U.S. patent application number 13/230658 was filed with the patent office on 2012-01-05 for amplified bioassay.
This patent application is currently assigned to VYBION, INC.. Invention is credited to Lee A. HENDERSON, Leo B. KRIKSUNOV, Geoffrey WHEELOCK.
Application Number | 20120004141 13/230658 |
Document ID | / |
Family ID | 35943742 |
Filed Date | 2012-01-05 |
United States Patent
Application |
20120004141 |
Kind Code |
A1 |
KRIKSUNOV; Leo B. ; et
al. |
January 5, 2012 |
AMPLIFIED BIOASSAY
Abstract
A method for assaying a biological sample includes forming
sensitized microcapsules filled with unique oligomarkers, capturing
sensitized microcapsules in the presence of analytes, releasing
oligomarkers from microcapsules and detecting and measuring
oligomarkers to detect and quantify presence of analyte in
biological sample. Using encapsulated oligomarkers provides for an
amplified high sensitivity assay and using plurality of oligomarker
types provides for a multiplexed assay.
Inventors: |
KRIKSUNOV; Leo B.; (Ithaca,
NY) ; WHEELOCK; Geoffrey; (Ithaca, NY) ;
HENDERSON; Lee A.; (Ithaca, NY) |
Assignee: |
VYBION, INC.
Ithaca
NY
|
Family ID: |
35943742 |
Appl. No.: |
13/230658 |
Filed: |
September 12, 2011 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11162142 |
Aug 30, 2005 |
8029985 |
|
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13230658 |
|
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60606446 |
Sep 1, 2004 |
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Current U.S.
Class: |
506/17 ; 435/7.1;
436/501; 506/13; 506/16; 506/18 |
Current CPC
Class: |
C12Q 1/682 20130101;
B82Y 10/00 20130101; C12Q 1/682 20130101; B82Y 5/00 20130101; C12Q
2565/501 20130101; C12Q 2537/125 20130101; C12Q 2563/131
20130101 |
Class at
Publication: |
506/17 ; 506/16;
506/18; 506/13; 436/501; 435/7.1 |
International
Class: |
C40B 40/08 20060101
C40B040/08; G01N 33/566 20060101 G01N033/566; C40B 40/00 20060101
C40B040/00; C40B 40/06 20060101 C40B040/06; C40B 40/10 20060101
C40B040/10 |
Claims
1. A kit for assaying a biological sample potentially containing
two or more analytes, said kit comprising: (a) a stationary solid
phase support with immobilized receptors for binding thereto said
analytes and (b) two or more different types of sensitized
microcapsules, each different type of said sensitized microcapsules
having a surface membrane and an interior volume, said surface
membrane of said each different type of said sensitized
microcapsules having an incorporated receptor that specifically
binds to a particular one of said analytes and said interior volume
of said each different type of said sensitized microcapsules
includes a unique first member of a binding pair, each said unique
first member of a binding pair in said each different type of said
sensitized microcapsules is different and specifically binds a
second member of the binding pair.
2. The kit according to claim 1 further comprising: a disrupting
agent suitable to disrupt said sensitized microcapsules and release
each said unique first member of the binding pair from each
different type of said sensitized microcapsules when said analytes
are present in the biological sample.
3. The kit according to claim 1 further comprising: a device
comprising: means for immobilizing each said unique first member of
the binding pair released from said each different type of said
sensitized microcapsules in spatially separated locations; and
means for detecting each said unique first member of the binding
pair released from said each different type of said sensitized
microcapsules immobilized in said spatially separated
locations.
4. The kit according to claim 1, wherein said unique first member
of the binding pair comprises a tag.
5. The kit according to claim 4, wherein said tag is an
electrochemically detectable species, a colorimetrically detectable
species, a fluorescent species, an oligomarker, an oligonucleotide
molecule, or an oligonucleotide molecule conjugated to a
fluorophore tag, an electrochemical tag, a quantum dot tag, or a
dye tag.
6. The kit according to claim 3, wherein said means for detecting
each said unique first member of the binding pair are colorimetric,
fluorescent, or electrochemical means of detection.
7. The kit according to claim 1, wherein said unique first member
of the binding pair is an antigen, a hapten, an antibody, a
cytokine, a chemokine, an enzyme, a drug, a protein, a biomarker, a
DNA, or an RNA.
8. The kit according to claim 1, wherein said second member of the
binding pair is an antigen, a hapten, an antibody, a cytokine, a
chemokine, an enzyme, a drug, a protein, a biomarker, a DNA, or an
RNA.
9. The kit according to claim 3, wherein said unique first member
of the binding pair is an oligonucleotide molecule.
10. The kit according to claim 1, wherein said microcapsules are
liposomes or polymer based microspheres.
11. The kit according to claim 3, wherein said means for
immobilizing each said unique first member of the binding pair
released from said each different type of said microcapsules
comprises the second members of the binding pair which are
immobilized.
12. The kit according to claim 9, wherein said means for
immobilizing each said unique first member of the binding pair
released from said each different type of said sensitized
microcapsules comprises an array of immobilized oligonucleotides
complementary to said oligonucleotide molecules, wherein each said
oligonucleotide molecule released from said each different type of
said sensitized microcapsules is brought into contact with said
array and hybridizes with said immobilized oligonucleotides.
