U.S. patent application number 13/149970 was filed with the patent office on 2011-12-22 for dialysis device with multi-layer structure.
This patent application is currently assigned to Geneasys Pty Ltd. Invention is credited to Mehdi Azimi, Kia Silverbrook.
Application Number | 20110312673 13/149970 |
Document ID | / |
Family ID | 45327470 |
Filed Date | 2011-12-22 |
United States Patent
Application |
20110312673 |
Kind Code |
A1 |
Silverbrook; Kia ; et
al. |
December 22, 2011 |
DIALYSIS DEVICE WITH MULTI-LAYER STRUCTURE
Abstract
A dialysis device for dialysis of a fluid containing
constituents of different sizes, the dialysis device having a first
layer of material defining a first channel and a second channel,
the first channel configured for receiving the fluid which contains
constituents of different sizes, a second layer having a plurality
of apertures open to the first channel and at least one fluid
connection leading from the apertures to the second channel for
establishing fluid communication between the first channel and the
second channel, wherein, the apertures are sized in accordance with
a predetermined size threshold such that the constituents flowing
to the second channel are smaller constituents that are smaller
than the predetermined size threshold, and the constituents
retained in the first channel include larger constituents which are
larger than the predetermined size threshold.
Inventors: |
Silverbrook; Kia; (Rozelle,
AU) ; Azimi; Mehdi; (Rozelle, AU) |
Assignee: |
Geneasys Pty Ltd
|
Family ID: |
45327470 |
Appl. No.: |
13/149970 |
Filed: |
June 1, 2011 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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61356018 |
Jun 17, 2010 |
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61437686 |
Jan 30, 2011 |
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Current U.S.
Class: |
506/39 ; 210/335;
435/289.1; 435/306.1 |
Current CPC
Class: |
B01L 2300/0883 20130101;
Y10T 137/0391 20150401; B01L 3/502707 20130101; Y10T 436/203332
20150115; B01L 2300/1827 20130101; Y10T 436/107497 20150115; B01L
3/502738 20130101; C12Q 1/68 20130101; Y10T 137/0352 20150401; Y10T
137/2202 20150401; Y10T 137/1044 20150401; Y10T 436/11 20150115;
Y10T 436/143333 20150115; B01L 2400/0633 20130101; B01L 2400/0677
20130101; Y10T 137/2076 20150401; B01L 2300/10 20130101; B01L
2400/0406 20130101; B01L 2300/023 20130101; Y10T 436/145555
20150115; B01L 7/52 20130101; Y10T 436/25375 20150115; Y10T
436/173845 20150115; B01L 2300/0654 20130101; B01L 3/5027 20130101;
F16K 99/003 20130101; B01L 2200/10 20130101; B01L 2400/0688
20130101; Y10T 436/25 20150115; Y10T 137/206 20150401; B01L
2300/024 20130101; F16K 99/0036 20130101; B01L 2300/0636
20130101 |
Class at
Publication: |
506/39 ;
435/306.1; 435/289.1; 210/335 |
International
Class: |
C40B 60/12 20060101
C40B060/12; C12M 1/00 20060101 C12M001/00; B01D 29/00 20060101
B01D029/00; C12M 1/33 20060101 C12M001/33 |
Claims
1. A dialysis device for dialysis of a fluid containing
constituents of different sizes, the dialysis device comprising: a
first layer of material defining a first channel and a second
channel, the first channel configured for receiving the fluid which
contains constituents of different sizes; a second layer having a
plurality of apertures open to the first channel and at least one
fluid connection leading from the apertures to the second channel
for establishing fluid communication between the first channel and
the second channel; wherein, the apertures are sized in accordance
with a predetermined size threshold such that the constituents
flowing to the second channel are smaller constituents that are
smaller than the predetermined size threshold, and the constituents
retained in the first channel include larger constituents which are
larger than the predetermined size threshold.
2. The dialysis device according to claim 1 wherein the second
layer is a laminate with a roof layer, and the at least one fluid
connection is a series of adjacent channels enclosed by the roof
layer, the apertures being formed in the roof layer between the
first channel and the adjacent channels.
3. The dialysis device according to claim 2 wherein the first and
second channels, and the series of adjacent channels are configured
to fill with the sample by capillary action.
4. The dialysis device according to claim 3 further comprising a
bypass channel between the first channel and the second channel,
wherein each of the adjacent channels are configured to pin a
meniscus of the fluid that arrests capillary flow between the first
channel and the second channel, and the bypass channel between the
first channel and the second channel, the bypass channel joining
the second channel upstream of the series of adjacent channels and
is configured for uninterrupted capillary driven flow from the
first channel to the second channel, such that flow from the bypass
channel reaches the meniscus pinned at each of the adjacent
channels after the meniscus has formed to sequentially remove each
of the menisci and flow from the first channel to the second
channel is via the adjacent channels as well as the bypass
channel.
5. The dialysis device according to claim 4 wherein the sample is a
biological sample containing constituents of different sizes and
the adjacent channels and the bypass channel have apertures sized
in accordance with a predetermined size threshold such that the
constituents flowing to the second channel are smaller constituents
that are smaller than the predetermined size threshold, and the
constituents retained in the first channel include larger
constituents which are larger than the predetermined size
threshold.
6. The dialysis device according to claim 5 wherein the biological
sample is blood and the larger constituents include leukocytes and
the smaller constituents include erythrocytes.
7. The dialysis device according to claim 5 wherein the adjacent
channels are a series of parallel, adjacent channels extending
normal to the first channel and the second channel.
8. The dialysis device according to claim 6 further comprising a
waste reservoir for receiving constituents not required for
downstream processing or analysis.
9. The dialysis device according to claim 1 wherein the apertures
are holes with diameters less than 8 microns.
10. The dialysis device according to claim 6 wherein the smaller
constituents include targets such that processing of the smaller
constituents includes detection of the targets.
11. The dialysis device according to claim 10 wherein the targets
are target cells and further comprising a lysis section connected
to the target channel for lysing the target cells to release target
nucleic acid sequences therein.
12. The dialysis device according to claim 10 wherein the targets
are target nucleic acid sequences and further comprising a nucleic
acid amplification section for amplifying the target nucleic acid
sequences.
13. The dialysis device according to claim 12 further comprising a
hybridization section having an array of probes for hybridization
with the target nucleic acid sequences amplified by the nucleic
acid amplification section.
14. The dialysis device according to claim 13 wherein the probes
are configured to form probe-target hybrids with the target nucleic
acid sequences, the probe-target hybrids being
electrochemiluminescent in response to an electrical pulse.
15. The dialysis device according to claim 14 further comprising
CMOS circuitry for operative control of the nucleic acid
amplification section and the hybridization section, the CMOS
circuitry also having a photosensor for sensing
electrochemiluminescence emission from the probe-target
hybrids.
16. The dialysis device according to claim 15 wherein the
hybridization section has an array of hybridization chambers
containing the probes for hybridization with the target nucleic
acid sequences.
17. The dialysis device according to claim 16 wherein the
photosensor is an array of photodiodes positioned adjacent each of
the hybridization chambers respectively.
18. The dialysis device according to claim 17 wherein the CMOS
circuitry has a digital memory for storing data relating to the
processing of the fluid, the data including the probe details and
location of each of the probes in the array of hybridization
chambers.
19. The dialysis device according to claim 18 wherein the CMOS
circuitry has bond-pads for electrical connection to an external
device and is configured to convert output from the photodiodes
into a signal indicative of the probes that hybridized with the
target nucleic acid sequences, and provide the signal to the
bond-pads for transmission to the external device.
20. The dialysis device according to claim 19 further comprising a
plurality of reservoirs for holding liquid reagents for addition to
the sample.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to diagnostic devices that use
microsystems technologies (MST). In particular, the invention
relates to microfluidic and biochemical processing and analysis for
molecular diagnostics.
CO-PENDING APPLICATIONS
[0002] The following applications have been filed by the Applicant
which relate to the present application:
TABLE-US-00001 GBS001US GBS002US GBS003US GBS005US GBS006US
GSR001US GSR002US GAS001US GAS002US GAS003US GAS004US GAS006US
GAS007US GAS008US GAS009US GAS010US GAS012US GAS013US GAS014US
GAS015US GAS016US GAS017US GAS018US GAS019US GAS020US GAS021US
GAS022US GAS023US GAS024US GAS025US GAS026US GAS027US GAS028US
GAS030US GAS031US GAS032US GAS033US GAS034US GAS035US GAS036US
GAS037US GAS038US GAS039US GAS040US GAS041US GAS042US GAS043US
GAS044US GAS045US GAS046US GAS047US GAS048US GAS049US GAS050US
GAS054US GAS055US GAS056US GAS057US GAS058US GAS059US GAS060US
GAS061US GAS062US GAS063US GAS065US GAS066US GAS067US GAS068US
GAS069US GAS070US GAS080US GAS081US GAS082US GAS083US GAS084US
GAS085US GAS086US GAS087US GAS088US GAS089US GAS090US GAS091US
GAS092US GAS093US GAS094US GAS095US GAS096US GAS097US GAS098US
GAS099US GAS100US GAS101US GAS102US GAS103US GAS104US GAS105US
GAS106US GAS108US GAS109US GAS110US GAS111US GAS112US GAS113US
GAS114US GAS115US GAS117US GAS118US GAS119US GAS120US GAS121US
GAS122US GAS123US GAS124US GAS125US GAS126US GAS127US GAS128US
GAS129US GAS130US GAS131US GAS132US GAS133US GAS134US GAS135US
GAS136US GAS137US GAS138US GAS139US GAS140US GAS141US GAS142US
GAS143US GAS144US GAS146US GAS147US GRR001US GRR002US GRR003US
GRR004US GRR005US GRR006US GRR007US GRR008US GRR009US GRR010US
GVA001US GVA002US GVA004US GVA005US GVA006US GVA007US GVA008US
GVA009US GVA010US GVA011US GVA012US GVA013US GVA014US GVA015US
GVA016US GVA017US GVA018US GVA019US GVA020US GVA021US GVA022US
GHU001US GHU002US GHU003US GHU004US GHU006US GHU007US GHU008US
GWM001US GWM002US GDI001US GDI002US GDI003US GDI004US GDI005US
GDI006US GDI007US GDI009US GDI010US GDI011US GDI013US GDI014US
GDI015US GDI017US GDI019US GDI023US GDI028US GDI030US GDI039US
GDI040US GDI041US GPC001US GPC002US GPC003US GPC004US GPC005US
GPC006US GPC007US GPC008US GPC009US GPC010US GPC011US GPC012US
GPC014US GPC017US GPC018US GPC019US GPC023US GPC027US GPC028US
GPC029US GPC030US GPC031US GPC033US GPC034US GPC035US GPC036US
GPC037US GPC038US GPC039US GPC040US GPC041US GPC042US GPC043US
GLY001US GLY002US GLY003US GLY004US GLY005US GLY006US GIN001US
GIN002US GIN003US GIN004US GIN005US GIN006US GIN007US GIN008US
GMI001US GMI002US GMI005US GMI008US GLE001US GLE002US GLE003US
GLE004US GLE005US GLE006US GLE007US GLE008US GLE009US GLE010US
GLE011US GLE012US GLE013US GLE014US GLA001US GGA001US GGA003US
GRE001US GRE002US GRE003US GRE004US GRE005US GRE006US GRE007US
GCF001US GCF002US GCF003US GCF004US GCF005US GCF006US GCF007US
GCF008US GCF009US GCF010US GCF011US GCF012US GCF013US GCF014US
GCF015US GCF016US GCF020US GCF021US GCF022US GCF023US GCF024US
GCF025US GCF027US GCF028US GCF029US GCF030US GCF031US GCF032US
GCF033US GCF034US GCF035US GCF036US GCF037US GSA001US GSA002US
GSE001US GSE002US GSE003US GSE004US GDA001US GDA002US GDA003US
GDA004US GDA005US GDA006US GDA007US GPK001US GMO001US GMV001US
GMV002US GMV003US GMV004US GRD001US GRD002US GRD003US GRD004US
GPD001US GPD003US GPD004US GPD005US GPD006US GPD007US GPD008US
GPD009US GPD010US GPD011US GPD012US GPD013US GPD014US GPD015US
GPD016US GPD017US GAL001US GPA001US GPA003US GPA004US GPA005US
GSS001US GSL001US GCA001US GCA002US GCA003US
[0003] The disclosures of these co-pending applications are
incorporated herein by reference. The above applications have been
identified by their filing docket number, which will be substituted
with the corresponding application number, once assigned.
BACKGROUND OF THE INVENTION
[0004] Molecular diagnostics has emerged as a field that offers the
promise of early disease detection, potentially before symptoms
have manifested. Molecular diagnostic testing is used to detect:
[0005] Inherited disorders [0006] Acquired disorders [0007]
Infectious diseases [0008] Genetic predisposition to health-related
conditions.
[0009] With high accuracy and fast turnaround times, molecular
diagnostic tests have the potential to reduce the occurrence of
ineffective health care services, enhance patient outcomes, improve
disease management and individualize patient care. Many of the
techniques in molecular diagnostics are based on the detection and
identification of specific nucleic acids, both deoxyribonucleic
acid (DNA) and ribonucleic acid (RNA), extracted and amplified from
a biological specimen (such as blood or saliva). The complementary
nature of the nucleic acid bases allows short sequences of
synthesized DNA (oligonucleotides) to bond (hybridize) to specific
nucleic acid sequences for use in nucleic acid tests. If
hybridization occurs, then the complementary sequence is present in
the sample. This makes it possible, for example, to predict the
disease a person will contract in the future, determine the
identity and virulence of an infectious pathogen, or determine the
response a person will have to a drug.
Nucleic Acid Based Molecular Diagnostic Test
[0010] A nucleic acid based test has four distinct steps:
[0011] 1. Sample preparation
[0012] 2. Nucleic acid extraction
[0013] 3. Nucleic acid amplification (optional)
[0014] 4. Detection
[0015] Many sample types are used for genetic analysis, such as
blood, urine, sputum and tissue samples. The diagnostic test
determines the type of sample required as not all samples are
representative of the disease process. These samples have a variety
of constituents, but usually only one of these is of interest. For
example, in blood, high concentrations of erythrocytes can inhibit
the detection of a pathogenic organism. Therefore a purification
and/or concentration step at the beginning of the nucleic acid test
is often required.
[0016] Blood is one of the more commonly sought sample types. It
has three major constituents: leukocytes (white blood cells),
erythrocytes (red blood cells) and thrombocytes (platelets). The
thrombocytes facilitate clotting and remain active in vitro. To
inhibit coagulation, the specimen is mixed with an agent such as
ethylenediaminetetraacetic acid (EDTA) prior to purification and
concentration. Erythrocytes are usually removed from the sample in
order to concentrate the target cells. In humans, erythrocytes
account for approximately 99% of the cellular material but do not
carry DNA as they have no nucleus. Furthermore, erythrocytes
contain components such as haemoglobin that can interfere with the
downstream nucleic acid amplification process (described below).
Removal of erythrocytes can be achieved by differentially lysing
the erythrocytes in a lysis solution, leaving remaining cellular
material intact which can then be separated from the sample using
centrifugation. This provides a concentration of the target cells
from which the nucleic acids are extracted.
[0017] The exact protocol used to extract nucleic acids depends on
the sample and the diagnostic assay to be performed. For example,
the protocol for extracting viral RNA will vary considerably from
the protocol to extract genomic DNA. However, extracting nucleic
acids from target cells usually involves a cell lysis step followed
by nucleic acid purification. The cell lysis step disrupts the cell
and nuclear membranes, releasing the genetic material. This is
often accomplished using a lysis detergent, such as sodium dodecyl
sulfate, which also denatures the large amount of proteins present
in the cells.
[0018] The nucleic acids are then purified with an alcohol
precipitation step, usually ice-cold ethanol or isopropanol, or via
a solid phase purification step, typically on a silica matrix in a
column, resin or on paramagnetic beads in the presence of high
concentrations of a chaotropic salt, prior to washing and then
elution in a low ionic strength buffer. An optional step prior to
nucleic acid precipitation is the addition of a protease which
digests the proteins in order to further purify the sample.
[0019] Other lysis methods include mechanical lysis via ultrasonic
vibration and thermal lysis where the sample is heated to
94.degree. C. to disrupt cell membranes.
[0020] The target DNA or RNA may be present in the extracted
material in very small amounts, particularly if the target is of
pathogenic origin. Nucleic acid amplification provides the ability
to selectively amplify (that is, replicate) specific targets
present in low concentrations to detectable levels.
[0021] The most commonly used nucleic acid amplification technique
is the polymerase chain reaction (PCR). PCR is well known in this
field and comprehensive description of this type of reaction is
provided in E. van Pelt-Verkuil et al., Principles and Technical
Aspects of PCR Amplification, Springer, 2008.
[0022] PCR is a powerful technique that amplifies a target DNA
sequence against a background of complex DNA. If RNA is to be
amplified (by PCR), it must be first transcribed into cDNA
(complementary DNA) using an enzyme called reverse transcriptase.
Afterwards, the resulting cDNA is amplified by PCR.
[0023] PCR is an exponential process that proceeds as long as the
conditions for sustaining the reaction are acceptable. The
components of the reaction are:
[0024] 1. pair of primers--short single strands of DNA with around
10-30 nucleotides complementary to the regions flanking the target
sequence
[0025] 2. DNA polymerase--a thermostable enzyme that synthesizes
DNA
[0026] 3. deoxyribonucleoside triphosphates (dNTPs)--provide the
nucleotides that are incorporated into the newly synthesized DNA
strand
[0027] 4. buffer--to provide the optimal chemical environment for
DNA synthesis
[0028] PCR typically involves placing these reactants in a small
tube (.about.10-50 microlitres) containing the extracted nucleic
acids. The tube is placed in a thermal cycler; an instrument that
subjects the reaction to a series of different temperatures for
varying amounts of time. The standard protocol for each thermal
cycle involves a denaturation phase, an annealing phase, and an
extension phase. The extension phase is sometimes referred to as
the primer extension phase. In addition to such three-step
protocols, two-step thermal protocols can be employed, in which the
annealing and extension phases are combined. The denaturation phase
typically involves raising the temperature of the reaction to
90-95.degree. C. to denature the DNA strands; in the annealing
phase, the temperature is lowered to .about.50-60.degree. C. for
the primers to anneal; and then in the extension phase the
temperature is raised to the optimal DNA polymerase activity
temperature of 60-72.degree. C. for primer extension. This process
is repeated cyclically around 20-40 times, the end result being the
creation of millions of copies of the target sequence between the
primers.
[0029] There are a number of variants to the standard PCR protocol
such as multiplex PCR, linker-primed PCR, direct PCR, tandem PCR,
real-time PCR and reverse-transcriptase PCR, amongst others, which
have been developed for molecular diagnostics.
[0030] Multiplex PCR uses multiple primer sets within a single PCR
mixture to produce amplicons of varying sizes that are specific to
different DNA sequences. By targeting multiple genes at once,
additional information may be gained from a single test-run that
otherwise would require several experiments. Optimization of
multiplex PCR is more difficult though and requires selecting
primers with similar annealing temperatures, and amplicons with
similar lengths and base composition to ensure the amplification
efficiency of each amplicon is equivalent.
[0031] Linker-primed PCR, also known as ligation adaptor PCR, is a
method used to enable nucleic acid amplification of essentially all
DNA sequences in a complex DNA mixture without the need for
target-specific primers. The method firstly involves digesting the
target DNA population with a suitable restriction endonuclease
(enzyme). Double-stranded oligonucleotide linkers (also called
adaptors) with a suitable overhanging end are then ligated to the
ends of target DNA fragments using a ligase enzyme. Nucleic acid
amplification is subsequently performed using oligonucleotide
primers which are specific for the linker sequences. In this way,
all fragments of the DNA source which are flanked by linker
oligonucleotides can be amplified.
[0032] Direct PCR describes a system whereby PCR is performed
directly on a sample without any, or with minimal, nucleic acid
extraction. It has long been accepted that PCR reactions are
inhibited by the presence of many components of unpurified
biological samples, such as the haem component in blood.
Traditionally, PCR has required extensive purification of the
target nucleic acid prior to preparation of the reaction mixture.
With appropriate changes to the chemistry and sample concentration,
however, it is possible to perform PCR with minimal DNA
purification, or direct PCR. Adjustments to the PCR chemistry for
direct PCR include increased buffer strength, the use of
polymerases which have high activity and processivity, and
additives which chelate with potential polymerase inhibitors.
[0033] Tandem PCR utilises two distinct rounds of nucleic acid
amplification to increase the probability that the correct amplicon
is amplified. One form of tandem PCR is nested PCR in which two
pairs of PCR primers are used to amplify a single locus in separate
rounds of nucleic acid amplification. The first pair of primers
hybridize to the nucleic acid sequence at regions external to the
target nucleic acid sequence. The second pair of primers (nested
primers) used in the second round of amplification bind within the
first PCR product and produce a second PCR product containing the
target nucleic acid, that will be shorter than the first one. The
logic behind this strategy is that if the wrong locus were
amplified by mistake during the first round of nucleic acid
amplification, the probability is very low that it would also be
amplified a second time by a second pair of primers and thus
ensures specificity.
[0034] Real-time PCR, or quantitative PCR, is used to measure the
quantity of a PCR product in real time. By using a
fluorophore-containing probe or fluorescent dyes along with a set
of standards in the reaction, it is possible to quantitate the
starting amount of nucleic acid in the sample. This is particularly
useful in molecular diagnostics where treatment options may differ
depending on the pathogen load in the sample.
