U.S. patent application number 11/282211 was filed with the patent office on 2006-04-13 for surface assembly for immobilizing dna capture probes in genetic assays using enzymatic reactions to generate signal in optical bio-discs and methods relating thereto.
Invention is credited to John Francis Gordon, Ramon Magpantay Valencia, Martin Elisabeth Werner.
Application Number | 20060078935 11/282211 |
Document ID | / |
Family ID | 26967159 |
Filed Date | 2006-04-13 |
United States Patent
Application |
20060078935 |
Kind Code |
A1 |
Werner; Martin Elisabeth ;
et al. |
April 13, 2006 |
Surface assembly for immobilizing DNA capture probes in genetic
assays using enzymatic reactions to generate signal in optical
bio-discs and methods relating thereto
Abstract
The invention relates to methods and systems for use of
bio-discs in quantifying an amount or concentration of one or more
substances that are present in a biological sample. The system
includes an optical disc having one or more samples deposited
thereon, an optical element configured to emit and divert radiation
onto the samples and components for measuring spectral change of
the compound and determining an amount or concentration of one or
more target substances. The bio-disc is prepared using a method
including providing a membrane that is or can be dimensional to fit
within a channel of the bio-disc, applying a reagent on the
membrane which is configured to allow the reagent to be released
from the membrane into a solution in contact with the membrane, and
depositing the membrane in a channel of the bio-disc.
Inventors: |
Werner; Martin Elisabeth;
(Aliso Viejo, CA) ; Valencia; Ramon Magpantay;
(Aliso Viejo, CA) ; Gordon; John Francis; (Irvine,
CA) |
Correspondence
Address: |
KNOBBE MARTENS OLSON & BEAR LLP
2040 MAIN STREET
FOURTEENTH FLOOR
IRVINE
CA
92614
US
|
Family ID: |
26967159 |
Appl. No.: |
11/282211 |
Filed: |
November 18, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10150702 |
May 17, 2002 |
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11282211 |
Nov 18, 2005 |
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60292110 |
May 18, 2001 |
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60313917 |
Aug 21, 2001 |
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Current U.S.
Class: |
435/6.12 ;
427/2.11; 435/287.2; 435/6.15 |
Current CPC
Class: |
C40B 60/14 20130101;
B01J 2219/00578 20130101; C40B 50/14 20130101; G01N 35/00069
20130101; B01L 3/5027 20130101; B01J 2219/00585 20130101; B01J
2219/00637 20130101; B01J 2219/00722 20130101; B01J 2219/00648
20130101; B01L 2400/0409 20130101; B82Y 30/00 20130101; Y10S
977/924 20130101; B01J 2219/0063 20130101; C40B 40/06 20130101;
B01J 2219/00617 20130101; B01L 3/545 20130101; B01J 2219/00707
20130101; B01J 2219/00274 20130101; B01J 19/0046 20130101; B01J
2219/00695 20130101; B01J 2219/00576 20130101; B01L 2300/0806
20130101; B01J 2219/00317 20130101; B01J 2219/00725 20130101; B01J
2219/005 20130101; B01J 2219/00596 20130101; B01J 2219/00536
20130101; B01J 2219/00675 20130101; B01J 2219/00641 20130101; B01L
3/5025 20130101; B01J 2219/00605 20130101; B01J 2219/00659
20130101; B01J 2219/00702 20130101; B01L 3/502753 20130101; B01L
3/502707 20130101; B01J 2219/0061 20130101; B01J 2219/00689
20130101; B01J 2219/00621 20130101 |
Class at
Publication: |
435/006 ;
435/287.2; 427/002.11 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68; G01N 1/28 20060101 G01N001/28; C12M 1/34 20060101
C12M001/34; B05D 3/00 20060101 B05D003/00 |
Claims
1. A method of preparing a bio-disc having at least one channel,
the method comprising: providing a membrane that is or can be
dimensioned to fit within the at least one channel of the bio-disc;
applying one or more reagents on the membrane, wherein the membrane
is configured to allow the one or more reagents to be released from
the membrane into a solution placed in contact with the one or more
reagents on the membrane; and depositing the membrane in one of the
at least one channels of the bio-disc.
2. The method of claim 1, wherein the bio-disc comprises a
semi-reflective layer having a thickness of less than about 400
.ANG..
3. The method of claim 1, wherein the bio-disc comprises a
semi-reflective layer having a thickness of between about 100 and
300 .ANG..
4. The method of claim 1, wherein the bio-disc comprises a
plurality of channels and a plurality of membranes are deposited in
the plurality of channels.
5. The method of claim 1, wherein the reagents on the membrane are
allowed to dry before the depositing step is performed.
6. The method of claim 1, wherein the one or more reagents comprise
one or more enzymes.
7. The method of claim 1, wherein providing a membrane comprises
providing a membrane that is or can be dimensioned to fit within a
side chamber of the bio-disc that is in fluid communication with
the at least one channel.
8. The method of claim 1, wherein the membrane is a bibulous
hydrophilic material.
9. The method of claim 1, wherein the membrane comprises
hydrophilic polyethersulfone.
10. The method of claim 1, wherein the applying is performed using
a pipette.
11. An apparatus for quantifying an optical density change in
calorimetric assays, the apparatus comprising: an optical disc
having one or more compounds deposited thereon, wherein the one or
more compounds change one or more spectral characteristics in the
presence of a target substance so that a spectral change by each of
the one or more compounds is a function of a concentration of the
target substance brought into contact with each of the one or more
compounds; an optical element configured to emit and direct
radiation so that the radiation is incident on the compounds; a
detector configured to measure an indication of the spectral change
for each of the one or more compounds; a computing device
responsive to the detector and configured to determine an amount or
concentration of one or more target substances.
12. A method of quantifying an amount of one or more analytes
present in a biological sample, the method comprising: providing an
optical disc having one or more reagents located on or in one or
more analysis zones of the optical disc; introducing a sample onto
or into the optical disc so that the sample contacts the one or
more reagents on the optical disc; incubating the optical disc for
a period of time; quantifying a spectral change in at least one
portion of the disc resulting from introduction of the sample; and
determining an amount of the one or more analytes present in the
sample based upon results from the quantifying step.
13. The method of claim 12, wherein the sample comprises one or
more of a body fluid sample, an agricultural product sample, a food
sample, a waste product sample, and an environmental sample.
14. A method of quantifying an amount of one or more analytes
present in a biological sample, the method comprising: depositing
one or more reagents onto respective one or more analysis zones on
an optical disc; applying a sample onto the optical disc so that
the one or more reagents is brought into contact with the sample;
incubating the optical disc for a period of time; emitting
radiation having a known wavelength so that the radiation is
incident upon the sample; quantifying an amount of radiation
transmitted through the sample in contact with each of the one or
more reagents; and determining an amount of the one or more
analytes present in sample based upon results from the quantifying
step.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of U.S. Provisional
Applications Ser. No. 60/292,110, filed May 18, 2001, and Ser. No.
60/313,917, filed Aug. 21, 2001, and is a continuation of U.S.
application Ser. No. 10/150,702, filed May 17, 2002, the contents
of which are hereby incorporated by reference in their
entireties.
BACKGROUND OF THE INVENTION
[0002] The present invention relates to methods and systems for the
detection of specific sequences of nucleic acids or
oligonucleotides, including deoxyribonucleic acids (DNA) and
ribonucleic acids (RNA). It relates more particularly to a
conjugated enzyme based assay system utilizing reflective and/or
transmissive optical discs for detection of specific sequences of
nucleic acids.
[0003] Assay systems utilizing optical discs have been described.
See, for example, Virtanen, U.S. Pat. No. 6,030,581 entitled
"Laboratory in a Disk". Such systems have enormous potential in the
field of medicine, for diagnostic and other clinical assays, as
well as in fields such as environmental testing and the like.
Nonetheless, there remains a continuing need to develop assays that
are faster, more efficient, and more economical.
[0004] Commonly assigned U.S. application Ser. No. 10/035,836
discloses a bead based DNA assay developed for the optical disc
platform. Although such assays are qualitatively reproducible,
quantitation of bead binding through different methods showed
relatively high variations.
[0005] Enzyme assays have been widely used in a microtiter plate
format. Commonly assigned U.S. Provisional Application Ser. No.
60/353,017, entitled "Data Capture and Signal Processing for
Colorimetric and Fluorescent Assays as Implemented on Reflective
Optical Analysis Discs", filed on Jan. 29, 2002, discloses an
enzyme assay implemented in an optical bio-disc system. In this
assay, the reactants are not immobilized on the disc surface, and
the reporter is not localized. This format is very useful for the
detection of small molecules, but is not easily adapted to
macromolecules, such as antibodies and DNA.
[0006] Assays that detect the presence of specific sequences of
nucleic acids have a number of applications. For example, nucleic
acid detection systems are used to test for the presence of
specific disease causing agents, such as viruses or bacteria, in
biological samples taken from patients. Nucleic acid detection
systems are also used to test water and soil samples for specific
microorganisms. Indeed, nucleic acid testing can be used to
identify particular strains or types of a microorganism, which may
have important implications for the appropriate response or
treatment. Nucleic acid testing is also helpful in monitoring
agricultural products as, for example, in testing for the presence
of genetically modified crop products. As is well known, nucleic
acid testing has important forensic applications as well.
[0007] What is needed, therefore, is a rapid, efficient, and
economical assay system for testing various samples for specific
nucleic acid sequences that also provides reproducible quantitation
of the target nucleic acid.
SUMMARY OF THE INVENTION
[0008] This invention relates to identification of at least one
target DNA or RNA that may exist in a sample and test methods
relating thereto. The invention is further directed to an optical
bio disc used to test a sample of DNA or RNA for a target DNA or
RNA of a prescribed sequence. The bio disc includes a flow channel
having target or capture zones, a return channel in fluid
communication therewith, and in some embodiments a mixing chamber
in fluid communication with the flow channel. The bio disc may be
implemented on an optical disc including an information encoding
format such as CD, CD R, DVD, DVD-R, any standard optical disc
format, or a modified version thereof. Methods of manufacturing the
optical bio disc according to the present invention are also
provided.
[0009] A bio disc drive assembly is employed to rotate the disc,
read and process any encoded information stored on the disc, and
analyze the DNA samples in the flow channel of the bio disc. The
bio disc drive is thus provided with a motor for rotating the bio
disc, a controller for controlling the rate of rotation of the
disc, a processor for processing return signals form the disc, and
an analyzer for analyzing the processed signals. The rotation rate
is variable and may be closely controlled both as to speed,
direction, and time of rotation.
[0010] The bio disc drive assembly may also be utilized to write
information to the bio disc either before, during, or after the
test material in the flow channel and target zones is interrogated
by a read beam of the drive and analyzed by the analyzer. The bio
disc may include encoded information for controlling the rotation
rate of the disc, providing data acquisition and processing
information specific to the type of DNA or RNA test to be
conducted, for displaying the results on a monitor associated with
the bio drive, and/or saving the results on a hard drive, floppy
disc, on the bio-disc itself, or on any other recordable media. A
reflective disc format suitable for use in the present invention is
disclosed in commonly assigned U.S. Provisional Application
60/249,391 entitled "Optical Disc Assembly for Performing
Microscopy and Spectroscopy Using Optical Disc Drive," hereby
incorporated by reference in its entirety.
[0011] In an alternative embodiment, a transmissive disc format may
be used in which the interrogation beam is transmitted through the
target zone and detected by a top detector. Such a transmissive
disc format is disclosed in commonly assigned U.S. Pat. No.
6,327,013 and in commonly assigned U.S. Provisional Applications
Nos. 60/293,917; 60/303,437; and 60/323,405, entitled "Optical
Discs and Assemblies for Detection of Microscopic Structures Using
Focal Zone Control," hereby incorporated by reference in their
entireties.
