U.S. patent application number 12/169618 was filed with the patent office on 2008-10-30 for method of preparing nucleic acids for detection.
This patent application is currently assigned to Nanosphere, Inc.. Invention is credited to William Cork, Jennifer Hollenstein, Christopher Khoury, Christian Kronshage, Tim Patno, Mark Weber, Tom Westberg.
Application Number | 20080268458 12/169618 |
Document ID | / |
Family ID | 34590724 |
Filed Date | 2008-10-30 |
United States Patent
Application |
20080268458 |
Kind Code |
A1 |
Patno; Tim ; et al. |
October 30, 2008 |
Method of Preparing Nucleic Acids for Detection
Abstract
A method is provided for preparing a test sample for detecting a
predetermined target nucleic acid. The method includes the steps of
providing a test probe comprising an oligonucleotide attached to a
nanoparticle and providing a hybridization unit containing the test
sample and the test probe, wherein said hybridization unit further
includes a target sample substrate and a distribution manifold
coupled to a first side of the substrate. The method further
includes the steps of clamping a processing fluids manifold to the
distribution manifold of the hybridization unit, denaturing the
test sample and preparing the test sample for detecting the
predetermined target nucleic acid by pumping a plurality of
processing fluids between the processing fluids source manifold and
distribution manifold to hybridize the test probe and predetermined
target nucleic acid to the target sample substrate, to wash the
hybridized sample and to amplify a detectable parameter of the
hybridized sample.
Inventors: |
Patno; Tim; (Barrington,
IL) ; Hollenstein; Jennifer; (Grayslake, IL) ;
Kronshage; Christian; (Round Lake, IL) ; Khoury;
Christopher; (Chicago, IL) ; Weber; Mark;
(Algonquin, IL) ; Westberg; Tom; (Gurnee, IL)
; Cork; William; (Lake Bluff, IL) |
Correspondence
Address: |
GREGORY T. PLETTA;Nanosphere, Inc.
4088 Commerical Avenue
Northbrook
IL
60062-1829
US
|
Assignee: |
Nanosphere, Inc.
Northbrook
IL
|
Family ID: |
34590724 |
Appl. No.: |
12/169618 |
Filed: |
July 8, 2008 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
10703368 |
Nov 7, 2003 |
7396677 |
|
|
12169618 |
|
|
|
|
Current U.S.
Class: |
435/6.16 |
Current CPC
Class: |
B01L 2300/0877 20130101;
C12Q 1/6806 20130101; B01L 2200/027 20130101; B01L 2200/10
20130101; C12Q 2565/519 20130101; B01L 2400/0487 20130101; C12Q
2563/155 20130101; B01L 3/5025 20130101; B01L 7/525 20130101; G01N
35/1097 20130101; B01L 3/5027 20130101; B01L 9/527 20130101; C12Q
1/6806 20130101 |
Class at
Publication: |
435/6 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68 |
Claims
1. A method of preparing a test sample for purposes of detecting a
predetermined target nucleic acid, such method comprising the steps
of: (a) providing a test probe comprising an oligonucleotide
attached to a nanoparticle; (b) providing a hybridization unit
containing the test sample and the test probe, wherein said
hybridization unit further comprises a target sample substrate and
a distribution manifold coupled to a first side of the substrate;
(c) clamping a processing fluids manifold to the distribution
manifold of the hybridization unit; (d) denaturing the test sample;
and (e) preparing the test sample for detecting the predetermined
target nucleic acid by pumping a plurality of processing fluids
between the processing fluids source manifold and distribution
manifold to hybridize the test probe and predetermined target
nucleic acid to the target sample substrate, to wash the hybridized
sample and to amplify a detectable parameter of the hybridized
sample.
2. The method of preparing the test sample as in claim 1 further
comprising disposing a hybridization solution into a sample well of
the hybridization unit.
3. The method of preparing the test sample as in claim 2 further
comprising disposing the test probe into the sample well.
4. The method of preparing the test sample as in claim 1 further
comprising disposing the test sample in the sample well.
5. The method of preparing the test sample as in claim 4 wherein
the step of denaturing the test sample further comprises heating
the sample well.
6. The method of preparing the test sample as in claim 5 further
comprising disposing an oligonucleotide having a sequence
complementary to a first portion of a genetic sequence of the
predetermined target nucleic acid within a hybridization zone of
the hybridization unit.
7. The method of preparing the test sample as in claim 6 wherein
the step of disposing the oligonucleotide within a hybridization
zone of the hybridization unit further comprises connecting the
oligonucleotide to the target sample substrate.
8. The method of preparing the test sample as in claim 7 further
comprising defining the oligonucleotide attached to the
nanoparticle as having a genetic sequence complementary to a second
portion of the genetic sequence of the predetermined target nucleic
acid.
9. The method of preparing the test sample as in claim 8 further
comprising drawing a content of the sample well from the sample
well into the hybridization zone using a fluid coupled through a
first port of the processing fluids manifold.
10. The method of preparing the test sample as in claim 9 further
comprising chilling the content of the sample well as the content
is drawn into the hybridization zone.
11. The method of preparing the test sample as in claim 9 further
comprising hybridizing the probe and predetermined nucleic acid
with the oligonucleotide connected to the target sample substrate
by shuttling the content of the well through the hybridization zone
a predetermined number of times.
12. The method of preparing the test sample as in claim 11 further
comprising flushing the hybridized probe, predetermined nucleic
acid and first oligonucleotide by introducing wash fluid through a
second port and discharging wash fluid through the first port.
13. The method of preparing the test sample as in claim 12 further
comprising amplifying optical characteristics of the hybridized
probe, predetermined nucleic acid and first oligonucleotide by
introducing a plating solution through a second port and
discharging spent plating solution through the first port.