[0035] Reverse-transcriptase PCR (RT-PCR) is used to amplify DNA
from RNA. Reverse transcriptase is an enzyme that reverse
transcribes RNA into complementary DNA (cDNA), which is then
amplified by PCR. RT-PCR is widely used in expression profiling, to
determine the expression of a gene or to identify the sequence of
an RNA transcript, including transcription start and termination
sites. It is also used to amplify RNA viruses such as human
immunodeficiency virus or hepatitis C virus.
[0036] Isothermal amplification is another form of nucleic acid
amplification which does not rely on the thermal denaturation of
the target DNA during the amplification reaction and hence does not
require sophisticated machinery. Isothermal nucleic acid
amplification methods can therefore be carried out in primitive
sites or operated easily outside of a laboratory environment. A
number of isothermal nucleic acid amplification methods have been
described, including Strand Displacement Amplification,
Transcription Mediated Amplification, Nucleic Acid Sequence Based
Amplification, Recombinase Polymerase Amplification, Rolling Circle
Amplification, Ramification Amplification, Helicase-Dependent
Isothermal DNA Amplification and Loop-Mediated Isothermal
Amplification.
[0037] Isothermal nucleic acid amplification methods do not rely on
the continuing heat denaturation of the template DNA to produce
single stranded molecules to serve as templates for further
amplification, but instead rely on alternative methods such as
enzymatic nicking of DNA molecules by specific restriction
endonucleases, or the use of an enzyme to separate the DNA strands,
at a constant temperature.
[0038] Strand Displacement Amplification (SDA) relies on the
ability of certain restriction enzymes to nick the unmodified
strand of hemi-modified DNA and the ability of a 5'-3'
exonuclease-deficient polymerase to extend and displace the
downstream strand. Exponential nucleic acid amplification is then
achieved by coupling sense and antisense reactions in which strand
displacement from the sense reaction serves as a template for the
antisense reaction. The use of nickase enzymes which do not cut DNA
in the traditional manner but produce a nick on one of the DNA
strands, such as N. Alw1, N. BstNB1 and Mly1, are useful in this
reaction. SDA has been improved by the use of a combination of a
heat-stable restriction enzyme (Ava1) and heat-stable
Exo-polymerase (Bst polymerase). This combination has been shown to
increase amplification efficiency of the reaction from 10.sup.8
fold amplification to 10.sup.10 fold amplification so that it is
possible using this technique to amplify unique single copy
molecules.
[0039] Transcription Mediated Amplification (TMA) and Nucleic Acid
Sequence Based Amplification (NASBA) use an RNA polymerase to copy
RNA sequences but not corresponding genomic DNA. The technology
uses two primers and two or three enzymes, RNA polymerase, reverse
transcriptase and optionally RNase H (if the reverse transcriptase
does not have RNase activity). One primer contains a promoter
sequence for RNA polymerase. In the first step of nucleic acid
amplification, this primer hybridizes to the target ribosomal RNA
(rRNA) at a defined site. Reverse transcriptase creates a DNA copy
of the target rRNA by extension from the 3' end of the promoter
primer. The RNA in the resulting RNA:DNA duplex is degraded by the
RNase activity of the reverse transcriptase if present or the
additional RNase H. Next, a second primer binds to the DNA copy. A
new strand of DNA is synthesized from the end of this primer by
reverse transcriptase, creating a double-stranded DNA molecule. RNA
polymerase recognizes the promoter sequence in the DNA template and
initiates transcription. Each of the newly synthesized RNA
amplicons re-enters the process and serves as a template for a new
round of replication.
[0040] In Recombinase Polymerase Amplification (RPA), the
isothermal amplification of specific DNA fragments is achieved by
the binding of opposing oligonucleotide primers to template DNA and
their extension by a DNA polymerase. Heat is not required to
denature the double-stranded DNA (dsDNA) template. Instead, RPA
employs recombinase-primer complexes to scan dsDNA and facilitate
strand exchange at cognate sites. The resulting structures are
stabilised by single-stranded DNA binding proteins interacting with
the displaced template strand, thus preventing the ejection of the
primer by branch migration. Recombinase disassembly leaves the 3'
end of the oligonucleotide accessible to a strand displacing DNA
polymerase, such as the large fragment of Bacillus subtilis Pol I
(Bsu), and primer extension ensues. Exponential nucleic acid
amplification is accomplished by the cyclic repetition of this
process.
[0041] Helicase-dependent amplification (HDA) mimics the in vivo
system in that it uses a DNA helicase enzyme to generate
single-stranded templates for primer hybridization and subsequent
primer extension by a DNA polymerase. In the first step of the HDA
reaction, the helicase enzyme traverses along the target DNA,
disrupting the hydrogen bonds linking the two strands which are
then bound by single-stranded binding proteins. Exposure of the
single-stranded target region by the helicase allows primers to
anneal. The DNA polymerase then extends the 3' ends of each primer
using free deoxyribonucleoside triphosphates (dNTPs) to produce two
DNA replicates. The two replicated dsDNA strands independently
enter the next cycle of HDA, resulting in exponential nucleic acid
amplification of the target sequence.
[0042] Other DNA-based isothermal techniques include Rolling Circle
Amplification (RCA) in which a DNA polymerase extends a primer
continuously around a circular DNA template, generating a long DNA
product that consists of many repeated copies of the circle. By the
end of the reaction, the polymerase generates many thousands of
copies of the circular template, with the chain of copies tethered
to the original target DNA. This allows for spatial resolution of
target and rapid nucleic acid amplification of the signal. Up to
10.sup.12 copies of template can be generated in 1 hour.
Ramification amplification is a variation of RCA and utilizes a
closed circular probe (C-probe) or padlock probe and a DNA
polymerase with a high processivity to exponentially amplify the
C-probe under isothermal conditions.
[0043] Loop-mediated isothermal amplification (LAMP), offers high
selectivity and employs a DNA polymerase and a set of four
specially designed primers that recognize a total of six distinct
sequences on the target DNA. An inner primer containing sequences
of the sense and antisense strands of the target DNA initiates
LAMP. The following strand displacement DNA synthesis primed by an
outer primer releases a single-stranded DNA. This serves as
template for DNA synthesis primed by the second inner and outer
primers that hybridize to the other end of the target, which
produces a stem-loop DNA structure. In subsequent LAMP cycling one
inner primer hybridizes to the loop on the product and initiates
displacement DNA synthesis, yielding the original stem-loop DNA and
a new stem-loop DNA with a stem twice as long. The cycling reaction
continues with accumulation of 10.sup.9 copies of target in less
than an hour. The final products are stem-loop DNAs with several
inverted repeats of the target and cauliflower-like structures with
multiple loops formed by annealing between alternately inverted
repeats of the target in the same strand.
[0044] After completion of the nucleic acid amplification, the
amplified product must be analysed to determine whether the
anticipated amplicon (the amplified quantity of target nucleic
acids) was generated. The methods of analyzing the product range
from simply determining the size of the amplicon through gel
electrophoresis, to identifying the nucleotide composition of the
amplicon using DNA hybridization.
[0045] Gel electrophoresis is one of the simplest ways to check
whether the nucleic acid amplification process generated the
anticipated amplicon. Gel electrophoresis uses an electric field
applied to a gel matrix to separate DNA fragments. The negatively
charged DNA fragments will move through the matrix at different
rates, determined largely by their size. After the electrophoresis
is complete, the fragments in the gel can be stained to make them
visible. Ethidium bromide is a commonly used stain which fluoresces
under UV light.
[0046] The size of the fragments is determined by comparison with a
DNA size marker (a DNA ladder), which contains DNA fragments of
known sizes, run on the gel alongside the amplicon. Because the
oligonucleotide primers bind to specific sites flanking the target
DNA, the size of the amplified product can be anticipated and
detected as a band of known size on the gel. To be certain of the
identity of the amplicon, or if several amplicons have been
generated, DNA probe hybridization to the amplicon is commonly
employed.
[0047] DNA hybridization refers to the formation of double-stranded
DNA by complementary base pairing. DNA hybridization for positive
identification of a specific amplification product requires the use
of a DNA probe around 20 nucleotides in length. If the probe has a
sequence that is complementary to the amplicon (target) DNA
sequence, hybridization will occur under favourable conditions of
temperature, pH and ionic concentration. If hybridization occurs,
then the gene or DNA sequence of interest was present in the
original sample.
[0048] Optical detection is the most common method to detect
hybridization. Either the amplicons or the probes are labelled to
emit light through fluorescence or electrochemiluminescence. These
processes differ in the means of producing excited states of the
light-producing moieties, but both enable covalent labelling of
nucleotide strands. In electrochemiluminescence (ECL), light is
produced by luminophore molecules or complexes upon stimulation
with an electric current. In fluorescence, it is illumination with
excitation light which leads to emission.
[0049] Fluorescence is detected using an illumination source which
provides excitation light at a wavelength absorbed by the
fluorescent molecule, and a detection unit. The detection unit
comprises a photosensor (such as a photomultiplier tube or
charge-coupled device (CCD) array) to detect the emitted signal,
and a mechanism (such as a wavelength-selective filter) to prevent
the excitation light from being included in the photosensor output.
The fluorescent molecules emit Stokes-shifted light in response to
the excitation light, and this emitted light is collected by the
detection unit. Stokes shift is the frequency difference or
wavelength difference between emitted light and absorbed excitation
light.
[0050] ECL emission is detected using a photosensor which is
sensitive to the emission wavelength of the ECL species being
employed. For example, transition metal-ligand complexes emit light
at visible wavelengths, so conventional photodiodes and CCDs are
employed as photosensors. An advantage of ECL is that, if ambient
light is excluded, the ECL emission can be the only light present
in the detection system, which improves sensitivity.
[0051] Microarrays allow for hundreds of thousands of DNA
hybridization experiments to be performed simultaneously.
Microarrays are powerful tools for molecular diagnostics with the
potential to screen for thousands of genetic diseases or detect the
presence of numerous infectious pathogens in a single test. A
microarray consists of many different DNA probes immobilized as
spots on a substrate. The target DNA (amplicon) is first labelled
with a fluorescent or luminescent molecule (either during or after
nucleic acid amplification) and then applied to the array of
probes. The microarray is incubated in a temperature controlled,
humid environment for a number of hours or days while hybridization
between the probe and amplicon takes place. Following incubation,
the microarray must be washed in a series of buffers to remove
unbound strands. Once washed, the microarray surface is dried using
a stream of air (often nitrogen). The stringency of the
hybridization and washes is critical. Insufficient stringency can
result in a high degree of nonspecific binding. Excessive
stringency can lead to a failure of appropriate binding, which
results in diminished sensitivity. Hybridization is recognized by
detecting light emission from the labelled amplicons which have
formed a hybrid with complementary probes.
[0052] Fluorescence from microarrays is detected using a microarray
scanner which is generally a computer controlled inverted scanning
fluorescence confocal microscope which typically uses a laser for
excitation of the fluorescent dye and a photosensor (such as a
photomultiplier tube or CCD) to detect the emitted signal. The
fluorescent molecules emit Stokes-shifted light (described above)
which is collected by the detection unit.
[0053] The emitted fluorescence must be collected, separated from
the unabsorbed excitation wavelength, and transported to the
detector. In microarray scanners, a confocal arrangement is
commonly used to eliminate out-of-focus information by means of a
confocal pinhole situated at an image plane. This allows only the
in-focus portion of the light to be detected. Light from above and
below the plane of focus of the object is prevented from entering
the detector, thereby increasing the signal to noise ratio. The
detected fluorescent photons are converted into electrical energy
by the detector which is subsequently converted to a digital
signal. This digital signal translates to a number representing the
intensity of fluorescence from a given pixel. Each feature of the
array is made up of one or more such pixels. The final result of a
scan is an image of the array surface. The exact sequence and
position of every probe on the microarray is known, and so the
hybridized target sequences can be identified and analysed
simultaneously.
[0054] More information regarding fluorescent probes can be found
at: http://www.premierbiosoft.com/tech_notes/FRET_probe.html and
http://www.invitrogen.com/site/us/en/home/References/Molecular-Probes-The-
-Handbook/Technical-Notes-and-Product-Highlights/Fluorescence-Resonance-En-
ergy-Transfer-FRET.html
Point-Of-Care Molecular Diagnostics
[0055] Despite the advantages that molecular diagnostic tests
offer, the growth of this type of testing in the clinical
laboratory has been slower than expected and remains a minor part
of the practice of laboratory medicine. This is primarily due to
the complexity and costs associated with nucleic acid testing
compared with tests based on methods not involving nucleic acids.
The widespread adaptation of molecular diagnostics testing to the
clinical setting is intimately tied to the development of
instrumentation that significantly reduces the cost, provides a
rapid and automated assay from start (specimen processing) to
finish (generating a result) and operates without major
intervention by personnel.
[0056] A point-of-care technology serving the physician's office,
the hospital bedside or even consumer-based, at home, would offer
many advantages including: [0057] rapid availability of results
enabling immediate facilitation of treatment and improved quality
of care. [0058] ability to obtain laboratory values from testing
very small samples. [0059] reduced clinical workload. [0060]
reduced laboratory workload and improved office efficiency by
reducing administrative work. [0061] improved cost per patient
through reduced length of stay of hospitalization, conclusion of
outpatient consultation at the first visit, and reduced handling,
storing and shipping of specimens. [0062] facilitation of clinical
management decisions such as infection control and antibiotic
use.
Lab-On-A-Chip (LOC) Based Molecular Diagnostics
[0063] Molecular diagnostic systems based on microfluidic
technologies provide the means to automate and speed up molecular
diagnostic assays. The quicker detection times are primarily due to
the extremely low volumes involved, automation, and the
low-overhead inbuilt cascading of the diagnostic process steps
within a microfluidic device. Volumes in the nanoliter and
microliter scale also reduce reagent consumption and cost.
Lab-on-a-chip (LOC) devices are a common form of microfluidic
device. LOC devices have MST structures within a MST layer for
fluid processing integrated onto a single supporting substrate
(usually silicon). Fabrication using the VLSI (very large scale
integrated) lithographic techniques of the semiconductor industry
keeps the unit cost of each LOC device very low. However,
controlling fluid flow through the LOC device, adding reagents,
controlling reaction conditions and so on necessitate bulky
external plumbing and electronics. Connecting a LOC device to these
external devices effectively restricts the use of LOC devices for
molecular diagnostics to the laboratory setting. The cost of the
external equipment and complexity of its operation precludes
LOC-based molecular diagnostics as a practical option for
point-of-care settings.
[0064] In view of the above, there is a need for a molecular
diagnostic system based on a LOC device for use at
point-of-care.
SUMMARY OF THE INVENTION
[0065] Accordingly, the present invention provides a dialysis
device for dialysis of a fluid containing constituents of different
sizes, the dialysis device comprising:
[0066] a first layer of material defining a first channel and a
second channel, the first channel configured for receiving the
fluid which contains constituents of different sizes;
[0067] a second layer having a plurality of apertures open to the
first channel and at least one fluid connection leading from the
apertures to the second channel for establishing fluid
communication between the first channel and the second channel;
wherein,
[0068] the apertures are sized in accordance with a predetermined
size threshold such that the constituents flowing to the second
channel are smaller constituents that are smaller than the
predetermined size threshold, and the constituents retained in the
first channel include larger constituents which are larger than the
predetermined size threshold.
[0069] Preferably, the second layer is a laminate with a roof
layer, and the at least one fluid connection is a series of
adjacent channels enclosed by the roof layer, the apertures being
formed in the roof layer between the first channel and the adjacent
channels.
[0070] Preferably, the first and second channels, and the series of
adjacent channels are configured to fill with the sample by
capillary action.
[0071] Preferably, the dialysis device also has a bypass channel
between the first channel and the second channel, wherein each of
the adjacent channels are configured to pin a meniscus of the fluid
that arrests capillary flow between the first channel and the
second channel, and the bypass channel between the first channel
and the second channel, the bypass channel joining the second
channel upstream of the series of adjacent channels and is
configured for uninterrupted capillary driven flow from the first
channel to the second channel, such that flow from the bypass
channel reaches the meniscus pinned at each of the adjacent
channels after the meniscus has formed to sequentially remove each
of the menisci and flow from the first channel to the second
channel is via the adjacent channels as well as the bypass
channel.
[0072] Preferably, the sample is a biological sample containing
constituents of different sizes and the adjacent channels and the
bypass channel have apertures sized in accordance with a
predetermined size threshold such that the constituents flowing to
the second channel are smaller constituents that are smaller than
the predetermined size threshold, and the constituents retained in
the first channel include larger constituents which are larger than
the predetermined size threshold.
[0073] Preferably, the biological sample is blood and the larger
constituents include leukocytes and the smaller constituents
include erythrocytes.
[0074] Preferably, the adjacent channels are a series of parallel,
adjacent channels extending normal to the first channel and the
second channel.
[0075] Preferably, the dialysis device also has a waste reservoir
for receiving constituents not required for downstream processing
or analysis.
[0076] Preferably, the apertures are holes with diameters less than
8 microns.
[0077] Preferably, the smaller constituents include targets such
that processing of the smaller constituents includes detection of
the targets.
[0078] Preferably, the dialysis device also has a lysis section
connected to the target channel for lysing the target cells to
release target nucleic acid sequences therein.
[0079] Preferably, the dialysis device also has a nucleic acid
amplification section for amplifying the target nucleic acid
sequences.
[0080] Preferably, the dialysis device also has a hybridization
section having an array of probes for hybridization with the target
nucleic acid sequences amplified by the nucleic acid amplification
section.
[0081] Preferably, the probes are configured to form probe-target
hybrids with the target nucleic acid sequences, the probe-target
hybrids being electrochemiluminescent in response to an electrical
pulse.
[0082] Preferably, the dialysis device also has CMOS circuitry for
operative control of the nucleic acid amplification section and the
hybridization section, the CMOS circuitry also having a photosensor
for sensing electrochemiluminescence emission from the probe-target
hybrids.
[0083] Preferably, the hybridization section has an array of
hybridization chambers containing the probes for hybridization with
the target nucleic acid sequences.
[0084] Preferably, the photosensor is an array of photodiodes
positioned adjacent each of the hybridization chambers
respectively.
[0085] Preferably, the CMOS circuitry has a digital memory for
storing data relating to the processing of the fluid, the data
including the probe details and location of each of the probes in
the array of hybridization chambers.
[0086] Preferably, the CMOS circuitry has bond-pads for electrical
connection to an external device and is configured to convert
output from the photodiodes into a signal indicative of the probes
that hybridized with the target nucleic acid sequences, and provide
the signal to the bond-pads for transmission to the external
device.
[0087] Preferably, the dialysis device also has a plurality of
reservoirs for holding liquid reagents for addition to the
sample.
[0088] The easily usable, mass-producible, and inexpensive
microfluidic multichip assembly accepts a biochemical sample, uses
a dialysis chip for separating the cells of different dimensions,
and separately processes the nucleic acid content of the cells
separated based on their dimensions. The dialysis chip
functionality extract additional information from the sample and
increases the sensitivity, signal-to-noise ratio, and dynamic range
of the assay system.
[0089] The microfluidic multichip assembly provides for higher
modularity. Surface-micromachined chips in the assembly would each
be much smaller and disproportionately cheaper that a monolithic
chip capable of providing the total functionality of the assembly.
Alternatively a large yet cost-effective dialysis chip can provide
a high level of dialysis capacity providing for further increased
sensitivity, signal-to-noise ratio, and dynamic range for the assay
system.
[0090] Functionally optimal, less expensive fabrication process is
utilized to fabricate each surface-micromachined chip constituent
of the microfluidic multichip assembly, ie, the bioMST-only
dialysis chip skips all of the CMOS process steps and uses an
inexpensive glass wafer.
BRIEF DESCRIPTION OF THE DRAWINGS
[0091] Preferred embodiments of the present invention will now be
described by way of example only with reference to the accompanying
drawings, in which:
[0092] FIG. 1 shows a test module and test module reader configured
for fluorescence detection;
[0093] FIG. 2 is a schematic overview of the electronic components
in the test module configured for fluorescence detection;
[0094] FIG. 3 is a schematic overview of the electronic components
in the test module reader;
[0095] FIG. 4 is a schematic representation of the architecture of
the LOC device;
[0096] FIG. 5 is a perspective of the LOC device;
[0097] FIG. 6 is a plan view of the LOC device with features and
structures from all layers superimposed on each other;
[0098] FIG. 7 is a plan view of the LOC device with the structures
of the cap shown in isolation;
[0099] FIG. 8 is a top perspective of the cap with internal
channels and reservoirs shown in dotted line;
[0100] FIG. 9 is an exploded top perspective of the cap with
internal channels and reservoirs shown in dotted line;
[0101] FIG. 10 is a bottom perspective of the cap showing the
configuration of the top channels;
[0102] FIG. 11 is a plan view of the LOC device showing the
structures of the CMOS+MST device in isolation;
[0103] FIG. 12 is a schematic section view of the LOC device at the
sample inlet;
[0104] FIG. 13 is an enlarged view of Inset AA shown in FIG. 6;
[0105] FIG. 14 is an enlarged view of Inset AB shown in FIG. 6;
[0106] FIG. 15 is an enlarged view of Inset AE shown in FIG.