13. A kit for assaying a biological sample potentially containing
at least one analyte, said kit comprising: (a) a first sensitized
microcapsule, said first sensitized microcapsule having a surface
membrane and an interior volume, said surface membrane of said
first sensitized microcapsule having an incorporated receptor that
specifically binds to said analyte and said interior volume of said
first sensitized microcapsule includes a unique first member of a
binding pair that specifically binds a second member of the binding
pair; (b) an immobilized binder of said analyte; (c) a second
sensitized microcapsule filled with tags, said second sensitized
microcapsule having a surface membrane and an interior volume, said
surface membrane of said second sensitized microcapsule having an
incorporated second member of the binding pair for specifically
binding to said first member of the binding pair; and (d) an
immobilized second member of the binding pair for specifically
binding to said first member of the binding pair.
14. The kit according to claim 13 further comprising: a disrupting
agent suitable to disrupt said first sensitized microcapsule and
release said first member of the binding pair from said first
sensitized microcapsule when said analyte is present in the
biological sample and a disrupting agent suitable to disrupt said
second sensitized microcapsule and releasing said tags when said
analyte is present in the biological sample.
15. The kit according to claim 13 further comprising: release said
first member of the binding pair from the first sensitized
microcapsule when said analyte is present in the biological sample
and to disrupt said second sensitized microcapsule and release said
tags when said analyte is present in the biological sample.
16. The kit according to claim 13, wherein said tags are
electrochemically detectable species, colorimetrically detectable
species, fluorescent species, oligomarkers, oligonucleotide
molecules, or oligonucleotide molecules conjugated to a fluorophore
tag, an electrochemical tag, a quantum dot tag, or a dye tag.
17. The kit according to claim 13, wherein said first member of the
binding pair is an antigen, a hapten, an antibody, a cytokine, a
chemokine, an enzyme, a drug, a protein, a biomarker, a DNA, or an
RNA.
18. The kit according to claim 13, wherein said second member of
the binding pair is an antigen, a hapten, an antibody, a cytokine,
a chemokine, an enzyme, a drug, a protein, a biomarker, a DNA, or
an RNA.
19. The kit according to claim 13, wherein said first member of the
binding pair is an oligonucleotide molecule.
20. The kit according to claim 13, wherein said microcapsules are
liposomes or polymer based microspheres.
21. The kit according to claim 19, wherein said immobilized second
member of the binding pair comprises oligonucleotides immobilized
on an array which are complementary to said oligonucleotide
molecules.
Description
[0001] This application is a continuation of U.S. patent
application Ser. No. 11/162,142, filed Aug. 30, 2005, and claims
the benefit of U.S. Provisional Patent Application Ser. No.
60/606,446, filed Sep. 1, 2004, each of which is hereby
incorporated by reference in its entirety.
FIELD OF THE INVENTION
[0002] The present invention relates to a method for detecting one
or more analytes such as determining the presence and/or
concentration of antigens, antibodies, polynucleic acids, and like
agents in biological fluids. More particularly, the invention
relates to a multiplexing amplified bioassay.
BACKGROUND OF THE INVENTION
[0003] There is an increasing need for rapid, reliable, inexpensive
"in the field" methods for detecting and measuring pathogens and
biomarkers in living organisms as well as pollutants and
contaminants in the environment and in food sources. Immunoassays
comprise one category of specific binding bioassays, which
generally rely on the affinity of naturally occurring receptors or
antibodies for specific compounds. The specific binding pairs
employed in immunoassays are either an antigen or a hapten, and the
antibody produced in immune response to the antigen or hapten.
Another type of bioassay relies on hybridization binding reaction
between complementary single strands of DNA or RNA.
[0004] Bioassays are of great importance because of their
specificity toward analytes present in complex mixtures, and their
high sensitivity. Most bioassays involve the use of a fluorescent,
chemiluminescent, electrochemiluminescent, enzyme, electrochemical,
or radioactive tag on an immunoreactive species which serves as an
indicator that an immunospecific reaction has occurred.
[0005] Applying a different classification, immunoassays can be
divided into two broad categories of non-amplified assays and
amplified assays. Non-amplified assays most often involve the use
of a tag, such as fluorescent tag, chemical species tag,
electrochemical tag, radioactive label, or the like on an
immunoreactive species which serves as an indicator that an
immunospecific reaction has occurred. Only one tag per occurrence
of immunospecific binding is being activated or released for
subsequent detection by optical, chemical, or electrochemical means
or by detection of radiation. One of the main disadvantages of the
non-amplified assays is low sensitivity of assays.
[0006] Amplified assays involve amplification of each binding act
between the analyte and the immunoreagent. For example,
enzyme-linked immunosorbant assays (ELISAs) involve the use of an
enzyme covalently coupled to an immunoreactive reagent to serve as
an indicator that an immunospecific reaction has occurred. The
enzyme is linked to a secondary reagent, which is added to the
assay after the initial immunochemical interaction between the
analyte and the immunospecific group, such as an antibody. The
enzyme is capable of catalyzing a number of concurrencies of
color-changing reaction, generating several hundred turnover events
of such reaction in a reasonable period of time. The sensitivity of
ELISA is due to the number of turnover events the enzyme is capable
of during an incubation period with a substrate that is cleaved to
a colored product. While ELISA can be extremely sensitive, it is
frequently a very time-consuming assay which is difficult to use in
the field.
