U.S. patent application number 13/150027 was filed with the patent office on 2011-12-22 for loc device for genetic analysis with dialysis, chemical lysis, incubation and tandem nucleic acid amplification.
This patent application is currently assigned to Geneasys Pty Ltd. Invention is credited to Mehdi Azimi, Geoffrey Richard Facer, Alireza Moini, Kia Silverbrook.
Application Number | 20110312568 13/150027 |
Document ID | / |
Family ID | 45327470 |
Filed Date | 2011-12-22 |
United States Patent
Application |
20110312568 |
Kind Code |
A1 |
Moini; Alireza ; et
al. |
December 22, 2011 |
LOC DEVICE FOR GENETIC ANALYSIS WITH DIALYSIS, CHEMICAL LYSIS,
INCUBATION AND TANDEM NUCLEIC ACID AMPLIFICATION
Abstract
A lab-on-a-chip (LOC) device for genetic analysis of a
biological sample, the LOC device having an inlet for receiving the
sample, a supporting substrate, a dialysis section for separating
cells larger than a predetermined threshold in the sample from
smaller constituents, whereby the cells larger than a predetermined
threshold include target cells containing genetic material for
analysis, a plurality of reagent reservoirs, a lysis section
downstream of the dialysis section for lysing the target cells to
release the genetic material therein, the lysis section being in
fluid communication with one of the reagent reservoirs containing a
lysis reagent for lysing the target cells in the lysis section, an
incubation section downstream of the lysis section, the incubation
section being in fluid communication with one of the reagent
reservoirs containing enzymes for enzymatic reaction with the
genetic material, a first nucleic acid amplification section
downstream of the incubation section for amplifying first nucleic
acid sequences in the genetic material, and, a second nucleic acid
amplification section downstream of the first nucleic acid
amplification section for amplifying second nucleic acid sequences
in the amplicon from the first nucleic acid amplification section,
wherein, the dialysis section, the lysis section, the incubation
section, the first nucleic acid amplification section and the
second nucleic acid amplification section are all supported on the
supporting substrate.
Inventors: |
Moini; Alireza; (Rozelle,
AU) ; Silverbrook; Kia; (Rozelle, AU) ; Azimi;
Mehdi; (Rozelle, AU) ; Facer; Geoffrey Richard;
(Rozelle, AU) |
Assignee: |
Geneasys Pty Ltd
|
Family ID: |
45327470 |
Appl. No.: |
13/150027 |
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: |
506/16 ;
435/287.2 |
Current CPC
Class: |
Y10T 436/173845
20150115; Y10T 436/203332 20150115; B01L 2400/0633 20130101; B01L
2400/0688 20130101; Y10T 137/0391 20150401; B01L 3/5027 20130101;
C12Q 1/68 20130101; B01L 2300/0636 20130101; Y10T 137/1044
20150401; B01L 2400/0677 20130101; F16K 99/0036 20130101; Y10T
137/2202 20150401; Y10T 436/11 20150115; Y10T 436/25375 20150115;
B01L 3/502707 20130101; B01L 2300/10 20130101; Y10T 137/206
20150401; Y10T 137/0352 20150401; Y10T 137/2076 20150401; B01L
2200/10 20130101; B01L 2300/023 20130101; Y10T 436/107497 20150115;
Y10T 436/25 20150115; B01L 2300/024 20130101; B01L 7/52 20130101;
B01L 2300/0654 20130101; B01L 3/502738 20130101; Y10T 436/145555
20150115; B01L 2300/0883 20130101; Y10T 436/143333 20150115; B01L
2300/1827 20130101; F16K 99/003 20130101; B01L 2400/0406
20130101 |
Class at
Publication: |
506/16 ;
435/287.2 |
International
Class: |
C40B 40/06 20060101
C40B040/06; C12M 1/34 20060101 C12M001/34 |
Claims
1. A lab-on-a-chip (LOC) device for genetic analysis of a
biological sample, the LOC device comprising: an inlet for
receiving the sample; a supporting substrate; a dialysis section
for separating cells larger than a predetermined threshold in the
sample from smaller constituents, whereby the cells larger than a
predetermined threshold include target cells containing genetic
material for analysis; a plurality of reagent reservoirs; a lysis
section downstream of the dialysis section for lysing the target
cells to release the genetic material therein, the lysis section
being in fluid communication with one of the reagent reservoirs
containing a lysis reagent for lysing the target cells in the lysis
section; an incubation section downstream of the lysis section, the
incubation section being in fluid communication with one of the
reagent reservoirs containing enzymes for enzymatic reaction with
the genetic material; a first nucleic acid amplification section
downstream of the incubation section for amplifying first nucleic
acid sequences in the genetic material; and, a second nucleic acid
amplification section downstream of the first nucleic acid
amplification section for amplifying second nucleic acid sequences
in the amplicon from the first nucleic acid amplification section;
wherein, the dialysis section, the lysis section, the incubation
section, the first nucleic acid amplification section and the
second nucleic acid amplification section are all supported on the
supporting substrate.
