-42
phosphor would however be sensitive to the 600nm emissions from
the chemiluminescent substrate. A photomultiplier tube could
discriminate between the light emissions from the two phosphors
in the manner described in the preceding paragraph.
The use of mixed phosphors in these and other
combination systems permits an unlimited variety of-comparisons
and discriminations. For example, one can discriminate between
distinct groups of macromolecules in a single sample or compare
3 1
against an internal standard. Other possibilities will be
readily apparent to those skilled in the art.
The method of the invention may serve as either a
detection method, a quantitation method or both. It may be
utilized in assays or other determinations in a variety of
configurations and arrangements, which will be readily apparent
to those skilled in the art. In general, the invention is
applicable to any procedure for detecting or quantifying an
immobilized macromolecule or portion of a macromolecule. It is
of particular interest in imaging spatial arrays of
macromolecules, since it can be conducted in a manner which
provides localized information. Such imaging is of value for
spatial arrays generated by a variety of laboratory procedures,
including electropherograms, chromatograms, dot blots, and any
other arrangement of separated solutes or species.
The term "immobilized" is used in this specification
to denote retention of a species in a fixed location on a non-
liquid surface or matrix, in a manner by which the species will
not become dislodged or unattached upon contact with the
liquid-phase chemiluminescent substrate. The non-liquid
surface or matrix may be a slab gel such as a polyacrylamide or
agarose gel, a blotting membrane such as nitrocellulose or
derivatized nylon, or a solid surface such as coated glass or a
plastic microtiter plate.
Selective tagging of the macromolecule of interest
with the label may be achieved by any of the various means
known in the biochemical art. The attachment may be a covalent
bond, an affinity-type bond, a hydrophobic interaction, a
hybridization-type interaction or any other means of
attachment. Selectivity may be inherent in the means of
attachment, as in covalent, hydrophobic and hybridization
interactions, or it may be the result of immunological-type
binding specificity or other specific binding behavior.
Preferred interactions are hybridization interactions such as
the use of DNA or RNA probes, and specific binding
interactions, such as antigen-antibody interactions and avidin-
biotin interactions.
T
In these preferred interactions, the label is
conjugated to an appropriate binding member which binds to the
immobilized macromolecule. The latter is thus "tagged" with
the label selectively, i.e., to the exclusion of the surface or
matrix itself and of the other macromolecules which lack the
specific binding characteristics involved in the attraction.
The conjugate is formed in the conventional manner through a
covalent bond, including in some cases a cross-linker when one
is desired or beneficial.
The method of the present invention is performed by
immersing the solid phase on which the macromolecules of
interest are immobilized in the liquid-phase substrate, the
macromolecules being tagged with the labels described above so
that the chemiluminescent reaction between the label and the
substrate occurs and light energy is emitted. The liquid
substrate, with the solid phase thus immersed in it, is exposed
to the surface of the phosphor screen, and held so for a
sufficient length of time to record or detect the emission
pattern. This is preferably done through a transparent liquid-
retaining barrier, with the solid phase and the screen insufficient proximity that the latter forms a well-defined image
closely representative of the source array on the solid phase.
A convenient arrangement is one in which the solid
phase is placed in a shallow enclosed receptacle filled with
the substrate solution such that the macromolecule pattern
faces a transparent wall of the receptacle with a thin layer of
the solution in between. The screen is held against the
opposite side of the wall for the length of time referred to
above. Minimum and optimal durations are matters of routine
experimentation which will be readily apparent to or
determinable by those skilled in the art.
This invention has utility in a wide range of assays
and laboratory procedures. Notable examples are protein
assays, antibody assays, screening procedures, dilution
studies, DNA sequencing, and gene mapping. Other applications
will be readily apparent to those skilled in the art.
Turning now to the drawings, FIG. l is a
representation of one method of causing a chemiluminescent
reaction to occur in accordance with the invention and
receiving the resulting emissions on a receptor material, and
FIG. 2 is a diagram of an arrangement of components for
stimulating the receptor material to emit signals corresponding
to the emission it has received, and for sensing the signals
and converting them to readable form.
FIG. 1 depicts a support 11 on which macromolecular
species are immobilized. As indicated above, this may be a
slab gel, a filter membrane, or a solid surface, depending on
the type of procedure being conducted. The macromolecular
species 12 itself is localized on the support surface in a
distinct planar array, and has been tagged with the label 13.
The entire support is placed in a transparent receptacle, such
as a liquid-tight plastic bag 14, filled with the
chemiluminescent substrate solution 15. As indicated in the
general description above, the label is preferably an enzyme
label, and the substrate solution preferably contains enough
substrate to cause emissions to continue for at least 24 hours.