[0012] Development of a DNA based assay for CD, CD R, DVD, DVD-R,
or any standard optical disc format and variations thereof
according to the present invention, includes attachment of
conjugated enzymes to the disc surface as a detection method. These
enzymes are selected so as to yield, in the presence of a suitable
substrate, reaction products that an interrogation beam of the
drive can "see" or detect by a change in surface reflectivity or
transmittance caused by the reaction products.
[0013] The enzymes are bound to the disc surface through binding
agents including, for example, Streptavidin and biotin. A capture
probe is attached to the disc in a capture zone, while a
biotinylated target is allowed to hybridize with the capture probe.
Once the target is hybridized with its respective capture probe, a
Streptavidin conjugated enzyme introduced into the capture zone and
allowed to bind to the biotin on the target. In this manner, the
enzyme is attached to a disc surface. In a subsequent
centrifugation (or wash) step, all unbound enzyme is removed.
Substrate appropriate for the bound enzyme is added, and the
enzymatic reaction is allowed to take place. The enzyme reaction
products deposit on the disc surface at or near the bound enzyme,
where they can be detected and quantitatively measured to provide
both a qualitative and quantitative measurement of the analyte of
interest.
[0014] In an alternative embodiment of the present invention, the
enzymes are bound to the disc surface through DNA hybridization. A
capture probe is attached to the disc, while the Streptavidin
conjugated enzyme is attached to a biotinylated signal probe. Each
of these probes is complementary to a different portion of the
target sequence, but are not complementary to each other. In the
presence of a target sequence, both capture and signal probes
hybridize with the target. In this manner, the enzyme is attached
to a disc surface within the capture zone. In a subsequent
centrifugation (or wash) step, all unbound enzyme is removed.
Substrate appropriate for the bound enzyme is added, and the
enzymatic reaction is allowed to take place. The enzyme reaction
products deposit on the disc surface at or near the bound enzyme,
where they can be detected and quantitatively measured by a beam of
electromagnetic radiation to provide both a qualitative and
quantitative measurement of the analyte of interest.
[0015] The DNA capture probe can be bound to an active layer,
bio-layer, or binding layer by passive adhesion or adsorption,
electrostatic interaction (using a positively charged active
layer), or covalent binding, achieved by using an activated active
layer and a modified DNA wherein the modified DNA can covalently
bind onto the active layer. For example, an aminated DNA can
covalently bind onto a polystyrene co-maleic-anhydride active
layer. This active layer may be formed from a variety of media
including nitrocellulose, polystyrene, polycarbonate, gold,
activated glass, modified glass, or modified media. The modified
media includes anhydride groups, activated carboxylate groups, or
carboxylic acid aldehyde groups.
[0016] After DNA hybridization, a neutravidin- or
streptavidin-conjugated enzyme as, for example, alkaline
phosphatase or horseradish peroxidase, is bound to the
biotinlylated DNA as described above. Then a solution of enzyme
substrate is added and reacts with the enzyme to form a reaction
product in the form of insoluble pellets or precipitate,
fluorescent, and/or colored product. The pellets stay localized on
the reaction spots where the DNA probe was applied, even after
centrifugation of the disc.
[0017] The specific enzymatic reaction products can be detected
using different methods. These methods include microscopic
analysis, measurement of fluorescence signal on the disc surface
using a FluorImager (Molecular Dynamics), or detection of insoluble
reaction product using an optical disc reader. For example, an
event counting software useful for reaction product detection in a
optical disc reader is disclosed in commonly assigned U.S.
Provisional Application 60/291,233 entitled "Variable Sampling
Control for Rendering Pixelization of Analysis Results in Optical
Bio-disc Assembly and Apparatus Relating Thereto," hereby
incorporated by reference in its entirety. As discussed below in
conjunction with FIG. 37 and in Example 5, the signal from an
enzymatic reaction product, in one embodiment of the present
invention, is concentration dependent using the event counting
software to quantitate the data, thus making this a quantitative
detection method. Moreover, a fluorescent enzymatic product may be
detected by a fluorescent type disc reader while a colored product
(chromagen) can be detected using a transmissive or reflective disc
set-up described below. Alternatively, the signal detection and
quantitation may be carried out using other methods of quantitation
in conjunction the optical bio-disc reader with appropriate
software. For example, the transmissive optical disc format may be
used to quantify changes in light transmission or scattering as a
result of generation of an enzymatic reaction product or
reporter.
[0018] The DNA assay according to the present invention may be
implemented in an open disc format as well as in a micro channel.
In the open disc format, the reagents are spotted directly on the
disc surface. Unbound reagents are removed by washing the disc. In
the micro channel format, the capture probe binding is initially
performed on an open disc substrate. After attaching the DNA
capture probes, the channel is assembled by affixing adhesive and a
cover disc or cap. Subsequent steps are performed in the closed
channel which is filled with liquids such as buffer solutions,
enzyme solutions, and DNA test samples which are analyzed for the
presence of a target sequence.
Brief Overview of the Assay
[0019] In the assay according to the present invention, an enzyme
reaction is used to detect the presence of an analyte (DNA or RNA)
in a microchannel on an optical bio-disc. The analyte is
immobilized on a capture layer on the surface, and the signal that
is generated is localized and specific, as, for example, by the
formation of an insoluble product of the enzyme reaction. The
signal can be in the form of a pellet, a fluorescent product,
and/or a colored product, and can be detected and quantified by an
optical bio-disc reader utilized in conjunction with the inventions
hereof. This assay is thus quantitative in nature. In addition, the
formation of the pellet in one embodiment hereof is facilitated by
a layer of nitrocellulose on the disc surface, which supports
binding of the capture layer and formation and retention of the
pellet.
Analytes
[0020] The present invention is directed to the detection and
analysis of target nucleic acid sequences present in test samples.
Target nucleic acids suitable for use with the present invention
include both deoxyribonucleic acid (DNA) and ribonucleic acid
(RNA), including mRNA, rRNA, hnRNA and tRNA.
[0021] Target nucleic acid may be used directly from a biological
sample, but preferably is simplified prior to testing via
polymerase chain reaction (PCR) or isothermal amplification to
generate amplicons. If using PCR for amplification, RNA may first
be reverse transcribed into DNA using techniques well known in the
art. Target nucleic acid may be single stranded or double stranded.
If double stranded, the nucleic acid may be denatured prior to
hybridization with capture DNA.
[0022] In one embodiment of the present invention, primers labeled
with biotin are used in PCR reactions to yield biotin-labeled
target DNA amplicons, which are then tested in the bio-disc assay
as described below. Amplicons of various lengths are suitable for
use in the present invention, with a preferred length from about 20
bases (or base pairs) to about 4000 bases (or base pairs), more
preferably from about 200 to about 400 bases or base pairs.
[0023] The present invention may be used to detect specific nucleic
acid sequences in a wide variety of biological samples, including
but not limited to bodily fluids such as whole blood, serum,
plasma, saliva, urine, lymph, spinal fluid, tears, mucous, semen
and the like, agricultural products, food items, waste products,
environmental samples, such as soil and water samples, or any other
sample containing, or suspected of containing specific nucleic acid
sequences of interest. For example, the present invention may be
used to detect the presence of particular strains of
microorganisms, such as viruses or bacteria, in body fluids or
environmental samples, by detecting the presence of particular
nucleic acid sequences in the sample. In another example, the
present invention may be used to detect the presence of genetically
modified agricultural products in food items. In yet another
example, the present invention may be used to identify specific
pulmonary infectious agents using a microarray format. Other uses
of the present invention will be apparent to those of skill in the
art.
Capture DNA
[0024] Capture DNA oligonucleotides, or probes, are immobilized
onto a bio-disc and are hybridized to target DNA or RNA to thereby
"capture" the target nucleic acid in the target zone for detection.
Capture DNA may be single stranded or partially double stranded
near the attachment point to the active layer on the bio-disc. One
preferred embodiment of the capture DNA includes a partially double
stranded DNA. The double strand us located at the reactive end of
the probe, such as the amino end, because the double strand has
been found to more effectively project the capture probe erectly or
upwardly from the active layer as compared to ssDNA in some
instances. An extension or spacer including, for example, ssDNA and
PEG, may be employed so as to increase the hybridization efficiency
of the capture probe with the target. The sequence of the capture
DNA is selected so as to hybridize directly with target DNA or RNA,
thereby forming a complex comprising capture DNA, target DNA or RNA
and conjugated enzyme bound thereto as depicted below in FIGS.
33A-33G and 35A-35F.
[0025] In an alternative embodiment described below, signal DNA is
also be present in this complex. In this embodiment, the signal DNA
sequence is complementary to a portion of the target DNA or RNA,
but is not complementary to the capture DNA. As one portion of the
target DNA hybridizes with capture DNA, while a different portion
hybridizes with signal DNA, a complex forms in which the target DNA
acts as a "bridge" between the capture DNA and signal DNA as
described below in conjunction with FIGS. 34A-34H.
Signal DNA
[0026] In one embodiment of the present invention, target DNA
hybridizes directly with capture DNA bound to the active layer of a
bio-disc. In an alternative embodiment, target DNA is used as a
"bridge" between signal DNA and the capture DNA. In the alternative
embodiment, the sequence of the signal DNA is selected so as to
contain a region that is complementary to the target DNA, but which
contains no sequence complementarity with the capture DNA, such
that the signal DNA will not form a complex with the capture DNA in
the absence of target DNA. The target DNA contains a first region
of complementary sequence to the capture DNA, permitting
hybridization of the first region of the target DNA to the capture
DNA, and a second region of complementary sequence to the signal
DNA, permitting hybridization of the signal DNA to the second
region of the target DNA, thereby linking or bridging the signal
DNA to the capture DNA.
[0027] The target DNA may be of any length suitable to effectively
immobilize itself and the signal DNA to the capture DNA. Typically,
signal DNA and capture DNA are from about 10 bases to about 100
bases in length, preferably from about 20 bases to about 60 bases
in length. Typically, the target DNA amplicons have an overlap of
from about 20 bases to about 40 bases with the signal DNA and an
overlap of from about 20 bases to about 40 bases with the capture
DNA. Preferably, the target DNA amplicon has a GC (guanine and
cytosine) content greater than 50%, within the areas of overlap
between the capture probe, target, and signal probe. Although one
skilled in the art will appreciate that GC content and length of
the target DNA amplicon may be modulated to effectuate stable
hybridization to the signal and capture DNA.
[0028] In one embodiment of the present invention, signal DNA or
the target DNA is labeled with an affinity agent, such as biotin,
to permit binding to conjugated enzymes via biotin/streptavidin
interactions with streptavidin-conjugated enzymes.
Capture Layer Preparation
[0029] Capture DNA probes are bound to the surface of the disc
through non-covalent adsorption to a layer of nitrocellulose, which
is spin-coated on the disc. The layer of nitrocellulose can be
applied to different types and surfaces of discs. After attaching
the capture probes, microchannels may be assembled and prepared for
sample application as shown and described below in conjunction with
FIGS. 2A-2C, and 3A-3D.
Blocking Non-specific DNA, RNA and Protein Adsorption
[0030] After the disc is assembled, the channels are blocked with a
DNA/protein blocking solution to prevent non-specific binding of
target nucleic acid, signal probes and/or enzymes on the target
zone. The blocking solution may be a buffer containing, for
example, bovine serum albumin (BSA), salmon sperm DNA, and/or
Denhardt's solution.
Sample Application
[0031] When a sample is injected into the microchannel, any target
RNA or DNA present in the sample binds to the capture probe through
hybridization. In one embodiment, target DNA is generated in an
amplification reaction using biotinylated primers, resulting in
biotinylation of the target DNA. Following hybridization, unbound
amplicon DNA is removed with a wash step.
[0032] In another embodiment of the assay, the target RNA or DNA is
not directly biotinylated. Rather, a biotinylated signal DNA probe
is used. In this embodiment, target DNA is amplified by PCR or
isothermal amplification using non-biotinylated primers (target RNA
is similarly generated by isothermal amplification). The target DNA
is then hybridized to a biotinylated signal DNA probe.