2. The LOC device according to claim 1 wherein the first nucleic
acid amplification section is a first polymerase chain reaction
(PCR) section and the second nucleic acid amplification section is
a second PCR section.
3. The LOC device according to claim 2 wherein the first PCR
section has a first set of primer pairs for annealing to a first
set of complementary nucleic acid sequences, and the second PCR
section has a second set of primer pairs for annealing to a second
set of complementary nucleic acid sequences, the first set of
complementary nucleic acid sequences being different to the second
set of complementary nucleic acid sequences.
4. The LOC device according to claim 3 wherein the first PCR
section and the second PCR section are configured to operate with
different amplification parameters, the amplification parameters
being at least one of: reverse-transcriptase type; polymerase type;
deoxyribonucleoside triphosphate concentrations; buffer solution;
thermal cycle time; thermal cycle repetitions; and, temperature
during a particular phase of PCR.
5. The LOC device according to claim 4 further comprising a
hybridization section downstream of the second PCR section that has
an array of probes for hybridization with target nucleic acid
sequences and a photosensor for detecting hybridization of any
probes within the array.
6. The LOC device according to claim 3 wherein the dialysis section
has a first channel in fluid communication with the inlet at an
upstream end, a second channel in fluid communication with the
waste channel at a down stream end, and a plurality of apertures
that are smaller than the target cells and larger than the smaller
constituents, the second channel being in fluid communication with
the first channel via the apertures such that the target cells are
retained in the first channel while the smaller constituents flow
into the second channel.
7. The LOC device according to claim 6 wherein the first channel
and the second channel are configured to fill with the sample by
capillary action.
8. The LOC device according to claim 1 wherein the lysis section
has an active valve for retaining the target cells in the lysis
section during lysis such that capillary driven flow to the
incubation section resumes upon opening the active valve.
9. The LOC device according to claim 1 wherein the first nucleic
acid amplification section is a first isothermal nucleic acid
amplification section and the second nucleic acid amplification
section is a second isothermal nucleic acid amplification
section.
10. The LOC device according to claim 1 wherein the reagent
reservoirs each have a surface tension valve for retaining reagents
therein, the surface tension valve having a meniscus anchor for
pinning a meniscus of the reagent until contact with the sample
flow removes the meniscus to allow the reagent to flow from the
reagent reservoir.
11. The LOC device according to claim 4 further comprising CMOS
circuitry, a temperature sensor and a microsystems technology (MST)
layer which incorporates the first and second PCR sections, wherein
the CMOS circuitry is positioned between the supporting substrate
and the MST layer, the CMOS circuitry being configured to use the
temperature sensor output for feedback control of the first and
second PCR sections.
12. The LOC device according to claim 11 wherein the first PCR
section has a PCR microchannel for thermally cycling the sample,
the PCR microchannel defining a flow-path with a cross sectional
area transverse to the flow less than 100,000 square microns.
13. The LOC device according to claim 12 wherein the PCR
microchannel has at least one elongate heater element extending
parallel to the PCR microchannel.
14. The LOC device according to claim 13 wherein the PCR section
has a plurality of elongate PCR chambers each formed by respective
sections of the PCR microchannel, the PCR microchannel having a
serpentine configuration formed by a series of wide meanders, each
of the wide meanders being a channel section that forms one of the
elongate PCR chambers.
15. The LOC device according to claim 1 wherein the incubation
section has a heater for heating the genetic material and the
enzymes to a predetermined enzymatic reaction temperature.