[0007] Another type of an amplified assay is
electrochemiluminescence (ECL) based assay, where an
electrochemical tag is covalently coupled to an immunoreactive
reagent and reacts electrochemically to emit light signal to serve
as an indicator that an immunospecific reaction has occurred. Yet
another type of an amplified assay is an assay based on method of
immunoanalysis which combines immobilized immunochemistry with the
technique of flow injection analysis, and employs microscopic
spherical microcapsules or sacs, such as animal erythrocytes,
polymer microcapsules, liposomes, or similar structures as carriers
of detectable reagents. For example, liposomes, or lipid vesicles,
can be modified on their surface with analytical reagents, and
carry in their internal volume a large number of fluorescent or
electroactive tags. After the immunospecific reaction has occurred,
the liposomes are disrupted or lysed by the contact with the
liposome lysing agent and release a large amount of tags per each
binding act. The presence of tags is then detected by chemical,
optical, or electrochemical means.
[0008] Liposomes have previously been reported as useful components
for amplified immunoassays. For example, McConnell et al., U.S.
Pat. No. 3,887,698, describe the use of liposomes containing stable
free radicals in an electron paramagnetic resonance monitored
immunoassay. Mandle et al., U.S. Pat. No. 4,372,745, describe the
use of liposomes as fluorescer containing microcapsules, useful in
an immunoassay. This assay requires the use of a detergent such as,
Triton X-100 to break the liposomes and release the fluorescent
compound. Liposomes have also been employed as a tags carrier in an
immunoassay described by Ullman et al., U.S. Pat. No. 4,193,983.
Tags used in this assay included fluorescers, enzymes and
chemiluminescent compounds.
[0009] Cole, U.S. Pat. No. 4,342,826, describes an immunoassay
method which utilizes antigen-marked, enzyme-encapsulated liposomes
which are immunospecifically ruptured in the presence of the
cognate antibody and an active complement. The assay utilizes the
homogeneous phase reaction between the antibody and complement to
release the enzyme tag. U.S. Pat. Nos. 6,248,596; 6,159,745;
6,086,748; 5,958,791; 5,789,154; 5,756,362; 5,753,519; and
5,389,523; by Durst and co-authors, further developed
Liposome-enhanced amplified immunoassays and test devices for
implementation of these assays. The technology described by Durst
et al. has a limited multiplexing potential.
[0010] The disadvantages of both ECL and Liposome-based assays is
the possibility to detect only one type of pathogen or
environmental contaminant in each sample. The simultaneous
detection of many pathogens or contaminants in a multiplexing assay
format is difficult as only very limited number of uniquely
detectable amplification tags compatible with the detection means
are available. Typically only one tag is available, such as
specific fluorescent, electrochemical, chemical, or radioactive
tag.
[0011] At the same time, ELISA assays can detect multiple pathogens
or environmental contaminants on a specially prepared multiplexing
plates, but ELISA is a very time-consuming assay which is difficult
to use in the field.
[0012] US Patent Application 20040009944 by Tam et al. describes
methylated oligonucleotides made immunostimulatory in vivo, by
encapsulation of the nucleic acid in a lipid particle. This
technology applies to drug development.
[0013] US Patent applications 20040110220 and 20040072231 by Mirkin
et al. describe using custom oligonucleotides for multiplexing
assays employing gold nanoparticles with attached oligonucleotides,
which are later released and detected. Conjugating oligonucleotides
to particles is a complicated process. There are limitations in the
amount and size of oligonucleotides which can be conjugated to gold
particles.
[0014] US Patent application 20030013091 by Dimitrov describes
capturing a target pathogen with a long piece of single strand
complementary DNA which has many repeating short oligonucleotide
sequences. After that Dimitrov proposes to add fluorescent labels
attached to complementary short oligonucleotide sequences. The
labels hybridize to the long piece of DNA and thus create
amplification. This technology has limited amplification potential.
Multiplexing is also complicated as discerning many fluorescent
labels from each other is necessary.
[0015] In many applications, there is a need to perform
multiplexing assays for many pathogens, biomarkers, and/or
environmental contaminants simultaneously, due to time constraints
and limited amount of analyte sample available. The optimal assay
system should be fast, reliable, highly sensitive, and
quantitative.
BRIEF DESCRIPTION OF THE INVENTION
[0016] In order to aid in the understanding of the present
invention, the following terms as used herein and in the claims
have the following meanings.
[0017] Analyte--the compound or composition to be measured, which
may be DNA or RNA molecule, antigen, hapten, or antibody. For
example, in a preferred embodiment, the analyte may be either an
antigen or an antibody.
[0018] Tag--any functional group of a compound capable of ready
detection, including chemiluminescent species,
electrochemiluminescent groups, colorogenic agents, dyes,
fluorogenic agents, and electrochemically active species. Typical
tag compounds are fluorescent dyes. These compounds include water
soluble derivatives of fluorescein such as carboxyfluorescein and
calcein.
[0019] Receptor--any compound or composition capable of
specifically recognizing and binding another molecule. Natural
receptors include immunospecific compounds, such as antibodies,
antigens, enzymes, lectins, and the like, as well as complementary
single stranded DNA or RNA molecules. For example, in a preferred
embodiment, the receptor for an antigen is an antibody, while the
receptor for an antibody is either an anti-antibody or, preferably,
that antibody's cognate antigen.
[0020] Oligonucleotide markers, or oligomarkers--short chains of
single stranded DNA or RNA, ranging from about 5 to about 100
oligonucleotides, preferably from about 10 to about 30
oligonucleotides. Oligomarkers can also include a tag which is
conjugated to the oligonucleotide molecule.
[0021] Sensitized microcapsules--microscopic sac-like structures or
microcapsules, having an interior volume defined by a membrane,
capable of carrying a large number of oligomarkers, and sensitized
with a linked or conjugated receptor on their surface to specific
analytes, such as antigens, proteins, DNA fragments, and the like.
Sensitized microcapsules are capable of binding to analyte.