13;
[0107] FIG. 16 is a partial perspective illustrating the laminar
structure of the LOC device within Inset AE;
[0108] FIG. 17 is a partial perspective illustrating the laminar
structure of the LOC device within Inset AE;
[0109] FIG. 18 is a partial perspective illustrating the laminar
structure of the LOC device within Inset AE;
[0110] FIG. 19 is a partial perspective illustrating the laminar
structure of the LOC device within Inset AE;
[0111] FIG. 20 is a partial perspective illustrating the laminar
structure of the LOC device within Inset AE;
[0112] FIG. 21 is a partial perspective illustrating the laminar
structure of the LOC device within Inset AE;
[0113] FIG. 22 is schematic section view of the lysis reagent
reservoir shown in FIG. 21;
[0114] FIG. 23 is a partial perspective illustrating the laminar
structure of the LOC device within Inset AB;
[0115] FIG. 24 is a partial perspective illustrating the laminar
structure of the LOC device within Inset AB;
[0116] FIG. 25 is a partial perspective illustrating the laminar
structure of the LOC device within Inset AI;
[0117] FIG. 26 is a partial perspective illustrating the laminar
structure of the LOC device within Inset AB;
[0118] FIG. 27 is a partial perspective illustrating the laminar
structure of the LOC device within Inset AB;
[0119] FIG. 28 is a partial perspective illustrating the laminar
structure of the LOC device within Inset AB;
[0120] FIG. 29 is a partial perspective illustrating the laminar
structure of the LOC device within Inset AB;
[0121] FIG. 30 is a schematic section view of the amplification mix
reservoir and the polymerase reservoir;
[0122] FIG. 31 show the features of a boiling-initiated valve in
isolation;
[0123] FIG. 32 is a schematic section view of the boiling-initiated
valve taken through line 33-33 shown in FIG. 31;
[0124] FIG. 33 is an enlarged view of Inset AF shown in FIG.
15;
[0125] FIG. 34 is a schematic section view of the upstream end of
the dialysis section taken through line 35-35 shown in FIG. 33;
[0126] FIG. 35 is an enlarged view of Inset AC shown in FIG. 6;
[0127] FIG. 36 is a further enlarged view within Inset AC showing
the amplification section;
[0128] FIG. 37 is a further enlarged view within Inset AC showing
the amplification section;
[0129] FIG. 38 is a further enlarged view within Inset AC showing
the amplification section;
[0130] FIG. 39 is a further enlarged view within Inset AK shown in
FIG. 38;
[0131] FIG. 40 is a further enlarged view within Inset AC showing
the amplification chamber;
[0132] FIG. 41 is a further enlarged view within Inset AC showing
the amplification section;
[0133] FIG. 42 is a further enlarged view within Inset AC showing
the amplification chamber;
[0134] FIG. 43 is a further enlarged view within Inset AL shown in
FIG. 42;
[0135] FIG. 44 is a further enlarged view within Inset AC showing
the amplification section;
[0136] FIG. 45 is a further enlarged view within Inset AM shown in
FIG. 44;
[0137] FIG. 46 is a further enlarged view within Inset AC showing
the amplification chamber;
[0138] FIG. 47 is a further enlarged view within Inset AN shown in
FIG. 46;
[0139] FIG. 48 is a further enlarged view within Inset AC showing
the amplification chamber;
[0140] FIG. 49 is a further enlarged view within Inset AC showing
the amplification chamber;
[0141] FIG. 50 is a further enlarged view within Inset AC showing
the amplification section;
[0142] FIG. 51 is a schematic section view of the amplification
section;
[0143] FIG. 52 is an enlarged plan view of the hybridization
section;
[0144] FIG. 53 is a further enlarged plan view of two hybridization
chambers in isolation;
[0145] FIG. 54 is schematic section view of a single hybridization
chamber;
[0146] FIG. 55 is an enlarged view of the humidifier illustrated in
Inset AG shown in FIG. 6;
[0147] FIG. 56 is an enlarged view of Inset AD shown in FIG.
52;
[0148] FIG. 57 is an exploded perspective view of the LOC device
within Inset AD;
[0149] FIG. 58 is an enlarged plan view of the humidity sensor
shown in Inset AH of FIG. 6;
[0150] FIG. 59 is a schematic showing part of the photodiode array
of the photo sensor;
[0151] FIG. 60 is an enlarged view of the evaporator shown in Inset
AP of FIG. 55;
[0152] FIG. 61 is a diagram of linker-primed PCR;
[0153] FIG. 62 is a schematic representation of a test module with
a lancet;
[0154] FIG. 63 is a diagrammatic representation of the architecture
of LOC variant VII;
[0155] FIG. 64 is a plan view of LOC variant VIII with features and
structures from all layers superimposed on each other;
[0156] FIG. 65 is an enlarged view of Inset CA shown in FIG.
64;
[0157] FIG. 66 is a partial perspective illustrating the laminar
structure of LOC variant VIII within Inset CA shown in FIG. 64;
[0158] FIG. 67 is an enlarged view of Inset CE shown in FIG.
65;
[0159] FIG. 68 is a diagrammatic representation of the architecture
of LOC variant VIII;
[0160] FIG. 69 is a schematic illustration of the architecture of
LOC variant XIV;
[0161] FIG. 70 is a schematic illustration of the architecture of
LOC variant XLI;
[0162] FIG. 71 is a schematic illustration of the architecture of
LOC variant XLIII;
[0163] FIG. 72 is a schematic illustration of the architecture of
LOC variant XLIV;
[0164] FIG. 73 is a schematic illustration of the architecture of
LOC variant XLVII;
[0165] FIG. 74 is a diagrammatic representation of the architecture
of LOC variant XI;
[0166] FIG. 75 is a circuit diagram of the differential imager;
[0167] FIG. 76 illustrates the reactions occurring during an
electrochemiluminescence (ECL) process;
[0168] FIG. 77 schematically illustrates three different anode
configurations;
[0169] FIG. 78 is a schematic partial cross-section of the anode
and cathode in the hybridization chamber;
[0170] FIG. 79 schematically illustrates an anode in a ring
geometry around the peripheral edge of a photodiode;
[0171] FIG. 80 schematically illustrates an anode in a ring
geometry within the peripheral edge of a photodiode;
[0172] FIG. 81 schematically illustrates an anode with a series of
fingers to increase the length of its lateral edges;
[0173] FIG. 82 schematically illustrates the use of a transparent
anode to maximise surface area coupling and ECL signal
detection;
[0174] FIG. 83 schematically illustrates the use of an anode
affixed to the roof of the hybridization chamber to maximise
surface area coupling and ECL signal detection;
[0175] FIG. 84 schematically illustrates an anode interdigitated
with a cathode;
[0176] FIG. 85 shows a test module and test module reader
configured for use with ECL detection;
[0177] FIG. 86 is a schematic overview of the electronic components
in the test module configured for use with ECL detection;
[0178] FIG. 87 shows a test module and alternative test module
readers;
[0179] FIG. 88 shows a test module and test module reader along
with the hosting system housing various databases;
[0180] FIG. 89 is a plan view of LOC variant L showing all the
features superimposed on each other and showing the locations of
Insets GA to GL;
[0181] FIG. 90 is an enlarged view of Inset GD shown in FIG.
89;
[0182] FIG. 91 is an enlarged view of Inset GG shown in FIG.
89;
[0183] FIG. 92 is an enlarged view of Inset GH shown in FIG.
89;
[0184] FIG. 93 is a diagram of an electrochemiluminescence
resonance energy transfer probe in a closed configuration;
[0185] FIG. 94 is a diagram of an electrochemiluminescence
resonance energy transfer probe in an open and hybridized
configuration;
[0186] FIG. 95 is a diagram of a primer-linked, luminescent linear
probe during the initial round of amplification;
[0187] FIG. 96 is a diagram of a primer-linked, luminescent linear
probe during a subsequent amplification cycle;
[0188] FIGS. 97A to 97F diagrammatically illustrate thermal cycling
of a luminescent primer-linked stem-and-loop probe;
[0189] FIG. 98 schematically illustrates a negative control
luminescent probe in its stem-and-loop configuration;
[0190] FIG. 99 schematically illustrates the negative control
luminescent probe of FIG. 98 in its open configuration;
[0191] FIG. 100 schematically illustrates a positive control
luminescent probe in its stem-and-loop configuration;
[0192] FIG. 101 schematically illustrates the positive control
luminescent probe of FIG. 100 in its open configuration;
[0193] FIG. 102 is a diagrammatic representation of the
architecture of a microfluidic device with dialysis device, LOC
device, interconnecting cap and electrochemiluminescence (ECL)
detection;
[0194] FIG. 103 is a perspective view of a microfluidic device with
dialysis device, LOC device and interconnecting cap;
[0195] FIG. 104 is a plan view showing the features of the dialysis
device of the microfluidic device and showing the location of Inset
JA;
[0196] FIG. 105 is an enlarged view of Inset JA shown in FIG.
104;
[0197] FIG. 106 is a plan view showing the features of the LOC
device of the microfluidic device and showing the locations of
Insets JB to JJ;
[0198] FIG. 107 is an enlarged view of Inset JB shown in FIG.
106;
[0199] FIG. 108 is an enlarged view of Inset JC shown in FIG.
106;
[0200] FIG. 109 is an enlarged view of Inset JD shown in FIG.
106;
[0201] FIG. 110 is an enlarged view of Inset JE shown in FIG.
106;
[0202] FIG. 111 is an enlarged view of Inset JF shown in FIG.
106;
[0203] FIG. 112 is an enlarged view of Inset JG shown in FIG.
106;
[0204] FIG. 113 is an enlarged view of Inset JH shown in FIG.
106;
[0205] FIG. 114 is an enlarged view of Inset JJ shown in FIG.
106;
[0206] FIG. 115 is an enlarged view of the hybridization chamber of
LOC variant L;
[0207] FIG. 116 is an enlarged view of the hybridization chamber
array of LOC variant L showing the distribution of calibration
chambers;
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
Overview
[0208] This overview identifies the main components of a molecular
diagnostic system that incorporates embodiments of the present
invention. Comprehensive details of the system architecture and
operation are set out later in the specification.
[0209] Referring to FIGS. 1, 2, 3, 85 and 86, the system has the
following top level components:
[0210] Test modules 10 and 11 are the size of a typical USB memory
key and very cheap to produce. Test modules 10 and 11 each contain
a microfluidic device, typically in the form of a lab-on-a-chip
(LOC) device 30 preloaded with reagents and typically more than
1000 probes for the molecular diagnostic assay (see FIGS. 1 and
85). Test module 10 schematically shown in FIG. 1 uses a
fluorescence-based detection technique to identify target
molecules, while test module 11 in FIG. 85 uses an
electrochemiluminescence-based detection technique. The LOC device
30 has an integrated photosensor 44 for fluorescence or
electrochemiluminescence detection (described in detail below).
Both test modules 10 and 11 use a standard Micro-USB plug 14 for
power, data and control, both have a printed circuit board (PCB)
57, and both have external power supply capacitors 32 and an
inductor 15. The test modules 10 and 11 are both single-use only
for mass production and distribution in sterile packaging ready for
use.
[0211] The outer casing 13 has a macroreceptacle 24 for receiving
the biological sample and a removable sterile sealing tape 22,
preferably with a low tack adhesive, to cover the macroreceptacle
prior to use. A membrane seal 408 with a membrane guard 410 forms
part of the outer casing 13 to reduce dehumidification within the
test module while providing pressure relief from small air pressure
fluctuations. The membrane guard 410 protects the membrane seal 408
from damage.
[0212] Test module reader 12 powers the test module 10 or 11 via
Micro-USB port 16. The test module reader 12 can adopt many
different forms and a selection of these are described later. The
version of the reader 12 shown in FIGS. 1, 3 and 85 is a smart
phone embodiment. A block diagram of this reader 12 is shown in
FIG. 3. Processor 42 runs application software from program storage
43. The processor 42 also interfaces with the display screen 18 and
user interface (UI) touch screen 17 and buttons 19, a cellular
radio 21, wireless network connection 23, and a satellite
navigation system 25. The cellular radio 21 and wireless network
connection 23 are used for communications. Satellite navigation
system 25 is used for updating epidemiological databases with
location data. The location data can, alternatively, be entered
manually via the touch screen 17 or buttons 19. Data storage 27
holds genetic and diagnostic information, test results, patient
information, assay and probe data for identifying each probe and
its array position. Data storage 27 and program storage 43 may be
shared in a common memory facility. Application software installed
on the test module reader 12 provides analysis of results, along
with additional test and diagnostic information.
[0213] To conduct a diagnostic test, the test module 10 (or test
module 11) is inserted into the Micro-USB port 16 on the test
module reader 12. The sterile sealing tape 22 is peeled back and
the biological sample (in a liquid form) is loaded into the sample
macroreceptacle 24. Pressing start button 20 initiates testing via
the application software. The sample flows into the LOC device 30
and the on-board assay extracts, incubates, amplifies and
hybridizes the sample nucleic acids (the target) with
presynthesized hybridization-responsive oligonucleotide probes. In
the case of test module 10 (which uses fluorescence-based
detection), the probes are fluorescently labelled and the LED 26
housed in the casing 13 provides the necessary excitation light to
induce fluorescence emission from the hybridized probes (see FIGS.
1 and 2). In test module 11 (which uses electrochemiluminescence
(ECL) detection), the LOC device 30 is loaded with ECL probes
(discussed above) and the LED 26 is not necessary for generating
the luminescent emission. Instead, electrodes 860 and 870 provide
the excitation electrical current (see FIG. 86). The emission
(fluorescent or luminescent) is detected using a photosensor 44
integrated into CMOS circuitry of each LOC device. The detected
signal is amplified and converted to a digital output which is
analyzed by the test module reader 12. The reader then displays the
results.
[0214] The data may be saved locally and/or uploaded to a network
server containing patient records. The test module 10 or 11 is
removed from the test module reader 12 and disposed of
appropriately.
[0215] FIGS. 1, 3 and 85 show the test module reader 12 configured
as a mobile phone/smart phone 28. In other forms, the test module
reader is a laptop/notebook 101, a dedicated reader 103, an ebook
reader 107, a tablet computer 109 or desktop computer 105 for use
in hospitals, private practices or laboratories (see FIG. 87). The
reader can interface with a range of additional applications such
as patient records, billing, online databases and multi-user
environments. It can also be interfaced with a range of local or
remote peripherals such as printers and patient smart cards.
[0216] Referring to FIG. 88, the data generated by the test module
10 can be used to update, via the reader 12 and network 125, the
epidemiological databases hosted on the hosting system for
epidemiological data 111, the genetic databases hosted on the
hosting system for genetic data 113, the electronic health records
hosted on the hosting system for electronic health records (EHR)
115, the electronic medical records hosted on the hosting system
for electronic medical records (EMR) 121, and the personal health
records hosted on the hosting system for personal health records
(PHR) 123. Conversely, the epidemiological data hosted on the
hosting system for epidemiological data 111, the genetic data
hosted on the hosting system for genetic data 113, the electronic
health records hosted on the hosting system for electronic health
records (EHR) 115, the electronic medical records hosted on the
hosting system for electronic medical records (EMR) 121, and the
personal health records hosted on the hosting system for personal
health records (PHR) 123, can be used to update, via network 125
and the reader 12, the digital memory in the LOC 30 of the test
module 10.
[0217] Referring back to FIGS. 1, 2, 85 and 86 the reader 12 uses
battery power in the mobile phone configuration. The mobile phone
reader contains all test and diagnostic information preloaded. Data
can also be loaded or updated via a number of wireless or contact
interfaces to enable communications with peripheral devices,
computers or online servers. A Micro-USB port 16 is provided for
connection to a computer or mains power supply for battery
recharge.
[0218] FIG. 62 shows an embodiment of the test module 10 used for
tests that only require a positive or negative result for a
particular target, such as testing whether a person is infected
with, for example, H1N1 Influenza A virus. Only a purpose built USB
power/indicator-only module 47 is adequate. No other reader or
application software is necessary. An indicator 45 on the USB
power/indicator-only module 47 signals positive or negative
results. This configuration is well suited to mass screening.
[0219] Additional items supplied with the system may include a test
tube containing reagents for pre-treatment of certain samples,
along with spatula and lancet for sample collection. FIG. 62 shows
an embodiment of the test module incorporating a spring-loaded,
retractable lancet 390 and lancet release button 392 for
convenience. A satellite phone can be used in remote areas.
Test Module Electronics
[0220] FIGS. 2 and 86 are block diagrams of the electronic
components in the test modules 10 and 11, respectively. The CMOS
circuitry integrated in the LOC device 30 has a USB device driver
36, a controller 34, a USB-compatible LED driver 29, clock 33,
power conditioner 31, RAM 38 and program and data flash memory 40.
These provide the control and memory for the entire test module 10
or 11 including the photosensor 44, the temperature sensors 170,
the liquid sensors 174, and the various heaters 152, 154, 182, 234,
together with associated drivers 37 and 39 and registers 35 and 41.
Only the LED 26 (in the case of test module 10), external power
supply capacitors 32 and the Micro-USB plug 14 are external to the
LOC device 30. The LOC devices 30 include bond-pads for making
connections to these external components. The RAM 38 and the
program and data flash memory 40 have the application software and
the diagnostic and test information (Flash/Secure storage, e.g. via
encryption) for over 1000 probes. In the case of test module 11
configured for ECL detection, there is no LED 26 (see FIGS. 85 and
86). Data is encrypted by the LOC device 30 for secure storage and
secure communication with an external device. The LOC devices 30
are loaded with electrochemiluminescent probes and the
hybridization chambers each have a pair of ECL excitation
electrodes 860 and 870.
[0221] Many types of test modules 10 are manufactured in a number
of test forms, ready for off-the-shelf use. The differences between
the test forms lie in the on board assay of reagents and
probes.
[0222] Some examples of infectious diseases rapidly identified with
this system include: [0223] Influenza--Influenza virus A, B, C,
Isavirus, Thogotovirus [0224] Pneumonia--respiratory syncytial
virus (RSV), adenovirus, metapneumovirus, Streptococcus pneumoniae,
Staphylococcus aureus [0225] Tuberculosis--Mycobacterium
tuberculosis, bovis, africanum, canetti, and microti [0226]
Plasmodium falciparum, Toxoplasma gondii and other protozoan
parasites [0227] Typhoid--Salmonella enterica serovar typhi [0228]
Ebola virus [0229] Human immunodeficiency virus (HIV) [0230] Dengue
Fever--Flavivirus [0231] Hepatitis (A through E) [0232] Hospital
acquired infections--for example Clostridium difficile, Vancomycin
resistant Enterococcus, and Methicillin resistant Staphylococcus
aureus [0233] Herpes simplex virus (HSV) [0234] Cytomegalovirus
(CMV) [0235] Epstein-Barr virus (EBV) [0236] Encephalitis--Japanese
Encephalitis virus, Chandipura virus [0237] Whooping
cough--Bordetella pertussis [0238] Measles--paramyxovirus [0239]
Meningitis--Streptococcus pneumoniae and Neisseria meningitidis
[0240] Anthrax--Bacillus anthracis
[0241] Some examples of genetic disorders identified with this
system include: [0242] Cystic fibrosis [0243] Haemophilia [0244]
Sickle cell disease [0245] Tay-Sachs disease [0246]
Haemochromatosis [0247] Cerebral arteriopathy [0248] Crohn's
disease [0249] Polycistic kidney disease [0250] Congential heart
disease [0251] Rett syndrome
[0252] A small selection of cancers identified by the diagnostic
system include: [0253] Ovarian [0254] Colon carcinoma [0255]
Multiple endocrine neoplasia [0256] Retinoblastoma [0257] Turcot
syndrome
[0258] The above lists are not exhaustive and the diagnostic system
can be configured to detect a much greater variety of diseases and
conditions using nucleic acid and proteomic analysis.
Detailed Architecture of System Components
LOC Device
[0259] The LOC device 30 is central to the diagnostic system. It
rapidly performs the four major steps of a nucleic acid based
molecular diagnostic assay, i.e. sample preparation, nucleic acid
extraction, nucleic acid amplification, and detection, using a
microfluidic platform. The LOC device also has alternative uses,
and these are detailed later. As discussed above, test modules 10
and 11 can adopt many different configurations to detect different
targets. Likewise, the LOC device 30 has numerous different
embodiments tailored to the target(s) of interest. One form of the
LOC device 30 is LOC device 301 for fluorescent detection of target
nucleic acid sequences in the pathogens of a whole blood sample.
For the purposes of illustration, the structure and operation of
LOC device 301 is now described in detail with reference to FIGS. 4
to 26 and 27 to 57.
[0260] FIG. 4 is a schematic representation of the architecture of
the LOC device 301. For convenience, process stages shown in FIG. 4
are indicated with the reference numeral corresponding to the
functional sections of the LOC device 301 that perform that process
stage. The process stages associated with each of the major steps
of a nucleic acid based molecular diagnostic assay are also
indicated: sample input and preparation 288, extraction 290,
incubation 291, amplification 292 and detection 294. The various
reservoirs, chambers, valves and other components of the LOC device
301 will be described in more detail later.
[0261] FIG. 5 is a perspective view of the LOC device 301. It is
fabricated using high volume CMOS and MST (microsystems technology)
manufacturing techniques. The laminar structure of the LOC device
301 is illustrated in the schematic (not to scale) partial section
view of FIG. 12. The LOC device 301 has a silicon substrate 84
which supports the CMOS+MST chip 48, comprising CMOS circuitry 86
and an MST layer 87, with a cap 46 overlaying the MST layer 87. For
the purposes of this patent specification, the term `MST layer` is
a reference to a collection of structures and layers that process
the sample with various reagents. Accordingly, these structures and
components are configured to define flow-paths with characteristic
dimensions that will support capillary driven flow of liquids with
physical characteristics similar to those of the sample during
processing. In light of this, the MST layer and components are
typically fabricated using surface micromachining techniques and/or
bulk micromachining techniques. However, other fabrication methods
can also produce structures and components dimensioned for
capillary driven flows and processing very small volumes. The
specific embodiments described in this specification show the MST
layer as the structures and active components supported on the CMOS
circuitry 86, but excluding the features of the cap 46. However,
the skilled addressee will appreciate that the MST layer need not
have underlying CMOS or indeed an overlying cap in order for it to
process the sample.