14. The method of preparing the test sample as in claim 13 further
comprising defining the plating solution as being a silver
solution.
15. A method of preparing a plurality of test samples for purposes
of detecting predetermined target nucleic acids, such method
comprising the steps of: (a) providing a hybridization unit
containing the plurality of test samples, said hybridization unit
further comprising a target samples substrate and a distribution
manifold coupled to a first side of the substrate said target
samples; (b) clamping a processing fluids manifold to the
distribution manifold of the hybridization unit; (c) denaturing the
test samples; and (d) preparing the plurality of test samples for
detecting the predetermined nucleic acids by pumping a plurality of
processing fluids between the processing fluids source manifold and
distribution manifold to hybridize the predetermined target nucleic
acid to the target samples substrate, to wash the hybridized
samples and to amplify a detectable parameter of the hybridized
samples.
16. The method of preparing the test sample as in claim 15 further
comprising disposing the plurality of test samples in a plurality
of respective sample wells.
17. The method of preparing the test sample as in claim 16 wherein
the step of denaturing the test samples further comprises heating a
sample well of the plurality of respective sample wells.
18. The method of preparing the test sample as in claim 17 further
comprising disposing a liquid test probe into a first well of the
plurality of sample wells.
19. The method of preparing the test sample as in claim 18 further
comprising disposing an first oligonucleotide having a sequence
complementary to a first portion of a sequence of a nucleic acid of
the predetermined nucleic acids within a hybridization zone of the
hybridization unit.
20. The method of preparing the test sample as in claim 19 wherein
the step of disposing the first oligonucleotide within a
hybridization zone of the hybridization unit further comprises
connecting an end of the oligonucleotide to the target samples
substrate.
21. The method of preparing the test sample as in claim 20 wherein
the test probe further comprises a nanoparticle and a second
oligonucleotide disposed on a surface of the nanoparticle, said
second oligonucleotide having a sequence complementary to a second
portion of the sequence of the nucleic acid of the predetermined
nucleic acids.
22. The method of preparing the test sample as in claim 21 further
comprising drawing a content of a well of the plurality of wells
from the well into the hybridization zone using a fluid coupled
through a first port of the processing fluids manifold.
23. The method of preparing the test sample as in claim 22 further
comprising cooling the content of the sample well of the sample
wells as the content is drawn into the hybridization zone.
24. The method of preparing the test sample as in claim 22 further
comprising hybridizing the probe and predetermined nucleic acid
with the first oligonucleotide by shuttling the content of the well
through the hybridization zone a predetermined number of times to
mix the probe and predetermined nucleic acid with the first
oligonucleotide.
25. The method of preparing the test sample as in claim 24 further
comprising flushing the hybridized probe, predetermined nucleic
acid and first oligonucleotide by introducing wash fluid through a
second port and discharging wash fluid through the first port.
26. The method of preparing the test sample as in claim 25 further
comprising amplifying optical characteristics of the hybridized
probe, predetermined nucleic acid and first oligonucleotide by
introducing a plating solution through a second port and
discharging spent plating solution through the first port.
27. The method of preparing the test sample as in claim 26 further
comprising defining the plating solution as being a silver
solution.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional application of U.S.
application Ser. No. 10/703,368, filed on Nov. 7, 2003, now U.S.
Pat. No. 7,396,677, the disclosure of which is hereby incorporated
herein by reference.
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] The field of the invention relates to biological testing and
more particularly to detecting nucleic acids.
[0004] 2. Description of the Related Art
[0005] Methods of detecting nucleic acids are generally known. In
fact, there are a number of methods available for detecting
specific nucleic acid sequences.
[0006] Known methods include those based upon electrophoresis,
polymerase chain reaction (PCR) processes, various hybridization
techniques, and a number of other techniques. While these methods
are effective, they are all time consuming, costly and subject to
significant human error.
[0007] For example, one manufacturer makes a microfluidics system
that hybridizes a sample to a chip followed by staining of the
chip. The hybridization process takes approximately 12 hours.
Staining takes approximately 1.5 hours to complete.
[0008] Another supplier provides a system that relies upon a single
nucleotide polymorphism (SNP) technique. This system uses a
microchip for performing multiple assays. Probes are added to a
cartridge and the particles move based on charge in an electric
field. A detection system may be used for analyzing the cartridges
after hybridization with the sample DNA.
[0009] Still another supplier provides a device called a
LightCycler that combines PCR amplification and DNA detection into
one process. The LightCycler can use one of two processes for
detection. The first process relies upon PCR and hybridization. The
second process relies upon PCR and dye and melting curve
analysis.
[0010] The development of reliable methods for detecting and
sequencing nucleic acids is critical to the diagnosis of genetic,
bacterial and viral diseases. Because of the importance of health
care and disease prevention, a need exists for quicker and cheaper
methods of identifying nucleic acids.
SUMMARY OF THE INVENTION
[0011] A method is provided for preparing a test sample for
detecting a predetermined target nucleic acid. The method includes
the steps of providing a test probe comprising an oligonucleotide
attached to a nanoparticle and providing a hybridization unit
containing the test sample and the test probe, wherein said
hybridization unit further includes a target sample substrate and a
distribution manifold coupled to a first side of the substrate. The
method further includes the steps of clamping a processing fluids
manifold to the distribution manifold of the hybridization unit,
denaturing the test sample and preparing the test sample for
detecting the predetermined target nucleic acid by pumping a
plurality of processing fluids between the processing fluids source
manifold and distribution manifold to hybridize the test probe and
predetermined target nucleic acid to the target sample substrate,
to wash the hybridized sample and to amplify a detectable parameter
of the hybridized sample.