16. The LOC device according to claim 5 further comprising a
hybridization chamber array for containing the probes such that the
probes within each hybridization chamber are configured to
hybridize with one of the target nucleic acid sequences.
17. The LOC device according to claim 16 wherein the photosensor is
an array of photodiodes positioned in registration with the
hybridization chambers.
18. The LOC device according to claim 16 wherein the CMOS circuitry
has a digital memory for storing hybridization data from the
photosensor output and a data interface for transmission of the
hybridization data to an external device.
19. The LOC device according to claim 16 wherein the first PCR
section has an active valve for retaining liquid in the first PCR
section during thermal cycling and allowing flow to the first
hybridization chamber array in response to an activation signal
from the CMOS circuitry.
20. The LOC device according to claim 19 wherein the active valve
is a boiling-initiated valve with a meniscus anchor configured to
pin a meniscus that arrests capillary driven flow of the liquid,
and a heater for boiling the liquid to unpin the meniscus from the
meniscus anchor such that capillary driven flow resumes.
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 GDI016US 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 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-No-
tes-and-Product-Highlights/Fluorescence-Resonance-Energy-Transfer-FRET.htm-
l
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 lab-on-a-chip
(LOC) device for genetic analysis of a biological sample, the LOC
device comprising:
[0066] an inlet for receiving the sample;
[0067] a supporting substrate;
[0068] a dialysis section for separating cells larger than a
predetermined threshold in the sample from smaller constituents,
whereby the cells larger than a predetermined threshold include
target cells containing genetic material for analysis;
[0069] a plurality of reagent reservoirs;
[0070] a lysis section downstream of the dialysis section for
lysing the target cells to release the genetic material therein,
the lysis section being in fluid communication with one of the
reagent reservoirs containing a lysis reagent for lysing the target
cells in the lysis section;
[0071] an incubation section downstream of the lysis section, the
incubation section being in fluid communication with one of the
reagent reservoirs containing enzymes for enzymatic reaction with
the genetic material;
[0072] a first nucleic acid amplification section downstream of the
incubation section for amplifying first nucleic acid sequences in
the genetic material; and,
[0073] a second nucleic acid amplification section downstream of
the first nucleic acid amplification section for amplifying second
nucleic acid sequences in the amplicon from the first nucleic acid
amplification section; wherein,
[0074] the dialysis section, the lysis section, the incubation
section, the first nucleic acid amplification section and the
second nucleic acid amplification section are all supported on the
supporting substrate.
[0075] Preferably, the first nucleic acid amplification section is
a first polymerase chain reaction (PCR) section and the second
nucleic acid amplification section is a second PCR section.
[0076] Preferably, the first PCR section has a first set of primer
pairs for annealing to a first set of complementary nucleic acid
sequences, and the second PCR section has a second set of primer
pairs for annealing to a second set of complementary nucleic acid
sequences, the first set of complementary nucleic acid sequences
being different to the second set of complementary nucleic acid
sequences.
[0077] Preferably, the first PCR section and the second PCR section
are configured to operate with different amplification parameters,
the amplification parameters being at least one of:
[0078] reverse-transcriptase type;
[0079] polymerase type;
[0080] deoxyribonucleoside triphosphate concentrations;
[0081] buffer solution;
[0082] thermal cycle time;
[0083] thermal cycle repetitions; and,
[0084] temperature during a particular phase of PCR.
[0085] Preferably, the LOC device also has a hybridization section
downstream of the second PCR section that has an array of probes
for hybridization with target nucleic acid sequences and a
photosensor for detecting hybridization of any probes within the
array.
[0086] Preferably, the dialysis section has a first channel in
fluid communication with the inlet at an upstream end, a second
channel in fluid communication with the waste channel at a down
stream end, and a plurality of apertures that are smaller than the
target cells and larger than the smaller constituents, the second
channel being in fluid communication with the first channel via the
apertures such that the target cells are retained in the first
channel while the smaller constituents flow into the second
channel.
[0087] Preferably, the first channel and the second channel are
configured to fill with the sample by capillary action.
[0088] Preferably, the lysis section has an active valve for
retaining the target cells in the lysis section during lysis such
that capillary driven flow to the incubation section resumes upon
opening the active valve.