[0262] The overall dimensions of the LOC device shown in the
following figures are 1760 .mu.m.times.5824 .mu.m. Of course, LOC
devices fabricated for different applications may have different
dimensions.
[0263] FIG. 6 shows the features of the MST layer 87 superimposed
with the features of the cap. Insets AA to AD, AG and AH shown in
FIG. 6 are enlarged in FIGS. 13, 14, 35, 56, 55 and 58,
respectively, and described in detail below for a comprehensive
understanding of each structure within the LOC device 301. FIGS. 7
to 10 show the features of the cap 46 in isolation while FIG. 11
shows the CMOS+MST device 48 structures in isolation.
Laminar Structure
[0264] FIGS. 12 and 22 are sketches that diagrammatically show the
laminar structure of the CMOS+MST device 48, the cap 46 and the
fluidic interaction between the two. The figures are not to scale
for the purposes of illustration. FIG. 12 is a schematic section
view through the sample inlet 68 and FIG. 22 is a schematic section
through the reservoir 54. As best shown in FIG. 12, the CMOS+MST
device 48 has a silicon substrate 84 which supports the CMOS
circuitry 86 that operates the active elements within the MST layer
87 above. A passivation layer 88 seals and protects the CMOS layer
86 from the fluid flows through the MST layer 87.
[0265] Fluid flows through both the cap channels 94 and the MST
channels 90 (see for example FIGS. 7 and 16) in the cap layer 46
and MST channel layer 100, respectively. Cell transport occurs in
the larger channels 94 fabricated in the cap 46, while biochemical
processes are carried out in the smaller MST channels 90. Cell
transport channels are sized so as to be able to transport cells in
the sample to predetermined sites in the MST channels 90.
Transportation of cells with sizes greater than 20 microns (for
example, certain leukocytes) requires channel dimensions greater
than 20 microns, and therefore a cross sectional area transverse to
the flow of greater than 400 square microns. MST channels,
particularly at locations in the LOC where transport of cells is
not required, can be significantly smaller.
[0266] It will be appreciated that cap channel 94 and MST channel
90 are generic references and particular MST channels 90 may also
be referred to as (for example) heated microchannels or dialysis
MST channels in light of their particular function. MST channels 90
are formed by etching through a MST channel layer 100 deposited on
the passivation layer 88 and patterned with photoresist. The MST
channels 90 are enclosed by a roof layer 66 which forms the top
(with respect to the orientation shown in the figures) of the
CMOS+MST device 48.
[0267] Despite sometimes being shown as separate layers, the cap
channel layer 80 and the reservoir layer 78 are formed from a
unitary piece of material. Of course, the piece of material may
also be non-unitary. This piece of material is etched from both
sides in order to form a cap channel layer 80 in which the cap
channels 94 are etched and the reservoir layer 78 in which the
reservoirs 54, 56, 58, 60 and 62 are etched. Alternatively, the
reservoirs and the cap channels are formed by a micromolding
process. Both etching and micromolding techniques are used to
produce channels with cross sectional areas transverse to the flow
as large as 20,000 square microns, and as small as 8 square
microns.
[0268] At different locations in the LOC device, there can be a
range of appropriate choices for the cross sectional area of the
channel transverse to the flow. Where large quantities of sample,
or samples with large constituents, are contained in the channel, a
cross-sectional area of up to 20,000 square microns (for example, a
200 micron wide channel in a 100 micron thick layer) is suitable.
Where small quantities of liquid, or mixtures without large cells
present, are contained in the channel, a very small cross sectional
area transverse to the flow is preferable.
[0269] A lower seal 64 encloses the cap channels 94 and the upper
seal layer 82 encloses the reservoirs 54, 56, 58, 60 and 62.
[0270] The five reservoirs 54, 56, 58, 60 and 62 are preloaded with
assay-specific reagents. In the embodiment described here, the
reservoirs are preloaded with the following reagents, but other
reagents can easily be substituted: [0271] reservoir 54:
anticoagulant with option to include erythrocyte lysis buffer
[0272] reservoir 56: lysis reagent [0273] reservoir 58: restriction
enzymes, ligase and linkers (for linker-primed PCR (see FIG. 61,
extracted from T. Stachan et al., Human Molecular Genetics 2,
Garland Science, NY and London, 1999)) [0274] reservoir 60:
amplification mix (dNTPs, primers, buffer) and [0275] reservoir 62:
DNA polymerase.
[0276] The cap 46 and the CMOS+MST layers 48 are in fluid
communication via corresponding openings in the lower seal 64 and
the roof layer 66. These openings are referred to as uptakes 96 and
downtakes 92 depending on whether fluid is flowing from the MST
channels 90 to the cap channels 94 or vice versa.
LOC Device Operation
[0277] The operation of the LOC device 301 is described below in a
step-wise fashion with reference to analysing pathogenic DNA in a
blood sample. Of course, other types of biological or
non-biological fluid are also analysed using an appropriate set, or
combination, of reagents, test protocols, LOC variants and
detection systems. Referring back to FIG. 4, there are five major
steps involved in analysing a biological sample, comprising sample
input and preparation 288, nucleic acid extraction 290, nucleic
acid incubation 291, nucleic acid amplification 292 and detection
and analysis 294.
[0278] The sample input and preparation step 288 involves mixing
the blood with an anticoagulant 116 and then separating pathogens
from the leukocytes and erythrocytes with the pathogen dialysis
section 70. As best shown in FIGS. 7 and 12, the blood sample
enters the device via the sample inlet 68. Capillary action draws
the blood sample along the cap channel 94 to the reservoir 54.
Anticoagulant is released from the reservoir 54 as the sample blood
flow opens its surface tension valve 118 (see FIGS. 15 and 22). The
anticoagulant prevents the formation of clots which would block the
flow.
[0279] As best shown in FIG. 22, the anticoagulant 116 is drawn out
of the reservoir 54 by capillary action and into the MST channel 90
via the downtake 92. The downtake 92 has a capillary initiation
feature (CIF) 102 to shape the geometry of the meniscus such that
it does not anchor to the rim of the downtake 92. Vent holes 122 in
the upper seal 82 allows air to replace the anticoagulant 116 as it
is drawn out of the reservoir 54.
[0280] The MST channel 90 shown in FIG. 22 is part of a surface
tension valve 118. The anticoagulant 116 fills the surface tension
valve 118 and pins a meniscus 120 to the uptake 96 to a meniscus
anchor 98. Prior to use, the meniscus 120 remains pinned at the
uptake 96 so the anticoagulant does not flow into the cap channel
94. When the blood flows through the cap channel 94 to the uptake
96, the meniscus 120 is removed and the anticoagulant is drawn into
the flow.
[0281] FIGS. 15 to 21 show Inset AE which is a portion of Inset AA
shown in FIG. 13. As shown in FIGS. 15, 16 and 17, the surface
tension valve 118 has three separate MST channels 90 extending
between respective downtakes 92 and uptakes 96. The number of MST
channels 90 in a surface tension valve can be varied to change the
flow rate of the reagent into the sample mixture. As the sample
mixture and the reagents mix together by diffusion, the flow rate
out of the reservoir determines the concentration of the reagent in
the sample flow. Hence, the surface tension valve for each of the
reservoirs is configured to match the desired reagent
concentration.
[0282] The blood passes into a pathogen dialysis section 70 (see
FIGS. 4 and 15) where target cells are concentrated from the sample
using an array of apertures 164 sized according to a predetermined
threshold. Cells smaller than the threshold pass through the
apertures while larger cells do not pass through the apertures.
Unwanted cells, which may be either the larger cells withheld by
the array of apertures 164 or the smaller cells that pass through
the apertures, are redirected to a waste unit 76 while the target
cells continue as part of the assay.
[0283] In the pathogen dialysis section 70 described here, the
pathogens from the whole blood sample are concentrated for
microbial DNA analysis. The array of apertures is formed by a
multitude of 3 micron diameter holes 164 fluidically connecting the
input flow in the cap channel 94 to a target channel 74. The 3
micron diameter apertures 164 and the dialysis uptake holes 168 for
the target channel 74 are connected by a series of dialysis MST
channels 204 (best shown in FIGS. 15 and 21). Pathogens are small
enough to pass through the 3 micron diameter apertures 164 and fill
the target channel 74 via the dialysis MST channels 204. Cells
larger than 3 microns, such as erythrocytes and leukocytes, stay in
the waste channel 72 in the cap 46 which leads to a waste reservoir
76 (see FIG. 7).
[0284] Other aperture shapes, sizes and aspect ratios can be used
to isolate specific pathogens or other target cells such as
leukocytes for human DNA analysis. Greater detail on the dialysis
section and dialysis variants is provided later.
[0285] Referring again to FIGS. 6 and 7, the flow is drawn through
the target channel 74 to the surface tension valve 128 of the lysis
reagent reservoir 56. The surface tension valve 128 has seven MST
channels 90 extending between the lysis reagent reservoir 56 and
the target channel 74. When the menisci are unpinned by the sample
flow, the flow rate from all seven of the MST channels 90 will be
greater than the flow rate from the anticoagulant reservoir 54
where the surface tension valve 118 has three MST channels 90
(assuming the physical characteristics of the fluids are roughly
equivalent). Hence the proportion of lysis reagent in the sample
mixture is greater than that of the anticoagulant.
[0286] The lysis reagent and target cells mix by diffusion in the
target channel 74 within the chemical lysis section 130. A
boiling-initiated valve 126 stops the flow until sufficient time
has passed for diffusion and lysis to take place, releasing the
genetic material from the target cells (see FIGS. 6 and 7). The
structure and operation of the boiling-initiated valves are
described in greater detail below with reference to FIGS. 31 and
32. Other active valve types (as opposed to passive valves such as
the surface tension valve 118) have also been developed by the
Applicant which may be used here instead of the boiling-initiated
valve. These alternative valve designs are also described
later.
[0287] When the boiling-initiated valve 126 opens, the lysed cells
flow into a mixing section 131 for pre-amplification restriction
digestion and linker ligation.
[0288] Referring to FIG. 13, restriction enzymes, linkers and
ligase are released from the reservoir 58 when the flow unpins the
menisci at the surface tension valve 132 at the start of the mixing
section 131. The mixture flows the length of the mixing section 131
for diffusion mixing. At the end of the mixing section 131 is
downtake 134 leading into the incubator inlet channel 133 of the
incubation section 114 (see FIG. 13). The incubator inlet channel
133 feeds the mixture into a serpentine configuration of heated
microchannels 210 which provides an incubation chamber for holding
the sample during restriction digestion and ligation of the linkers
(see FIGS. 13 and 14).
[0289] FIGS. 23, 24, 25, 26, 27, 28 and 29 show the layers of the
LOC device 301 within Inset AB of FIG. 6. Each figure shows the
sequential addition of layers forming the structures of the
CMOS+MST layer 48 and the cap 46. Inset AB shows the end of the
incubation section 114 and the start of the amplification section
112. As best shown in FIGS. 14 and 23, the flow fills the
microchannels 210 of the incubation section 114 until reaching the
boiling-initiated valve 106 where the flow stops while diffusion
takes place. As discussed above, the microchannel 210 upstream of
the boiling-initiated valve 106 becomes an incubation chamber
containing the sample, restriction enzymes, ligase and linkers. The
heaters 154 are then activated and held at constant temperature for
a specified time for restriction digestion and linker ligation to
occur.
[0290] The skilled worker will appreciate that this incubation step
291 (see FIG. 4) is optional and only required for some nucleic
acid amplification assay types. Furthermore, in some instances, it
may be necessary to have a heating step at the end of the
incubation period to spike the temperature above the incubation
temperature. The temperature spike inactivates the restriction
enzymes and ligase prior to entering the amplification section 112.
Inactivation of the restriction enzymes and ligase has particular
relevance when isothermal nucleic acid amplification is being
employed.
[0291] Following incubation, the boiling-initiated valve 106 is
activated (opened) and the flow resumes into the amplification
section 112. Referring to FIGS. 31 and 32, the mixture fills the
serpentine configuration of heated microchannels 158, which form
one or more amplification chambers, until it reaches the
boiling-initiated valve 108. As best shown in the schematic section
view of FIG. 30, amplification mix (dNTPs, primers, buffer) is
released from reservoir 60 and polymerase is subsequently released
from reservoir 62 into the intermediate MST channel 212 connecting
the incubation and amplification sections (114 and 112
respectively).
[0292] FIGS. 35 to 51 show the layers of the LOC device 301 within
Inset AC of FIG. 6. Each figure shows the sequential addition of
layers forming the structures of the CMOS+MST device 48 and the cap
46. Inset AC is at the end of the amplification section 112 and the
start of the hybridization and detection section 52. The incubated
sample, amplification mix and polymerase flow through the
microchannels 158 to the boiling-initiated valve 108. After
sufficient time for diffusion mixing, the heaters 154 in the
microchannels 158 are activated for thermal cycling or isothermal
amplification. The amplification mix goes through a predetermined
number of thermal cycles or a preset amplification time to amplify
sufficient target DNA. After the nucleic acid amplification
process, the boiling-initiated valve 108 opens and flow resumes
into the hybridization and detection section 52. The operation of
boiling-initiated valves is described in more detail later.
[0293] As shown in FIG. 52, the hybridization and detection section
52 has an array of hybridization chambers 110. FIGS. 52, 53, 54 and
56 show the hybridization chamber array 110 and individual
hybridization chambers 180 in detail. At the entrance to the
hybridization chamber 180 is a diffusion barrier 175 which prevents
diffusion of the target nucleic acid, probe strands and hybridized
probes between the hybridization chambers 180 during hybridization
so as to prevent erroneous hybridization detection results. The
diffusion barriers 175 present a flow-path-length that is long
enough to prevent the target sequences and probes diffusing out of
one chamber and contaminating another chamber within the time taken
for the probes and nucleic acids to hybridize and the signal to be
detected, thus avoiding an erroneous result.
[0294] Another mechanism to prevent erroneous readings is to have
identical probes in a number of the hybridization chambers. The
CMOS circuitry 86 derives a single result from the photodiodes 184
corresponding to the hybridization chambers 180 that contain
identical probes. Anomalous results can be disregarded or weighted
differently in the derivation of the single result.
[0295] The thermal energy required for hybridization is provided by
CMOS-controlled heaters 182 (described in more detail below). After
the heater is activated, hybridization occurs between complementary
target-probe sequences. The LED driver 29 in the CMOS circuitry 86
signals the LED 26 located in the test module 10 to illuminate.
These probes only fluoresce when hybridization has occurred thereby
avoiding washing and drying steps that are typically required to
remove unbound strands. Hybridization forces the stem-and-loop
structure of the FRET probes 186 to open, which allows the
fluorophore to emit fluorescent energy in response to the LED
excitation light, as discussed in greater detail later.
Fluorescence is detected by a photodiode 184 in the CMOS circuitry
86 underlying each hybridization chamber 180 (see hybridization
chamber description below). The photodiodes 184 for all
hybridization chambers and associated electronics collectively form
the photosensor 44 (see FIG. 59). In other embodiments, the
photosensor may be an array of charge coupled devices (CCD array).
The detected signal from the photodiodes 184 is amplified and
converted to a digital output which is analyzed by the test module
reader 12. Further details of the detection method are described
later.
Additional Details for the LOC Device
Modularity of the Design
[0296] The LOC device 301 has many functional sections, including
the reagent reservoirs 54, 56, 58, 60 and 62, the dialysis section
70, lysis section 130, incubation section 114, and amplification
section 112, valve types, the humidifier and humidity sensor. In
other embodiments of the LOC device, these functional sections can
be omitted, additional functional sections can be added or the
functional sections can be used for alternative purposes to those
described above.
[0297] For example, the incubation section 114 can be used as the
first amplification section 112 of a tandem amplification assay
system, with the chemical lysis reagent reservoir 56 being used to
add the first amplification mix of primers, dNTPs and buffer and
reagent reservoir 58 being used for adding the reverse
transcriptase and/or polymerase. A chemical lysis reagent can also
be added to the reservoir 56 along with the amplification mix if
chemical lysis of the sample is desired or, alternatively, thermal
lysis can occur in the incubation section by heating the sample for
a predetermined time. In some embodiments, an additional reservoir
can be incorporated immediately upstream of reservoir 58 for the
mix of primers, dNTPs and buffer if there is a requirement for
chemical lysis and a separation of this mix from the chemical lysis
reagent is desired.
[0298] In some circumstances it may be desirable to omit a step,
such as the incubation step 291. In this case, a LOC device can be
specifically fabricated to omit the reagent reservoir 58 and
incubation section 114, or the reservoir can simply not be loaded
with reagents or the active valves, if present, not activated to
dispense the reagents into the sample flow, and the incubation
section then simply becomes a channel to transport the sample from
the lysis section 130 to the amplification section 112. The heaters
are independently operable and therefore, where reactions are
dependent on heat, such as thermal lysis, programming the heaters
not to activate during this step ensures thermal lysis does not
occur in LOC devices that do not require it. The dialysis section
70 can be located at the beginning of the fluidic system within the
microfluidic device as shown in FIG. 4 or can be located anywhere
else within the microfluidic device. For example, dialysis after
the amplification phase 292 to remove cellular debris prior to the
hybridization and detection step 294 may be beneficial in some
circumstances. Alternatively, two or more dialysis sections can be
incorporated at any location throughout the LOC device. Similarly,
it is possible to incorporate additional amplification sections 112
to enable multiple targets to be amplified in parallel or in series
prior to being detected in the hybridization chamber arrays 110
with specific nucleic acid probes. For analysis of samples like
whole blood, in which dialysis is not required, the dialysis
section 70 is simply omitted from the sample input and preparation
section 288 of the LOC design. In some cases, it is not necessary
to omit the dialysis section 70 from the LOC device even if the
analysis does not require dialysis. If there is no geometric
hindrance to the assay by the existence of a dialysis section, a
LOC with the dialysis section 70 in the sample input and
preparation section can still be used without a loss of the
required functionality.
[0299] Furthermore, the detection section 294 may encompass
proteomic chamber arrays which are identical to the hybridization
chamber arrays but are loaded with probes designed to conjugate or
hybridize with sample target proteins present in non-amplified
sample instead of nucleic acid probes designed to hybridize to
target nucleic acid sequences.
[0300] It will be appreciated that the LOC devices fabricated for
use in this diagnostic system are different combinations of
functional sections selected in accordance with the particular LOC
application. The vast majority of functional sections are common to
many of the LOC devices and the design of additional LOC devices
for new application is a matter of compiling an appropriate
combination of functional sections from the extensive selection of
functional sections used in the existing LOC devices.
[0301] Only a small number of the LOC devices are shown in this
description and some more are shown schematically to illustrate the
design flexibility of the LOC devices fabricated for this system.
The person skilled in the art will readily recognise that the LOC
devices shown in this description are not an exhaustive list and
many additional LOC designs are a matter of compiling the
appropriate combination of functional sections.
Sample Types
[0302] LOC variants can accept and analyze the nucleic acid or
protein content of a variety of sample types in liquid form
including, but not limited to, blood and blood products, saliva,
cerebrospinal fluid, urine, semen, amniotic fluid, umbilical cord
blood, breast milk, sweat, pleural effusion, tear, pericardial
fluid, peritoneal fluid, environmental water samples and drink
samples. Amplicon obtained from macroscopic nucleic acid
amplification can also be analysed using the LOC device; in this
case, all the reagent reservoirs will be empty or configured not to
release their contents, and the dialysis, lysis, incubation and
amplification sections will be used solely to transport the sample
from the sample inlet 68 to the hybridization chambers 180 for
nucleic acid detection, as described above.
[0303] For some sample types, a pre-processing step is required,
for example semen may need to be liquefied and mucus may need to be
pre-treated with an enzyme to reduce the viscosity prior to input
into the LOC device.
Sample Input
[0304] Referring to FIGS. 1 and 12, the sample is added to the
macroreceptacle 24 of the test module 10. The macroreceptacle 24 is
a truncated cone which feeds into the inlet 68 of the LOC device
301 by capillary action. Here it flows into the 64 .mu.m
wide.times.60 .mu.m deep cap channel 94 where it is drawn towards
the anticoagulant reservoir 54, also by capillary action.
Reagent Reservoirs
[0305] The small volumes of reagents required by the assay systems
using microfluidic devices, such as LOC device 301, permit the
reagent reservoirs to contain all reagents necessary for the
biochemical processing with each of the reagent reservoirs having a
small volume. This volume is easily less than 1,000,000,000 cubic
microns, in the vast majority of cases less than 300,000,000 cubic
microns, typically less than 70,000,000 cubic microns and in the
case of the LOC device 301 shown in the drawings, less than
20,000,000 cubic microns.
Dialysis Section
[0306] Referring to FIGS. 15 to 21, 33 and 34, the pathogen
dialysis section 70 is designed to concentrate pathogenic target
cells from the sample. As previously described, a plurality of
apertures in the form of 3 micron diameter holes 164 in the roof
layer 66 filter the target cells from the bulk of the sample. As
the sample flows past the 3 micron diameter apertures 164,
microbial pathogens pass through the holes into a series of
dialysis MST channels 204 and flow back up into the target channel
74 via 16 .mu.m dialysis uptake holes 168 (see FIGS. 33 and 34).