Signal Generation
[0033] In one embodiment, a solution of streptavidin- or
neutravidin-conjugated enzyme, such as horseradish peroxidase, is
injected into the microchannel, where the enzyme binds to the
biotinylated amplicon or signal probe via the streptavidin- or
neutravidin-biotin interactions. Excess enzyme is removed through a
wash step and the microchannel is filled with a solution of an
enzyme substrate, such as TMB (3,3,5,5 tetramethylbenzidine in
stable peroxide buffer), that is converted to an insoluble product,
becomes luminescent or fluorescent, changes color through the
enzyme reaction, or otherwise generates a detectable signal. In one
embodiment, the enzyme reaction product is an insoluble precipitate
that adheres to the active layer, forming a detectable precipitate
as described below in conjunction with FIGS. 21 and 22A-22D.
[0034] In another embodiment, the conjugated enzyme is first dried
onto a pad or membrane, which is deposited into a side chamber in
fluid communication with the microchannel as illustrated in FIG. 4,
below. A buffer solution is introduced into the side chamber via an
input port, causing the enzyme to be released and travel into the
microchannel where it can interact with the biotinylated DNA.
Substrate is then introduced as above. Materials useful for the pad
or membrane include filter paper, cellulose acetate,
nitrocellulose, glass fiber, hydrophilic polyether sulfone, nylon,
cellulose and the like.
Enzymes and Substrates
[0035] Enzymes useful in the practice of the present invention
include any enzyme that may be adapted to interact with a specific
nucleic acid probe, as, for example, through the interaction of an
enzyme conjugated with streptavidin- or neutravidin- and with a
biotin labeled DNA. The enzyme produces a detectable signal in the
presence of a suitable substrate. For example, conjugated
horseradish peroxidase (HRP; Pierce, Rockford, Ill.) may be used
with the substrate 3,3,5,5-tetramethylbenzidine (TMB; Calbiochem
cat. no. 613548, CAS-54827-17-7) in the presence of hydrogen
peroxide to produce an insoluble precipitate. Horseradish
peroxidase can also be used in conjunction with CN/DAB
(4-chloronaphthol/3,3'-diaminobenzidine, tetrahydrochloride), 4-CN
(4-chloro-1-napthol), AEC (3-amino-9-ethyl carbazol) and DAB
(3,3-diaminobenzidine tetrahydrochloride) to form insoluble
precipitates. Similarly, the enzyme alkaline phosphatase (AP) can
be used with the substrate bromochloroindolylphosphate in the
practice of the present invention. Other suitable enzyme/substrate
combinations will be apparent to those of skill in the art.
Disc Implementation
[0036] The assays and methods of the present invention may be
advantageously implemented on an analysis disc, modified optical
disc, or bio-disc. The bio-disc may include a flow channel having
target or capture zone, a return channel in fluid communication
therewith, and in some embodiments a mixing chamber and/or a side
chamber in fluid communication with the flow channel.
[0037] The bio-disc may be implemented on an optical disc including
an standard information encoding format such as CD, CD-R, DVD,
DVD-R or a modified version thereof. The bio-disc may include
encoded information for performing, controlling, and
post-processing the test or assay. For example, such encoded
information may be directed to controlling the rotation rate of the
disc. Depending on the test, assay, or investigational protocol,
the rotation rate may be variable with intervening or consecutive
sessions of acceleration, constant speed, and deceleration. These
sessions may be closely controlled both as to speed and time of
rotation to provide, for example, mixing, agitation, or separation
of fluids and suspensions with agents, reagents, DNA, RNA or
antibodies.
Drive Implementation
[0038] A bio-disc drive assembly or reader may be employed to
rotate the disc, read and process any encoded information stored on
the disc, and analyze the samples in the flow channel of the
bio-disc. The bio-disc drive is thus provided with a motor for
rotating the bio-disc, a controller for controlling the rate of
rotation of the disc, a processor for processing return signals
form the disc, and an analyzer for analyzing the processed signals.
The drive or disc may include software specifically developed for
performing the assays disclosed herein.
[0039] The rotation rate of the motor is controlled to achieve the
desired rotation of the disc. The bio-disc drive assembly may also
be utilized to write information to the bio-disc either before,
during, or after the test material in the flow channel and target
zone is interrogated by the read beam of the drive and analyzed by
the analyzer. The bio-disc may include encoded information for
controlling the rotation rate of the disc, providing data
acquisition, and processing, reporting and recording information
specific to the type of genetic test to be conducted, and for
displaying the results on a display monitor associated with the
bio-drive in accordance with the assay methods relating hereto.
BRIEF DESCRIPTION OF THE DRAWINGS
[0040] FIG. 1 is a pictorial representation of a bio-disc system
according to the present invention;
[0041] FIG. 2A is an exploded perspective view of a reflective
bio-disc;
[0042] FIG. 2B is a top plan view of the optical bio-disc 110
illustrated in FIG. 2A, with the reflective layer 142 on the cap
portion 116 shown as transparent;
[0043] FIG. 2C is an enlarged perspective view of the optical
bio-disc 110 according to one embodiment of the present invention
having a portion of the various layers thereof, cut away to
illustrate a partial sectional view of each principle, layer,
substrate, coating, or membrane;
[0044] FIG. 3A is an exploded perspective view of the principle
structural elements of a transmissive bio-disc;
[0045] FIG. 3B is an enlarged perspective view of the substrate of
the transmissive bio-disc;
[0046] FIG. 3C is a top plan view of the transmissive bio-disc;
[0047] FIG. 3D is an enlarged perspective view of the transmissive
bio-disc;
[0048] FIG. 4 is an enlarged top plan view of an alternative
embodiment of a fluidic circuit having a side chamber;
[0049] FIG. 5 is a perspective and block diagram representation
illustrating the system of FIG. 1 in more detail;
[0050] FIG. 6 is a cross sectional view of a reflective
bio-disc;
[0051] FIG. 7 is a cross sectional view of a transmissive
bio-disc;
[0052] FIG. 8 is a cross sectional view taken across the tracks of
the reflective bio-disc embodiment;
[0053] FIG. 9 is a cross sectional view taken across the tracks of
the transmissive bio-disc embodiment;
[0054] FIG. 10 is a longitudinal cross-section of the reflective
bio-disc embodiment shown in FIG. 8;
[0055] FIG. 11 is a longitudinal cross-section of the transmissive
bio-disc embodiment shown in FIG. 9;
[0056] FIG. 12 illustrates an embodiment of the optical bio-disc
that utilizes a reflective open-face or open-disc format;
[0057] FIG. 13 illustrates an embodiment of the optical bio-disc
that utilizes a transmissive open-face or open-disc format;
[0058] FIG. 14 is an alternate sectional view of the disc
illustrated in FIG. 12 taken longitudinally along one of the tracks
or grooves;
[0059] FIG. 15 is an alternate sectional view of the disc
illustrated in FIG. 13 taken longitudinally along one of the tracks
or grooves;
[0060] FIG. 16 is an enlarged detailed partial cross sectional view
showing the active layer and the substrate of the bio-disc, with
capture DNA and target DNA;
[0061] FIG. 17 is an enlarged view similar to FIG. 16, shown after
the introduction of enzymes;
[0062] FIG. 18 is an enlarged detailed partial cross sectional view
showing an alternate method of introducing the target DNA, in which
the enzyme is pre-conjugated to the target DNA;
[0063] FIG. 19 is a detailed partial cross sectional view showing
the active layer and the substrate of the present bio-disc
according to an embodiment utilizing a signal DNA attached to
enzymes;
[0064] FIG. 20 is a detailed partial cross sectional view similar
to FIG. 19, showing the signal DNA/target DNA or RNA/capture DNA
complex formed at the target zone, with bound enzymes;
[0065] FIG. 21 is a detailed partial cross sectional view of a
reaction, showing the active layer, the substrate, target RNA or
DNA 174 hybridized to the capture DNA, and the introduction of
enzyme substrate, resulting in enzyme/substrate reactions by
enzymes bound to the target DNA or RNA;
[0066] FIGS. 22A-22D are detailed partial cross sectional views of
a target zone showing a method according to the present invention
for detecting or determining the presence of target RNA or DNA in a
sample by pellet formation through an enzyme-substrate reaction in
conjunction with the optical bio-disc;
[0067] FIG. 23 is a longitudinal cross sectional view (cut along
the tracking grooves) of the mixing chamber of the reflective
bio-disc showing the inlet port;
[0068] FIG. 24 is a longitudinal cross sectional view (cut along
the tracking grooves) of the mixing chamber of the transmissive
bio-disc showing the inlet port;
[0069] FIG. 25 is a longitudinal cross sectional view (cut along
the tracking grooves) of a mixing chamber of a reflective bio-disc
containing conjugated enzyme;
[0070] FIG. 26 is a longitudinal cross sectional view (cut along
the tracking grooves) of a mixing chamber of a transmissive
bio-disc containing conjugated enzyme;
[0071] FIG. 27 is a longitudinal cross sectional view of a
reflective bio-disc, similar to FIG. 6, which shows the capture DNA
attached to the active layer within the target zone;
[0072] FIG. 28 is a longitudinal cross sectional view of a
reflective bio-disc, similar to FIG. 6, which shows the flow
channel and target zone after hybridization of the target DNA with
the capture DNA;
[0073] FIG. 29 is a longitudinal cross sectional view of a
reflective bio-disc, similar to FIG. 6, which shows enzymes bound
to the target DNA hybridized to the capture DNA;
[0074] FIG. 30 is a longitudinal cross sectional view of a
transmissive bio-disc, similar to FIG. 7, which shows the capture
DNA attached to the active layer within the target zone;
[0075] FIG. 31 is a longitudinal cross sectional view of a
transmissive bio-disc, similar to FIG. 7, which shows the flow
channel and target zone after hybridization of the target DNA with
the capture DNA;
[0076] FIG. 32 is a longitudinal cross sectional view of a
transmissive bio-disc, similar to FIG. 7, which shows enzymes bound
to the target DNA hybridized to the capture DNA;
[0077] FIGS. 33A-33G show a longitudinal cross-section of a flow
channel, illustrating a method according to the present invention
for detecting or determining the presence of target DNA in
conjunction with the optical bio-disc;
[0078] FIGS. 34A-34H show a longitudinal cross-section of a flow
channel, illustrating another method according to the present
invention for detecting or determining the presence of target DNA
in conjunction with the optical bio-disc, utilizing signal DNA;
[0079] FIGS. 35A-35F show a longitudinal cross-section of a flow
channel, illustrating yet another method according to the present
invention for detecting or determining the presence of target DNA
in conjunction with the optical bio-disc, in which enzymes with the
associated binding agent are pre-loaded in a mixing chamber;
[0080] FIG. 36 is an example of data output collected using an
optical disc reader and its respective software;
[0081] FIG. 37 is a series of pictorial representations
illustrating image detection according to one embodiment of the
present invention; and
[0082] FIG. 38 is a schematic overview of one embodiment of the
present invention useful in detecting and quantifying genetically
modified material.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT
[0083] Further objects of the present invention together with
additional features contributing thereto and advantages accruing
therefrom will be apparent from the following description of
preferred embodiments of the present invention which are shown in
the accompanying drawing figures with like reference numerals
indicating like components throughout.
[0084] FIG. 1 is a perspective view of an optical bio-disc 110 or
111 according to the present invention. The present optical
bio-disc 110 or 111 is shown in conjunction with an optical disc
drive 112 and a display monitor 114.
Reflective Disc
[0085] FIG. 2A is an exploded perspective view of the principle
structural elements of a reflective optical bio-disc 110 or a
reflective zone optical bio-disc (hereinafter "reflective disc")
that may be used in the present invention. The principle structural
elements include a cap portion 116, an adhesive member 118, and a
substrate 120.