[0022] The present invention provides an amplified immunoassay or
DNA assay which combines immunocapture or hybridization-based
capture with the technique of flow injection analysis and employs
microscopic sac-like structures, or microcapsules, such as animal
erythrocytes, polymer microcapsules, liposomes, and the like, to
carry a large number of unique oligonucleotide markers. Said
specific markers are released after the immunospecific reaction or
hybridization reaction has occurred. The specific oligomarkers are
then captured by complementary oligonucleotide targets for
quantification of the analyte.
[0023] The microcapsules are modified on their surface with
receptors (sensitization), and carry in their internal volume
unique oligomarkers. After the immunospecific reaction or
hybridization reaction has occurred, the microcapsules are
disrupted, open, or lysed by contact with the chemical agent,
changes in media environment, such as pH or addition of surfactant,
or physical effects, including electric impulse, heat, and the
like, and release a large amount of oligomarkers per each binding
act. The presence of markers is then detected by binding or
hybridizing to complementary oligonucleotide molecules situated
spatially apart from each other on an inert substrate.
[0024] A very large number of unique oligomarkers can be created by
varying sequences of oligonucleotides, thus enabling multiplexing
of the present assay. A large number of sensitized microcapsules
specific to various analytes can be created, each type of specific
sensitized microcapsule containing unique oligomarkers. The
oligomarkers are molecules comprising a fragment of single strand
DNA or RNA with a unique sequence. In another embodiment, the
oligomarker is formed from a fragment of single strand DNA and a
tag attached to it.
[0025] This invention further provides for amplified multiplexing
assay capable of simultaneous detection and quantification of more
than one pathogen, biomarker, or contaminant. The invention further
provides for amplified multiplexing assay for detection, analysis,
and quantification of biologically important species, particles,
cells, molecules, and functional groups in biological fluids, in
the environment, on solid surfaces including tissue samples, and in
experimental environments performing drug discovery research.
[0026] The invention further improves the amplified bioassays by
using primary microcapsules encapsulating oligomarkers and
secondary microcapsules encapsulating oligomarkers with tags or
tags alone for detecting and amplifying the markers released by
primary microcapsules.
BRIEF DESCRIPTION OF DRAWINGS
[0027] FIG. 1 illustrates the structure of sensitized microcapsules
and the structure of oligomarkers (including SEQ ID NO: 1)
according to present invention.
[0028] FIG. 2 illustrates the first step of the assay according to
present invention, wherein the sensitized microcapsules are bought
into contact with the analyte and immobilized receptors.
[0029] FIG. 3 illustrates embodiment for the first step of the
assay according to present invention, wherein the sensitized
microcapsules are bought into contact with the analyte and
immobilized receptors.
[0030] FIG. 4 illustrates capturing of the analyte by the
sensitized microcapsules and immobilized receptors.
[0031] FIG. 5 illustrates embodiment of capturing of the analyte by
the sensitized microcapsules and immobilized receptors.
[0032] FIG. 6 illustrates disrupting of the sensitized
microcapsules and release of oligomarkers.
[0033] FIG. 7 illustrates quantitative capture and measurement of
the oligomarkers released from disrupted or lysed
microcapsules.
[0034] FIG. 8 illustrates capturing oligomarkers by hybridizing to
immobilized complementary capture fragments of DNA or RNA.
[0035] FIG. 9 illustrates the multiplexing aspect of the bioassay
according to present invention, wherein there are at least two
different types of sensitized microcapsules containing different
oligomarkers.
[0036] FIG. 10 illustrates embodiment of the amplified multiplexing
assay, wherein receptor groups are single strand DNA or RNA capture
molecules binding the analyte via hybridization.
[0037] FIG. 11 illustrates disruption of immobilized sensitized
microcapsules release of the oligomarkers which are then brought
into contact with spatially separated immobilized single stranded
arrays of complementary DNA or RNA fragments.
[0038] FIG. 12 illustrates how oligomarkers hybridize to the
immobilized complementary single strand DNA or RNA receptors.
[0039] FIG. 13 illustrates a schematic diagram of a planar
microfluidic device.
[0040] FIG. 14 illustrates hybridization and measurement area of
the microfluidic device.
[0041] FIG. 15 illustrates embodiment of the bioassay in a well on
a test plate.
[0042] FIG. 16 illustrates embodiment of the present invention
utilizing two types of sensitized microcapsules sequentially.
[0043] FIG. 17 illustrates how the oligomarkers released by a
primary microcapsule are captured by surface immobilized
complementary strand of DNA and a secondary microcapsule.
[0044] FIG. 18 presents a flow chart illustrating the sequence of
the bioassay steps according to principles of the present
invention.
DETAILED DESCRIPTION OF THE INVENTION
[0045] The assay of the present invention will be illustrated by
referring to the assay for one particular entity, e.g. an antigen.
The general principles and techniques described herein for assaying
an antigen can then be applied to assay for other species such as,
for instance antibodies, haptens, DNA, RNA, etc.
[0046] Referring now to FIG. 1, the present invention employs
microscopic sac-like structures or microcapsules 110, such as
animal erythrocytes, polymer microcapsules, liposomes, and the
like, to carry a large number of unique oligomarkers 120.
[0047] Oligomarkers 120 shown in FIG. 1 are short chains of single
stranded DNA or RNA. For illustration, the oligomarker 120 is shown
in FIG. 1 as a 20-mer oligonucleotide comprising of twenty
nucleotides CTCTCTCTCTCTCTCTCTCT (SEQ ID NO: 1). The oligomarkers
according to present invention are ranging from about 5 to about
100 oligonucleotides, preferably from about 10 to about 30
oligonucleotides.