The remainder of the sample (erythrocytes and so on) stay in the
cap channel 94. Downstream of the pathogen dialysis section 70, the
cap channel 94 becomes the waste channel 72 leading to the waste
reservoir 76. For biological samples of the type that generate a
substantial amount of waste, a foam insert or other porous element
49 within the outer casing 13 of the test module 10 is configured
to be in fluid communication with the waste reservoir 76 (see FIG.
1).
[0307] The pathogen dialysis section 70 functions entirely on
capillary action of the fluid sample. The 3 micron diameter
apertures 164 at the upstream end of the pathogen dialysis section
70 have capillary initiation features (CIFs) 166 (see FIG. 33) so
that the fluid is drawn down into the dialysis MST channel 204
beneath. The first uptake hole 198 for the target channel 74 also
has a CIF 202 (see FIG. 15) to avoid the flow simply pinning a
meniscus across the dialysis uptake holes 168.
[0308] The small constituents dialysis section 682 schematically
shown in FIG. 70 can have a similar structure to the pathogen
dialysis section 70. The small constituents dialysis section
separates any small target cells or molecules from a sample by
sizing (and, if necessary, shaping) apertures suitable for allowing
the small target cells or molecules to pass into the target channel
and continue for further analysis. Larger sized cells or molecules
are removed to a waste reservoir 766. Thus, the LOC device 30 (see
FIGS. 1 and 85) is not limited to separating pathogens that are
less than 3 .mu.m in size, but can be used to separate cells or
molecules of any size desired.
Lysis Section
[0309] Referring back to FIGS. 7, 11 and 13, the genetic material
in the sample is released from the cells by a chemical lysis
process. As described above, a lysis reagent from the lysis
reservoir 56 mixes with the sample flow in the target channel 74
downstream of the surface tension valve 128 for the lysis reservoir
56. However, some diagnostic assays are better suited to a thermal
lysis process, or even a combination of chemical and thermal lysis
of the target cells. The LOC device 301 accommodates this with the
heated microchannels 210 of the incubation section 114. The sample
flow fills the incubation section 114 and stops at the
boiling-initiated valve 106. The incubation microchannels 210 heat
the sample to a temperature at which the cellular membranes are
disrupted.
[0310] In some thermal lysis applications, an enzymatic reaction in
the chemical lysis section 130 is not necessary and the thermal
lysis completely replaces the enzymatic reaction in the chemical
lysis section 130.
Boiling-Initiated Valve
[0311] As discussed above, the LOC device 301 has three
boiling-initiated valves 126, 106 and 108. The location of these
valves is shown in FIG. 6. FIG. 31 is an enlarged plan view of the
boiling-initiated valve 108 in isolation at the end of the heated
microchannels 158 of the amplification section 112.
[0312] The sample flow 119 is drawn along the heated microchannels
158 by capillary action until it reaches the boiling-initiated
valve 108. The leading meniscus 120 of the sample flow pins at a
meniscus anchor 98 at the valve inlet 146. The geometry of the
meniscus anchor 98 stops the advancing meniscus to arrest the
capillary flow. As shown in FIGS. 31 and 32, the meniscus anchor 98
is an aperture provided by an uptake opening from the MST channel
90 to the cap channel 94. Surface tension in the meniscus 120 keeps
the valve closed. An annular heater 152 is at the periphery of the
valve inlet 146. The annular heater 152 is CMOS-controlled via the
boiling-initiated valve heater contacts 153.
[0313] To open the valve, the CMOS circuitry 86 sends an electrical
pulse to the valve heater contacts 153. The annular heater 152
resistively heats until the liquid sample 119 boils. The boiling
unpins the meniscus 120 from the valve inlet 146 and initiates
wetting of the cap channel 94. Once wetting the cap channel 94
begins, capillary flow resumes. The fluid sample 119 fills the cap
channel 94 and flows through the valve downtake 150 to the valve
outlet 148 where capillary driven flow continues along the
amplification section exit channel 160 into the hybridization and
detection section 52. Liquid sensors 174 are placed before and
after the valve for diagnostics.
[0314] It will be appreciated that once the boiling-initiated
valves are opened, they cannot be re-closed. However, as the LOC
device 301 and the test module 10 are single-use devices,
re-closing the valves is unnecessary.
Incubation Section and Nucleic Acid Amplification Section
[0315] FIGS. 6, 7, 13, 14, 23, 24, 25, 35 to 45, 50 and 51 show the
incubation section 114 and the amplification section 112. The
incubation section 114 has a single, heated incubation microchannel
210 etched in a serpentine pattern in the MST channel layer 100
from the downtake opening 134 to the boiling-initiated valve 106
(see FIGS. 13 and 14). Control over the temperature of the
incubation section 114 enables enzymatic reactions to take place
with greater efficiency. Similarly, the amplification section 112
has a heated amplification microchannel 158 in a serpentine
configuration leading from the boiling-initiated valve 106 to the
boiling-initiated valve 108 (see FIGS. 6 and 14). These valves
arrest the flow to retain the target cells in the heated incubation
or amplification microchannels 210 or 158 while mixing, incubation
and nucleic acid amplification takes place. The serpentine pattern
of the microchannels also facilitates (to some extent) mixing of
the target cells with reagents.
[0316] In the incubation section 114 and the amplification section
112, the sample cells and the reagents are heated by the heaters
154 controlled by the CMOS circuitry 86 using pulse width
modulation (PWM). Each meander of the serpentine configuration of
the heated incubation microchannel 210 and amplification
microchannel 158 has three separately operable heaters 154
extending between their respective heater contacts 156 (see FIG.
14) which provides for the two-dimensional control of input heat
flux density. As best shown in FIG. 51, the heaters 154 are
supported on the roof layer 66 and embedded in the lower seal 64.
The heater material is TiAl but many other conductive metals would
be suitable. The elongate heaters 154 are parallel with the
longitudinal extent of each channel section that forms the wide
meanders of the serpentine shape. In the amplification section 112,
each of the wide meanders can operate as separate PCR chambers via
individual heater control.
[0317] The small volumes of amplicon required by the assay systems
using microfluidic devices, such as LOC device 301, permit low
amplification mixture volumes for amplification in amplification
section 112. This volume is easily less than 400 nanoliters, in the
vast majority of cases less than 170 nanoliters, typically less
than 70 nanoliters and in the case of the LOC device 301, between 2
nanoliters and 30 nanoliters.
Increased Rates of Heating and Greater Diffusive Mixing
[0318] The small cross section of each channel section increases
the heating rate of the amplification fluid mix. All the fluid is
kept a relatively short distance from the heater 154. Reducing the
channel cross section (that is the amplification microchannel 158
cross section) to less than 100,000 square microns achieves
appreciably higher heating rates than that provided by more
`macro-scale` equipment. Lithographic fabrication techniques allow
the amplification microchannel 158 to have a cross sectional area
transverse to the flow-path less than 16,000 square microns which
gives substantially higher heating rates. Feature sizes on the
order of 1 micron are readily achievable with lithographic
techniques. If very little amplicon is needed (as is the case in
the LOC device 301), the cross sectional area can be reduced to
less than 2,500 square microns. For diagnostic assays with 1,000 to
2,000 probes on the LOC device, and a requirement of `sample-in,
answer out` in less than 1 minute, a cross sectional area
transverse to the flow of between 400 square microns and 1 square
micron is adequate.
[0319] The heater element in the amplification microchannel 158
heats the nucleic acid sequences at a rate more than 80 Kelvin (K)
per second, in the vast majority of cases at a rate greater than
100 K per second. Typically, the heater element heats the nucleic
acid sequences at a rate more than 1,000 K per second and in many
cases, the heater element heats the nucleic acid sequences at a
rate more than 10,000 K per second. Commonly, based on the demands
of the assay system, the heater element heats the nucleic acid
sequences at a rate more than 100,000 K per second, more than
1,000,000 K per second more than 10,000,000 K per second, more than
20,000,000 K per second, more than 40,000,000 K per second, more
than 80,000,000 K per second and more than 160,000,000 K per
second.
[0320] A small cross-sectional area channel is also beneficial for
diffusive mixing of any reagents with the sample fluid. Before
diffusive mixing is complete, diffusion of one liquid into the
other is greatest near the interface between the two. Concentration
decreases with distance from the interface. Using microchannels
with relatively small cross sections transverse to the flow
direction, keeps both fluid flows close to the interface for more
rapid diffusive mixing. Reducing the channel cross section to less
than 100,000 square microns achieves appreciably higher mixing
rates than that provided by more `macro-scale` equipment.
Lithographic fabrication techniques allows microchannels with a
cross sectional area transverse to the flow-path less than 16000
square microns which gives significantly higher mixing rates. If
small volumes are needed (as is the case in the LOC device 301),
the cross sectional area can be reduced to less than 2500 square
microns. For diagnostic assays with 1000 to 2000 probes on the LOC
device, and a requirement of `sample-in, answer out` in less than 1
minute, a cross sectional area transverse to the flow of between
400 square microns and 1 square micron is adequate.
Short Thermal Cycle Times
[0321] Keeping the sample mixture proximate to the heaters, and
using very small fluid volumes allows rapid thermal cycling during
the nucleic acid amplification process. Each thermal cycle (i.e.
denaturing, annealing and primer extension) is completed in less
than 30 seconds for target sequences up to 150 base pairs (bp)
long. In the vast majority of diagnostic assays, the individual
thermal cycle times are less than 11 seconds, and a large
proportion are less than 4 seconds. LOC devices 30 with some of the
most common diagnostic assays have thermal cycles time between 0.45
seconds to 1.5 seconds for target sequences up to 150 bp long.
Thermal cycling at this rate allows the test module to complete the
nucleic acid amplification process in much less than 10 minutes;
often less than 220 seconds. For most assays, the amplification
section generates sufficient amplicon in less than 80 seconds from
the sample fluid entering the sample inlet. For a great many
assays, sufficient amplicon is generated in 30 seconds.
[0322] Upon completion of a preset number of amplification cycles,
the amplicon is fed into the hybridization and detection section 52
via the boiling-initiated valve 108.
Hybridization Chambers
[0323] FIGS. 52, 53, 54, 56 and 57 show the hybridization chambers
180 in the hybridization chamber array 110. The hybridization and
detection section 52 has a 24.times.45 array 110 of hybridization
chambers 180, each with hybridization-responsive FRET probes 186,
heater element 182 and an integrated photodiode 184. The photodiode
184 is incorporated for detection of fluorescence resulting from
the hybridization of a target nucleic acid sequence or protein with
the FRET probes 186. Each photodiode 184 is independently
controlled by the CMOS circuitry 86. Any material between the FRET
probes 186 and the photodiode 184 must be transparent to the
emitted light. Accordingly, the wall section 97 between the probes
186 and the photodiode 184 is also optically transparent to the
emitted light. In the LOC device 301, the wall section 97 is a thin
(approximately 0.5 micron) layer of silicon dioxide.
[0324] Incorporation of a photodiode 184 directly beneath each
hybridization chamber 180 allows the volume of probe-target hybrids
to be very small while still generating a detectable fluorescence
signal (see FIG. 54). The small amounts permit small volume
hybridization chambers. A detectable amount of probe-target hybrid
requires a quantity of probe, prior to hybridization, which is
easily less than 270 picograms (corresponding to 900,000 cubic
microns), in the vast majority of cases less than 60 picograms
(corresponding to 200,000 cubic microns), typically less than 12
picograms (corresponding to 40,000 cubic microns) and in the case
of the LOC device 301 shown in the accompanying figures, less than
2.7 picograms (corresponding to a chamber volume of 9,000 cubic
microns). Of course, reducing the size of the hybridization
chambers allows a higher density of chambers and therefore more
probes on the LOC device. In LOC device 301, the hybridization
section has more than 1,000 chambers in an area of 1,500 microns by
1,500 microns (i.e. less than 2,250 square microns per chamber).
Smaller volumes also reduce the reaction times so that
hybridization and detection is faster. An additional advantage of
the small amount of probe required in each chamber is that only
very small quantities of probe solution need to be spotted into
each chamber during production of the LOC device. Embodiments of
the LOC device according to the invention can be spotted using a
probe solution volume of 1 picoliter or less.
[0325] After nucleic acid amplification, boiling-initiated valve
108 is activated and the amplicon flows along the flow-path 176 and
into each of the hybridization chambers 180 (see FIGS. 52 and 56).
An end-point liquid sensor 178 indicates when the hybridization
chambers 180 are filled with amplicon and the heaters 182 can be
activated.
[0326] After sufficient hybridization time, the LED 26 (see FIG. 2)
is activated. The opening in each of the hybridization chambers 180
provides an optical window 136 for exposing the FRET probes 186 to
the excitation radiation (see FIGS. 52, 54 and 56). The LED 26 is
illuminated for a sufficiently long time in order to induce a
fluorescence signal from the probes with high intensity. During
excitation, the photodiode 184 is shorted. After a pre-programmed
delay 300 (see FIG. 2), the photodiode 184 is enabled and
fluorescence emission is detected in the absence of the excitation
light. The incident light on the active area 185 of the photodiode
184 (see FIG. 54) is converted into a photocurrent which can then
be measured using CMOS circuitry 86.
[0327] The hybridization chambers 180 are each loaded with probes
for detecting a single target nucleic acid sequence. Each
hybridization chambers 180 can be loaded with probes to detect over
1,000 different targets if desired. Alternatively, many or all the
hybridization chambers can be loaded with the same probes to detect
the same target nucleic acid repeatedly. Replicating the probes in
this way throughout the hybridization chamber array 110 leads to
increased confidence in the results obtained and the results can be
combined by the photodiodes adjacent those hybridization chambers
to provide a single result if desired. The person skilled in the
art will recognise that it is possible to have from one to over
1,000 different probes on the hybridization chamber array 110,
depending on the assay specification.
Hybridization Chambers with Electrochemiluminescence Detection
[0328] FIGS. 78, 90, 115 and 116 show the hybridization chambers
180 used in an ECL variant of the LOC device, LOC variant L 729. In
this embodiment of the LOC device, a 24.times.45 array 110 of
hybridization chambers 180, each with hybridization-responsive ECL
probes 237, is positioned in registration with a corresponding
array of photodiodes 184 integrated into the CMOS. In a similar
fashion to the LOC devices configured for fluorescence detection,
each photodiode 184 is incorporated for detection of ECL resulting
from the hybridization of a target nucleic acid sequence or protein
with an ECL probe 237. Each photodiode 184 is independently
controlled by the CMOS circuitry 86. Again, the transparent wall
section 97 between the probes 186 and the photodiode 184 is
transparent to the emitted light.
[0329] A photodiode 184 closely adjacent each hybridization chamber
180 allows the amount of probe-target hybrids to be very small
while still generating a detectable ECL signal (see FIG. 78). The
small amounts permit small volume hybridization chambers. A
detectable amount of probe-target hybrid requires a quantity of
probe, prior to hybridization, which is easily less than 270
picograms (corresponding to a chamber volume of 900,000 cubic
microns), in the vast majority of cases less than 60 picograms
(corresponding to 200,000 cubic microns), typically less than 12
picograms (corresponding to 40,000 cubic microns) and in the case
of the LOC device shown in the drawings less than 2.7 picograms
(corresponding to a chamber volume of 9,000 cubic microns). Of
course, reducing the size of the hybridization chambers allows a
higher density of chambers and therefore more probes on the LOC
device. In the LOC device shown, the hybridization section has more
than 1,000 chambers in an area of 1,500 microns by 1,500 microns
(i.e. less than 2,250 square microns per chamber). Smaller volumes
also reduce the reaction times so that hybridization and detection
is faster. An additional advantage of the small amount of probe
required in each chamber is that only very small quantities of
probe solution need be spotted into each chamber during production
of the LOC device. In the case of the LOC device shown in the
drawings, the required amount of probe can be spotted using a
solution volume of 1 picoliter or less.
[0330] After nucleic acid amplification, the boiling-initiated
valve 108 is activated and the amplicon flows along the flow-path
176 and into each of the hybridization chambers 180 (see FIGS. 52
and 116). An end-point liquid sensor 178 indicates when the
hybridization chambers 180 are filled with amplicon so that the
heaters 182 can be activated.
[0331] After sufficient hybridization time, the photodiode 184 is
enabled ready for collection of the ECL signal. Then the ECL
excitation drivers 39 (see FIG. 86) activate the ECL electrodes 860
and 870 for a predetermined length of time. The photodiode 184
remains active for a short time after cessation of the ECL
excitation current to maximize the signal-to-noise ratio. For
example, if the photodiode 184 remains active for five times the
decay lifetime of the luminescent emission, then the signal will
have decayed to less than one percent of the initial value. The
incident light on the photodiode 184 is converted into a
photocurrent which can then be measured using CMOS circuitry
86.
Humidifier and Humidity Sensor
[0332] Inset AG of FIG. 6 indicates the position of the humidifier
196. The humidifier prevents evaporation of the reagents and probes
during operation of the LOC device 301. As best shown in the
enlarged view of FIG. 55, a water reservoir 188 is fluidically
connected to three evaporators 190. The water reservoir 188 is
filled with molecular biology-grade water and sealed during
manufacturing. As best shown in FIGS. 55 and 60, water is drawn
into three downtakes 194 and along respective water supply channels
192 by capillary action to a set of three uptakes 193 at the
evaporators 190. A meniscus pins at each uptake 193 to retain the
water. The evaporators have annular shaped heaters 191 which
encircle the uptakes 193. The annular heaters 191 are connected to
the CMOS circuitry 86 by the conductive columns 376 to the top
metal layer 195 (see FIG. 37). Upon activation, the annular heaters
191 heat the water causing evaporation and humidifying the device
surrounds.
[0333] The position of the humidity sensor 232 is also shown in
FIG. 6. However, as best shown in the enlarged view of Inset AH in
FIG. 58, the humidity sensor has a capacitive comb structure. A
lithographically etched first electrode 296 and a lithographically
etched second electrode 298 face each other such that their teeth
are interleaved. The opposed electrodes form a capacitor with a
capacitance that can be monitored by the CMOS circuitry 86. As the
humidity increases, the permittivity of the air gap between the
electrodes increases, so that the capacitance also increases. The
humidity sensor 232 is adjacent the hybridization chamber array 110
where humidity measurement is most important to slow evaporation
from the solution containing the exposed probes.
Feedback Sensors
[0334] Temperature and liquid sensors are incorporated throughout
the LOC device 301 to provide feedback and diagnostics during
device operation. Referring to FIG. 35, nine temperature sensors
170 are distributed throughout the amplification section 112
Likewise, the incubation section 114 also has nine temperature
sensors 170. These sensors each use a 2.times.2 array of bipolar
junction transistors (BJTs) to monitor the fluid temperature and
provide feedback to the CMOS circuitry 86. The CMOS circuitry 86
uses this to precisely control the thermal cycling during the
nucleic acid amplification process and any heating during thermal
lysis and incubation.
[0335] In the hybridization chambers 180, the CMOS circuitry 86
uses the hybridization heaters 182 as temperature sensors (see FIG.
56). The electrical resistance of the hybridization heaters 182 is
temperature dependent and the CMOS circuitry 86 uses this to derive
a temperature reading for each of the hybridization chambers
180.
[0336] The LOC device 301 also has a number of MST channel liquid
sensors 174 and cap channel liquid sensors 208. FIG. 35 shows a
line of MST channel liquid sensors 174 at one end of every other
meander in the heated microchannel 158. As best shown in FIG. 37,
the MST channel liquid sensors 174 are a pair of electrodes formed
by exposed areas of the top metal layer 195 in the CMOS structure
86. Liquid closes the circuit between the electrodes to indicate
its presence at the sensor's location.
[0337] FIG. 25 shows an enlarged perspective of cap channel liquid
sensors 208. Opposing pairs of TiAl electrodes 218 and 220 are
deposited on the roof layer 66. Between the electrodes 218 and 220
is a gap 222 to hold the circuit open in the absence of liquid. The
presence of liquid closes the circuit and the CMOS circuitry 86
uses this feedback to monitor the flow.
Gravitational Independence
[0338] The test modules 10 are orientation independent. They do not
need to be secured to a flat stable surface in order to operate.
Capillary driven fluid flows and a lack of external plumbing into
ancillary equipment allow the modules to be truly portable and
simply plugged into a similarly portable hand held reader such as a
mobile telephone. Having a gravitationally independent operation
means the test modules are also accelerationally independent to all
practical extents. They are resistant to shock and vibration and
will operate on moving vehicles or while the mobile telephone is
being carried around.
Dialysis Variants
[0339] Dialysis Section with Flow Channel to Prevent Trapped Air
Bubbles
[0340] Described below is an embodiment of the LOC device referred
to as LOC variant VIII 518 and shown in FIGS. 64, 65, 66 and 67.
This LOC device has a dialysis section that fills with the fluid
sample without leaving air bubbles trapped in the channels. LOC
variant VIII 518 also has an additional layer of material referred
to as an interface layer 594. The interface layer 594 is positioned
between the cap channel layer 80 and the MST channel layer 100 of
the CMOS+MST device 48. The interface layer 594 allows a more
complex fluidic interconnection between the reagent reservoirs and
the MST layer 87 without increasing the size of the silicon
substrate 84.
[0341] Referring to FIG. 65, the bypass channel 600 is designed to
introduce a time delay in the fluid sample flow from the interface
waste channel 604 to the interface target channel 602. This time
delay allows the fluid sample to flow through the dialysis MST
channel 204 to the dialysis uptake 168 where it pins a meniscus.