[0086] The cap portion 116 includes an inlet port 122, a vent port
124, and, optionally, an enzyme buffer port 141. The cap portion
116 may be formed from polycarbonate and is preferably coated with
a thin reflective surface 146 on the bottom thereof as viewed from
the perspective of FIG. 2A. The reflective surface 146 is best
illustrated in FIG. 2C.
[0087] In a preferred embodiment, trigger markings 126 are included
on the reflective surface. Trigger markings 126 may include a clear
window in all three layers of the bio-disc, an opaque area, or a
reflective or semi-reflective area encoded with information that
sends data to a processor 166, as shown in FIG. 5, that in turn
interacts with the operative functions of the interrogation or
incident beam 152, shown in FIGS. 3B and 5.
[0088] The second element shown in FIG. 2A is an adhesive member
118 having fluidic circuits or U-channels 128 formed therein. The
fluidic circuits 128 are formed by stamping or cutting the member
to remove plastic film and form the shapes as indicated. Each of
the fluidic circuits 128 includes a flow channel 130 and a return
channel 132.
[0089] Some of the fluidic circuits 128 illustrated in FIG. 2A
include a mixing chamber 134. Two different types of mixing
chambers 134 are illustrated. The first is a symmetric mixing
chamber 136 which is symmetrically formed relative to the flow
channel 130. The second is an off-set mixing chamber 138. The
off-set mixing chamber 138 is formed to one side of the flow
channel 130 as indicated. In one embodiment, some of the fluidic
circuits 128 include a side chamber 139 in fluid communication with
the flow channel 130.
[0090] The third element illustrated in FIG. 2A is a substrate 120
including target or capture zone 140. The substrate 120 is
preferably made of polycarbonate and has a reflective layer 142
deposited on the top thereof, best illustrated in FIG. 2C. The
target zones 140 are formed by removing the reflective layer 142 in
the indicated shape or, alternatively, in any desired shape.
Alternatively, the target zone 140 may be formed by a masking
technique that includes masking the target zone 140 area before
applying the reflective layer 142. The reflective layer 142 may be
formed from a metal such as aluminum or gold.
[0091] FIG. 2B is a top plan view of the reflective optical
bio-disc 110 illustrated in FIG. 2A with the reflective surface 146
on the cap portion 116 shown as transparent to reveal the fluidic
circuits 128, the target zones 140, and trigger markings 126
situated within the disc.
[0092] Referring now to FIG. 2C, there is shown an enlarged
perspective view of the reflective optical bio-disc 110 according
to one embodiment of the present invention, having a portion of the
various layers thereof, cut away to illustrate a partial sectional
view of each principle, layer, substrate, coating, and membrane.
FIG. 2C shows the substrate 120 that is coated with the reflective
layer 142. An active layer 144 is applied over the reflective layer
142. In the preferred embodiment, the active layer 144 is formed
from nitrocellulose. Alternatively, polystyrene, polycarbonate,
gold, activated glass, modified glass, or a modified polystyrene,
for example, polystyrene-co-maleic anhydride, may be used. As
illustrated in this embodiment, the plastic adhesive member 118 is
applied over the active layer 144. The exposed section of the
plastic adhesive member 118 illustrates the cut out or stamped
U-shaped form that creates the fluidic circuits 128. The final
principle structural layer in this embodiment of the present
bio-disc is the cap portion 116. The cap portion 116 includes the
reflective surface 146 on the bottom thereof. The reflective
surface 146 may be made from a metal such as aluminum or gold.
Transmissive Disc
[0093] FIGS. 3A-3D below illustrate the transmissive disc
embodiment of the present invention. Specifically, FIG. 3A shows an
exploded perspective view of the principle structural elements of
the transmissive type of optical bio-disc 111 similar to the
reflective disc 110 of FIGS. 2A-2C. FIG. 3A is an example of a
transmissive optical bio-disc 111 (hereinafter, "transmissive
disc") that may be used in the present invention. The principle
structural elements include the cap portion 116, the adhesive
member 118, and the substrate layer 120. The cap portion 116
includes the inlet port 122 and the vent port 124; optionally, may
also include a buffer input port 141, similar to that shown in FIG.
2A. The cap portion 116 may be formed from a polycarbonate layer.
Optional trigger markings 126 may be included on the surface of cap
116. Trigger markings 126 may include a clear window in all three
layers of the bio-disc, an opaque area, or a reflective or
semi-reflective area encoded with information that sends data to
the processor 166, shown in FIG. 5, which in turn interacts with
the operative functions of the interrogation beam 152, FIGS. 3B and
5.
[0094] The second element shown in FIG. 3A is the adhesive member
118 having fluidic circuits or U-channels 128 formed therein. The
fluidic circuits 128 are formed by stamping or cutting the membrane
to remove plastic film and form the shapes as indicated. Each of
the fluidic circuits 128 includes the flow channel 130 and the
return channel 132. Some of the fluidic circuits 128 illustrated in
FIG. 3A include the mixing chamber 134; optionally, they may
include a side chamber 139. Two different types of mixing chambers
134 are illustrated. The first is the symmetric mixing chamber 136
which is symmetrically formed relative to the flow channel 130. The
second is the off-set mixing chamber 138. The off-set mixing
chamber 138 is formed to one side of the flow channel 130 as
indicated.
[0095] The third element illustrated in FIG. 3A is the substrate
120 which may include the target or capture zone 140. The substrate
120 is preferably made of polycarbonate and has the thin
semi-reflective layer 143 deposited on the top thereof, as shown in
FIG. 3B. The semi-reflective layer 143 on the substrate 120 of FIG.
3B is significantly thinner than the reflective layer 142 on the
substrate 120 of FIG. 2C. The thinner semi-reflective layer 143
allows for some transmission of the interrogation beam 152 through
the structural layers of the transmissive disc as shown in FIG. 3B.
The thin semi-reflective layer 143 may be formed from a metal such
as aluminum or gold.
[0096] Referring next to FIG. 3B, there is shown an enlarged
perspective view of the substrate 120 of the embodiment of the
transmissive optical bio-disc 111 illustrated in FIG. 3A. In the
preferred embodiment, the thin semi-reflective layer 143 of the
transmissive disc illustrated in FIGS. 3B and 3D is approximately
100-300 Angstroms thick and does not exceed 400 Angstroms. This
thinner semi-reflective layer 143 allows a portion of the incident
or interrogation beam 152 to penetrate and pass through the thin
semi-reflective layer 143 to be detected by a top detector 158,
shown in FIG. 5, while some of the light is reflected back. Table
1, below, shows the reflective and transmissive characteristics
relative to thickness for a gold reflective layer. The gold film
layer is fully reflective at a thickness greater than 800
Angstroms, while the threshold density for transmission of light
through the gold film is approximately 400 Angstroms.
TABLE-US-00001 TABLE 1 Thickness Thickness (Angstroms) (nm)
Reflectance Transmittance 0 0 0.0505 0.9495 50 5 0.1683 0.7709 100
10 0.3981 0.5169 150 15 0.5873 0.3264 200 20 0.7142 0.2057 250 25
0.7959 0.1314 300 30 0.8488 0.0851 350 35 0.8836 0.0557 400 40
0.9067 0.0368 450 45 0.9222 0.0244 500 50 0.9328 0.0163 550 55
0.9399 0.0109 600 60 0.9448 0.0073 650 65 0.9482 0.0049 700 70
0.9505 0.0033 750 75 0.9520 0.0022 800 80 0.9531 0.0015
[0097] With reference now to FIG. 3C, there is presented a top plan
view of the transmissive optical bio-disc 111 illustrated in FIGS.
3A and 3B with the transparent cap portion 116 revealing the
fluidic channels 128, the trigger markings 126, and the target or
capture zone 140 as situated within the disc. The fluidic circuits
128 may include a side chamber 139 with the buffer input port
141.
[0098] Referring next to FIG. 3D, there is illustrated an enlarged
perspective view of the transmissive optical bio-disc 111
embodiment of the present invention. The disc 111 is illustrated
with a portion of the various layers thereof cut away to illustrate
a partial sectional view of each principle, layer, substrate,
coating, or membrane. FIG. 3D illustrates the transmissive disc
format with the clear cap portion 116, the thin semi-reflective
layer 143 on the substrate 120, and trigger markings 126. Trigger
markings 126 include opaque material placed on the top portion of
the cap, clear, non-reflective windows etched on the thin
reflective layer 143 of the disc, or any mark that absorbs or does
not reflect the signal coming from the trigger detector 160, shown
in FIG. 5.
[0099] FIG. 3D also shows target zones 140 formed by marking the
designated area in the indicated shape or, alternatively, in any
desired shape. Markings to indicate target zone 140 may be made on
the thin semi-reflective layer 143 on the substrate 120 or on the
bottom portion of the substrate 120 (under the disc). In a
preferred embodiment of the present invention, the active layer 144
is formed from nitrocellulose. Alternatively, polystyrene,
polycarbonate, gold, activated glass, modified glass, or a modified
polystyrene, for example, polystyrene-co-maleic anhydride, may be
used.
[0100] As illustrated in this embodiment, the plastic adhesive
member 118 is applied over the active layer 144. The exposed
section of the plastic adhesive member 118 illustrates the cut out
or stamped U-shaped form that creates the fluidic circuits 128. The
final principle structural layer in this embodiment of the present
bio-disc 110 is the clear, non-reflective cap portion 116 that
includes the inlet 122, vent port 124, and, optionally, the buffer
port (not shown) as described above in FIG. 3C.
[0101] Referring now to FIG. 4, there is illustrated an enlarged
top plan view of a fluidic circuit 128 of the reflective disc 110
or transmissive disc 111 having a side chamber 139, the buffer
inlet port 141, and the target zones 140 situated within the disc.
In this embodiment, a membrane or pad 145, onto which conjugated
enzyme may be dried on, is deposited in the side chamber 139. In
this embodiment, an enzyme buffer is added to the side chamber 139
via a buffer inlet port 141 to solubilize the enzyme, which then
enters the flow channel 130 where it can interact with the
biotinylated target DNA or signal DNA bound within the target zones
140.
[0102] With reference now to FIG. 5, there is a representation in
perspective and block diagram the optical bio-disc system showing
optical components 148, a light source 150 that produces an
incident or interrogation beam 152, a return beam 154, and a
transmitted beam 156. In the case of the reflective bio-disc, the
return beam 154 is reflected from the cap portion 116 of the
reflective bio-disc 110. In this embodiment of the present
invention, the return beam 154 is detected and analyzed for the
presence of signal agents by a bottom detector 157.
[0103] In the transmissive bio-disc 111, on the other hand, the
transmitted beam 156 is detected by a top detector 158 and is
analyzed for the presence of signal agents. In this embodiment, a
photo detector may be used as a top detector 158.
[0104] FIG. 5 also shows a hardware trigger mechanism that includes
the trigger markings 126 on the disc and a trigger detector 160.
The hardware triggering mechanism is used in both reflective
bio-discs 110 (FIG. 2C) and transmissive bio-discs 111 (FIG. 3D).
The triggering mechanism allows the processor 166 to collect data
only when the interrogation beam 152 is on a respective target
zone. Alternatively, a software triggering system may also be used
to control the data acquisition parameters. The software trigger
uses the bottom detector to signal the processor 166 to collect
data as soon as the interrogation beam 152 hits the edge of a
respective target zone 140 or fluidic circuit 130. Further details
relating to triggering methods, data aquistion, and disc drive
mechanisms in optical disc systems is disclosed in, for example,
commonly assigned co-pending U.S. patent application Ser. No.
10/043,688, entitled "Optical Disc Analysis System Including
Related Methods For Biological and Medical Imaging" filed Jan. 10,
2002, which is incorporated herein by reference in its
entirety.