[0089] Preferably, the first nucleic acid amplification section is
a first isothermal nucleic acid amplification section and the
second nucleic acid amplification section is a second isothermal
nucleic acid amplification section.
[0090] Preferably, the reagent reservoirs each have a surface
tension valve for retaining reagents therein, the surface tension
valve having a meniscus anchor for pinning a meniscus of the
reagent until contact with the sample flow removes the meniscus to
allow the reagent to flow from the reagent reservoir.
[0091] Preferably, the LOC device also has CMOS circuitry, a
temperature sensor and a microsystems technology (MST) layer which
incorporates the first and second PCR sections, wherein the CMOS
circuitry is positioned between the supporting substrate and the
MST layer, the CMOS circuitry being configured to use the
temperature sensor output for feedback control of the first and
second PCR sections.
[0092] Preferably, the first PCR section has a PCR microchannel for
thermally cycling the sample, the PCR microchannel defining a
flow-path with a cross sectional area transverse to the flow less
than 100,000 square microns.
[0093] Preferably, the PCR microchannel has at least one elongate
heater element extending parallel to the PCR microchannel.
[0094] Preferably, the PCR section has a plurality of elongate PCR
chambers each formed by respective sections of the PCR
microchannel, the PCR microchannel having a serpentine
configuration formed by a series of wide meanders, each of the wide
meanders being a channel section that forms one of the elongate PCR
chambers.
[0095] Preferably, the incubation section has a heater for heating
the genetic material and the enzymes to a predetermined enzymatic
reaction temperature.
[0096] Preferably, the LOC device also has a hybridization chamber
array for containing the probes such that the probes within each
hybridization chamber are configured to hybridize with one of the
target nucleic acid sequences.
[0097] Preferably, the photosensor is an array of photodiodes
positioned in registration with the hybridization chambers.
[0098] Preferably, the CMOS circuitry has a digital memory for
storing hybridization data from the photosensor output and a data
interface for transmission of the hybridization data to an external
device.
[0099] Preferably, the first PCR section has an active valve for
retaining liquid in the first PCR section during thermal cycling
and allowing flow to the first hybridization chamber array in
response to an activation signal from the CMOS circuitry.
[0100] Preferably, the active valve is a boiling-initiated valve
with a meniscus anchor configured to pin a meniscus that arrests
capillary driven flow of the liquid, and a heater for boiling the
liquid to unpin the meniscus from the meniscus anchor such that
capillary driven flow resumes.
[0101] The easily usable, mass-producible, and inexpensive genomic
analysis LOC device accepts a biological sample through its sample
receptacle, uses its dialysis section to separate the leukocytes
contained in the sample, lyses the leukocytes in its chemical lysis
chamber to release the leukocytes' genetic material, preprocesses
the sample's genetic material in its incubation section, amplifies
target genetic sequences, and analyzes the sample's nucleic acid
sequences via hybridization with oligonucleotide probes with
sensing via its integral imaging array, utilizing reagents stored
in the LOC device's reagent reservoirs.
[0102] The dialysis section functionality extracts additional
information from the sample and increases the sensitivity,
signal-to-noise ratio, and dynamic range of the assay system. The
dialysis section being integral to the device, provides for the low
system component-count and simple manufacturing procedures, leading
into an inexpensive assay system.
[0103] The lysing process extracts analytical and diagnostic
targets from cells in the sample and provides for follow-on
processing and analysis of the targets. The lysis subunit being
integral to the device, provides for simple assay procedures, low
system component-count, and simple system manufacturing procedures,
leading into an inexpensive assay system.
[0104] In the incubation section the genetic material undergo
various types of preprocessing, like DNA restriction and ligation
of adaptor primers, to provide optimal or necessary conditions for
the subsequent analytical stages, increasing the informational
content of the analytical outcomes and increasing the sensitivity,
signal-to-noise-ration, and reliability of the assay system.
[0105] The amplification of target genetic sequences increases the
sensitivity and signal-to-noise ratio of the assay system.
Furthermore, the tandem amplification chambers allow piecewise
partial optimization of the earlier cycles and later cycles of the
amplification process, with the consequent increase in assay
sensitivity, signal-to-noise-ratio, and reliability.