With a capillary initiation feature (CIF) 202 at the uptake from
the bypass channel 600 to the interface target channel 602, the
sample fluid fills the interface target channel 602 from a point
upstream of all the dialysis uptakes 168 from the dialysis MST
channels 204.
[0342] Without the bypass channel 600, the interface target channel
602 still starts filling from the upstream end, but eventually the
advancing meniscus reaches and passes over an uptake belonging to
an MST channel that has not yet filled, leading into air entrapment
at that point. Trapped air reduces the sample flow rate through the
leukocyte dialysis section 328.
Nucleic Acid Amplification Variants
Parallel PCR
[0343] Several variants of the LOC device have multiple
amplification sections operating in parallel. For example, LOC
variant VII 492 shown in FIG. 63 has parallel amplification
sections 112.1 to 112.4 which allow multiple nucleic acid
amplification assays to be performed simultaneously.
[0344] LOC variant XI 746 shown in FIG. 74 also has parallel
amplification sections 112.1 to 112.4, but additionally has
parallel incubation sections 114.1 to 114.4, so that the sample can
be processed differently prior to amplification. Other LOC
variants, such as LOC XIV 641 shown schematically in FIG. 69,
demonstrate that the number of parallel amplification sections can
be "X" number, which is only limited by the size of the LOC device.
A larger LOC device can be made to accommodate greater numbers of
parallel amplification sections.
[0345] Separate amplification sections can be configured to run on
different cycle times and/or temperatures for particular target
sizes or particular amplification mix constituents. With several
amplification sections running in parallel, the LOC device can
operate a multiplex nucleic acid amplification process or a uniplex
amplification process in each of the sections. In multiplex nucleic
acid amplification, more than one target sequence is amplified
using more than one pair of primers. A parallel nucleic acid
amplification system with "m" chambers can run the equivalent of an
n-plex amplification where n=n(1)+n(2)+ . . . +n(i)+ . . . +n(m),
with n(i) being the number of different primer pairs used in the
multiplex amplification being run in chamber "i", bearing in mind
that the SNR (signal-to-noise ratio) in the parallel amplification
system is higher than the n-plex amplification run in a single
chamber system. Under the special case where n(i)=1, the
amplification in chamber "i" becomes just a uniplex
amplification.
Direct PCR
[0346] Traditionally, PCR requires extensive purification of the
target DNA prior to preparation of the reaction mixture. However,
with appropriate changes to the chemistry and sample concentration,
it is possible to perform nucleic acid amplification with minimal
DNA purification, or direct amplification. When the nucleic acid
amplification process is PCR, this approach is called direct PCR.
In LOC devices where nucleic acid amplification is performed at a
controlled, constant temperature, the approach is direct isothermal
amplification. Direct nucleic acid amplification techniques have
considerable advantages for use in LOC devices, particularly
relating to simplification of the required fluidic design.
Adjustments to the amplification chemistry for direct PCR or direct
isothermal amplification include increased buffer strength, the use
of polymerases which have high activity and processivity, and
additives which chelate with potential polymerase inhibitors.
Dilution of inhibitors present in the sample is also important.
[0347] To take advantage of direct nucleic acid amplification
techniques, the LOC device designs incorporate two additional
features. The first feature is reagent reservoirs (for example
reservoir 58 in FIG. 8) which are appropriately dimensioned to
supply a sufficient quantity of amplification reaction mix, or
diluent, so that the final concentrations of sample components
which might interfere with amplification chemistry are low enough
to permit successful nucleic acid amplification. The desired
dilution of non-cellular sample components is in the range of
5.times. to 20.times.. Different LOC structures, for example the
pathogen dialysis section 70 in FIG. 4, are used when appropriate
to ensure that the concentration of target nucleic acid sequences
is maintained at a high enough level for amplification and
detection. In this embodiment, further illustrated in FIG. 6, a
dialysis section which effectively concentrates pathogens small
enough to be passed into the amplification section 292 is employed
upstream of the sample extraction section 290, and rejects larger
cells to a waste receptacle 76. In another embodiment, a dialysis
section is used to selectively deplete proteins and salts in blood
plasma while retaining cells of interest.
[0348] The second LOC structural feature which supports direct
nucleic acid amplification is design of channel aspect ratios to
adjust the mixing ratio between the sample and the amplification
mix components. For example, to ensure dilution of inhibitors
associated with the sample in the preferred 5.times.-20.times.
range through a single mixing step, the length and cross-section of
the sample and reagent channels are designed such that the sample
channel, upstream of the location where mixing is initiated,
constitutes a flow impedance 4.times.-19.times. higher than the
flow impedance of the channels through which the reagent mixture
flows. Control over flow impedances in microchannels is readily
achieved through control over the design geometry. The flow
impedance of a microchannel increases linearly with the channel
length, for a constant cross-section. Importantly for mixing
designs, flow impedance in microchannels depends more strongly on
the smallest cross-sectional dimension. For example, the flow
impedance of a microchannel with rectangular cross-section is
inversely proportional to the cube of the smallest perpendicular
dimension, when the aspect ratio is far from unity.
Reverse-Transcriptase PCR (RT-PCR)
[0349] Where the sample nucleic acid species being analysed or
extracted is RNA, such as from RNA viruses or messenger RNA, it is
first necessary to reverse transcribe the RNA into complementary
DNA (cDNA) prior to PCR amplification. The reverse transcription
reaction can be performed in the same chamber as the PCR (one-step
RT-PCR) or it can be performed as a separate, initial reaction
(two-step RT-PCR). In the LOC variants described herein, a one-step
RT-PCR can be performed simply by adding the reverse transcriptase
to reagent reservoir 62 along with the polymerase and programming
the heaters 154 to cycle firstly for the reverse transcription step
and then progress onto the nucleic acid amplification step. A
two-step RT-PCR could also be easily achieved by utilizing the
reagent reservoir 58 to store and dispense the buffers, primers,
dNTPs and reverse transcriptase and the incubation section 114 for
the reverse transcription step followed by amplification in the
normal way in the amplification section 112.
Isothermal Nucleic Acid Amplification
[0350] For some applications, isothermal nucleic acid amplification
is the preferred method of nucleic acid amplification, thus
avoiding the need to repetitively cycle the reaction components
through various temperature cycles but instead maintaining the
amplification section at a constant temperature, typically around
37.degree. C. to 41.degree. C. A number of isothermal nucleic acid
amplification methods have been described, including Strand
Displacement Amplification (SDA), Transcription Mediated
Amplification (TMA), Nucleic Acid Sequence Based Amplification
(NASBA), Recombinase Polymerase Amplification (RPA),
Helicase-Dependent isothermal DNA Amplification (HDA), Rolling
Circle Amplification (RCA), Ramification Amplification (RAM) and
Loop-mediated Isothermal Amplification (LAMP), and any of these, or
other isothermal amplification methods, can be employed in
particular embodiments of the LOC device described herein.
[0351] In order to perform isothermal nucleic acid amplification,
the reagent reservoirs 60 and 62 adjoining the amplification
section will be loaded with the appropriate reagents for the
specified isothermal method instead of PCR amplification mix and
polymerase. For example, for SDA, reagent reservoir 60 contains
amplification buffer, primers and dNTPs and reagent reservoir 62
contains an appropriate nickase enzyme and Exo-DNA polymerase. For
RPA, reagent reservoir 60 contains the amplification buffer,
primers, dNTPs and recombinase proteins, with reagent reservoir 62
containing a strand displacing DNA polymerase such as Bsu.
Similarly, for HDA, reagent reservoir 60 contains amplification
buffer, primers and dNTPs and reagent reservoir 62 contains an
appropriate DNA polymerase and a helicase enzyme to unwind the
double stranded DNA strand instead of using heat. The skilled
person will appreciate that the necessary reagents can be split
between the two reagent reservoirs in any manner appropriate for
the nucleic acid amplification process.
[0352] For amplification of viral nucleic acids from RNA viruses
such as HIV or hepatitis C virus, NASBA or TMA is appropriate as it
is unnecessary to first transcribe the RNA to cDNA. In this
example, reagent reservoir 60 is filled with amplification buffer,
primers and dNTPs and reagent reservoir 62 is filled with RNA
polymerase, reverse transcriptase and, optionally, RNase H.
[0353] For some forms of isothermal nucleic acid amplification it
may be necessary to have an initial denaturation cycle to separate
the double stranded DNA template, prior to maintaining the
temperature for the isothermal nucleic acid amplification to
proceed. This is readily achievable in all embodiments of the LOC
device described herein, as the temperature of the mix in the
amplification section 112 can be carefully controlled by the
heaters 154 in the amplification microchannels 158 (see FIG.
14).
[0354] Isothermal nucleic acid amplification is more tolerant of
potential inhibitors in the sample and, as such, is generally
suitable for use where direct nucleic acid amplification from the
sample is desired. Therefore, isothermal nucleic acid amplification
is sometimes useful in LOC variant XLIII 673, LOC variant XLIV 674
and LOC variant XLVII 677, amongst others, shown in FIGS. 71, 72
and 73, respectively. Direct isothermal amplification may also be
combined with one or more pre-amplification dialysis steps 70, 686
or 682 as shown in FIGS. 71 and 73 and/or a pre-hybridization
dialysis step 682 as indicated in FIG. 72 to help partially
concentrate the target cells in the sample before nucleic acid
amplification or remove unwanted cellular debris prior to the
sample entering the hybridization chamber array 110, respectively.
The person skilled in the art will appreciate that any combination
of pre-amplification dialysis and pre-hybridization dialysis can be
used.
[0355] Isothermal nucleic acid amplification can also be performed
in parallel amplification sections such as those schematically
represented in FIGS. 63, 68 and 69, multiplexed and some methods of
isothermal nucleic acid amplification, such as LAMP, are compatible
with an initial reverse transcription step to amplify RNA.
Photodiode
[0356] FIG. 54 shows the photodiode 184 integrated into the CMOS
circuitry 86 of the LOC device 301. The photodiode 184 is
fabricated as part of the CMOS circuitry 86 without additional
masks or steps. This is one significant advantage of a CMOS
photodiode over a CCD, an alternate sensing technology which could
be integrated on the same chip using non-standard processing steps,
or fabricated on an adjacent chip. On-chip detection is low cost
and reduces the size of the assay system. The shorter optical path
length reduces noise from the surrounding environment for efficient
collection of the fluorescence signal and eliminates the need for a
conventional optical assembly of lenses and filters.
[0357] Quantum efficiency of the photodiode 184 is the fraction of
photons impinging on its active area 185 that are effectively
converted to photo-electrons. For standard silicon processes, the
quantum efficiency is in the range of 0.3 to 0.5 for visible light,
depending on process parameters such as the amount and absorption
properties of the cover layers.
[0358] The detection threshold of the photodiode 184 determines the
smallest intensity of the fluorescence signal that can be detected.
The detection threshold also determines the size of the photodiode
184 and hence the number of hybridization chambers 180 in the
hybridization and detection section 52 (see FIG. 52). The size and
number of chambers are technical parameters that are limited by the
dimensions of the LOC device (in the case of the LOC device 301,
the dimensions are 1760 .mu.m.times.5824 .mu.m) and the real estate
available after other functional modules such as the pathogen
dialysis section 70 and amplification section(s) 112 are
incorporated.
[0359] For standard silicon processes, the photodiode 184 detects a
minimum of 5 photons. However, to ensure reliable detection, the
minimum can be set to 10 photons. Therefore with the quantum
efficiency range being 0.3 to 0.5 (as discussed above), the
fluorescence emission from the probes should be a minimum of 17
photons but 30 photons would incorporate a suitable margin of error
for reliable detection.
Electrochemiluminescence as an Alternative Detection Method
[0360] Electrochemiluminescence (ECL) involves the generation of
species at electrode surfaces that then undergo electron-transfer
reactions to form excited states that emit light.
Electrochemiluminescence differs from normal chemiluminescence in
that formation of the excited species relies on oxidation or
reduction of the luminophore or a coreactant at an electrode.
Coreactants, in this context, are additional reagents added to the
ECL solution which enhance the efficiency of ECL emission. In
normal chemiluminescence, the excited species form purely through
mixing of suitable reagents. The emitting atom or complex is
traditionally referred to as a luminophore. In brief, ECL relies on
generating an excited state of the luminophore, at which point a
photon will be emitted. As with any such process, it is possible
for an alternate path to be taken from the excited state which does
not lead to the desired light emission (i.e. quenching).
[0361] Embodiments of the test module that use ECL instead of
fluorescence detection do not require an excitation LED. Electrodes
are fabricated within the hybridization chambers to provide the
electrical pulse for ECL generation and the photons are detected
using the photosensor 44. The duration and voltage of the
electrical pulse are controlled; in some embodiments, control over
the current is used as an alternative to controlling the
voltage.
Luminophore and Quencher
[0362] The ruthenium complex, [Ru(bpy).sub.3].sup.2+, described
previously for use as a fluorescent reporter in the probes, can
also be used as a luminophore in an ECL reaction in the
hybridization chambers, with TPrA (tri-n-propylamine
(CH.sub.3CH.sub.2--CH.sub.2).sub.3N) as the coreactant. Coreactant
ECL has the benefit that luminophores are not consumed after photon
emission and the reagents are available for the process to repeat.
Furthermore, the [Ru(bpy).sub.3].sup.2+/TPrA ECL system provides
good signal levels at physiologically relevant conditions of pH in
aqueous solutions. Alternative coreactants which can produce
equivalent or better results than TPrA with ruthenium complexes are
N-butyldiethanolamine and 2-(dibutylamino)ethanol.
[0363] FIG. 76 illustrates the reactions occurring during an ECL
process in which [Ru(bpy).sub.3].sup.2+ is the luminophore 864 and
TPrA is the coreactant 866. ECL emission 862 in the
[Ru(bpy).sub.3].sup.2+/TPrA ECL system follows the oxidation of
both Ru(bpy).sub.3.sup.2+ and TPrA at the anode 860. The reactions
are as follows:
Ru(bpy).sub.3.sup.2+-e.sup.-.fwdarw.Ru(bpy).sub.3.sup.3+ (1)
TPrA-e.sup.-.fwdarw.[TPrA.sup.].sup.+.fwdarw.TPrA.sup.+H.sup.+
(2)
Ru(bpy).sub.3.sup.3++TPrA.sup..fwdarw.Ru(bpy).sub.3.sup.2++products
(3)
Ru(bpy).sub.3.sup.2+.fwdarw.Ru(bpy).sub.3.sup.2++hv (4)
[0364] The wavelength of the emitted light 862 is around 620 nm and
the anode potential is around 1.1 V with respect to a Ag/AgCl
reference electrode. For the [Ru(bpy).sub.3].sup.2+/TPrA ECL
system, either the Black Hole Quencher, BHQ 2, or Iowa Black RQ
described previously, would be a suitable quencher. In the
embodiments described here, the quencher is a functional moiety
which is initially attached to the probe, but other embodiments are
possible in which the quencher is a separate molecule free in
solution.
Hybridization Probes for ECL Detection
[0365] FIGS. 93 and 94 show the hybridization-responsive ECL probes
237. These are often referred to as molecular beacons and are
stem-and-loop probes, generated from a single strand of nucleic
acid, that luminesce upon hybridization to complementary nucleic
acids. FIG. 93 shows a single ECL probe 237 prior to hybridization
with a target nucleic acid sequence 238. The probe has a loop 240,
stem 242, a luminophore 864 at the 5' end, and a quencher 248 at
the 3' end. The loop 240 consists of a sequence complementary to
the target nucleic acid sequence 238. Complementary sequences on
either side of the probe sequence anneal together to form the stem
242.
[0366] In the absence of a complementary target sequence, the probe
remains closed as shown in FIG. 93. The stem 242 keeps the
luminophore-quencher pair in close proximity to each other, such
that significant resonant energy transfer can occur between them,
substantially eliminating the ability of the luminophore to emit
light after electrochemical excitation.
[0367] FIG. 94 shows the ECL probe 237 in an open or hybridized
configuration. Upon hybridization to a complementary target nucleic
acid sequence 238, the stem-and-loop structure is disrupted, the
luminophore 864 and quencher 248 are spatially separated, thus
restoring the ability of the luminophore 864 to emit light. The ECL
emission 862 is optically detected as an indication that the probe
has hybridized.
[0368] The probes hybridize with very high specificity with
complementary targets, since the stem helix of the probe is
designed to be more stable than a probe-target helix with a single
nucleotide that is not complementary. Since double-stranded DNA is
relatively rigid, it is sterically impossible for the probe-target
helix and the stem helix to coexist.
Primer-Linked ECL Probes
[0369] Primer-linked stem-and-loop probes and primer-linked linear
probes, otherwise known as scorpion probes, are an alternative to
molecular beacons and can be used for real-time and quantitative
nucleic acid amplification in the LOC device. Real-time
amplification is performed directly in the hybridization chambers
of the LOC device. The benefit of using primer-linked probes is
that the probe element is physically linked to the primer, thus
only requiring a single hybridization event to occur during the
nucleic acid amplification rather than separate hybridizations of
the primers and probes being required. This ensures that the
reaction is effectively instantaneous and results in stronger
signals, shorter reaction times and better discrimination than when
using separate primers and probes. The probes (along with
polymerase and the amplification mix) would be deposited into the
hybridization chambers 180 during fabrication and there would be no
need for an amplification section on the LOC device. Alternatively,
the amplification section is left unused or used for other
reactions.
Primer-Linked Linear ECL Probes
[0370] FIGS. 95 and 96 show a primer-linked linear ECL probe 693
during the initial round of nucleic acid amplification and in its
hybridized configuration during subsequent rounds of nucleic acid
amplification, respectively. Referring to FIG. 95, the
primer-linked linear ECL probe 693 has a double-stranded stem
segment 242. One of the strands incorporates the primer linked
probe sequence 696 which is homologous to a region on the target
nucleic acid 696 and is labelled on its 5' end with luminophore
864, and linked on its 3' end to an oligonucleotide primer 700 via
an amplification blocker 694. The other strand of the stem 242 is
labelled at its 3 end with a quencher molecule 248. After the
initial round of nucleic acid amplification has completed, the
probe can loop around and hybridize to the extended strand with
the, now, complementary sequence 698. During the initial round of
nucleic acid amplification, the oligonucleotide primer 700 anneals
to the target DNA 238 (see FIG. 95) and is then extended, forming a
DNA strand containing both the probe sequence and the amplification
product. The amplification blocker 694 prevents the polymerase from
reading through and copying the probe region 696. Upon subsequent
denaturation, the extended oligonucleotide primer 700/template
hybrid is dissociated and so is the double stranded stem 242 of the
primer-linked linear probe, thus releasing the quencher 248. Once
the temperature decreases for the annealing and extension steps,
the primer linked probe sequence 696 of the primer-linked linear
ECL probe curls around and hybridizes to the amplified
complementary sequence 698 on the extended strand and light
emission is detected indicating the presence of the target DNA.
Non-extended primer-linked linear ECL probes retain their
double-stranded stem and light emission remains quenched. This
detection method is particularly well suited for fast detection
systems as it relies on a single-molecule process.
[0371] Primer-Linked Stem-And-Loop ECL Probes
[0372] FIGS. 97A to 97F show the operation of a primer-linked
stem-and-loop ECL probe 705. Referring to FIG. 97A, the
primer-linked stem-and-loop ECL probe 705 has a stem 242 of
complementary double-stranded DNA and a loop 240 which incorporates
the probe sequence. One of the stem strands 708 is labelled at its
5' end with luminophore 864. The other strand 710 is labelled with
a 3'-end quencher 248 and carries both the amplification blocker
694 and oligonucleotide primer 700. During the initial denaturation
phase (see FIG. 97B), the strands of the target nucleic acid 238
separate, as does the stem 242 of the primer-linked stem-and-loop
ECL probe 705. When the temperature cools for the annealing phase
(see FIG. 97C), the oligonucleotide primer 700 on the primer-linked
stem-and-loop ECL probe 705 hybridizes to the target nucleic acid
sequence 238. During extension (see FIG. 97D), the complement 706
to the target nucleic acid sequence 238 is synthesized forming a
DNA strand containing both the probe sequence 705 and the amplified
product. The amplification blocker 694 prevents the polymerase from
reading through and copying the probe region 705. When the probe
next anneals, following denaturation (see FIG. 97E), the probe
sequence of the loop segment 240 of the primer-linked stem-and-loop
probe (see FIG. 97F) anneals to the complementary sequence 706 on
the extended strand. This configuration leaves the luminophore 864
relatively remote from the quencher 248, resulting in a significant
increase in light emission.
ECL Control Probes
[0373] The hybridization chamber array 110 includes some
hybridization chambers 180 with positive and negative ECL control
probes used for assay quality control. FIGS. 98 and 99
schematically illustrate negative control ECL probes 786 without a
luminophore, and FIGS. 100 and 101 are sketches of positive control
ECL probes 787 without a quencher. The positive and negative
control ECL probes have a stem-and-loop structure like the ECL
probes described above. However, an ECL signal 862 (see FIG. 94)
will always be emitted from positive control ECL probes 787 and no
ECL signal 862 is ever emitted from negative control ECL probes
786, regardless of whether the probes hybridize into an open
configuration or remain closed.
[0374] Referring to FIGS. 98 and 99, the negative control ECL probe
786 has no luminophore (and may or may not have a quencher 248).