[0105] FIG. 5 also illustrates a drive motor 162 and a controller
164 for controlling the rotation of the optical bio-disc 110 or
111. FIG. 5 further illustrates the processor 166 and analyzer 168
implemented for processing the return 154 and transmitted beams
156.
[0106] Referring now to FIG. 6, there is shown a cross sectional
view of one embodiment of the reflective optical bio-disc 110
according to the present invention. FIG. 6 also shows the substrate
120 and the reflective layer 142. In this embodiment, the substrate
120 is smooth. FIG. 6 also illustrates the active layer 144 applied
over the reflective layer 142. As shown in FIG. 6, the target zone
140 is formed by removing an area or portion of the reflective
layer 142 at a desired location or, alternatively, by masking the
desired area prior to applying the reflective layer 142. As further
illustrated in FIG. 6, the plastic adhesive member 118 is applied
over the active layer 144. FIG. 6 also shows the cap portion 116
and the reflective surface 146 associated therewith. Thus when the
cap portion 116 is applied to the plastic adhesive member 118
including the desired cut-out shapes, the flow channel 130 is
thereby formed.
[0107] Reference in now made to FIG. 7 that shows a cross sectional
view of an embodiment of the transmissive bio-disc 111 according to
the present invention. FIG. 7 illustrates the clear cap portion 116
and the thin semi-reflective layer 143 on the substrate 120 of the
transmissive disc 111. The substrate 120 in this embodiment is
smooth. FIG. 7 also shows the active layer 144 applied over the
thin semi-reflective layer 143. In the preferred embodiment, the
transmissive disc has the thin semi-reflective layer 143 made from
a metal such as aluminum or gold, approximately 100 to 300
Angstroms thick, not exceeding 400 Angstroms. This thin
semi-reflective layer 143 allows some portion of the incident or
interrogation beam 152 to penetrate and pass through the disc to be
detected by a top detector 158, while some portion of the light is
reflected back along the same path as the incident beam but in the
opposite direction as described above. The reflected light or
return beam is used for tracking of the light source along the
disc. In the disc embodiment illustrated in FIG. 7, a defined
target zone 140 may or may not be present. Target zone 140 may be
created by direct markings made on the thin semi-reflective layer
143 on the substrate 120. These marking may be done using silk
screening or any equivalent method.
[0108] With reference now to FIG. 8, there is illustrated a cross
sectional view taken across the tracks of the reflective disc 10
embodiment of the bio-disc according to the present invention. FIG.
8 includes the substrate 120 and the reflective layer 142. The
substrate 120 in this embodiment includes a series of grooves 170.
The grooves 170 are in the form of a spiral extending from near the
center of the disc toward the outer edge. The grooves 170 are
implemented so that the interrogation beam 152 may track along the
spiral grooves 170 on the disc. This type of groove 170 is known as
a "wobble groove." The groove 170 is formed by a bottom portion
having undulating or wavy side walls. A raised or elevated portion
separates adjacent grooves 170 in the spiral. The reflective layer
142 applied over the grooves 170 in this embodiment is, as
illustrated, conformal in nature.
[0109] FIG. 8 also shows the active layer 144 applied over the
reflective layer 142. As shown in FIG. 8, the target zone 140 is
formed by removing an area or portion of the reflective layer 142
at a desired location or, alternatively, by masking the desired
area prior to applying the reflective layer 142. As further
illustrated in FIG. 8, the plastic adhesive member 118 is applied
over the active layer 144. FIG. 8 also shows the cap portion 116
and the reflective surface 146 associated therewith. Thus, when the
cap portion 116 is applied to the plastic adhesive member 118
including the desired cut-out shapes, the flow channel 130 is
thereby formed.
[0110] Referring to FIG. 9, there is depicted a cross sectional
view taken across the tracks of the transmissive disc 111
embodiment, as described above in conjunction with FIG. 7. FIG. 9
illustrates the substrate 120 and the thin semi-reflective layer
143. In the preferred embodiment, the transmissive disc has the
thin semi-reflective layer 143 made from a metal such as aluminum
or gold, which is approximately 100 to 300 Angstroms thick and does
not exceed 400 Angstroms. This thin semi-reflective layer 143
allows the incident or interrogation beam 152 to penetrate and pass
through the disc (transmitted beam 156) to be detected by the top
detector 158, while some of the light is reflected back. The
thickness of the thin semi-reflective layer 143 is determined by
the minimum amount of reflected light required by the disc reader
to maintain its tracking ability.
[0111] The substrate 120 in this embodiment includes a series of
grooves 170. The grooves 170 are in the form of a spiral extending
from near the center of the disc toward the outer edge and are
implemented so that the interrogation beam 152 may track along the
spiral.
[0112] FIG. 9 also shows the active layer 144 applied over the thin
semi-reflective layer 143. As further illustrated in FIG. 9, the
plastic adhesive member 118 is applied over the active layer 144.
FIG. 9 also shows the cap portion 116 without a reflective surface.
Thus, when the cap is applied to the plastic adhesive member 118
including the desired cut-out shapes, the flow channel 130 is
thereby formed and the incident beam is allowed to pass
therethrough substantially unreflected.
[0113] FIGS. 10 and 11 shows longitudinal cross-sections of the
embodiments shown in FIGS. 8 and 9, respectively, containing all
components mentioned in FIGS. 8 and 9. Grooves 170 are not seen in
this illustration since the sections are cut along the grooves 170.
This section also shows the presence of the narrow flow channels
130 that are perpendicular to the grooves 170.
Open Disc Formats
[0114] Referring to FIG. 12, there is illustrated an embodiment of
the present optical bio-disc 110 that utilizes a reflective
open-face or open-disc format. In this embodiment, the substrate
120 is implemented as a distal layer relative to the interrogation
beam 152. The reflective layer 142, showing tracking grooves 170,
is next provided as illustrated. The bottom layer or proximal layer
relative to the beam in this embodiment is provided by the active
layer 144.
[0115] In this embodiment, a capture DNA 172 may be depended
downwardly when the disc is loaded in the drive (FIG. 1). In this
open-face format, the other assay reactants are brought into
proximity with the capture DNA 172 by a variety of different
methods which include, for example, depositing a test sample on the
disc with a pipette. In this embodiment, the target zones 140 are
simply formed by the application of a small volume of capture DNA
172 solution to the active layer 144 to form clusters of capture
DNA 172 in desired locations on the active layer 144 as
illustrated.
[0116] Referring next to FIG. 13, there is shown an alternate
embodiment of the open-face or open-disc optical bio-disc. In this
transmissive disc 111 embodiment, the substrate 120 is implemented
as a proximal layer relative to the interrogation beam 152. The
thin semi-reflective layer 143, showing tracking grooves 170, is
next provided as illustrated. The thin semi-reflective layer 143 is
relatively thinner than the reflective layer 142 described in FIG.
12 to allow transmission of some desired percentage of the incident
beam, while some portion is reflected back to facilitate tracking
on the disc. Detection of the transmitted beam 156 is then carried
out by the top detector 158 as discussed above in conjunction with
FIGS. 5 and 7. The top layer or distal layer relative to the
interrogation beam in this embodiment is provided by the active
layer 144. In this embodiment, the capture DNA 172 may be oriented
upward when the disc is loaded in the drive (FIG. 1).
[0117] In this transmissive (or semi-reflective) open-face format,
the assay reactants are brought into proximity with the capture DNA
172 by a variety of different methods which include, for example,
depositing a test sample on the disc with a pipette. In this
alternative embodiment, the target zones 140 are simply formed by
the application of a small volume of capture DNA 172 solution to
the active layer 144 to form clusters of capture DNA 172 in desired
locations on the active layer 144 as illustrated. Detection of the
beam carrying information about the analyte for this embodiment is
achieved by use of a top detector 158.
[0118] FIGS. 14 and 15 show longitudinal cross-sections of the
embodiments shown in FIGS. 12 and 13, respectively, containing all
components mentioned in FIGS. 12 and 13. Grooves 170 are not seen
in this illustration since the sections are cut along the grooves
170.
Attaching Capture DNA, Target DNA, and Conjugated Enzyme
[0119] Referring to FIG. 16, there is portrayed an enlarged
detailed partial cross sectional view showing the active layer 144
and the substrate 120 of the present bio-disc 110 or 111. FIG. 16
also illustrates the capture DNA 172 attached to the active layer
144 in the target zone. In this embodiment, the capture DNA 172
binds onto the active layer 144 through passive adhesion. However,
the capture DNA 172 may also be bound to the active layer by
covalent binding as discussed above. As indicated, the capture DNA
172 is situated within the target zone. The bond between the
capture DNA 172 and the active layer 144 is sufficient so that the
capture DNA 172 remains attached to the active layer 144 within the
target zone 140 when the disc is rotated.
[0120] FIG. 16 also depicts the target DNA 174. In this embodiment
of the present invention, the target DNA 174 includes an affinity
agent 176, such as, for example, biotin. The capture DNA is
selected such that a portion of the capture DNA sequence is
complementary to a potion of the sequence of the target DNA,
allowing hybridization between the capture and target DNA as the
target DNA flows toward the capture DNA 172 as shown.
[0121] Referring next to FIG. 17, there is shown an enlarged view
similar to FIG. 16, showing the introduction of enzymes 178. As
illustrated in FIG. 17, the enzymes 178 are conjugated with a
binding agent 180 that includes receptors 182. The binding agent
180 includes streptavidin, neutravidin and the like. In this
embodiment of the present invention, the target DNA 174 hybridizes
with capture DNA 172 and the affinity agent 176 links with the
receptor 182 of the binding agent 180 to anchor the enzyme within
the target zone.
[0122] Referring now to FIG. 18, there is shown an alternate method
of introducing the enzyme 178. FIG. 18 illustrates the enzyme
pre-conjugated to the target DNA 174 via interaction between an
affinity agent 176, such as biotin, on the target DNA and a binding
agent 180, conjugated to the enzyme 178. The binding agent 180
includes affinity agent 176. The binding agent 180 may be, for
example, streptavidin or neutravidin. In this embodiment, the
pre-conjugated enzyme-DNA complex hybridizes directly to the
capture DNA probes, which are attached to the active layer 144 in
the target zone thereby imobilize the enzyme within the target
zone.
Using a Signal DNA to Attach Conjugated Enzyme to the Target
Zone
[0123] With reference to FIG. 19, there is illustrated a detailed
partial cross sectional view showing the active layer 144 and the
substrate 120 of the present bio-disc according to the embodiment
utilizing the enzyme 178 attached to a signal DNA 184. In this
embodiment, biotinylated signal DNA 184 is linked to the enzyme 178
via the binding agent 180 and the receptors 182 associated
therewith. In this method, the signal DNA 184 is non-complementary
to the capture DNA 172, while the target RNA or DNA 174 contains
separate sequences that are complementary to the signal DNA 184 and
the capture DNA 172. In this embodiment, the target RNA or DNA 174
acts as a "bridge" to attach the signal DNA 184 to the capture DNA
172, as shown in FIG. 20. This places the enzymes 178 in the target
zone when the target RNA or DNA 174 is present. In this embodiment,
the signal DNA 184 may be pre-conjugated with the enzyme prior to
hybridizing with the target DNA. Alternatively, biotinylated signal
DNA can be hybridized to the target DNA first, then exposed to the
conjugated enzyme.
The Enzyme-Substrate Reaction and the Formation of an Insoluble
Precipitate
[0124] Referring to FIG. 21, there is shown a detailed partial
cross sectional view of a target zone including the active layer
144, the substrate 120, and target RNA or DNA 174 hybridized to the
capture DNA 172. The target RNA or DNA 174 may contain an affinity
agent 176 such as biotin; alternatively, the target nucleic acid
can be hybridized to a signal DNA 184 with an affinity agent 176.
Thus, the enzyme 178 may be attached directly to the target RNA or
DNA 174 or to signal DNA 184 as described in FIGS. 16-20. FIG. 21
also shows the addition of an enzyme substrate 186.