[0106] The probe hybridization section provides for analysis of the
targets via hybridization. The integrated probe hybridization
section provides for an easily usable, mass-producible, and
inexpensive integrated solution with low system
component-count.
[0107] The integrated image sensor obviates the need for an
expensive external imaging system and provides for a
mass-producible inexpensive integrated solution with low system
component-count that is a compact, light, and highly portable
system. The integrated image sensor increases the readout
sensitivity by benefiting from large angle of light collection and
obviates the need for optical components in the optical collection
train.
[0108] The reagent reservoirs, being integral to the LOC device and
holding the assay's total reagent requirements, provide for the low
system component-count and simple manufacturing procedures, leading
into an inexpensive assay system.
BRIEF DESCRIPTION OF THE DRAWINGS
[0109] Preferred embodiments of the present invention will now be
described by way of example only with reference to the accompanying
drawings, in which:
[0110] FIG. 1 shows a test module and test module reader configured
for fluorescence detection;
[0111] FIG. 2 is a schematic overview of the electronic components
in the test module configured for fluorescence detection;
[0112] FIG. 3 is a schematic overview of the electronic components
in the test module reader;
[0113] FIG. 4 is a schematic representation of the architecture of
the LOC device;
[0114] FIG. 5 is a perspective of the LOC device;
[0115] FIG. 6 is a plan view of the LOC device with features and
structures from all layers superimposed on each other;
[0116] FIG. 7 is a plan view of the LOC device with the structures
of the cap shown in isolation;
[0117] FIG. 8 is a top perspective of the cap with internal
channels and reservoirs shown in dotted line;
[0118] FIG. 9 is an exploded top perspective of the cap with
internal channels and reservoirs shown in dotted line;
[0119] FIG. 10 is a bottom perspective of the cap showing the
configuration of the top channels;
[0120] FIG. 11 is a plan view of the LOC device showing the
structures of the CMOS+MST device in isolation;
[0121] FIG. 12 is a schematic section view of the LOC device at the
sample inlet;
[0122] FIG. 13 is an enlarged view of Inset AA shown in FIG. 6;
[0123] FIG. 14 is an enlarged view of Inset AB shown in FIG. 6;
[0124] FIG. 15 is an enlarged view of Inset AE shown in FIG.
13;
[0125] FIG. 16 is a partial perspective illustrating the laminar
structure of the LOC device within Inset AE;
[0126] FIG. 17 is a partial perspective illustrating the laminar
structure of the LOC device within Inset AE;
[0127] FIG. 18 is a partial perspective illustrating the laminar
structure of the LOC device within Inset AE;
[0128] FIG. 19 is a partial perspective illustrating the laminar
structure of the LOC device within Inset AE;
[0129] FIG. 20 is a partial perspective illustrating the laminar
structure of the LOC device within Inset AE;
[0130] FIG. 21 is a partial perspective illustrating the laminar
structure of the LOC device within Inset AE;
[0131] FIG. 22 is schematic section view of the lysis reagent
reservoir shown in FIG. 21;
[0132] FIG. 23 is a partial perspective illustrating the laminar
structure of the LOC device within Inset AB;
[0133] FIG. 24 is a partial perspective illustrating the laminar
structure of the LOC device within Inset AB;
[0134] FIG. 25 is a partial perspective illustrating the laminar
structure of the LOC device within Inset AI;
[0135] FIG. 26 is a partial perspective illustrating the laminar
structure of the LOC device within Inset AB;
[0136] FIG. 27 is a partial perspective illustrating the laminar
structure of the LOC device within Inset AB;
[0137] FIG. 28 is a partial perspective illustrating the laminar
structure of the LOC device within Inset AB;
[0138] FIG. 29 is a partial perspective illustrating the laminar
structure of the LOC device within Inset AB;
[0139] FIG. 30 is a schematic section view of the amplification mix
reservoir and the polymerase reservoir;
[0140] FIG. 31 show the features of a boiling-initiated valve in
isolation;
[0141] FIG. 32 is a schematic section view of the boiling-initiated
valve taken through line 33-33 shown in FIG. 31;
[0142] FIG. 33 is an enlarged view of Inset AF shown in FIG.