Hence, whether the target nucleic acid sequence 238 hybridizes with
the probe as shown in FIG. 99, or the probe remains in its stem 242
and loop 240 configuration as shown in FIG. 98, the ECL signal is
negligible. Alternatively, the negative control ECL probe could be
designed so that it always remains quenched. For example, by having
an artificial probe (loop) sequence 240 that will not hybridize to
any nucleic acid sequence within the sample under investigation,
the stem 242 of the probe molecule will re-hybridize to itself and
the luminophore and quencher will remain in close proximity and no
appreciable ECL signal will be detected. This negative control
would account for any low level emission that may occur if the
quenching is not complete.
[0375] Conversely, the positive control ECL probe 787 is
constructed without a quencher as illustrated in FIGS. 100 and 101.
Nothing quenches the ECL emission 862 from the luminophore 864
regardless of whether the positive control probe 787 hybridizes
with the target nucleic acid sequence 238.
[0376] FIGS. 91 and 92 show another possibility for constructing a
positive control chamber. In this case, the calibration chambers
382 which are sealed from the amplicon (or any flow containing
target molecules) can be filled with the ECL luminophore solution
such that a positive signal is always detected at the electrode
[0377] Similarly, the control chambers can be negative control
chambers because the lack of inlets prevents any targets from
reaching the probes such that an ECL signal is never detected.
[0378] FIG. 52 shows a possible distribution of the positive and
negative control probes (378 and 380 respectively) throughout the
hybridization chamber array 110. For ECL, positive and negative
control ECL probes 786 and 787 would replace control fluorescent
probes 378 and 380, respectively. The control probes are placed in
hybridization chambers 180 along a line extending diagonally across
the hybridization chamber array 110. However, the arrangement of
the control probes within the array is arbitrary (as is the
configuration of the hybridization chamber array 110).
Calibration Chambers for ECL Detection
[0379] The non-uniformity of the electrical characteristic of the
photodiode 184, response to any ambient light present at the sensor
array, and light originating at other locations in the array,
introduce background noise and offset into the output signal. This
background is removed from each output signal by calibration
chambers 382 in the hybridization chamber array 110 which either do
not contain any probes, contain probes that have no ECL
luminophore, or contain probes with a luminophore and quencher
configured such that quenching is always expected to occur. The
number and arrangement of the calibration chambers 382 throughout
the hybridization chamber array is arbitrary. However, the
calibration is more accurate if photodiodes 184 are calibrated by a
calibration chamber 382 that is relatively proximate. Referring to
FIG. 116, the hybridization chamber array 110 has one calibration
chamber 382 for every eight hybridization chambers 180. That is, a
calibration chamber 382 is positioned in the middle of every three
by three square of hybridization chambers 180. In this
configuration, the hybridization chambers 180 are calibrated by a
calibration chamber 382 that is immediately adjacent.
[0380] FIG. 75 shows a differential imager circuit 788 used to
substract the signal from the photodiode 184 corresponding to the
calibration chamber 382 as a result of the applied electrical
pulse, from the ECL signal from the surrounding hybridization
chambers 180. The differential imager circuit 788 samples the
signal from the pixel 790 and a "dummy" pixel 792. Signals arising
from ambient light in the region of the chamber array are also
subtracted. The signals from the pixel 790 are small (i.e. close to
dark signal), and without a reference to a dark level it is hard to
differentiate between the background and a very small signal.
[0381] During use, the "read_row" 794 and "read_row_d" 795 are
activated and M4 797 and MD4 801 transistors are turned on.
Switches 807 and 809 are closed such that the outputs from the
pixel 790 and "dummy" pixel 792 are stored on pixel capacitor 803
and dummy pixel capacitor 805 respectively. After the pixel signals
have been stored, switches 807 and 809 are deactivated. Then the
"read_col" switch 811 and dummy "read_col" switch 813 are closed,
and the switched capacitor amplifier 815 at the output amplifies
the differential signal 817.
ECL Levels and Signal Efficiency
[0382] The normal metric of efficiency in ECL is the number of
photons obtained per "Faradaic" electron, i.e. per electron which
participates in the electrochemistry. The ECL efficiency is denoted
.phi..sub.ECL:
.phi. ECL = .intg. 0 t I .tau. ( N A F ) .intg. 0 t i .tau. ( 5 )
##EQU00001##
where I is the intensity in photons per second, i is the current in
amperes, F is Faraday's constant, and N.sub.A is Avogadro's
constant.
Efficiency of Coreactant ECL
[0383] Annihilation ECL in deoxygenated, aprotic solutions (e.g.
nitrogen-flushed acetonitrile solutions) is simple enough to allow
efficiency measurements, and the consensus value of .phi..sub.ECL
is around 5%. Coreactant systems, however, have been generally
declared to be beyond meaningful direct measurements of efficiency.
Instead, emission intensity is related by scaling to
easily-prepared standard solutions such as Ru(bpy).sub.3.sup.2+,
measured in the same format. The literature (see for example J. K.
Leland and M. J. Powell, J. Electrochem. Soc., 137, 3127 (1990),
and R. Pyati and M. M. Richter, Annu. Rep. Prog. Chem. C, 103,
12-78 (2007)) indicates that (without enhancers such as
surfactants), the efficiency of Ru(bpy).sub.3.sup.2+ ECL with TPrA
coreactants peaks at levels comparable to the 5% seen for
annihilation ECL in acetonitrile (e.g. 2% efficiency; see I.
Rubinstein & A. J. Bard, J. Am. Chem. Soc., 103 512-516
(1981)).
ECL Potentials
[0384] The voltage at the working electrode for the
Ru(bpy).sub.3.sup.2+/TPrA system is approximately +1.1 V (generally
measured in the literature with respect to a reference Ag/AgCl
electrode). Voltages this high shorten electrode lifetimes but this
is not an issue for single-use devices such as the LOC device used
in the present diagnostic system.
[0385] The ideal voltage between the anode and cathode depends on
the combination of solution components and electrode materials.
Selecting the correct voltage can require compromising between the
highest signal levels, reagent and electrode stability, and the
activation of undesired side reactions such as electrolysis of the
water in the chamber. In tests on buffered aqueous
Ru(bpy).sub.3].sup.2+/coreactant solution and platinum electrodes,
the ECL emission is maximized at 2.1-2.2 V (depending on the
coreactant choice). Emission intensities drop to <75% of the
peak values for voltages below 1.9 V and above 2.6 V, and to
<50% of the peak values for voltages below 1.7 V and above 2.8
V. A preferred anode-cathode voltage difference for ECL operation
in such systems is therefore 1.7-2.8 V, with the range 1.9-2.6 V
being particularly preferred. This allows maximization of the
emission intensity as a function of voltage, while avoiding
voltages at which significant gas evolution at the electrodes is
observed.
ECL Emission Wavelength
[0386] The wavelength of the emitted light 862 from ECL has an
intensity peak at around 620 nm (measured in air or vacuum), and
the emission spans a relatively broad wavelength range. Significant
emission occurs at wavelengths from around 550 nm to 700 nm.
Furthermore, the peak emission wavelength can vary by .about.10%
due to changes in the chemical environment around the active
species. The LOC device embodiments described here, which
incorporate no wavelength-specific filters, have two advantages for
capturing signals with such a broad and variable spectrum. The
first advantage is sensitivity: any wavelength filter reduces light
transmission, even within its pass band, so efficiency is improved
by not including a filter. The second advantage is flexibility:
adjustment of filter pass bands is not required after minor reagent
changes, and the signals are less dependent on minor differences in
non-target components of the input sample.
Solution Volume Participating in ECL
[0387] ECL relies on the availability of luminophore (and
coreactant) in solution. However, as illustrated in FIG. 78, the
excited species 868 are generated only in the solution 872 near the
electrodes 860 and 870. The parameter boundary layer depth in the
models presented here, is the depth of the layer of solution 872
around the electrode 860 in which the excited species 868 are
generated.
[0388] This is a simplification, since solution dynamics can drive
the available concentration upward or downward: [0389] Increased
availability: diffusion and electrophoretic effects will allow
exchange with more of the solution. [0390] Decreased availability:
reagents can adsorb onto the electrodes and may become unavailable
to the ECL process.
[0391] For a boundary layer depth value of 0.5 .mu.m, the following
observations are made:
[0392] ECL is observed in experiments where conjugation to magnetic
beads with diameters up to 4.5 .mu.m is used to attract the
luminophore 864 to the anode 860.
[0393] Ru(bpy).sub.3.sup.2+/TPrA ECL emission 862 as a function of
electrode spacing, for interdigitated electrode arrays, was found
to be maximised at a 0.8 .mu.m electrode spacing. The requirement
for a coreactant 866 in aqueous solutions 872 can be lifted when
electrode spacings are .about.2 .mu.m. This indicates that the
excited species 868 diffuse multiple microns, which implies
diffusive exchange on a similar scale for the species in the ground
state.
Steady State and Pulsed Operation
[0394] During pulsed activation of the electrodes 860 and 870, the
intensity of the ECL emission 862 (see FIG. 94) is generally higher
than the intensity of the emission 862 from steady-state activation
of the electrodes. Accordingly, the activation signal to the
electrodes 860 and 870 is pulse-width modulated (PWM) by the CMOS
circuitry 86 (see FIG. 83).
Reagent Recycling and Species Lifetime
[0395] The Ru complex is not consumed in the
Ru(bpy).sub.3.sup.2+/TPrA ECL system, so the intensity of emission
862 does not reduce with successive reaction cycles. The lifetime
of the rate-limiting step is approximately 0.2 milliseconds giving
a total reaction recycling time of approximately 1 millisecond.
Electrophoretic Effects and Other Constraints
[0396] Given the complexity of the solutions in the hybridization
chamber, a large number of phenomena take place when the ECL
voltage is turned on. Electrophoresis of macromolecules, ohmic
conduction, and capacitive effects from small ion migration occur
simultaneously.
[0397] Electrophoresis of the oligonucleotides (probes and
amplicon) can complicate the detection of probe-target hybrids, as
DNA is highly negatively charged and attracted to the anode 860.
The time scale for this motion is typically short (in the order of
milliseconds). Electrophoretic effects are strong even though the
voltages are moderate (.about.1 V), because the separation between
the anode 860 and cathode 870 is small.
[0398] Electrophoresis enhances the ECL emission 862 in some
embodiments of the LOC device and degrades the emission in others.
This is addressed by increasing or decreasing the electrode spacing
to get the associated increases or decreases in electrophoretic
effect. Interdigitation of the anode 860 and the cathode 870 above
the photodiode 184 represents the extreme case of minimizing this
separation. Such an arrangement produces ECL, even in the absence
of a coreactant 866 at carbon electrodes 860 and 870.
Ohmic Heating (DC Current)
[0399] The current required to maintain an ECL voltage of
.about.2.2 V, is determined as follows with reference to the ECL
cell 874 schematically illustrated in FIG. 79.
[0400] The DC current through the chamber is determined by two
resistances: the interface resistance R.sub.i between the
electrodes 860 and 870 and the bulk of the solution, and the
solution resistance R.sub.s which is derived from the bulk solution
resistivity and conduction path geometry. For solutions with ionic
strengths relevant to the conditions in LOC devices, the chamber
resistance is dominated by interfacial resistances at the
electrodes 860 and 870, and R.sub.s can be neglected.
[0401] The effect of the interfacial resistance is estimated by
scaling measurements of macroscopic current flow through similar
solutions for the electrode geometries in the LOC devices.
[0402] Macroscopic measurements of current density through a
similar solution, at platinum electrodes, were taken. Consistent
with the worst-case (high current) approach being taken, overall
ionic strength and ECL reactant concentrations in the test solution
were higher than those used in the LOC devices. The anode area was
smaller than the cathode area, and was surrounded by a cathode with
comparable area in a ring geometry. For an anode consisting of a
circle 2 mm in diameter, the current measured was 1.1 mA, giving a
current density of 350 A/m.sup.2.
[0403] In the heating model, the electrode area is for the square
ring geometry schematically illustrated in FIG. 79. The anode is a
ring with width 1 .mu.m and thickness 1 .mu.m. The surface area is
196 square microns, and therefore the calculated current I=69
nA.
[0404] The heating (power=V.sup.2/R) was modelled for the worst
case in which all the heat goes into raising the temperature of the
water in the chamber. This leads to heating of chamber contents at
5.8.degree. C./s, at a voltage difference of 2.2 V, if no allowance
for heat removal by the bulk of the LOC device is made.
[0405] Heating of the chambers by .about.20.degree. C. can cause
denaturation of most hybridization probes. For highly specific
probes intended for mutation detection, it is preferable to further
restrict heating to 4.degree. C. or less. With this level of
temperature stability, single base mismatch-sensitive
hybridization, using appropriately designed sequences, becomes
feasible. This allows the detection of mutations and allelic
differences at the level of single nucleotide polymorphisms. Hence
the DC current is applied to the electrodes 860 and 870 for 0.69 s,
to limit the heating to 4.degree. C.
[0406] A current of .about.69 nA passing through the chamber is far
more than can be accommodated as Faradaic current by the ECL
species at micromolar concentrations. Therefore, low-duty-cycle
pulsing of the electrodes 860 and 870 to further reduce heating (to
1.degree. C. or less) while maintaining sufficient ECL emission
862, does not introduce complications associated with reagent
depletion. In other embodiments, the current is reduced to 0.1 nA
which removes the need for pulsed activation of the electrodes.
Even at currents as low as 0.1 nA, the ECL emission 862 is
luminophore-limited.
Chamber and Electrode Geometry
Maximizing Optical Coupling Between ECL Luminescence and
Photosensor
[0407] The immediate chemical precursors of ECL luminescence are
generated within nanometres of the working electrode. Referring
again to FIG. 78, light emission (the excited species 868)
generally occurs within microns or less of that location. Hence the
volume immediately adjacent to the working electrode (anode 860) is
visible to the corresponding photodiode 184 of the photosensor 44.
Accordingly, the electrodes 860 and 870 are directly adjacent the
active surface area 185 of the corresponding photodiode 184 in the
photosensor 44. Furthermore, the anode 860 is shaped to increase
the length of its lateral periphery `seen` by the photodiode 184.
This aims to maximize the volume of excited species 868 that can be
detected by the underlying photodiode 184.
[0408] FIG. 77 schematically illustrates three embodiments of the
anode 860. A comb structure anode 878 has the advantage that the
parallel fingers 880 can be interdigitated with the fingers of a
cathode 870. The interdigitated configuration is shown in FIG. 84,
and in a partial view of a LOC layout in FIGS. 90 and 92. The
interdigitated configuration provides a uniform dielectric gap 876
(see FIG. 78) that is relatively narrow (1 to 2 microns) and the
interdigitated comb structure is relatively simple for the
lithographic fabrication process. As discussed above, a relatively
narrow dielectric gap 876 between the electrodes 860 and 870
obviates the need for a coreactant in some solutions 872, as the
excited species 868 will diffuse between anode and cathode. The
removal of the requirement for a coreactant removes the potential
chemical impact of the coreactant on the various assay chemistries
and provides a wider range of possible assay options.
[0409] Referring again to FIG. 77, some embodiments of the anode
860 have a serpentine configuration 882. To achieve high periphery
length while maintaining tolerance against fabrication errors, it
is convenient to form wide, rectangular meanders 884.
[0410] The anode may have a more complex configuration 886 if
necessary or desirable. For example, it may have a crenulated
section 888, a branched structure 890, or a combination of the two.
Partial views of LOC designs incorporating a branched structure 890
are shown in FIGS. 115 and 116. The more complicated configurations
such as 886 provide a long length of lateral periphery, and are
best suited to solution chemistries where a coreactant is employed
since patterning a closely-spaced opposing cathode is more
difficult.
Electrode Thickness
[0411] Generally, ECL cells involve a planar working electrode
which is viewed externally. Also, traditional microfabrication
techniques for metal layers tend to lead to planar structures with
metal thicknesses of approximately 1 micron. As has been indicated
earlier, and shown schematically in FIGS. 77, 80 and 81, increasing
the length of lateral periphery enhances the coupling between the
ECL emission and the photodiode 184.
[0412] A second strategy to further increase the efficiency of
collection of emitted light 862 (see FIG. 94) by the photodiode 184
is to increase the thickness of the anode 860. This is shown
schematically in FIG. 78. The part of the participating volume 892
adjacent to the walls of the working electrode is the region most
efficiently coupled to the photodiode 184. Therefore, for a given
width of working electrode 860, the overall collection efficiency
of the emitted light 862 can be improved by increasing the
thickness of the electrodes. Further, since high current carrying
capacity is not required, the width of the working electrode 860 is
reduced as far as is practical. The thickness of the electrodes 860
and 870 can not increase without restrictions. Noting that the
feature and separation sizes of the electrodes are likely to be of
the order of 1 micron, and that liquid filling makes gaps which are
wider than they are deep unfavourable, the optimum practical
thickness for the electrodes is 0.25 micron to 2 microns.
Electrode Spacing
[0413] The spacing between the electrodes 860 and 870 is important
for the quality of signals in LOC devices, particularly in
embodiments where the electrodes are interdigitated. In embodiments
where the anode 860 is a branched structure such as shown in FIG.
77 and FIG. 81, the spacing between adjacent elements can also be
important. ECL emission efficiency, and the collection efficiency
of the emitted light, should both be maximised.
[0414] Generation of ECL emission tends to favour electrode
spacings on the order of one micron or less. Small spacings are
particularly attractive when performing ECL in the absence of a
coreactant. The fact that the spacing can be comparable to the
wavelength of the emitted light 862 is of limited importance.
Therefore, in many embodiments where the emitted light 862 (see
FIG. 94) is measured at a location which does not require that the
light have passed between the electrodes 860 and 870, making the
electrode spacing as small as practical is often the goal. In
embodiments where the emitted light 862 must pass between the
electrodes 860 and 870, however, it becomes necessary to move
beyond considering just the ECL emission process, and consider the
wave properties of light.
[0415] The wavelength of the emitted light 862 from ECL of
Ru(bpy).sub.3.sup.2+ is around 620 nm, and therefore 460 nm (0.46
microns) in water. In embodiments where the photodiode 184 and the
ECL excited species 868 are on different sides of the electrode
structure, and the electrode structure is metallic, the emitted
light 862 must pass through a gap between elements of the metallic
structures. If this gap is comparable to the wavelength of the
light, diffraction generally reduces the intensity of propagating
light which reaches the photodiode 184. In cases where the emitted
light 862 is incident on the gap at large angles, however,
evanescent mode coupling can be harnessed to improve the strength
of collected signals. Two measures are taken in the LOC devices to
enhance the efficiency of coupling between the photodiode 184 and
the emitted light 862.
[0416] First, the separation between metallic elements is not
reduced below approximately the wavelength of the emitted light in
water, i.e. approximately 0.4 microns. When combined with other
observations regarding small separations between interdigitated
electrodes, this indicates an optimal range for the electrode
spacing of 0.4 to 2 microns.
[0417] Second, the distance from the gap between elements to the
photodiode 184 is minimised. In the LOC device embodiments
described here, this indicates that the total thickness of layers
between the electrodes 860 and 870 and the photodiode 184 be one
micron or less. In embodiments where multiple layers are present
between the electrodes and the photodiode, arranging their
thicknesses to be quarter-wave or three-quarter wave layers has the
further benefit of suppressing reflection of the emitted light
862.
Electrode Models
[0418] FIG. 78 is a schematic partial cross-section of the
electrodes 860 and 870 in the hybridization chamber. The volume
around the lateral periphery of the anode 860 occupied by the
excited species 868, is sometimes referred to as the participating
volume 892. The occluded region 894 above the anode 860 is ignored
because its optical coupling to the photodiode 184 is
negligible.
[0419] A technique for determining whether a particular electrode
configuration provides a foundation for the level of ECL emission
862 for the underlying photodiode 184 is set out below with
reference to FIGS. 79, 80 and 81.
[0420] FIG. 79 is a ring geometry in which the anode 860 is around
the edge of photodiode 184. In FIG. 80, the anode 860 is positioned
within the periphery of the photodiode 184. FIG. 81 shows a more
complex configuration in which the anode 860 has a series of
parallel fingers 880 to increase the length of its lateral
edges.
[0421] For all of the above configurations, the model calculations
are as follows. For a participating volume 892 of solution
V.sub.ECL, the total effective number of emitters N.sub.em is:
N.sub.em=N.sub.lum.tau..sub.p/.tau..sub.ECL=V.sub.ECLC.sub.LN.sub.A.tau.-
.sub.p/.tau..sub.ECL (6)
where the participating number of luminophores
N.sub.lum=V.sub.ECLC.sub.L N.sub.A, .tau..sub.ECL is the lifetime
of the ECL process, C.sub.L is the luminophore concentration,
.tau..sub.p is the pulse duration, and N.sub.A is Avogadro's
number.
[0422] The number of isotropically emitted photons N.sub.phot
is:
N.sub.phot=.phi..sub.ECL N.sub.em (7)
where .phi..sub.ECL is the ECL efficiency, defined as the average
number of photons emitted by the ECL reaction of a single
luminophore.
[0423] The signal count of electrons, S, from the photodiode is
then
S=N.sub.phot.phi..sub.o.phi..sub.q, (8)
where .phi..sub.o is the optical coupling efficiency (the number of
photons absorbed by the photodiode 184) and .phi..sub.q is the
photodiode quantum efficiency. The signal is therefore:
S = V ECL C L N A .tau. p .tau. ECL .phi. ECL .phi. o .phi. q ( 9 )
##EQU00002##
[0424] For FIGS. 79 and 80 electrode configurations, .phi..sub.o
is:
.phi..sub.o=(25% photons which are directed towards the photodiode
184).times.(10% of photons which are not reflected) i.e.,
.phi..sub.o=2.5% for configurations shown in FIGS. 79 and 80
[0425] For the electrode configuration of FIG. 81, 50% of photons
are emitted in a direction pointing towards the photodiode 184, but
the absorption efficiency as a function of angle is unchanged,
so
.phi..sub.o=(50% photons which are directed towards the
photodiode).times.(10% of photons which are not reflected) i.e.,
.phi..sub.o=5% for the configuration of FIG. 81.