Enzyme-substrate reaction 194 occurs as soon as the substrate comes
in contact with the enzyme 178. The resulting enzyme-substrate
reaction 194 produces a signal that is detectable by a disc type
reader. The signal generated may consist of precipitate formation,
enzyme substrate luminescence, and/or enzyme substrate color change
or formation.
[0125] FIGS. 22A-22D illustrate a method according to the present
invention for detecting or determining the presence of target RNA
or DNA 174 in a sample by precipitate formation through an
enzyme-substrate reaction 194 in conjunction with the optical
bio-discs 110 or 111 according to the present invention. FIGS.
22A-22D are detailed partial cross sectional views of a target zone
showing the active layer 144, the substrate 120, and target RNA or
DNA 174 hybridized to the capture DNA 172. The target RNA or DNA
174 may contain an affinity agent 176 or, alternatively, can be
hybridized to a signal DNA containing an affinity agent. The enzyme
can then be attached to either the target RNA or DNA 174 or signal
DNA 184 as described above in conjunction with FIGS. 16-20.
[0126] Referring now to FIG. 22A, there is depicted the formation
of an insoluble product 188 by the enzyme-substrate reaction 194
(FIG. 21). FIG. 22B further illustrates more massive amounts of
insoluble product 188 formed by the enzyme reaction, which fill the
capture or target zone.
[0127] Referring next to FIG. 22C, there is shown the insoluble
product 188 aggregating and forming insoluble pellets or
precipitates 190, which are deposited within the target zone. The
active layer 144 may facilitate aggregation and deposition of the
insoluble products 188, resulting in the formation of pellets
comprising precipitates 190 that adhere to the active layer.
[0128] Next, FIG. 22D shows an expanded view of the complete
aggregation of the insoluble product 188 forming large precipitate
particles 190, relative to the DNA deposited on the target zone.
These large aggregated particles or pellets 190 can then be
detected using a disc reader.
Target Detection Methods
[0129] FIG. 23 is a longitudinal cross sectional view (cut along
the tracking grooves 170) of the mixing chamber 134 of the
reflective disc 110 showing the inlet port 122. FIG. 23 includes
the substrate 120, the reflective layer 142 and the active layer
144 applied over the reflective layer 142. As further illustrated
in FIG. 23, the plastic adhesive member 118 is applied over the
active layer 144. FIG. 23 also shows the cap portion 116 and the
reflective surface 146 associated therewith. Thus when the cap is
applied to the plastic adhesive member 118 including the desired
cut-out shapes, the flow channel is thereby formed, including, in
this embodiment, the mixing chamber 134.
[0130] FIG. 24 is a longitudinal cross sectional view (cut along
the tracking grooves 170) of the mixing chamber 134 of the
transmissive disc 111 showing the inlet port 122. FIG. 24 is an
alternate embodiment to the reflective disc 110 illustrated in FIG.
23, wherein a transmissive disc format is utilized as shown in FIG.
7. FIG. 24 illustrates the transmissive disc format with the clear
cap portion 116 and the thin semi-reflective layer 143 on the
substrate 120, as discussed in FIG. 7. FIG. 24 also shows the
active layer 144 applied over the thin semi-reflective layer 143.
The thin semi-reflective layer 143 may be made from a metal such as
aluminum or gold which is approximately 100-300 Angstroms thick,
allowing the incident or interrogation beam to penetrate and pass
through the disc and thus be detected by the top detector 158.
[0131] FIG. 25 is a longitudinal cross sectional view (cut along
the tracking grooves 170) of a mixing chamber 134 of the reflective
disc 110 showing an inlet port 122. Similar to FIG. 23, the
principle elements of the reflective disc as described in FIG. 2A
are also present. FIG. 25 further illustrates the enzymes 178, each
conjugated with the binding agent 180, pre-loaded into the mixing
chamber 134.
[0132] FIG. 26 is a longitudinal cross sectional view (cut along
the tracking grooves 170) of the mixing chamber 134 of the
transmissive disc 111 showing the inlet port 122. Similar to FIG.
24, the principle elements of the reflective disc 110 as described
in FIG. 3A are also present. In this alternative embodiment, the
enzymes 178, each conjugated with the binding agent 180, are
pre-loaded into the mixing chamber 134.
[0133] Referring next to FIGS. 27-29, there are illustrated
longitudinal cross sectional views of the reflective optical
bio-disc 110, similar to FIG. 6, which contains all components for
the reflective disc 110 as discussed for FIG. 6. FIG. 27
illustrates the capture DNA 172 attached to the active layer 144
within the target zone 140. In this embodiment, capture DNA 172
attaches to the active layer 144 by either passive adhesion or
covalent bonding. Application of a small volume of capture DNA 172
solution to the active layer 144 forms clusters of capture DNA 172
within the area of the target zone 140, as illustrated.
[0134] FIG. 28 shows the flow channel 130 and target zone 140 after
hybridization of the target DNA 174 with the capture DNA 172. In
addition, FIG. 28 shows the affinity agent 176 on the target RNA or
DNA 174 or, alternatively, on signal DNA, as employed in the
present invention and discussed above in conjunction with FIGS. 19
and 20. In one embodiment of the present invention, the affinity
agent 176 includes biotin or any equivalent affinity agent.
[0135] FIG. 29 shows enzymes 178 bound to the hybridized capture
DNA/target DNA. These enzymes are conjugated with binding agent 180
that binds to an affinity agent 176, either directly on the target
DNA or on signal DNA hybridized to the target RNA or DNA.
[0136] With reference now to FIGS. 30-32, there are shown yet
another longitudinal cross sectional views of the transmissive
optical bio-disc 111, similar to FIG. 7, which contains all
components for the transmissive disc as discussed for FIG. 7. In
this embodiment, the capture DNA 172 is attached to the active
layer 144 within the target zone either by passive adhesion or
covalent bonding. Application of a small volume of capture DNA 172
solution to the active layer 144 forms clusters of capture DNA 172
within the area of the target zone 140 as illustrated in FIG.
30.
[0137] FIG. 31 shows the flow channel 130 and target zone after
hybridization of target RNA or DNA with the capture DNA. In
addition, FIG. 31 shows the affinity agent 176 on the target RNA or
DNA or, alternatively, on signal DNA. In one embodiment of the
present invention, the affinity agent 176 includes biotin or any
equivalent affinity agent.
[0138] FIG. 32 depicts the flow channel 130 and the target zone 140
after hybridization of target RNA or DNA with the capture DNA. In
addition, FIG. 32 shows enzymes 178 as employed in the present
invention. These enzymes 178 are conjugated with binding agent 180
that bind to the affinity agent 176 on the target DNA (or on the
signal DNA hybridized to the target RNA or DNA).
[0139] Referring now to FIGS. 33A-33G, there is illustrated a
method according to the present invention for detecting or
determining the presence of target DNA 174 in a test sample in
conjunction with the optical bio-disc according to the present
invention. As shown in FIGS. 33A-33G and discussed above with
reference to FIGS. 2 and 3, the optical bio-disc includes the cap
portion 116, the adhesive member 118 and the substrate 120. The
disc format may be either the reflective disc format 110 or the
transmissive disc format 111 with varying elements to each
respective cap portion 116 and substrate 120 as described above in
conjunction with FIGS. 2 and 3.
[0140] Although the disc composition between the different disc
formats may vary, the biochemical interactions remain the same. In
FIG. 33A, a pipette 192 is loaded with a test sample of DNA that
has been linked to affinity agent 176. The test sample is injected
or deposited into the flow channel 130 through the inlet or
injection port 122. As the flow channel 130 is further filled with
test sample, the target DNA 174 begin to flow or move down the flow
channel 130 as illustrated in FIGS. 33A and 33B. When target DNA
174 of a specific sequence is present in the test sample, the
target DNA 174 hybridizes with the capture DNA 172 as shown in FIG.
33B. In this manner, the target DNA 174 with its affinity agent 176
is retained within the target zone. Hybridization may be further
facilitated by adjusting the temperature of the disc or the flow
channel and/or ionic strength of the hybridization buffer to
optimize annealing of the nucleic acids.
[0141] After hybridization, the flow channel 130 may be washed to
clear the target zone 140 of any unattached DNA in the sample.
After removing the unattached DNA in the sample, enzymes 178 with
binding agent 180 are introduced in the channel, as shown in FIG.
33C. In one embodiment, enzymes are introduced via the input port
122, as shown in FIG. 33C. In an alternative embodiment, enzyme
buffer is introduced through an enzyme buffer port into a side
chamber (not shown), which is in fluid communication with the flow
channel and which contains a pad or membrane onto which enzyme has
been dried as described above in conjunction with FIG. 4. The
buffer solubilizes the enzyme, which then flows into the flow
channel 130.
[0142] As the flow channel 130 is filled with enzyme solution, the
enzymes 178 begin to flow or move down the flow channel 130 as
illustrated in FIGS. 33C and 33D. When the enzyme comes into close
proximity with the target DNA 174 hybridized in the target zone 140
with the capture probe, the enzymes 178 bind to the target DNA 174
via the interaction between the affinity agent 176 and the binding
agent 180, as illustrated in FIG. 33D.
[0143] After enzymes 178 bind to the affinity agent 176, the flow
channel 130 may be washed to clear the target zone 140 of any
unattached enzyme 178. As shown in FIG. 33E, upon removal of
unattached enzyme 178 in the solution, enzyme-reactive substrates
186 are then introduced in the channel as previously described with
reference to FIG. 21. As the flow channel 130 is filled with enzyme
substrate 186, the enzyme substrate 186 begin to flow or move down
the flow channel 130 as illustrated in FIG. 33E. When the substrate
comes in contact with the enzyme on the target DNA, the enzyme
substrate reaction 194 occurs, which results in the production of a
signal agent as described with reference to FIGS. 22A-22D. The
signal agent may be color production or luminophore production, or
it may be insoluble precipitate 190 formation as illustrated in
FIG. 33G and FIGS. 22A to 22D.
[0144] The interrogation beam 152 may then be scanned through the
target zone 140 to determine the presence of signal agents as
illustrated in FIGS. 33F and 33G. In the event no target DNA 174 is
present in the test sample, no enzyme substrate reaction 194 will
occur and the signal agents will not be present. In this case, when
the interrogation beam 152 is directed into the target zone 140, a
negative or baseline reading will result, thereby indicating that
no target DNA 174 was present in the sample.
[0145] Referring next to FIGS. 34A-34H, there is illustrated
another method according to the present invention for detecting or
determining the presence of target RNA or DNA 174, in a sample of
DNA or RNA, in conjunction with the optical bio-disc 110 or 111
according to the present invention. As shown in FIGS. 34A-34H and
discussed in FIGS. 2 and 3, the optical bio-disc 110 or 111
includes the cap portion 116, the adhesive member 118 and the
substrate 120. The disc format may be either the reflective disc
format 110 or the transmissive disc format 111 with varying
elements to each respective cap portion 116 and substrate 120 as
described in FIGS. 2 and 3. Although the disc composition of
different disc formats may vary, the biochemical interactions
remain the same.
[0146] In FIG. 34A, the pipette 192 is loaded with a test sample of
DNA or RNA. The test sample is injected or deposited into the flow
channel 130 through inlet or injection port 122. As the flow
channel 130 is further filled with test sample, the target RNA or
DNA 174 begin to flow or move down the flow channel 130 as
illustrated in FIG. 34A. When target RNA or DNA 174 of a specific
sequence is present in the test sample, the target RNA or DNA 174
hybridizes with the capture DNA 172, as shown in FIG. 34B.
Hybridization may be further facilitated by adjusting the
temperature of the disc and/or the flow channel and/or adjusting
the ionic strength of the hybridization buffer to optimize
annealing of the nucleic acids. The techniques for optimizing
oligonucleotide hybridization is well know in the art.