BRIEF DESCRIPTION OF THE DRAWINGS
[0012] FIG. 1 depicts a nucleic acid testing system in accordance
with an illustrated embodiment of the invention;
[0013] FIG. 2 depicts a hybridization unit that may be used with
the system of FIG. 1;
[0014] FIG. 3 depicts a manifold that may be used with the
hybridization unit of FIG. 2;
[0015] FIG. 4 depicts a gasket that may be used with the
hybridization unit of FIG. 2;
[0016] FIG. 5 is a schematic of controls that may be used to
control the sample processing unit of FIG. 1;
[0017] FIG. 6 depicts preparatory steps that may be used in
conjunction with the sample processing system of FIG. 1;
[0018] FIG. 7 depicts preparatory steps that may be used in
conjunction with the hybridization unit of FIG. 2;
[0019] FIG. 8 depicts loading steps that may occur when the
hybridization unit of FIG. 2 is loaded into the sample processing
system of FIG. 1;
[0020] FIG. 9 depicts operation of the heating/cooling unit of FIG.
8;
[0021] FIG. 10 depicts fluid flows in the hybridization unit of
FIG. 2;
[0022] FIG. 11 depicts a wash cycle that may be used with the
sample processing system of FIG. 1;
[0023] FIG. 12 depicts an amplification step that may be used with
the sample processing system of FIG. 1;
[0024] FIG. 13 depicts an amplification stop step that may be used
with the sample processing system of FIG. 1;
[0025] FIG. 14 depicts a flushing step that may be used with the
sample processing system of FIG. 1;
[0026] FIG. 15 depicts disassembly of the hybridization unit and
reading of the substrate within the optical reader of FIG. 1;
[0027] FIG. 16 is a flow chart of method steps that may be followed
by the sample processing system of FIG. 1;
[0028] FIG. 17 is a flow chart of a process control
application;
[0029] FIG. 18 depicts a distribution manifold that may be used
with the system of FIG. 1 under an alternative embodiment of the
invention;
[0030] FIG. 19 depicts an underside of the distribution manifold of
FIG. 18;
[0031] FIG. 20 depicts a gasket that may be used with the
distribution manifold of FIG. 18;
[0032] FIG. 21 depicts a fluid flow schematic for sample processing
under an alternate embodiment of the invention.
DETAILED DESCRIPTION OF AN ILLUSTRATED EMBODIMENT
[0033] FIG. 1 is a perspective view of a nucleic acid detection
system 10, shown generally in accordance with an illustrated
embodiment of the invention. The processing system 10 may be used
for the detection of any of a number of predetermined target
nucleic acids. In fact, any type of nucleic acid may be detected,
and the methods may be used for the diagnosis of disease and in
sequencing of nucleic acids. Examples of nucleic acids that can be
detected by the methods of the invention include genes (e.g., a
gene associated with a particular disease), viral RNA and DNA,
bacterial DNA, fungal DNA, cDNA, mRNA, RNA and DNA fragments,
oligonucleotides, synthetic oligonucleotides, modified
oligonucleotides, single-stranded and double-stranded nucleic
acids, natural and synthetic nucleic acids, etc. Examples of the
uses of the methods of detecting nucleic acids include: the
diagnosis and/or monitoring of viral diseases (e.g., human
immunodeficiency virus, hepatitis viruses, herpes viruses,
cytomegalovirus, and Epstein-Barr virus), bacterial diseases (e.g.,
tuberculosis, Lyme disease, H. pylori, Escherichia coli infections,
Legionella infections Mycoplasma infections, Salmonella
infections), sexually transmitted diseases (e.g., gonorrhea),
inherited disorders (e.g., cystic fibrosis, Duchene muscular
dystrophy, phenylketonuria, sickle cell anemia), and cancers (e.g.,
genes associated with the development of cancer); in forensics; in
DNA sequencing; for paternity testing; for cell line
authentication; for monitoring gene therapy; and for many other
purposes.
[0034] Included within the system 10 may be a sample processing
system 12 and an optical reader 14 for reading samples
automatically prepared by the sample processing system 12. The
optical reader 14 may be a model Verigene.RTM. ID made by
Nanosphere, Inc. of Northbrook, Ill.
[0035] The sample processing system 12 may include a controller 300
and a number of functionally distinct elements used for storage and
handling of processing solutions and samples. For example, the
processing system 12 may include one or more removable
hybridization units 20. The hybridization unit 20 may be used by
the processing system 12 as a processing vessel for detecting the
predetermined target nucleic acid(s).
[0036] The detection system 10 may also require a number of
processing solutions for preparing the nucleic acids for detection.
For example, the processing system 12 may require one or more
probes 22 and a hybridization buffer fluid (solution) 24. In
addition, a processing fluids package 18 may be provided that
includes a wash solution, sterile water, one or more amplifying
solutions (e.g., silver part A, silver part B, etc.) and a stop
solution.
[0037] The hybridization unit 20 (FIG. 2) may include at least
three functionally separate portions. A target sample substrate 42
of optically transparent glass may be provided as a base for
processing the predetermined nucleic acid. A distribution manifold
44 may be provided that contacts the substrate and that, together
with the substrate 42 and a silicone gasket 58, define the chambers
and passageways that allow flow of processing solutions through the
hybridization unit 20. Finally, a base 40 is provided that supports
the substrate 42.
[0038] The manifold 44 may be provided with a flange 43, 45 on
opposing sides that each contain a set of apertures 56 that
resiliently engages a complementary set of pegs 54 on the base. The
pegs 54 may be provided with a taper on the engagement side to
allow the flange to resiliently expand over and allow the apertures
56 to engage the pegs 54. The silicone gasket 58 (provided on the
engagement side of the manifold 44) allows the manifold to
resiliently engage with the substrate 42 and define a seal around a
periphery of chambers and passageways of the hybridization unit
20.