[0426] The participating volume 892 depends on the electrode
configuration, and details are presented in the corresponding
sections.
[0427] The input parameters for the calculations are listed in the
following:
TABLE-US-00002 TABLE 5 Input Parameters Parameter Value Comment
Luminophore concen- 2.89 .mu.M Probe concentration tration C.sub.L
calculated previously ECL recycling period 1 ms Combined lifetimes
of (lifetime) .tau..sub.ECL reaction steps for luminophore.
Boundary layer 0.5 .mu.m Effective volume (including depth D
diffusion and electro- phoresis) of solution participating in ECL
Duration of current 0.69 s Chosen to limit ohmic application
.tau..sub.p heating to 4.degree. C. (as described previously)
Chamber X dimension 28 .mu.m Chamber Y dimension 28 .mu.m Chamber
height Z 8 .mu.m Photodiode X dimension 16 .mu.m Photodiode Y
dimension 16 .mu.m Electrode thickness (i.e., 1 .mu.m exposed edge
height) Electrode layer minimum 1 .mu.m Process critical width and
gap dimension Electrode interfacial 350 A/m.sup.2 For ohmic heating
current density Solution volume 0.5 .OMEGA. m For ohmic heating
resistivity Voltage difference 2.2 V applied (working - counter
electrode)
Ring Geometry Around Periphery of Photodiode
[0428] Referring to FIG. 79, the anode 860 is a ring around the
edge of the photodiode 184. In this configuration, the
participating volume 892 is:
V.sub.ECL=4.times.[(layer beside the electrode
wall)+(quarter-cylinder above the electrode wall)]
[0429] Calculation results:
Photons generated from a 0.5 .mu.m boundary layer:
3.1.times.10.sup.5 Electron counts in photodiode:
2.3.times.10.sup.3
[0430] This signal is readily detectable by the underlying
photodiode 184 of the LOC device photosensor 44.
Additional Fingers to Increase Edge Length
[0431] Referring to FIG. 81, parallel fingers 880 are added across
the anode 860. Only horizontal edges shown in figure contribute to
the participating volume 892, to avoid double-counting the
perpendicular edges. The participating volume 892 is then:
V.sub.ECL=(8.times.2).times.[(layer beside the electrode
wall)+(quarter-cylinder above the electrode wall)]
[0432] Calculation results for FIG. 81 configuration:
Photons generated from a 0.5 .mu.m boundary layer:
1.1.times.10.sup.6 Electron counts in photodiode 184:
8.0.times.10.sup.3
[0433] This signal is easily detectable in the photodiode 184.
Complete Overlay
[0434] This configuration shown in FIG. 82 and FIG. 83 is included
as a limiting case of maximum surface area coupling. In practice,
90% or better coupling between the electrode surface area and the
active surface area 185 of the photodiode 184 achieves a nearly
optimal result, and even coupling of 50% of the photodiode active
surface area 185 to the electrode surface area provides most of the
benefit of the complete overlay configuration. Complete overlay can
be achieved in two embodiments: first, as indicated schematically
in FIG. 82, by employing a transparent anode 860, in a plane
parallel with that of the photodiode 184 and with an area matched
to that of the photodiode, and arranging the anode in immediate
proximity to the photodiode 184, such that emitted light 862 passes
through the anode and onto the photodiode. In a second embodiment
shown schematically in FIG. 83, the anode 860 is again parallel to
and registered with the photodiode area, but the solution 872 fills
a void between the anode 860 and the photodiode 184. For signal
modelling of a complete overlay configuration, the anode is assumed
to be a complete layer above the photodiode 184, with half of the
photons directed toward the photodiode 184 (absorption efficiency
still 10%).
Photons generated from a 0.5 .mu.m boundary layer:
7.7.times.10.sup.5 Electron counts in photodiode:
1.2.times.10.sup.4
[0435] It is possible to improve the signal and assay beyond the
above models by using surfactants and probe immobilization at the
anode.
Maximum Spacing Between ECL Probes and Photodiode
[0436] The on-chip detection of hybridization avoids the needs for
detection via confocal microscopy (see Background of the
Invention). This departure from traditional detection techniques is
a significant factor in the time and cost savings associated with
this system. Traditional detection requires imaging optics which
necessarily uses lenses or curved mirrors. By adopting non-imaging
optics, the diagnostic system avoids the need for a complex and
bulky optical train. Positioning the photodiode very close to the
probes has the advantage of extremely high collection efficiency:
when the thickness of the material between the probes and the
photodiode is on the order of 1 micron, the angle of collection of
emission light is up to 174.degree.. This angle is calculated by
considering light emitted from a probe at the centroid of the face
of the hybridization chamber closest to the photodiode, which has a
planar active surface parallel to that chamber face. The cone of
emission angles within which light is able to be absorbed by the
photodiode is defined as having the emitting probe at its vertex
and the corner of the sensor on the perimeter of its planar face.
For a 16 micron.times.16 micron sensor, the vertex angle of this
cone is 170.degree.; in the limiting case where the photodiode is
expanded so that its area matches that of the 28 micron.times.26.5
micron hybridization chamber, the vertex angle is 174.degree.. A
separation between the chamber face and the photodiode active
surface of 1 micron or less is readily achievable.
[0437] Employing a non-imaging optics scheme does require the
photodiode 184 to be very close to the hybridization chamber in
order to collect sufficient photons of fluorescence emission. The
maximum spacing between the photodiode and probes is determined as
follows.
[0438] Utilizing a ruthenium chelate luminophore and the electrode
configuration of FIG. 81, we calculated 27,000 photons being
absorbed by our 16 micron.times.16 micron sensor from the
respective hybridization chamber, to generate 8000 electrons
assuming a sensor quantum efficiency of 30%. In performing this
calculation we assumed that the light-collecting region of our
hybridization chamber has a base area which is the same as our
sensor area, one quarter of the total number of the hybridization
photons is angled so as to reach the sensor, and a conservative 10%
estimate for the proportion of photons which do not scatter away
from the sensor-dielectric interface. That is, the light gathering
efficiency of the optical system is .phi..sub.0=0.025.
[0439] More accurately we can write .phi..sub.0=[(base area of the
light-collecting region of the hybridization
chamber)/(photodetector area)][.OMEGA./4.pi.][10% absorbed], where
.OMEGA.=solid angle subtended by the photodetector at a
representative point on the base of the hybridization chamber. For
a right square pyramid geometry:
.OMEGA.=4 arcsin(a.sup.2/(4d.sub.0.sup.2+a.sup.2)), where
d.sub.0=distance between the chamber and the photodiode, and a is
the photodiode dimension.
[0440] Each hybridization chamber releases 1.1.times.10.sup.6
photons. The selected photodetector has a detection threshold of 17
photons, and for values of d.sub.0 greater than ten times the
sensor size (i.e., essentially normal incidence) the proportion of
photons not reflected at the sensor surface can be increased from
10% to 90%. Therefore, the minimum optical efficiency required
is:
.phi..sub.0=17/(1.1.times.10.sup.6.times.0.9)=1.72.times.10.sup.-5
[0441] The base area of the light-emitting region of the
hybridization chamber 180 is 29 micron.times.19.75 micron.
[0442] Solving for d.sub.0, we will get the maximum limiting
distance between the bottom of our hybridization chamber and our
photodetector to be d.sub.0=1600 microns. In this limit, the
collection cone angle as defined above is only 0.8.degree.. It
should be noted this analysis ignores the negligible effect of
refraction.
LOC Variants
[0443] The LOC device 301 described and illustrated above in full
is just one of many possible LOC device designs. Variations of the
LOC device that use different combinations of the various
functional sections described above will now be described and/or
shown as schematic flow-charts, from sample inlet to detection, to
illustrate some of the combinations possible. The flow-charts have
been divided, where appropriate, into sample input and preparation
stage 288, extraction stage 290, incubation stage 291,
amplification stage 292, pre-hybridization stage 293 and detection
stage 294. For all the LOC variants that are briefly described or
shown only in schematic form, the accompanying full layouts are not
shown for reasons of clarity and succinctness. Also in the
interests of clarity, smaller functional units such as liquid
sensors and temperature sensors are not shown but it will be
appreciated that these have been incorporated into the appropriate
locations in each of the following LOC device designs.
Microfluidic Device with Dialysis Device, LOC Device and
Interconnecting Cap
[0444] A microfluidic device 783 with dialysis device 784, LOC
device 785 and interconnecting cap 51 provides for greater
modularity and improved sensitivity. In contrast to LOC designs
which combine all the functions (see, for example, LOC variant 729
in FIG. 89), separating the dialysis function from the LOC device
allows different specialized dialysis devices 784 to be developed
that select for different targets. These specialized dialysis
devices 784 can be combined with a LOC device 785 and
interconnecting cap 51 to form a complete assay system.
Furthermore, different LOC devices 785 optimized for different
assay approaches can also be developed and coordinated with
different dialysis devices 784, thus providing an extremely
powerful and flexible approach to system development. It is also
possible to deploy the LOC device without a dialysis device for
certain applications, or to combine multiple LOC devices 785.
[0445] Each surface-micromachined chip constituent of the
microfluidic device 783 can be fabricated with the optimal and most
cost effective fabrication process. For example, the dialysis
device 784 requires no CMOS circuitry and so can be manufactured
utilizing less expensive materials and fewer process steps.
Furthermore, a larger and optimized dialysis device 784 provides
increased sensitivity, signal-to-noise ratio, and dynamic range for
the assay system.
[0446] As diagrammatically shown in FIG. 102, the microfluidic
device 783 prepares the sample 288, and then extracts 290,
incubates 291, amplifies 292, and detects 294 pathogenic DNA using
twelve separate amplification chambers (112.1 to 112.12). The
assembly employs multiple amplification chambers to increase assay
sensitivity and improve the signal-to-noise ratio. This LOC device
utilizes ECL in the hybridization chamber arrays 110.1 to 110.12 to
detect probe-target hybrids. The system modularity provides for
different dialysis devices 784 to be employed to detect other
targets, for example, leukocytes, erythrocytes, pathogens, or
molecules like free proteins or DNA within a liquid sample, and
although the current description describes pathogenic DNA
detection, the skilled worker will appreciate that the microfluidic
device 783 is not limited to detecting this one target only.
[0447] FIG. 103 shows the microfluidic device 783 with dialysis
device 784, LOC device 785, and interconnecting cap 51. The
interconnecting cap 51 consists of the reservoir layer 78, the cap
channel layer 80, and the interface layer 594. The interface layer
594 is positioned between the cap channel layer 80 and the MST
channel layer 100 of the CMOS+MST device 48. The interface layer
594 allows a more complex fluidic interconnection between the
reagent reservoirs and the MST layer 87 without increasing the size
of the silicon substrate 84. FIG. 104 superimposes the reservoirs,
the top channels, and the interface channels to illustrate the more
sophisticated plumbing achieved with the interface layer 594.
[0448] Referring to FIGS. 104 and 105, the sample (for example,
blood) enters the sample inlet 68 and capillary action draws it
along the cap channel 94 to the anticoagulant surface tension valve
118. As best shown in FIG. 105, the anticoagulant surface tension
valve 118 has two interface channels 596 and 598 in the interface
layer 594. A reservoir-side interface channel 596 connects the
reservoir outlet with the downtakes 92 and a sample-side interface
channel 598 connects the uptakes 96 with the cap channel 94.
Anticoagulant from the reservoir 54 flows through the MST channels
90 via the reservoir-side interface channel 596 until the meniscus
is pinned at the uptakes 96. The sample flow along the cap channel
94 dips into the sample-side interface channel 598 to remove the
meniscus so that the anticoagulant combines with the blood sample
as it continues on to the pathogen dialysis section 70.
[0449] Referring to FIGS. 104 and 105, the pathogen dialysis
section 70 in this embodiment comprises an interface target channel
602 and an interface waste channel 604 fluidically coupled by a
plurality of apertures of a predetermined threshold size. The
aperture at the very upstream of the dialysis section 70 is
different from the apertures downstream of it; the downstream
apertures are holes with diameters of less than 8.0 microns
selected to allow targets to pass through to the interface target
channel 602. In the present embodiment the apertures are 3.0 micron
diameter holes 164 selected to allow pathogens to pass through to
the interface target channel 602. The pathogen dialysis section 70
is configured such that the sample flows through the channels and
apertures under capillary action.
[0450] Referring to FIGS. 104 and 105, the blood sample flows
through the cap channel 94 to the upstream end of the interface
waste cell channel 604. The interface waste cell channel 604 is
open to the 3.0 micron diameter apertures 164 leading to the
dialysis MST channels 204. Each of the dialysis MST channels 204
lead from the 3.0 micron diameter apertures 164 to respective
dialysis uptake holes 168. The dialysis uptake holes 168 are open
to the interface target channel 602. However the uptakes are
configured to pin a meniscus rather than allow capillary driven
flow to continue.
[0451] The pathogen dialysis section 70 incorporates a bypass
channel 600 for filling the flow channel structures without trapped
air bubbles. The bypass channel 600 at the very upstream end of the
pathogen dialysis section 70, has a CIF (capillary initiation
feature) 202 to promote capillary driven flow from the bypass
channel 600 into the interface target channel 602 (see FIGS. 104
and 105). The bypass channel also has a wide meander to lengthen
the flow-path from the interface waste cell channel 604 to the
interface target channel 602. The longer flow-path delays the
sample flow such that it fills the interface target channel 602
after the meniscus forms at the most upstream dialysis MST channel
204. The sample flow starts at the upstream end and unpins the
meniscus at each of the dialysis uptake holes 168 as the flow moves
downstream along the interface waste channel 602. This ensures all
the dialysis bottom channels fill with sample flow as the dialysis
section fills. Without the bypass channel 600, or dialysis uptakes
168 configured to pin a meniscus, some dialysis MST channels 204
may not fill. Similarly, an air bubble may form in the interface
target channel 602. In either case, flow through the dialysis
section can be substantially throttled.
[0452] The interface waste channel 604 feeds into the waste channel
72 and the interface target channel 602 feeds into the target
channel 74 (see FIGS. 104 and 105). Five dialysis sections are
joined in series by cap channels 72 and 74 to increase the
efficiency of the dialysis process. At the exit of the fifth
dialysis section, the sample flow with the target is drawn along
the target channel 74 in the cap channel layer away from the
dialysis device 784 and into the LOC device 785 by capillary driven
flow. The waste channel 72 flows to the waste reservoir 76 (see
FIG. 103).
[0453] The LOC device 785 can also be employed as a standalone
microfluidic device with an alternate cap suitable for a single
device, and in this configuration optional anticoagulant reservoir
55 can be used to supply anticoagulant. One example of this
standalone use of the LOC device 785 is its use for whole blood
analysis. Referring to FIGS. 106 and 107, the target flows into LOC
device 785 along cap channel 74. It passes via optional
anticoagulant surface tension valve 117, which is used to add the
contents of optional anticoagulant reservoir 55, and continues on
until it reaches the lysis surface tension valve 128. In the
configuration being described here, this optional reservoir and
surface tension valves are unused and do not affect the sample flow
or operation of the LOC device.
[0454] As with the anticoagulant surface tension valve 118
described above, the lysis surface tension valve 128 has a lysis
reservoir-side interface channel 606 and a lysis sample-side
interface channel 608 (see FIG. 107). Lysis reagent flows from the
reservoir 56 to the lysis reservoir-side interface channel 606 via
a cap channel 94. The reagent flows into the downtakes 92, through
the MST channels 90 to the uptakes 96 where the reagents pin a
meniscus (see FIG. 107). Sample flow from the target channel 74
fills the lysis sample-side interface channel 608. The sample flow
removes the menisci at the uptakes 96 and the lysis reagent
combines with the sample as it flows into the chemical lysis
section 130.
[0455] In the chemical lysis chamber section 130, the lysis reagent
diffusively mixes through the flow to lyse the target cells and
release the genetic material therein. The sample flow stops at the
mixing section exit valve 206. The mixing section exit valve is a
boiling-initiated valve 206. The liquid sensor 174 upstream of the
valve provides feedback that the sample flow is about to reach the
valve 206. If the CMOS circuitry 86 is programmed with a delay to
ensure the target cells are completely lysed, the liquid sensor
feedback initiates the delay period. After any delay period, the
boiling-initiated valve 206 is activated and the downstream liquid
sensor 174 registers that the flow has resumed along the MST
channel 90.
[0456] The lysed sample flow continues to the restriction enzyme,
ligase and linker surface tension valve 132. The operation of the
surface tension valve 132 is the same as described earlier for the
anticoagulant surface tension valve 118. Referring to FIG. 107,
when the lysed sample flow reaches the pinned-menisci at the
surface tension valve 132, restriction enzymes, ligase and linker
primers are released from the reservoir 58 and combine with the
sample flow. The sample then flows through MST channel 90 to the
heated microchannels of the incubation section 114. Referring to
FIGS. 107 and 108, the incubation section 114 is composed of a
serpentine microchannel 210 heated by heaters 154.
[0457] Referring to FIG. 108, the sample flow is stopped at the
incubator outlet valve 207 until sufficient time has passed. The
incubator outlet valve 207 is a boiling-initiated valve similar to
the mixing section outlet valve 206. The liquid sensor 174 at the
beginning of the incubation section, in conjunction with the flow
rate sensor 740 (see FIG. 112) and CMOS circuitry 86, initiates an
incubation time delay. After sufficient incubation, the incubator
outlet valve 207 activates and flow resumes flowing along the MST
incubation exit channel 630 to the polymerase surface tension valve
140 (see FIG. 109). Polymerase from the reservoir 62 combines with
the sample flow as it travels the serpentine path of the
amplification input channel 632.
[0458] Referring to FIGS. 108 and 109, the amplification input
channel 632 directs the sample flow past the twelve amplification
mix surface tension valves 138. Amplification mix in each of the
amplification mix reservoirs 60.1-60.12 (see FIG. 109) flows
through respective cap channels 94 to pin menisci at the
amplification mix surface tension valves 138. The sample flow opens
each of the surface tension valves in turn, and the amplification
mix from the respective amplification mix reservoirs 60.1-60.12
entrains with the sample flow into each of the twelve amplification
chambers 112.1-112.12. The LOC device has CMOS circuitry that
allows operative control of amplification section via temperature
sensors and heaters.
[0459] Referring to FIG. 110, each of the twelve amplification
chambers 112.1-112.12 has one of the amplification outlet valves
207, respectively. The amplification outlet valves 207 are
boiling-initiated valves like the incubator outlet valve 207. The
sample flow stops at each amplification outlet valve 207. After
amplification, the amplification outlet valves 207 open for the
amplicon to flow into the hybridization chamber arrays 110.1-110.12
containing probes configured to form probe-target hybrids with the
target nucleic acid sequences, in this case pathogenic DNA. The
sample is drawn along the flow-path 176 through each of the
separate arrays 110.1-110.12 and into the individual hybridization
chambers 180 via respective diffusion barrier inlets 175 (see FIG.
90 and FIG. 111).
[0460] Referring to FIGS. 91 and 111, when the sample flow reaches
the end-point liquid sensor 178, the hybridization heaters 182 are
activated after a time delay to enhance the generation of
probe-target hybrids. The flow rate sensor 740 (see FIG. 112) is
included in the pathogen incubation section 114 to determine the
time delay. After a suitable delay for hybridization, an excitation
current applied to the ECL electrodes 860 and 870 (see FIGS. 111
and 114) causes the probe-target hybrids to emit photons of light
that are detected with the photosensor 44 in the underlying CMOS
circuitry 86. The photosensor is comprised of an array of
photodiodes 184 positioned adjacent each of the hybridization
chambers.
[0461] FIGS. 113 and 114 show the calibration chambers 382. They
are used to calibrate the photodiodes 184 to adjust for system
noise and background levels as described elsewhere in this
specification. Also, positive ECL control probes 787 and negative
ECL control probes 786 are placed in some of the hybridization
chambers 180 for assay quality control. A variant of the
calibration chambers 382 with interdigitated ECL electrodes is
shown in FIG. 92.
[0462] A humidifier 196 and humidity sensor 232 are used to control
evaporation and condensation in the LOC device 785, particularly
the hybridization chamber array 110. FIG. 110 shows the major
components of the humidifier 196, the water reservoir 188 and
evaporators 190.
[0463] Referring to FIG. 109, the evaporation-based telltale 189
indicates whether the packaging of the device has been damaged
during storage and thus whether the integrity and reliability of
the microfluidic device has been compromised. During manufacture, a
small drop of liquid is applied to the liquid sensor 174 at the
centre of the evaporation-based telltale 189. If the package seal
is breached during storage then the drop of liquid will evaporate.
The presence or absence of the liquid can be detected with the
liquid sensor 174 and thus indicate the integrity of the seals on
the microfluidic device.
CONCLUSION
[0464] The devices, systems and methods described here facilitate
molecular diagnostic tests at low cost with high speed and at the
point-of-care.
The system and its components described above are purely
illustrative and the skilled worker in this field will readily
recognize many variations and modifications which do not depart
from the spirit and scope of the broad inventive concept.
* * * * *
References