[0147] After hybridization, the disc may be washed to clear the
target zone 140 of any unattached DNA or RNA sample. Upon removing
the unattached DNA or RNA in the sample, signal DNA probes 184
containing an affinity agent 176 are introduced into the flow
channel 130, as shown in FIG. 34B. As the flow channel 130 is
further filled with signal DNA 184, the signal DNA begins to flow
or move down the flow channel 130 as illustrated in FIGS. 34B and
34C. When signal DNA 184 comes in contact with the target RNA or
DNA 174, which is hybridized with the capture DNA 172 in the target
zone 140, the signal DNA 184 hybridizes with the target RNA or DNA
174 and is retained in the target zone as shown 34C and above in
FIG. 20. In this manner, the target RNA or DNA 174 and signal DNA
184 probe with its affinity agent 176 are retained within the
target zone 140. Hybridization may be further facilitated by
adjusting the temperature of the disc and/or the flow channel to
optimize annealing of the nucleic acids.
[0148] After hybridization, the flow channel 130 may be washed to
clear the target zone 140 of any unattached signal DNA 184 probes.
As shown in FIG. 34D, after the removal of the unattached signal
DNA 184 probes in the sample, enzymes 178 are introduced in the
channel. In one embodiment, enzymes are introduced via the input
port 122, as shown in FIG. 34D. In an alternative embodiment,
enzyme buffer is introduced through an enzyme buffer port into a
side chamber (not shown), which is in fluid communication with the
flow channel and which contains a pad or membrane onto which enzyme
has been dried as discussed above in conjunction with FIG. 4. The
buffer solubilizes the enzyme, which then flows into the flow
channel.
[0149] As the flow channel 130 is filled with enzyme solution, the
enzymes 178 begin to flow or move down the flow channel 130 as
illustrated in FIG. 34D. When the enzyme 178 comes into close
proximity with the hybridized signal DNA/target DNA/capture DNA
complex in the target zone 140, the enzymes 178 bind to the signal
DNA 184 via the interaction between the affinity agent 176 and
binding agent 180, best shown in FIG. 20 and as further illustrated
in FIG. 34E.
[0150] After enzyme binding, the flow channel 130 may be washed to
clear the target zone 140 of any unattached enzyme 178. After
removing unbound enzymes 178, the enzyme substrate 186 is
introduced in the channel, as illustrated in FIG. 34F. As the flow
channel 130 is filled with enzyme substrate 186, the enzyme
substrate 186 begin to flow or move down the flow channel 130. When
the enzyme substrate 186 comes in contact with the enzyme 178 bound
to the target zone 140 via the signal DNA/target DNA/capture DNA
complex, the enzyme substrate reaction 194 occurs, which results in
the production of a signal agent, as shown in FIG. 34G. The signal
agent may be a color production, luminophore production,
fluorescence, or the formation of insoluble precipitate 190 as
illustrated in FIG. 34H and FIGS. 22A to 22D.
[0151] The interrogation beam 152 is then scanned through the
target zone 140 to determine the presence of signal agents, as
illustrated in FIGS. 34G and 34H. If no target DNA 174 is present
in the test sample, no enzyme substrate reaction 194 will occur and
the signal agents will not be present. In this case, when the
interrogation beam 152 is directed into the target zone 140, a
negative or baseline reading will result, thereby indicating that
no target DNA was present in the sample.
[0152] With reference now to FIGS. 35A-35F, there is illustrated
yet another method according to the present invention for detecting
or determining the presence of target RNA or DNA 174 in a test
sample in conjunction with the optical bio-disc according to the
present invention. As shown in FIGS. 35A-35F and discussed with
reference to FIGS. 2 and 3, the optical bio-disc includes the cap
portion 116, the adhesive member 118 and substrate 120. The disc
format may be either the reflective disc format 110 or the
transmissive disc format 111 with varying elements to each
respective cap portion 116 and substrate 120 as described in FIGS.
2 and 3. The target RNA or DNA 174 may be detected using either
disc format. Although the disc composition of different disc
formats may vary, the biochemical interactions remain the same.
[0153] In FIG. 35A, enzymes 178 with the associated binding agent
180 are pre-loaded in a mixing chamber 134. In FIG. 35B, the
pipette 192 is loaded with a test sample solution containing DNA
that has been linked to affinity agent 176 or, alternatively, a
test sample solution of DNA or RNA that has been pre-hybridized
with a signal DNA probe 184 containing an affinity agent. The test
sample solution is injected or deposited into the flow channel 130
through inlet or injection port 122.
[0154] As the flow channel 130 is further filled with test sample
solution, the target RNA or DNA 174, the signal DNA 184 probe (if
present), and the enzymes 178 begin to flow or move down the flow
channel 130 as illustrated in FIG. 35B. FIG. 35B also shows the
binding of the pre-loaded enzymes 178 with the target DNA 174 or,
if present, with the signal DNA/target DNA complex, through the
interaction of the affinity agent and binding agent.
[0155] When target RNA or DNA 174 of a specific sequence is present
in the test sample, the target RNA or DNA 174 hybridizes with the
capture DNA 172 as shown in FIG. 35C. In this manner, the enzymes
178, attached to the target DNA 174 as described in FIG. 18 (or to
the target RNA or DNA/signal DNA complex as described in FIGS. 19
and 20), are retained within the target zone 140. Hybridization may
be further facilitated by heating the disc or a local area
thereof.
[0156] After hybridization, the flow channel 130 may be washed to
clear the target zone 140 of any unattached target RNA or DNA,
after which enzyme substrate 186 is introduced into the channel, as
shown in FIG. 35D. As the flow channel 130 is filled with enzyme
substrate 186, the enzyme substrate 186 begin to flow or move down
the flow channel 130. When the substrate comes in contact with the
enzyme 178, the enzyme substrate reaction 194 occurs, producing a
signal agent, as shown in FIG. 35E. The signal agent may be color
production or luminophore production. The signal agent may also be
the formation of a precipitate 190, as illustrated in FIG. 35F and
above in FIGS. 22A-22D.
[0157] The interrogation beam 152 is scanned through the target
zone 140 to determine the presence of signal agents, as illustrated
in FIGS. 35E and 35F. In the event no target DNA 174 is present in
the test sample, no enzyme substrate reaction 194 will occur and
the signal agents will not be present. In this case, when the
interrogation beam 152 is directed into the target zone 140, a
negative or baseline reading will result thereby indicating that no
target DNA 174 was present in the sample.
Data Generated from the Assay
[0158] FIG. 36 is an example of a data output collected using an
optical disc reader and its respective software. The output data
illustrated in FIG. 36 was collected using a reflective disc format
(FIGS. 2 and 6) with 8 target zones 140 and an event counting
software. The event counting software used for reaction product
detection in the optical disc reader is disclosed in the above
referenced commonly assigned U.S. Provisional Application
60/291,233. In this embodiment, the first two target zones were not
used in this experiment (counting from left to right). Target zone
3 was a blank (no capture probe) negative control. Target zone 4
contained a single strand DNA bound to the active layer 144 with an
affinity agent 176 attached, which served as the positive control.
Target zones 5, 6 and 7 contained capture DNA 172 attached to the
active layer 144, which was selected to be complementary to various
target DNA sequences: NPTII, CamV, and NosT, respectively. Target
zone 8 contained a mixture of all the three capture DNA 172 probes,
NPTII, CamV, and NosT, attached to the active layer 144.
Accordingly, target zone 8 will bind any or all of the three target
DNA molecules. In this test, NPTII showed the highest signal,
followed by NosT then CamV. The sample used in this test were
amplicons from a multiplex PCR amplification. Further details
relating to other aspects associated with data acquisition,
processing, collecting, and reporting are disclosed in, for
example, commonly assigned co-pending U.S. Provisional Patent
Application Ser. No. 60/291,233 entitled "Variable Sampling Control
For Rendering Pixelation of Analysis Results In Optical Bio-Disc
Assembly And Apparatus Relating Thereto" filed May 16, 2001, U.S.
Provisional Patent Application Ser. No. 60/348,767 entitled
"Optical Disc Analysis System Including Related Signal Processing
Methods and Software" filed Jan. 14, 2002, and U.S. Provisional
Patent Application Ser. No. 60/352,625 entitled "Logical Triggering
Methods and Apparatus for Use with Optical Analysis Discs and
Related Disc Drive Systems" filed Jan. 28, 2002, all of which are
incorporated herein by reference in their entirety.
[0159] Referring to FIG. 37, there is shown a series of pictorial
representations illustrating image detection according to one
embodiment of the present invention. These results demonstrate
microscopic micrographs of pellets/precipitate on the active layer
of the transmissive disc format as described above in conjunction
with FIG. 22D. Various concentrations (0 to 5 uM) of single
stranded DNA containing an affinity agent 176 were deposited onto
the active layer 144. Enzymes 178 with binding agent 180 were then
allowed to bind to the DNA on the active layer 144 via
affinity-binding agent interaction. The solution of enzyme
substrate 186 was then introduced and pellet formation was observed
in a concentration dependent manner.
[0160] The test results of any of the test methods described above
may be readily displayed on the display monitor 114 shown in FIG.
1. The optical bio-disc 110 or 111 according to the present
invention may include encoded software that interacts with the
drive, the controller 164, the processor 166, and the analyzer 168
as shown in FIGS. 1 and 5. The interactive software is implemented
to facilitate the methods described herein and display the results.
In the preferred embodiment, the software is used to quantify
signal differentiation based upon luminescence, color changes,
and/or pellet/precipitate formation.
Applications of the Present Invention
[0161] As will be appreciated, the enzyme-based assay for detecting
and analyzing nucleic acids in test samples has a number of
potential applications. For example, this assay can be used to test
for the presence of specific disease causing agents, such as
viruses or bacteria, in biological samples taken from patients.
Capture DNA probes specific for various disease causing organisms
can be deposited in specified target zones on a bio-disc in an
ordered array (or microarray). Multiplexed PCR reactions, using
mixed primer sets directed to these various disease causing
organisms, can be conducted on patient samples, and the resulting
amplicons used as target DNA probes in the bio-disc assay system.
Enzyme reactions can be monitored as described above to determine
which target zones in the ordered array produce a detectable
signal. These target zones can then be correlated to the specific
capture DNA probe deposited at that location, allowing clinicians
to quickly identify which target DNA molecules are amplified from
the patient sample and, accordingly, which disease causing organism
is likely present in the patient.
[0162] Similarly, the present invention can be used to test water
and soil samples for specific microorganisms and is also helpful in
monitoring agricultural products as, for example, in testing for
the presence of genetically modified organisms (GMO). For example,
crop samples can be amplified using primer sets specific for a
marker gene in a genetically modified plant and for intrinsic plant
genes, such as lectin or zein. The amplified target DNA can then be
used on bio-discs containing capture probes for both the marker
gene and for intrinsic plant genes. Calibration controls can also
be run simultaneously, in which known concentrations of the GMO are
amplified for use as target DNA. By comparing the reaction products
of the unknown sample with the calibration curve, the presence of
genetically modified organisms in a sample can be detected and
quantified.
[0163] An overview of how the present invention can be used in
detecting and quantifying GMOs is shown in FIG. 38. A crop product,
for example, corn, is suspected of containing some portion of
genetically modified corn. A marker gene is identified that is
specific for the genetically modified corn, and capture probes are
synthesized that are specific for that marker gene. Intrinsic plant
gene probes, such as those specific for the gene lectin, are also
synthesized.