[0048] Another embodiment of the oligomarker 122 shown in FIG. 1
includes functional group or tag 124 conjugated to the
oligonucleotide molecule. The functional group represents a
detectable tag and provides additional means for detecting and
quantifying oligomarkers. This tag group is suitable for detection
by electrochemical, optical, or other analytical means. The
preferred types of the tag 124 is a fluorescent group or a quantum
dot. Methods of conjugating tags to oligomarkers are known to these
skilled in the art.
[0049] The microcapsules 110 are further sensitized to specific
analytes, such as antigens, proteins, DNA fragments, and the like,
by providing a receptor 130, such as an immunospecific group, for
instance, antibody, which is linked or conjugated to the
microcapsule 110. The receptor is capable of specifically binding
to analyte of interest.
[0050] In summary, the sensitized microcapsule 100 shown in FIG. 1
comprises microcapsule 110, filled with unique oligomarkers 120,
and sensitized with at least one receptor 130.
[0051] In a preferred embodiment, liposomes are utilized as the
microcapsules 110. Liposomes are microscopic vesicles composed of
closed lipid bilayers. Due to their relatively simple composition
and their flexibility for chemical, physical and immunological
manipulations, liposomes are readily adapted for encapsulating a
range of different markers or tags. Forming microcapsules such as
liposomes, encapsulating tags or markers therein, and sensitizing
liposomes to bind with a specific analyte, such as antigen, is
known and described in the art, for example in U.S. patents by
Durst and co-authors cited above. These patents are incorporated
herein by reference.
[0052] The lipid vesicles or liposomes may be prepared from a wide
variety of lipids, including phospholipids, glycol lipids, and as
representative examples there may be mentioned lecithin,
spingomyelin, dipalmitoyl lecithin, distearoylphosphatidylcho line,
etc. The amphiphilic lipids employed for producing liposomes
generally have a hydrophilic group, such as a phosphato,
carboxylic, sulfato, or amino group, and a hydrophobic group, such
as saturated and unsaturated aliphatic hydrocarbons, and aliphatic
hydrocarbon groups substituted by one or more aromatic or
cycloaliphatic groups. The wall forming compounds for producing the
liposomes may further include a steroid component such as
cholesterol, cholestanol, and the like. The compounds for producing
liposomes are generally known in the art, and no further details in
this respect are deemed necessary for a complete understanding of
the present invention.
[0053] The liposomes may be produced by procedures generally
available in the art. For example, liposomes may be produced by a
reverse phase evaporation technique wherein the compound or
compounds used in producing liposomes are initially dissolved in an
organic phase, followed by addition of an aqueous phase and forming
of a homogeneous emulsion. After forming the emulsion, the organic
solvent is evaporated to form a gel like material, and such gel may
be converted to a liposome by agitation or dispersion in an aqueous
media.
[0054] Procedures for producing liposomes are described, for
example, in U.S. Pat. No. 4,241,046 and U.S. Pat. No.
4,342,828.
[0055] If a material is to be encapsulated in the liposome, such
material may be encapsulated in the liposome by including the
material in the aqueous solution in which the liposome is formed.
Alternatively, the material may be encapsulated into a previously
formed empty liposome (without material to be encapsulated) by the
procedure described in U.S. Pat. No. 4,539,376. The liposomes may
also be produced by the procedures disclosed in U.S. Pat. No.
4,522,803.
[0056] Other types of microcapsules besides liposomes can be
utilized, including microcapsules made of polymer materials,
biological materials, silicon and silicon compounds, as known in
the art.
[0057] Referring now to FIG. 2, the first step of the assay
according to the present invention is illustrated. The sensitized
microcapsule 100 containing oligomarkers is brought into a contact
with liquid sample containing analyte 150. The moving of sensitized
microcapsules is done in a moving carrier stream, such as aqueous
buffer solution. The analyte 150 is characterized by one or more
immunospecific groups or receptors shown as 160 and 170. In
addition, capture immunospecific groups or receptors 190 specific
to the analyte are immobilized on the surface 180, which is a
reaction enclosure, a channel in a microfluidic device, an inert
particle, magnetic bead, or an assay plate.
[0058] Immobilizing receptor groups 190 such as antibodies, on
inert solid surfaces, may be accomplished using any technique
available to the skilled artisan as known and described in the art.
Such techniques include, but are not limited to, adsorption,
absorption, ionic bonds, covalent bonds, hydrogen bonds, and the
like. Typically, glass and water insoluble polymers are used as the
support for the receptor. The support may be in any shape or form.
For example, flat objects such as glass slides, polymeric disks or
strips, the walls of a test tube, or widely available beads can be
employed as the support herein. The bonds between the receptor and
the support should be strong enough so that normal washing
procedures, or contact with aqueous solutions, including a test
serum, do not destroy the attachment means.
[0059] One suitable form of chemical binding is to provide bridges
of covalent character between the solid support and the receptor.
For this purpose the solid support is selected so that it contains
or can be provided with suitable reactive functional groups, for
example, amino groups, hydroxyl groups, and carboxyl groups, to
enable the receptor to be easily bound to the solid support.
Especially useful are bridges between the solid support and the
receptor having chemical bonds of a covalent nature.
[0060] The particular bridge between the receptor and the support
is not a critical part of the assay of this invention. The bridge
may be of any type, or a mixture of types, as its only purpose is
to prevent the receptor from being washed away from the support.
One of preferred immobilization methods involves binding
biotinylated receptor groups to a support through streptavidin.