15;
[0143] FIG. 34 is a schematic section view of the upstream end of
the dialysis section taken through line 35-35 shown in FIG. 33;
[0144] FIG. 35 is an enlarged view of Inset AC shown in FIG. 6;
[0145] FIG. 36 is a further enlarged view within Inset AC showing
the amplification section;
[0146] FIG. 37 is a further enlarged view within Inset AC showing
the amplification section;
[0147] FIG. 38 is a further enlarged view within Inset AC showing
the amplification section;
[0148] FIG. 39 is a further enlarged view within Inset AK shown in
FIG. 38;
[0149] FIG. 40 is a further enlarged view within Inset AC showing
the amplification chamber;
[0150] FIG. 41 is a further enlarged view within Inset AC showing
the amplification section;
[0151] FIG. 42 is a further enlarged view within Inset AC showing
the amplification chamber;
[0152] FIG. 43 is a further enlarged view within Inset AL shown in
FIG. 42;
[0153] FIG. 44 is a further enlarged view within Inset AC showing
the amplification section;
[0154] FIG. 45 is a further enlarged view within Inset AM shown in
FIG. 44;
[0155] FIG. 46 is a further enlarged view within Inset AC showing
the amplification chamber;
[0156] FIG. 47 is a further enlarged view within Inset AN shown in
FIG. 46;
[0157] FIG. 48 is a further enlarged view within Inset AC showing
the amplification chamber;
[0158] FIG. 49 is a further enlarged view within Inset AC showing
the amplification chamber;
[0159] FIG. 50 is a further enlarged view within Inset AC showing
the amplification section;
[0160] FIG. 51 is a schematic section view of the amplification
section;
[0161] FIG. 52 is an enlarged plan view of the hybridization
section;
[0162] FIG. 53 is a further enlarged plan view of two hybridization
chambers in isolation;
[0163] FIG. 54 is schematic section view of a single hybridization
chamber;
[0164] FIG. 55 is an enlarged view of the humidifier illustrated in
Inset AG shown in FIG. 6;
[0165] FIG. 56 is an enlarged view of Inset AD shown in FIG.
52;
[0166] FIG. 57 is an exploded perspective view of the LOC device
within Inset AD;
[0167] FIG. 58 is a diagram of a FRET probe in a closed
configuration;
[0168] FIG. 59 is a diagram of a FRET probe in an open and
hybridized configuration;
[0169] FIG. 60 is a graph of the intensity of an excitation light
over time;
[0170] FIG. 61 is a diagram of the excitation illumination geometry
of the hybridization chamber array;
[0171] FIG. 62 is a diagram of a Sensor Electronic Technology LED
illumination geometry;
[0172] FIG. 63 is an enlarged plan view of the humidity sensor
shown in Inset AH of FIG. 6;
[0173] FIG. 64 is a schematic section view of a leukocyte target
dialysis section;
[0174] FIG. 65 is a schematic showing part of the photodiode array
of the photo sensor;
[0175] FIG. 66 is a circuit diagram for a single photodiode;
[0176] FIG. 67 is a timing diagram for the photodiode control
signals;
[0177] FIG. 68 is an enlarged view of the evaporator shown in Inset
AP of FIG. 55;
[0178] FIG. 69 is a schematic section view through a hybridization
chamber with a detection photodiode and trigger photodiode;
[0179] FIG. 70 is a diagram of linker-primed PCR;
[0180] FIG. 71 is a schematic representation of a test module with
a lancet;
[0181] FIG. 72 is a diagrammatic representation of the architecture
of LOC variant VII;
[0182] FIG. 73 is a perspective of LOC variant VIII;
[0183] FIG. 74 is a plan view of LOC variant VIII with features and
structures from all layers superimposed on each other;
[0184] FIG. 75 is a plan view of LOC variant VIII with the
structures of the cap shown in isolation;
[0185] FIG. 76 is a bottom perspective of the cap channels for LOC
variant VIII;
[0186] FIG. 77 is a plan view of LOC variant VIII showing the
structures of the CMOS+MST device in isolation;
[0187] FIG. 78 is an enlarged view of Inset CA shown in FIG.