[0164] An optical bio-disc containing at least five flow channels
is prepared with five target zones in each flow channel (as will be
appreciated, these numbers may be varied depending on the
particular assay): two for the marker gene capture probe, two for
the lectin capture probe (as positive controls), and one with a
non-specific probe, as a negative control. Each of these flow
channels is depicted in FIG. 38. Five separate PCR reactions are
performed to produce target DNA using primer sets directed to both
the marker gene and the lectin gene. Four of the reactions are
performed using template DNA from calibration standards containing
known quantities of GMO (e.g., 0%, 0.1%, 0.5% or 1% GMO); the fifth
reaction is performed using the unknown sample. Each set of DNA
sample is hybridized to one of the flow channels and the enzyme
reaction is performed as described herein. The detectable signal,
as, for example, measured by the amount of an insoluble precipitate
or pellet formation, for the unknown sample is compared to those of
the calibration curve, allowing the presence (and amount) of GMO in
the unknown sample to be determined. If the signal in the unknown
sample is too high to permit quantification, it may be necessary to
repeat the procedure with dilutions of the unknown sample.
Optical Bio-discs with Equi-radial Fluidic Circuits
[0165] The optical bio-disc systems and methods for detecting
specific sequences of oligonucleotides, as described above, may be
readily implemented in an optical bio-disc with an equi-radial
fluidic circuit. Details regarding optical bio-dics with
equi-radial fluidic circuits is disclosed in, for example, commonly
assigned co-pending U.S. Provisional Patent Application Ser. No.
60/353,014 entitled "Optical Discs Including Equi-Radial and/or
Spiral Analysis Zones and Related Disc Drive Systems and Methods"
filed Jan. 29, 2002, which is incorporated herein by reference in
its entirety.
EXPERIMENTAL EXAMPLES
[0166] Having generally described the invention, the same will be
more readily understood through reference to the following
examples, which are provided by way of illustration, and are not
intended to be limiting of the present invention.
Example 1
Spin Coating the Bio-disc
[0167] Fresh polystyrene solution was prepared by adding 3 g
polystyrene pellets (Sigma cat. no. 182427; molecular
weight=280,000) to 310 ml toluene and stirring for 1 hour using a
teflon stir bar and a stir plate. After the polystyrene was
completely dissolved in the toluene, 68 ml reagent grade
isopropanol was slowly added while stirring.
[0168] A stock solution of nitrocellulose was prepared by diluting
a nitrocellulose collodium solution (4-8% in ethanol/diethylether,
Fluka cat. no. 09986, lot no. 389973/1 30299) 1:5 in reagent grade
ethanol. Prior to spin coating, the stock solution was diluted 1:10
with reagent grade ethanol and filtered using a 0.2 .mu.m syringe
filter.
[0169] A polycarbonate disc having a 200 Angstrom gold
semi-reflective layer (BTI Optical Bio-disk Set FDL21:E001308) was
placed on a "spin coater," or modified centrifuge, with the
reflective surface up. While rotating the disc on the spin coater,
the reflective surface was cleaned with reagent grade alcohol.
[0170] The spin coater was set to start spinning at 2500 rpm,
followed by acceleration to 4000 rpm within 10 seconds. During this
10-sec acceleration, a steady stream of polystyrene solution was
applied to the disc using a pasteur pipette, with the polystyrene
solution applied from the outer edge to the inner side in one
smooth stroke.
[0171] The spin coater speed was then adjusted to 1500 rpm, and the
diluted and filtered nitrocellulose solution was applied onto the
inner portion of the disc in a steady stream using a pasteur
pipette.
Example 2
Preparing the Bio-disk for the Enzyme Assay
[0172] The disc from Example 1 was placed on a CD assembler/spindle
with the nitrocellulose layer up. Between about 0.5 to 2.0 .mu.l of
1 .mu.M oligo probes (capture DNA) in 1 M NH4OAc were applied to
the disk at defined target zones. The droplets of capture DNA were
dried onto the nitrocellulose at 37.quadrature.C.
[0173] A cover disc containing U-shaped fluidic circuits (50 .mu.M
adhesive; Fraylock, DBL243a) was applied using a disk assembler
spindle, and the disc was run through a wringer to seal the two
disks.
Example 3
General Enzyme DNA Assay
[0174] DNA blocking solution (1% bovine serum albumin [BSA],
5.times. Denhardt's solution, 0.1 mg/ml salmon sperm DNA, 200 mM
KCl, 10 mM MgCl2, 50 mM Tris, pH 7.4) was degassed in a vacuum
desiccator and injected into the fluidic circuits of a bio-disc
prepared as in Example 2, taking care that no air bubbles remained
in the circuits. The bio-disc was then icubated at room temperature
for 30 to 60 minutes.
[0175] The DNA blocking solution was removed, and the fluidic
circuits washed with hybridization buffer (200 mM NaCl, 10 mM
MgCl2, 50 mM Tris, pH 7.4) injected into the fluidic circuits using
a syringe. PCR amplicons (target DNA amplified using biotinylated
primer sets, purified using the Qiagen QIAquick PCR Purification
Kit, cat. no. 28104, lot no. 10927932, and eluted using
hybridization buffer) were denatured at 95.quadrature.C for 5
minutes and immediately placed on ice for 5 minutes.
[0176] The denatured amplicons were added to the appropriate
fluidic circuits (10 .mu.l per fluidic circuit) and allowed to
hybridize for 1.5 to 2 hours at room temperature. Following
hybridization, the fluidic circuits were washed with hybridization
buffer using a syringe.
[0177] Neutravidin-Horseradish Peroxidase Conjugated enzyme (N-HRP;
Pierce product no. 31001, lot no. BK46404) was diluted 1:5000 in
hybridization buffer, and 12 .mu.l was applied to each fluidic
circuit. The disc was then incubated at room temperature for 15
minutes.
[0178] The fluidic circuits were then washed with hybridization
buffer using a syringe, and 12 .mu.l of TMB Substrate in Stable
hydrogen peroxide buffer (Calbiochem cat. no. 613548, lot B34202)
was added to each fluidic circuit. The enzyme reaction was allowed
to proceed for 5 mintues, after which the reaction was stopped by
flushing the fluidic circuits with distilled water using a
syringe.
[0179] Each fluidic circuit was sealed with tape, and the bio-disc
was then placed in a disc-reader, similar to that shown in FIG. 5,
and scanned with a 780 nm lightbeam, with the light transmitted
through the bio-disc at each target zone measured to detect changes
in the amplitude of the transmitted light.
Example 4
Enzyme DNA Assay Used to Identify Brucella Strains
[0180] A bio-disc with 6 target zones was prepared as in Example 2,
with 1.6 .mu.l of 10 .mu.M DNA oligonucleotides specific to one of
the Brucella strains applied to three of the target zones, as
indicated in Table 2, below. One target zone contained a mix of all
three Brucella species, one target zone contained biotinylated DNA
(positive control), and one target zone contained no capture DNA
(background).
[0181] Brucella sp. genomic DNA was subjected to PCR amplification
using forward/reverse primer sets directed to B. melitensis to
generate target B. melitensis amplicons. Each reaction contained 1
ng/.mu.l Brucella DNA, 0.2 .mu.M biotinylated forward and reverse
primers, 0.2 mM dNTPs, 0.05 U/.mu.l Taq polymerase, 3.0 mM
MgCl.sub.2 and 1.times.PCR buffer (Qiagen; 15 mM MgCl.sub.2). The
thermocycle conditions were: [0182] Step 1: 95.degree. C. for 12.5
minutes [0183] Step 2: 95.degree. C. for 0.5 minute [0184] Step 3:
57.degree. C. for 0.5 minute [0185] Step 4: 72.degree. C. for 0.5
minute [0186] Step 5: Repeat Steps 2-4 34 times [0187] Step 6:
72.degree. C. for 5.0 minutes
[0188] The enzyme DNA assay was performed as described in Example
3, both without the addition of the target B. melitensis amplicons
(results indicated in Table 2) and with the addition of purified
target B. melitensis amplicons (results indicated in Table 3).
Values from Tables 2 and 3 are event counts collected using an
optical disc reader using the reflective disc format. The counting
area was a rectangle, 940 .mu.m.times.2300 .mu.m; event count
amplitude was between 225-500. TABLE-US-00002 TABLE 2 No B.
melitensis Amplicons Positive Bkgd B. abortus B. melitensis B. suis
B. mix Control 234 3338 723 1635 624 74696 720 1906 343 401 630
106775 Avg. 477 2622 533 1018 627 90736 SD 344 1013 269 873 4 22683
CV 72% 39% 50% 86% 1% 25%
[0189] TABLE-US-00003 TABLE 3 Purified B. melitensis Amplicons
Positive Bkgd B. abortus B. melitensis B. suis B. mix Control 1179
5620 43185 2635 23020 65981 693 5783 35862 2636 20792 68436 754
4491 32250 2057 16263 81495 394 5299 43165 2756 22929 46564 Avg.
755 5298 38616 2521 20751 65619 SD 323 575 5467 315 3164 14413 CV
43% 11% 14% 12% 15% 22%
[0190] As shown above, the B. melitensis target zone produces a
much higher signal than the other Brucella species when the B.
melitensis amplicon is used in the present invention.
Example 5
Concentration Dependent Detection of NosT Amplicon in Genetically
Modified Plant Material
[0191] Bio-discs were prepared as in Example 2, with NosT 52-mer
oligonucleotide capture probes applied to the target zones. NosT is
a marker gene for genetically modified plant material.
[0192] GMO reference materials, consisting of soya bean powder
containing different mass fractions of powder from genetically
modified soya beans, were obtained from Fluka BioChemika, certified
reference material IRMM-410R (0, 0.1, 0.5, 1, 2, 5% Roundup Ready
Soya). DNA was extracted from each of the six reference materials,
using the WIZARD.RTM. Magnetic DNA Purification System for Food
(Promega, Madison, Wis.).
[0193] The extracted DNA was subjected to PCR amplification using
biotinylated forward/reverse primer sets directed to NosT to
generate biotinylated 280-mer target NosT amplicons. Each reaction
contained 50 ng extracted DNA, 0.2 .mu.M biotinylated forward and
reverse primers, 0.2 mM dNTPs, 0.05 U/.mu.l Taq polymerase, 3.0 mM
MgCl.sub.2 and 1.times. PCR buffer.
[0194] The enzyme assay was performed as in Example 3, using the
NosT amplicons as target probes. The results are shown in Table 4.
TABLE-US-00004 TABLE 4 % GMO i. Event Counts 0 70 0.1 160 0.5 520 1
627 2 651 5 925
[0195] As shown in Table 4, the detectable signal (event counts)
increased as the percentage of GMO material increased. Between 0%
and 1% GMO, there was a 90% correlation between the amount of GMO
material and the resulting signal.
CONCLUDING SUMMARY
[0196] All patents, patent applications, and other publications
mentioned in this specification are incorporated herein in their
entireties by reference.
[0197] While this invention has been described in detail with
reference to a certain preferred embodiments, it should be
appreciated that the present invention is not limited to those
precise embodiments. Rather, in view of the present disclosure that
describes the current best mode for practicing the invention, many
modifications and variations would present themselves to those of
skill in the art without departing from the scope and spirit of
this invention. For example, the optical bio-disc molecular
diagnostic-enzyme based detection methods and systems, of the
present invention, may be readily modified and implemented to test
for proteins, cells, cell surface markers, or any antigen in an
immunoassay format. In this immunoassay implementation of the
present invention, the capture probe may be an antibody having
specific affinity to an analyte. The analyte may be an antigen
including proteins, glycoproteins, cells, cell surface markers, or
any antigen. The enzyme would then be conjugated to a second
(signal) antibody, having an affinity to a different epitope on the
same antigen and no affinity for the capture probe, and the enzyme
substrate reaction and signal detection carried out as described
above. Alternatively, labeled or tagged signal antibodies may be
used as the reporter instead of the precipitate or pellet formation
of the enzyme-substrate system described above. Labels may include,
for example, fluorescent molecules and microparticles. The scope of
the invention is, therefore, indicated by the following claims
rather than by the foregoing description. All changes,
modifications, and variations coming within the meaning and range
of equivalency of the claims are to be considered within their
scope.
* * * * *