[0061] Referring now to FIG. 3, another embodiment of the present
invention is illustrated. The analyte 155 is single strand DNA or
RNA. The microcapsule 105 containing oligomarkers is sensitized
with a DNA or RNA fragment 125 complementary to analyte 155.
Capture DNA or RNA fragments 195, complementary to the analyte, are
immobilized on the surface 180. In one embodiment, biotinylated
capture DNA is bound to the surface through streptavidin.
[0062] Both embodiments illustrated in FIGS. 2 and 3 can be
employed in the present invention interchangeably, utilizing
immunospecific groups (antigens, antibodies, proteins, and the
like) or single strand DNA or RNA groups for sensitization of the
microcapsules as well as utilizing immunospecific groups (antigens,
antibodies, proteins, and the like) or DNA or RNA groups for
surface immobilized capture groups within the framework of the
present invention.
[0063] Referring now to FIG. 4, we illustrate the next step of the
present assay, after the sensitized microcapsule 100, containing
oligomarkers, was brought into a contact with analyte 150 and the
surface immobilized receptors. The analyte 150 is captured by the
surface immobilized receptors and is also captured by the
sensitized microcapsule 100. In one embodiment of this invention,
this capture step is performed sequentially, with the analyte first
being captured by the surface immobilized receptors, and the
sensitized microcapsule conjugating to the analyte thereafter. In
another embodiment, the sensitized microcapsule is first conjugated
to the analyte, and then the analyte, bound to the sensitized
microcapsule, is captured by the surface immobilized receptors. In
yet another embodiment of this invention, both capture steps are
performed at the same time, whereas the analyte is brought into
contact with both sensitized microcapsules and surface immobilized
receptors simultaneously.
[0064] The above description completes the analyte capture step
according to the present invention. The same capture step is
additionally illustrated by FIG. 5, with the sensitized
microcapsule 105 attached to the surface 180 through a bridge
formed by the captured analyte DNA or RNA 155. As a result of the
capture step, the analyte is immobilized on the surface together
with attached sensitized microcapsules.
[0065] After completing the capture step, the analyzed sample and
unbound microcapsules are removed, preferably by a washing step, so
that only the captured analyte with attached sensitized
microcapsules remain immobilized on the surface.
[0066] Referring now to FIG. 6, the next step of the assay
according to present invention is the step of disrupting of the
immobilized sensitized microcapsules and releasing the
oligomarkers. The microcapsules are disrupted by chemical,
enzymatic, or physical-chemical means, including effects of pH,
surfactant, temperature, or action of an enzyme. The preferred
method is lysis by changing the pH of the solution or by adding a
surfactant to the solution. The methods of disrupting of
microcapsules are known to these skilled in the art.
[0067] As illustrated in FIG. 6, after disrupting of the sensitized
microcapsules 100, the oligomarkers 122 are released. The
oligomarkers are shown with a tag facilitating their detection and
quantification. The amplification of the assay is a function of how
many of the oligomarkers are released per each immunospecific or
hybridization reaction binding act. Since oligomarkers are
relatively small, a large number of these markers can fit into one
sensitized microcapsule. If 1,000 oligomarkers can fit into a
sensitized microcapsule, the assay amplification factor is 1,000.
If 10,000 oligomarkers can fit into a sensitized microcapsule, the
assay amplification factor is 10,000.
[0068] Referring now to FIG. 7, the next step of the assay is
quantitative capture and measurement of the oligomarkers released
from disrupted or lysed microcapsules. The oligomarkers can be
captured and measured by a number of methods readily available to a
skilled artisan as known and described in the art. For example, the
oligomarkers 122 are brought into contact with complementary
fragments of single strand DNA or RNA 210 immobilized on an inert
surface 200.
[0069] Referring now to FIG. 8, the oligomarkers 122 are captured
by hybridizing to immobilized complementary capture fragments of
DNA or RNA 210.
[0070] The captured oligomarkers can now be quantified and detected
by readily available devices, including by optical means by
measuring fluorescence, the intensity of a dye, or the reflectivity
of quantum dots; electrochemically utilizing an interdigitated
electrode; utilizing electrochemiluminescence; using surface
plasmon resonance techniques; using quartz microbalance or
microcantilever techniques; and other techniques known in the
art.
[0071] Yet another method of capturing and measuring the
oligomarkers according to the present invention is by using DNA
arrays and similar devices, for example DNA array devices
manufactured by Affymetrix. In this embodiment, the oligomarkers
are captured on a DNA array containing complementary DNA sequences
and quantified as known in the art. Other methods of detecting the
oligomarkers, including detection of tag-free markets, are
piezoelectric, surface plasmon resonance, and colorimetry. An
appropriate dye, such as SYBR green, to detect double strand DNA
molecules formed by oligomarkers and complementary immobilized DNA
fragments, can also be employed. Such methods are known to these
skilled in the art and are readily available.
[0072] The following description will in more detail describe the
multiplexing aspect of the amplified assay of the present
invention.
[0073] Referring now to FIG. 9, the amplified multiplexing assay is
performed as follows. There are at least two different types of
sensitized microcapsules 100 and 101, containing oligomarkers 122
and 123 with different nucleotide sequences. In this embodiment,
and for illustration only, the oligomarker 122 is a 20-mer
oligonucleotide CTCTCTCTCTCTCTCTCTCT (SEQ ID NO: 1) with a tag, and
oligomarker 123 is a 20-mer oligonucleotide TTTTTTTTTTTTTTTTTTTT
(SEQ ID NO: 2) with a tag.