A phosphor screen 16 is placed over the plastic bag
15, directly above the immobilization pattern on the support,
and held in this position for a sufficient period of time to
receive sufficient emission to be detectable and yet show the
same spatial arrangement as the immobilization pattern on the
support. The phosphor in this illustration is one which traps
the energy of the emission, and releases it only when
stimulated with an external source such as infrared light.
Once the phosphors on the screen 16 are sufficiently
excited, the screen is removed from contact with the plastic
bag 14 and placed in the optical arrangement shown in FIG. 2.
To permit a full two-dimensional scan of the screen, the screen
is placed on a translating apparatus 21 which provides
translation along the x- and y-axes. The x-stage translator
component 22 and the y-stage translator component 23 of the
apparatus are driven by an x-y translator controller 24.
The phosphor screen 16 is stimulated by light energy
originating from an infrared laser 25 such as, for example, a
Nd:YAG laser emitting light at 1064nm. The beam leaving the
laser is collimated through collimating lenses 26 and deflected
~û43~
11
by a YAG mirror 27. The beam is then focused on the phosphor
screen 16 by a lens such as a 20x microscope objective 28
controlled by a z-axis micrometer 29. The translating
apparatus 21 causes the beam to scan the entire surface of the
screen.
Energy released from the phosphor screen by the
infrared stimulation is deflected by a cold mirror 30 through a
short pass filter 31 to a photomultiplier tube 32 powered by a
high voltage power supply 33. The signal from the
photomultiplier tube is directed to an oscilloscope 34 and a
high speed digitizing voltmeter 35. The voltmeter reading is
translated to a visual form by a graphics display 36 mediated
by computer 37. The graphics display 36 permits a full reading
and determination of the presence, location and amount of
macromolecule immobilized on the support phase 11 of ~IG. 1.
All components in this illustration may be supplied
by conventional equipment and instrumentation well known and
widely used in molecular biology laboratories. It is
emphasized once again that this arrangement merely illustrates
one method of practicing the invention. Others will readily
come to mind to the routineer seeking to adapt the concept to a
particular system, environment or available components.
The following example is offered for purposes of
illustration. It is intended neither to define nor limit the
invention in any manner.
EXAMPLE
A DNA strand was synthesized in a manner in which a
limited number of UTP nucleotides with biotin molecules
covalently attached were used in place of TTP nucleotides in a
random primer labelling reaction. A dilution series of the DNA
was then prepared, and a drop of each dilution was placed as a
dot on a Zeta-Probe cationized nylon membrane (Bio-Rad
Laboratories, Hercules, California). The resulting dots
contained 100pg, 10pg, lpg, 0.1pg and 0.01pg, respectively.
The membrane was then incubated with a steptavidin-AP
conjugate, with result that the DNA in all dots was labeled
* Trade-mark
h ~ 3 ~
with AP. The membrane was then placed in a plastic bag filled
with an a~ueous solution of 3-~2'-spiroadamantane)-4-methoxy-
4-(3"-phosphoryloxy)phenyl-1,2-dioxetane (AMPPD), which upon
undergoing chemiluminescence emits light at 470nm, at a
concentration of 0.2mM and a total volume of 5mL.
An x-ray film (Kodak X-omat) was then placed against
the plastic bag directly over the dots as shown in FIG. 1, and
thus exposed to the AMPPD for ten minutes. The film was then
developed by standard conventional x-ray film development
procedures. Observation of the screen indicated that images
corresponding to the dots representing 100pg, 10pg and lpg were
easily detected. An image of the 0.lpg dot could barely be
detected, while no image of the 0.01pg dot could be detected.
The same plastic bag and contents were then placed
against a Quantex Q-16 phosphor screen that had been prepared
on an alumina substrate, in the same manner as the x-ray film,
and likewise exposed for ten minutes. The screen is composed
of a base of strontium sulfide, with samarium and cerium oxide
as dopants, and barium sulfate and lithium fluoride as fluxes,
with infrared sensitivity of 1120nm to 1220nm, a light emission
curve peaking at 510nm, and is charged by light at 470nm.
After the exposure, the screen was subjected to infrared laser
scanning, using the apparatus depicted in FIG. 2. The results
indicated that the phosphor screen was capable of clearly
imaging the 100pg, lOpg, lpg and 0.lpg dots, but not the 0.01pg
dot.
The foregoing is offered primarily for purposes of
illustration. It will be readily apparent to those skilled in
the art that further variations, alternatives, substitutions
and the like may be made without departing from the spirit and
scope of the invention.
* Trade-marks