[0039] FIG. 3 depicts a simplified view of the manifold 44. FIG. 4
depicts the silicone gasket 58.
[0040] As shown in FIGS. 3 and 4, each hybridization unit 20 may
include four sample processing areas 100, 102, 104, 106 (FIG. 4).
Each processing area 100, 102, 104, 106 may include a hybridization
zone 140, 142, 144, 146 (FIG. 4), an associated sample well 108,
110, 112, 114 (FIG. 3), three processing ports 116, 118, 120; 122,
124, 126; 128, 130, 132; 134, 136, 138 (FIG. 3) associated with
each respective hybridization zone 140, 142, 144, 146 (FIG. 4) and
interconnecting passageways (shown disposed in the gasket in FIG.
4).
[0041] FIG. 4 shows a range of gasket depths that may be used in
conjunction with sample processing. It may be noted that the
varying depths may be used to minimize flow resistance in the
channels while maximizing fluid mixing and interaction among the
hybridizing elements within the hybridization chamber 140, 142,
144, 146.
[0042] In preparation for testing for a particular nucleic acid, a
first oligonucleotide or first group of oligonucleotides 46, 48,
50, 52 (FIG. 2) with a first predetermined genetic sequence may be
disposed on the substrate 42 (FIG. 2) within each of the
hybridization zones 140, 142, 144, 146 (FIG. 4). The first
oligonucleotides 46, 48, 50, 52 (FIG. 2) may have a genetic
sequence that is complementary to a first portion of the genetic
sequence of the predetermined target nucleic acid.
[0043] The probes 22 (FIG. 1) may be constructed of nanoparticles
with one or more strands of second oligonucleotides of a second
predetermined genetic sequence attached to the nanoparticles.
Nanoparticles useful in the practice of the invention may include
metal (e.g., gold, silver, copper, and platinum), semiconductor
(e.g., CdSe, CdS, and CdS or CdSe coated with ZnS) and magnetic
(e.g., ferromagnetite) colloidal materials. Other nanoparticles
useful in the practice of the invention include ZnS, ZnO,
TiO.sub.2, AgI, AgBr, HgI.sub.2, PbS, PbSe, ZnTe, CdTe,
In.sub.2S.sub.3, Cd.sub.3P.sub.2, Cd.sub.3As.sub.2, InAs, and GaAs.
The size of the nanoparticles is preferably from about 5 nm to
about 150 nm (mean diameter), more preferably from about 5 to about
50 nm, most preferably from about 10 to about 30 nm.
[0044] The nanoparticles, the second oligonucleotides or both are
functionalized in order to attach the oligonucleotides to the
nanoparticles. Such methods are known in the art. For instance,
oligonucleotides functionalized with alkanethiols at their
3'-termini or 5'-termini readily attach to gold nanoparticles.
[0045] The second oligonucleotides may have a sequence that is
complementary to a second portion of the genetic sequence of the
predetermined target nucleic acid. Preparation of the first and
second oligonucleotides and attachment to the respective particles
and substrate may be accomplished generally as described in U.S.
Pat. No. 6,417,340 assigned to the assignee of the present
invention and incorporated herein by reference.
[0046] In general, the test probe and test sample (that may or may
not contain the predetermined target nucleic acid) and a
hybridization fluid may be mixed in a sample well. The mixture may
be denatured and passed through the hybridization chamber.
Denaturing may be accomplished using any known process (e.g., heat,
chemical, etc.).
[0047] Within the hybridization chamber, the test probe and
predetermined nucleic acid may hybridize with the first
oligonucleotide. The optical characteristics of the hybridized
materials may be enhanced (e.g., plating a silver solution to the
nanoparticles of the hybrid). After enhancement, any hybridized
materials may be detected optically within the optical reader 14
(FIG. 1). In this case, the plating of the silver solution to the
gold nanoparticles of the hybrid amplifies the optical reflectivity
of the hybrid. The optical reflectivity may then be compared with a
threshold value to confirm the presence of the target nucleic
acid.
[0048] Alternatively, the detectable parameter may be resistance.
In this case, the silver plated to the gold nanoparticle within the
hybrid amplifies a current path through the hybrid. The resistance
may then be compared with a threshold value to confirm the presence
of the target nucleic acid.
[0049] Turning now to operation of the sample processing system 12
(FIG. 1), an explanation will now be provided of the controller 300
and the interaction of the controller 300 with the hybridization
unit 20. In this regard, FIG. 5 depicts the controller 300 and
various actuating elements used by the sample processing system 12
in processing samples within the hybridization unit 20.
[0050] Along the right side of FIG. 5 is shown a set of ports
labeled "SAMPLE WELL, B, A, C". The reference "SAMPLE WELL" may be
used to refer to large ports 108, 110, 112, 114 in FIG. 3.
Similarly, port B may be used to refer to smaller ports 116, 126,
128, 138, port A may be used to refer to ports 118, 124, 130, 136
and port C may be used to refer to ports 120, 122, 132, 134.
[0051] Processing of samples in sample processing areas 100, 102,
104, 106 (FIG. 4) may be assumed to be substantially identical. It
should be noted in this regard that while the processing may be
substantially identical for each sample processing area 100, 102,
104, 106, the target nucleic acid that is detected may be different
within each of the four areas 100, 102, 104, 106.
[0052] As shown in FIG. 5, the sample processing system 12 may
include a number of pumps 302, 304, 306, 308, 310, 312 and a vacuum
source 314. While any form of pump 302, 304, 306, 308, 310, 312 may
be used, it is contemplated that a positive displacement pump such
as a syringe pump may be used for reasons that will become apparent
from the description below.