[0074] Each of the sensitized microcapsules 100 and 101 is
sensitized to a different analyte utilizing receptor groups 170 and
171, such as immunospecific receptor groups. The sensitized
microcapsules are brought into contact with the analyzed sample
potentially containing at least two different analytes 150 and 151
and surface immobilized receptor groups 190 and 191 specific to
each analyte.
[0075] The analytes, if present in the analyzed sample, are then
captured by the sensitized microcapsules and surface immobilized
receptors. The non-captured analyte and non-captured sensitized
microcapsules are removed form the reaction area, preferably by a
simple washing step.
[0076] Referring now to FIG. 10, another embodiment of the present
amplified multiplexing assay is demonstrated whereas immobilized
receptor groups 192 and 193 utilized in the assay are single strand
DNA or RNA capture molecules binding the analyte 155 and 156 via
hybridization. The sensitized microcapsules 105 and 106 are in turn
being captured by the analyte through receptors 172 and 173,
similar to above FIGS. 3 and 5.
[0077] Referring now to FIG. 11, all immobilized microcapsules
shown in FIGS. 9 and 10 are disrupted, for example by lysis, and
the unique oligomarkers 122 and 123 contained in each type of the
microcapsule are released and brought into contact with spatially
separated immobilized single stranded arrays of complementary DNA
or RNA fragments 200 and 210.
[0078] Referring now to FIG. 12, the oligomarkers hybridize to the
immobilized complementary single strand DNA or RNA receptors 200
and 210. The spatial separation of the areas where the oligomarkers
are immobilized permits to readily quantify the oligomarkers by any
of the oligomarkers measurement techniques known in the art, as
described above.
[0079] One embodiment for the implementation of the assay according
to present invention is a planar microfluidic device. Methods of
building such devices are readily available to these skilled in the
art. Referring now to FIG. 13, a schematic diagram of the
microfluidic device is shown. Channels 500 formed in the planar
microfluidic device 505 enable movement of analyte samples and
reagent fluids along the device as shown by arrows. The analyzed
sample is introduced through a port 510 and moves in the direction
of capture area 520. The sensitized microcapsules reservoir 530
provides sensitized microcapsules which are moving through a
channel and also enter the capture area 520. The capture reactions
in the capture area 520 were detailed above, specifically in FIGS.
4, 5, 9, and 10.
[0080] The reservoir 550 provides a washing buffer which removes
all non-captured analyte and non-captured sensitized microcapsules,
which are washed away and removed into the disposal reservoir 570.
The lysing buffer reservoir 540 then supplies the lysing solution
which disrupts the captured sensitized microcapsules in the capture
area 520, resulting in the release of oligomarkers. The lysing
process occurring in the capture area 520 was illustrated above,
specifically in FIG. 6.
[0081] The oligomarkers released by lysing of the sensitized
microcapsules continue moving through the channel towards the
hybridization and measurement area 560. As it was illustrated in
the FIGS. 7, 8, 11, and 12, the oligomarkers are bound through the
hybridization process to immobilized complementary single stranded
DNA or RNA.
[0082] Referring now to FIG. 14, the hybridization and measurement
area 560 of FIG. 13 is shown in more detail. A number of spatially
separated areas 200, 210, etc. for hybridizing different
oligomarkers is shown, each corresponding to a unique oligomarker
sequence which in turn corresponds to a specific analyte. Following
the illustration of FIG. 12, area 600 in FIG. 14 will capture
specific oligomarkers 122, while area 210 will capture specific
oligomarkers 123.
[0083] The amount of the released and captured oligomarkers is
directly proportional to the amount of captured analyte.
Quantifiably detecting oligomarkers in each of the areas 200, 210,
etc., is readily performed by techniques known in the art and
described above.
[0084] Fluidic reservoirs 510, 530, 540, 550, and 570 of the
microfluidic device can be reusable, while the capture area 520 and
measurement area 560 can be adapted for single use only.
[0085] Referring now to FIG. 15, another embodiment of the
implementation of the assay according to present invention is in a
well on a test plate. A well 700 has an analyte capture area where
surface immobilized receptors 190, 191, 192, 193, etc.,
immunospecific or complementary to analytes of interest are
situated. Elsewhere in the well are situated a number of spatially
separated areas 200, 210, etc., for hybridizing different
oligomarkers, each corresponding to a unique marker sequence, which
in turn corresponds to a specific analyte.
[0086] The assay is performed by first sequentially or
simultaneously introducing the analyzed biological fluid sample and
sensitized microcapsules into the well and capturing the analytes
present in the sample and corresponding sensitized microcapsules.
After that the non-captured microcapsules and the rest of the
sample are removed, preferably by washing.
[0087] In the next step, a buffer with a specific pH or containing
a surfactant is introduced into the well and the captured
sensitized microcapsules are lysed. The released oligomarkers are
then hybridized in the spatially separated areas 200, 210, etc.,
and quantitatively measured by optical means. The preferred way of
measuring and quantifying oligomarkers is by measuring fluorescence
of fluorescent tags attached to oligomarkers.
[0088] Another embodiment of the implementation of the assay
according to present invention comprises an assay performed in two
wells on a test plate. The first well contains an analyte capture
area where surface immobilized receptors for capturing analytes of
interest are situated. The second well contains a number of
spatially separated areas for hybridizing different oligomarkers,
each corresponding to a unique oligomarker sequence which in turn
corresponds to a specific analyte. The assay includes the steps of
capturing the analytes and sensitized microcapsules and lysis of
sensitized microcapsules in the first well, transfer of the
released oligomarkers into the second well, binding or
hybridization of the oligomarkers in the second well in the
corresponding spatially separated areas, and quantifiable
measurement of the hybridized oligomarkers in the second well.
[0089] The measurement is performed by detecting the tags
conjugated to the oligomarkers, preferably by optical or
electrochemical means, as known in the art.
[0090] The oligomarkers according to this invention can be made
inexpensively in large quantities. Importantly, there are a high
number of different and unique oligomarkers that can be made for
this multiplexing assay by varying sequence, composition, and
length of the oligonucleotide chain of the oligomarker, as well as
type of the functional group or tag which makes the marker
detectable. The preferred embodiment of this invention utilizes
oligomarkers of the same length, for instance 20 base nucleotides
long, with variable nucleotide sequences.
[0091] Referring now to FIG. 16, yet another embodiment of the
present invention utilizes two types of sensitized microcapsules
sequentially. The primary sensitized microcapsule 800 contains
oligomarkers 810 without tags. After lysing of the primary
sensitized microcapsule 800, as illustrated by FIG. 16, the
oligomarkers are released.
[0092] Referring now to FIG. 17, the oligomarkers 810, released by
the primary sensitized microcapsule, are captured by the surface
immobilized complementary strand of DNA 840 and a secondary
sensitized microcapsule 820, which is sensitized with a single
strand oligonucleotide 850 complementary to the oligomarkers
released by the primary sensitized microcapsule. The secondary
sensitized microcapsule, in turn, contains easily detectable tags
830 which are released by lysing the secondary sensitized
microcapsule. The tags 830 are preferably fluorescent or
electrochemical tags and are quantifiably measured, preferably by
optical or electrochemical means.
[0093] In yet another embodiment of this invention, the secondary
sensitized microcapsules according to FIG. 17 contain oligomarkers
which are then captured via hybridization reaction and
quantitatively measured.
[0094] The embodiment of the invention utilizing primary and
secondary microcapsules enables additional significant
amplification of the assay. If the primary sensitized microcapsule
provides for amplification of 1000, and secondary sensitized
microcapsule provides for amplification of 1000, then the resulting
total assay amplification is 1,000,000.
[0095] Yet another embodiment of the present invention utilizes
principles of the present invention for the biological tissue
analysis or tissue typing, for example for detection of certain
proteins, cancer cells, and the like on tissue samples. In this
embodiment, the analytes are tissue components which are already
immobilized on tissue samples.
[0096] The sensitized microcapsules are sensitized with receptors
which are specific to certain tissue types or tissue proteins. Then
the sensitized microcapsules are brought into contact with tissue
samples and are captured by the tissue components and thus
immobilized on the tissue.
[0097] After this capture step, all non-captured sensitized
microcapsules are removed, preferably by a simple washing step.
After the washing step, the captured or immobilized microcapsules
are disrupted and the oligomarkers are released. The released
oligomarkers are then measured and quantified as described
above.
[0098] In yet another embodiment of the present invention, the
principles of the invention are utilized to facilitate drug
discovery. In this embodiment, prospective drug candidates, which
can be small molecule chemical entities, peptides, proteins, and
the like, are conjugated to microcapsules as receptors to form
sensitized microcapsules. The successful binding of the sensitized
microcapsules to the drug targets is then detected, with
amplification, by disrupting microcapsules and measuring the
released oligomarkers, as described above. Simultaneous testing of
many drug candidates and on many targets is possible. The capture
of the sensitized microcapsules is then used as indication of
prospective drug candidates propensity for binding to targets.
[0099] Referring now to FIG. 18, a flow chart illustrating the
principles of the present invention is presented. The main steps in
carrying out the amplified multiplexing bioassay are outlined.
[0100] In carrying out the assay an aqueous medium will normally be
employed. Other polar solvents may also be employed, usually
oxygenated organic solvents of from 1 to 6, more usually from 1 to
4 carbon atoms, including alcohols, ethers and the like. Usually
these cosolvents will be present in less than about 40 volume
percent, more usually in less than about 20 volume percent.
[0101] The assay of this invention is carried out in an aqueous
medium at a moderate pH, such as neutral pH, generally close to
optimum assay sensitivity, without the need for separation of the
assay components or products. The pH for the medium will usually be
in the range of about 4 to 10, more usually in the range of about 5
to 9, and preferably in the range of about 5.5 to 8.5. The pH is
chosen so as to maintain a significant level of specific binding by
the receptor while optimizing signal producing efficiency. In some
instances, a compromise will be made between these two
considerations. Various buffers may be used to achieve the desired
pH and to maintain the pH during the determination. Illustrative
buffers include borate, phosphate, carbonate, Tris, Tris HCI,
barbital and the like. The particular buffer employed is not
critical to this invention but in individual assays, one buffer may
be preferred over another.
[0102] From the foregoing description, one skilled in the art can
easily ascertain the essential characteristics of this invention
and, without departing from the spirit and scope thereof, can make
various changes and modifications of the invention to adapt it to
various usages and conditions. The present invention has been
described in detail, including the preferred embodiments thereof.
However, it will be appreciated that those skilled in the art, upon
consideration of the present disclosure, may make modifications and
improvements on this invention and still be within the scope and
spirit of this invention as set forth in the following claims.
[0103] Incorporation by Reference. The contents of all references
and patents cited herein are hereby incorporated by reference in
their entirety.
Sequence CWU 1
1
2120DNAArtificialOligomarkers 1ctctctctct ctctctctct
20220DNAArtificialOligomarkers 2tttttttttt tttttttttt 20
* * * * *