[0053] The syringe pumps may include a syringe body and a linear
actuator. The linear actuator may be programmed by the controller
300 to fill and empty at precisely controlled rates.
[0054] The routing of fluids to and from the pumps 302, 304, 306,
308, 310, 312 may be controlled by a number of multiport valves
316, 318, 320, 322, 324, 326. While any number of ports may be
used, it is believed that the four-port valves 316, 318, 320, 322,
324, 326 shown in FIG. 5 are particularly well adapted to the
purpose described below.
[0055] In this regard, the valves 316, 318, 320, 322, 324, 326 may
have ports labeled 1-4. A spool within the valves 316, 318, 320,
322, 324, 326 may allow any two opposing ports to be connected
together (e.g., port 1 may be connected to port 3 or port 2 may be
connected to port 4).
[0056] When used with syringe pumps 302, 304, 306, 308, 310, 312,
multiport valves 316, 318, 320, 322, 324, 326 allow a precise
amount of a selected fluid to be transferred at each stage of
processing. For example, with ports 1 and 3 of valve 322 connected
(as shown in FIG. 5), the syringe pump 308 may withdraw a precise
amount of water from the water container 334 on a fill portion of
the pump cycle. The multiport valve 322 may then be actuated to
connect ports 2 and 4. The water previously drawn into the syringe
pump 308 may now be discharged through port 4 of valve 322 and into
port A of the hybridization unit 20.
[0057] It may be noted that in some applications, the valves 316,
318, 320, 322, 324, 326 and containers 328, 330, 332, 334, 336, 338
may not be needed. For example, the valves and containers would not
be needed in cases where the total flow for each function is less
than the capacity of the syringe pump 302, 304, 306, 308, 310. In
these cases, the syringe pump may simply be replaced after each
testing procedure or after multiples of each testing procedure.
[0058] FIG. 21 illustrates fluid flow for sample processing under
an even more preferred embodiment of the invention. In the example
of FIG. 21, fluidic control is maintained without the use of valves
by utilizing pumps on the inlet and outlet ports to route fluids
down a specified path.
[0059] By introducing fluids via pump 1 and only withdrawing fluids
via pump 2, the fluid can be routed through hybridization chamber
and flow path A. Fluids can also be routed down multiple paths in
parallel by actuating the control pumps (2, 3, 4 or 5) for that
fluid path. Parallel fluid processing may be useful to reduce
processing time when high tolerance pumping is not required, such
as during washing and rinsing steps.
[0060] Other additional pumps on the inlet side (not shown in FIG.
21) will provide additional fluids. A system such as that shown in
FIG. 21 with 8 inlet pumps for 8 specific fluids can perform a
variety of nucleic acid tests. The type of tests can be selected by
the insertion of various fluids into the flow paths from the 8 pump
chambers.
[0061] Access to fluids inserted into the sample well by the user
is accomplished by pulling on the outlet pump(s) only. The sample
well is designed to easily collapse and block flow so that the
target sample will flow preferentially only out of the specific
sample well for the specific flow path and outlet pump desired.
[0062] FIGS. 6-16 show process steps that may be used in detecting
the predetermined nucleic acid. For example, Frame #1 of FIG. 6
shows the preliminary step of providing a reagent cartridge 18 and
a waste container 16. Frame #2 shows the loading of the cartridges
16, 18 into the sample processing system 12. Frame #3 shows the
closing of the door and references the fact that closing the door
causes a set of connection fittings to puncture the seals on the
reagent and waste containers. Alternatively, or in addition,
closing the door provides a signal to the controller which then
controls linear actuators to engage the pumps which provides
fluid(s) for processing. A bar code reader 340 (FIG. 8) may be
provided to read a bar code on the reagent cartridge to
automatically verify that the correct reagent cartridge has been
inserted.
[0063] FIG. 7 shows preparation of the hybridization unit 20. Frame
#1 shows a user opening a set of lids covering the four sample
wells 108, 110, 112, 114 (FIG. 3). The user than pipettes a
hybridization buffer into the four wells 108, 110, 112, 114 as
shown in Frame #2. The user then pipettes the probe 24 into the
four wells 108, 110, 112, 114, as shown in Frame #3. As a fourth
step, the user pipettes a sample that may contain the predetermined
target nucleic acid into the well 108, 110, 112, 114 as shown in
Frame #4. Finally, the user closes the lids on the wells 108, 110,
112, 114 as shown in Frame #5. Alternatively, the user may provide
only the predetermined target nucleic acid into the sample well or
a combination of the predetermined target nucleic acid and
hybridization buffer or predetermined target nucleic acid and probe
into the sample well.
[0064] FIG. 8 shows preparation and loading of the hybridization
unit 20 into the sample processing system 12. As a first step,
shown in Frame #1 of FIG. 8, the user may use a barcode reader 340
to identify the hybridization unit 20 to the system 12.
Alternatively, the bar code reader may be embedded inside the
loading door of the system and the bar code may be read when the
hybridization unit is loaded into the system.
[0065] To load the hybridization unit 20, the user may open a door
on the sample processing system 12. A spring-loaded receptacle that
catches fluid from a fluid manifold 72 of the processing system 12
is found extended to a fully forward position as shown in Frame 2
of FIG. 8. The user then pushes the hybridization unit 20 into the
sample processing system 12 as shown in Frame #3 and closes the
door (Frame #4).
[0066] Activation of the sample processing system 12 may occur by
closure of the door or by activating a START button 342 (FIG. 1).
In either case, activation of the system 12 causes the
hybridization unit 20 to be raised into contact with a processing
fluids manifold 72 and a heating/cooling block 60 to be raised into
contact with the hybridization unit 20 (FIG. 8, Frame #5). The
raising of the hybridization unit 20 and heating/cooling block 60
may be accomplished by a simple mechanical linkage connected to the
door or through a linear actuator coupled to an elevator
assembly.
[0067] The raising of the hybridization unit 20 creates a
fluid-transfer connection between the ports 116, 118, 120, 122,
124, 126, 128, 130, 132, 134, 136, 138 of the hybridization unit 20
and respective ports of the processing fluids manifold 72 and with
the pumps 1-7 of FIG. 21 or with respective valves 316, 318, 320,
322, 324, 326 and with pumps 302, 304, 306, 308, 310, 312, 314 of
the sample processing system 12 of FIG. 5. Similarly, the raising
of the heating/cooling block 60 causes a thermal transfer
connection between the hybridization unit 20 and the
heating/cooling block 60.
[0068] FIG. 9 depicts a preliminary processing step 400 (FIG. 16)
performed by the sample processing system 12. As shown, a first
heating element 62 and a second heating element 70 of the
heating/cooling block 60 connect to and heat the sample wells 108,
110, 112, 114 to a temperature for denaturing the samples (e.g.,
95.degree. C.) of the predetermined target nucleic acid. As used
herein, denature means to cause the tertiary structure of the
nucleic acid to unfold.
[0069] A first cooling element 64 and a second cooling element 68
function to cool the denatured samples as they are transferred from
the sample wells 108, 110, 112, 114 to hybridization chambers 140,
142, 144, 146. A third heating element 66 is located adjacent the
hybridization chambers 140, 142, 144, 146 to heat the samples to a
specified temperature for hybridization (e.g., 40.degree. C.).
[0070] Frame #1 of FIG. 10 depicts the heating of the samples in
the sample wells 108, 110, 112, 114 to the denaturing temperature
(e.g., 95.degree. C.). Frame #2 of FIG. 10 depicts loading 402
(FIG. 16) of the samples by transferring the samples through the
chill zone into the hybridization chambers 140, 142, 144, 146.
Transfer of the samples from the sample wells 108, 110, 112, 114
may be accomplished by activating the waste pump 312 with the waste
valve 324 in the position shown in FIG. 5. The transfer of the
samples across the chill zone may be accomplished by the controller
300 choosing a relatively slow rate of fluid transfer (e.g., 1
cc/min) as the pump 312 pulls fluid from port C to ensure proper
cooling of the samples as they pass over the chill zone.
[0071] It may be noted that to load the sample into the
hybridization zone 140, 142, 144, 146, the controller 300 may
retrieve and execute a set of valve and motor control parameters
(instructions) 346 (FIG. 5) from memory for controlling a linear
actuator of the pump 312. The parameters 346 may include a motor
identifier 348, a direction 350, a speed 352, a time 354 and a
valve position 356.
[0072] If the linear actuator has its own controller, then the
direction 350, speed 352 and time may be simply downloaded to the
controller for execution. If the controller is provided through the
use of special purpose programs within the controller 300, then
execution of the instructions may be provided from within the
controller 300.
[0073] It should be noted that (before loading of the samples) the
hybridization chambers 140, 142, 144 may initially have been filled
with air. As such, the fluid pulled from port C would be air. The
withdrawal of air from port C pulls the samples from the sample
wells 108, 110, 112, 114 into the hybridization chambers 140, 142,
144, 146.
[0074] As a final step in the process of loading the sample, the
controller 300 may reset the waste pump 310. Resetting the waste
pump 310 may mean retrieving a set of instructions 358 (FIG. 5)
from memory. The instructions 358 may contain an instruction 368
that causes the waste valve 326 forms a connection between ports 2
and 4. The instructions 358 may also contain a motor identifier
360, a direction 362, a speed 364 and a time 366 necessary to cause
the waste pump 310 to move to a fully discharged position.
[0075] It may be noted that the instructions for loading the sample
and for resetting the waste pump 310 and for performing the other
process steps described herein may be accomplished by a process
control application depicted in FIGS. 16 and 17. FIG. 16 may be
used to depict the overall functionality of the control application
and FIG. 17 may be used to depict the activity performed within the
individual blocks of FIG. 16.
[0076] With respect to execution of the control application,
activation of the START button 342 or closing the door brings the
hybridization unit 20 into contact with the manifold 72 and
heating/cooling block 60. Activation may also start a timer within
the controller 300 to detect completion of the denaturization
process 400. From the denaturization process 400, the control
application proceeds to the load sample process 402. As a first
step of the load sample process 402, the application 500 loads and
executes the load sample file 346. As a second step, the
application 500 loads and executes the reset pump files 358. In
each case, the application 500 positions the valves, loads actuator
positioning parameters and executes the positioning parameters.
Once each process is complete, the application 500 advances to the
next process step.
[0077] Frame #3 of FIG. 10 depicts hybridization of the sample and
probe with the oligonucleotide strands within hybridization chamber
140, 142, 144, 146. In this case, the controller 300 functions to
shuttle 404 the partially hybridized sample and probe back and
forth across the hybridization chamber 140, 142, 144, 146.
[0078] To shuttle the partially hybridized sample back and forth
across the hybridization chamber 140, 142, 144, 146, the
application 500 retrieves and execute a set of instructions 370,
372 that activate the wash pump 310 and waste pump 312 to move in
opposite directions. In this case, the instructions 370, 372 would
cause the wash valve 324 to form a connection between ports 2 and 4
and the waste valve 326 to form a connection between ports 1 and 3.
The shuttle forward instruction 370 may cause the wash pump 310 to
move a predefined distance towards an empty position and the waste
pump 312 to move a predefined distance towards a filled position.
When the wash pump 310 and waste pump 312 reach the predefined
distance, the application 500 would execute the shuttle reverse
instructions 372. The shuttle reverse instruction 372 may cause the
wash pump 310 to move a similar distance towards a full position
and the waste pump 312 to move a similar towards an empty position.
When the predetermined distances are reached, the application 500
may again execute the shuttle forward instructions 370.
[0079] Each time the application 500 executes the shuttle forward
instructions 370, a counter 374 is incremented 406. After each
increment, the value within the counter 374 may be compared 408
within a comparator 376 with a shuttle cycle limit value that
terminates the shuttling process after a predefined number of
cycles.
[0080] Since the pumps 310, 312 would initially contain air, the
reciprocal action of the pumps 310, 312 would simply push the
sample into and out of the passageways on either end of the
hybridization chamber 140, 142, 144, 146 with very little if any of
the partially hybridized sample entering either pump 310, 312.
Shuttling of the partially hybridized sample across the
hybridization zones 140, 142, 144, 146 may continue for a time
period determined by the identity and type of the sample (e.g.,
10-60 minutes).
[0081] Following hybridization of the sample and probe with the
oligonucleotide strands within the hybridization chamber 140, 142,
144, 146, the hybridized materials may be washed 410 as shown in
Frames #1 and #2 of FIG. 11. To wash the hybridized materials, the
controller 300 may execute a set of wash instructions 378 that may
concurrently activate the wash pump 310 and the waste pump 312. As
a first step, the instructions 378 may cause the wash valve 324 to
form a connection between ports 1 and 3. The wash pump 310 may then
be activated to draw water from a wash container 336.
[0082] Once the syringe pump 310 is full, the instructions 378 may
cause the valve 324 to form a connection between ports 2 and 4. The
waste valve 326 may also be moved to form a connection between
ports 1 and 3. The wash pump 310 and waste pump 312 may be
simultaneously activated to operate at the same rate. The wash pump
310 functions to push water into port A and the waste pump 312
functions to pull fluids out of port C.
[0083] When the syringe of the wash pump 310 reaches its empty
position, the waste pump 312 would reach its full position. At this
stage, the wash valve 324 may move to form a connection between
ports 1 and 3 and the waste valve 326 may move to form a connection
between ports 2 and 4. The wash pump 310 and waste pump 312 may
again be activated. In this case, the wash pump 310 now refills
from the wash container 336 and the waste pump 312 now discharges
into the waste container 338. The fill and empty process may repeat
for the number of cycles necessary to flush any un-hybridized
materials from the hybridization unit 20. A counter may be
incremented after each fill and empty cycle and a value within the
counter may be compared with a cycle limit within a comparator to
determine completion of the wash cycle.
[0084] Once the hybridized materials have been washed, a detectable
parameter of the hybridized materials may be amplified to allow
detection of the hybridization. The detectable parameter may be any
measurable quantity that indicates the presence or absence of the
hybridized materials. Under illustrated embodiments the optical or
conductive properties of the hybridized materials may be amplified
412 for purposes of detection. Amplification, in this case occurs
by plating a silver solution onto the nanoparticles of the
hybrid.
[0085] Amplification may occur by passing a silver A solution and a
silver B solution through the hybridization chamber 140, 142, 144,
146. To pass the silver A solution and silver B solutions through
the hybridization chamber, the controller 300 may execute a set of
instructions 380 that causes silver A valve 320 and the silver B
valve 316 to form a connection between ports 1 and 3. The silver A
pump 306 and silver B pump 302 may then be activated by the
instructions 380 to draw the silver A solution from the silver A
container 332 into the silver A pump 306 and the silver B solution
from the silver B container 328 into the silver B pump 302.
[0086] The silver A valve 320 and the silver B valve 316 may then
be instructed to form a connection between ports 2 and 4. The waste
valve 326 may be instructed to form a connection between ports 2
and 4. The instructions 380 may specify a discharge rate for silver
A pump 306 and the silver B pump 302 and the controller 300 may
activate the pumps 306, 302 to discharge at those rates. The silver
A pump 306 may discharge into port A and the silver B pump 302 may
discharge into port B. The instructions 380 may also specify an
intake rate for the waste pump 312 equal to an output of the silver
A pump 306 and silver B pump 302 and the controller 300 may
activate the waste pump 312 to withdraw fluid from the port C at
the selected rate. Once the silver A pump 306 and the silver B pump
302 have discharged their materials into the respective ports and
the waste pump 312 has been filled with fluid withdrawn from port
C, the valves 316, 320, 326 may again be moved under control of the
instructions 380. The silver A valve 320 and the silver B valve 316
may be positioned to again fill the silver A pump 306 and silver B
pump 302 with silver solutions. The waste valve 326 may be
positioned to discharge withdrawn materials into the waste
container 338. The fill and empty steps may be repeated by the
number of cycles necessary for sufficient amplification of the
hybridized materials again under the control of a counter and
comparator based upon a cycle limit value.
[0087] Once the amplification step has been completed, a stop
solution may be passed through the hybridization chambers 140, 142,
144, 146 as shown in FIG. 13 to stop amplification 414. In this
regard, a set of stop instruction may be executed by the controller
300 to position the stop valve 318 with a connection between ports
1 and 3. The stop pump 304 may be activated to fill the pump 304
from the stop solution container 330. The controller 300 under
control of the instructions 382 may then move the stop valve to
form a connection between ports 2 and 4 and the waste valve to form
a connection between ports 1 and 3. The controller 300 may then
select a discharge rate for the stop pump 304 and activate the stop
pump 304. The controller 300 may select the same withdrawal rate
for the waste pump 312 and simultaneously activate the waste pump
312 to pull the stop solution through the hybridization chamber
140, 142, 144, 146. The valves 318, 326 may be repositioned to
refill the stop pump 304 and empty the waste pump 312 and the
process may be repeated.
[0088] Under an even more preferred embodiment, the pumps would
never be refilled. In this case, the pump bodies are integrated
into a reagent cartridge that is simply replaced when empty.
[0089] Once the stop solution has been passed through the
hybridization chamber 140, 142, 144, 146, the hybridization chamber
140, 142, 144, 146 may be flushed 416 with dd water and vacuumed to
remove residual fluid as shown in FIG. 14.
[0090] To flush the hybridization chambers 140, 142, 144, 146, the
controller 300 operating under flush instructions 384 may move the
flush valve 322 to form a connection between ports 1 and 3 and
activate the flush pump 308 to fill with water from the water
container 334. The controller 300 may then reposition the flush
valve 322 to allow the flush pump 308 to discharge into port A and
reposition the waste valve 326 to withdraw fluid from port C. Once
the flush pump 308 is empty, the valves 322, 326 may be
repositioned to refill the flush pump 308 and empty the waste pump
312 and the process may be repeated.
[0091] Once flushing is complete, the controller 300 operating
under control of instructions 384 may activate the vacuum 314. The
vacuum 314 may pull any remaining fluids out of the hybridization
unit 20 by displacing the fluids with air pulled in through the
respective sample wells 108, 110, 112, 114.
[0092] Once any remaining fluids have been removed, the sample
processing unit 12 may unlock as shown in FIG. 15 and the
hybridization units 20 may be removed. The substrate 58 may be
removed from the hybridization unit 20 and placed in the optical
reader 14 where the optical characteristics of the hybridized
sample may be read.
[0093] In another illustrated embodiment of the invention (shown in
FIGS. 18-20), the distribution manifold 44 shown on the
hybridization unit 20 of FIG. 2 is replaced with a distribution 600
(shown as a complete hybridization unit 20 in FIG. 18). FIG. 19
shows a reverse view of the manifold 600. FIG. 20 shows a gasket
700 that may be used with the manifold 600 of FIGS. 18 and 19.
[0094] As with the manifold 44 of FIG. 2, the distribution manifold
600 of FIG. 18 has sample wells 602, 604, 606, 608 in opposing
corners. This distribution manifold 600 has four waste ports 610,
612, 614, 616 associated with a respective hybridization zone 708,
706, 704, 702 (FIG. 20). Also shown in FIG. 19 is a common fill
port. The manifold 600 of FIGS. 18, 19 and 20 is believed to be
particularly well adapted for use with the system of FIG. 21.
[0095] FIG. 19 shows an underside of the distribution manifold 600
of FIG. 18. As shown, each of the ports 602, 604, 606, 608, 610,
612, 614, 616, 618 of FIG. 18 has a corresponding feedthrough 602,
604, 606, 608, 610, 612, 614, 616, 618. It should also be noted
that the fill port 618 has a channel 620 disposed on a surface of
the distribution manifold 600 that terminates at four feedthrough
points 622, 624, 626, 628.
[0096] Turning now to the gasket 700 (FIG. 20), it may be noted
that the gasket 700 defines the hybridization chambers 702, 704,
706, 708 and a number of connecting channels. For example, the
first hybridization chamber 702 has a connecting channel that
connects the sample well 606, feedthrough 628 and the first end of
the hybridization chamber 702. The first hybridization chamber 702
also a connecting channel that connects a second end of the
hybridization chamber 702 to waste port 616.
[0097] The second hybridization chamber 704 has a connecting
channel that connects the sample well 608, feedthrough 622 and the
first end of the hybridization chamber 704. The second
hybridization chamber 704 also a connecting channel that connects a
second end of the hybridization chamber 704 to process port
614.
[0098] The third hybridization chamber 706 has a connecting channel
that connects the sample well 602, feedthrough 624 and the first
end of the hybridization chamber 706. The third hybridization
chamber 706 also a connecting channel that connects a second end of
the hybridization chamber 706 to process port 612.
[0099] Similarly, the fourth hybridization chamber 708 has a
connecting channel that connects the sample well 604, feedthrough
626 and the first end of the hybridization chamber 708. The second
hybridization chamber 708 also a connecting channel that connects a
second end of the hybridization chamber 708 to process port
610.
[0100] It should be noted that the fluid manifold 72 and pump
connections with the processing unit 12 may also be changed to
accommodate the distribution manifold 600. It may be noted in this
regard that port connections A and B in FIG. 5 would be combined
and connected to the respective process port 610, 612, 614, 616.
The waste port 618 in FIG. 18 would correspond to port C in FIG. 5.
In other regards, a hybridization unit 20 using the distribution
manifold 600 would operate substantially the same as described
above.
[0101] In another illustrated embodiment of the invention, the
manifold 72 may be provided with a connection to replaceable
cartridges for the hybridization buffer and/or probes. Under this
embodiment, the user would simply add the target sample to the test
wells and insert the hybridization unit 20 into the sample
processing system 12. The system 12 would add any missing elements
to the sample wells.
[0102] A specific embodiment of method and apparatus for processing
nucleic acid samples has been described for the purpose of
illustrating the manner in which the invention is made and used. It
should be understood that the implementation of other variations
and modifications of the invention and its various aspects will be
apparent to one skilled in the art, and that the invention is not
limited by the specific embodiments described. Therefore, it is
contemplated to cover the present invention and any and all
modifications, variations, or equivalents that fall within the true
spirit and scope of the basic underlying principles disclosed and
claimed herein.
* * * * *