74;
[0188] FIG. 79 is a partial perspective illustrating the laminar
structure of LOC variant VIII within Inset CA shown in FIG. 74;
[0189] FIG. 80 is a partial perspective illustrating the laminar
structure of LOC variant VIII within Inset CA shown in FIG. 74;
[0190] FIG. 81 is a partial perspective illustrating the laminar
structure of LOC variant VIII within Inset CA shown in FIG. 74;
[0191] FIG. 82 is a partial perspective illustrating the laminar
structure of LOC variant VIII within Inset CA shown in FIG. 74;
[0192] FIG. 83 is a partial perspective illustrating the laminar
structure of LOC variant VIII within Inset CA shown in FIG. 74;
[0193] FIG. 84 is a partial perspective illustrating the laminar
structure of LOC variant VIII within Inset CA shown in FIG. 74;
[0194] FIG. 85 is an enlarged view of Inset CB shown in FIG.
74;
[0195] FIG. 86 is a partial perspective illustrating the laminar
structure of LOC variant VIII within Inset CB shown in FIG. 74;
[0196] FIG. 87 is a partial perspective illustrating the laminar
structure of LOC variant VIII within Inset CB shown in FIG. 74;
[0197] FIGS. 88A and 88B show a partial perspective illustrating
the laminar structure of LOC variant VIII within Inset CB shown in
FIG. 74;
[0198] FIG. 89 is a partial perspective illustrating the laminar
structure of LOC variant VIII within Inset CB shown in FIG. 74;
[0199] FIG. 90 is a partial perspective illustrating the laminar
structure of LOC variant VIII within Inset CB shown in FIG. 74;
[0200] FIG. 91 is a partial perspective illustrating the laminar
structure of LOC variant VIII within Inset CB shown in FIG. 74;
[0201] FIG. 92 is an enlarged view of Inset CC shown in FIG.
74;
[0202] FIG. 93 is a partial perspective illustrating the laminar
structure of LOC variant VIII within Inset CC shown in FIG. 74;
[0203] FIG. 94 is a partial perspective illustrating the laminar
structure of LOC variant VIII within Inset CC shown in FIG. 74;
[0204] FIG. 95 is a partial perspective illustrating the laminar
structure of LOC variant VIII within Inset CC shown in FIG. 74;
[0205] FIGS. 96A and 96B show a partial perspective illustrating
the laminar structure of LOC variant VIII within Inset CC shown in
FIG. 74;
[0206] FIG. 97 is a partial perspective illustrating the laminar
structure of LOC variant VIII within Inset CC shown in FIG. 74;
[0207] FIG. 98 is a partial perspective illustrating the laminar
structure of LOC variant VIII within Inset CC shown in FIG. 74;
[0208] FIG. 99 is a partial perspective illustrating the laminar
structure of LOC variant VIII within Inset CC shown in FIG. 74;
[0209] FIG. 100 is an enlarged view of Inset CD shown in FIG.
74;
[0210] FIG. 101 is an enlarged perspective view of Inset CD shown
in FIG. 74;
[0211] FIG. 102 is an exploded perspective view of Inset CD shown
in FIG. 74;
[0212] FIG. 103 is an enlarged view of Inset CE shown in FIG.
78;
[0213] FIG. 104 is a diagrammatic representation of the
architecture of LOC variant VIII;
[0214] FIG. 105 is a schematic illustration of the architecture of
LOC variant XIV;
[0215] FIG. 106 is a schematic illustration of the architecture of
LOC variant XV;
[0216] FIG. 107 is a schematic illustration of the architecture of
LOC variant XVIII;
[0217] FIG. 108 is a schematic illustration of the architecture of
LOC variant XXII;
[0218] FIG. 109 is a schematic illustration of the architecture of
LOC variant XXIII;
[0219] FIG. 110 is a schematic illustration of the architecture of
LOC variant XXIV;
[0220] FIG. 111 is a schematic illustration of the architecture of
LOC variant XXV;
[0221] FIG. 112 is a schematic illustration of the architecture of
LOC variant XXVI;
[0222] FIG. 113 is a schematic illustration of the architecture of
LOC variant XXVII;
[0223] FIG. 114 is a schematic illustration of the architecture of
LOC variant XXVIII;
[0224] FIG. 115 is a schematic illustration of the architecture of
LOC variant XLI;
[0225] FIG. 116 is a schematic illustration of the architecture of
LOC variant XLII;
[0226] FIG. 117 is a schematic illustration of the architecture of
LOC variant XLIII;
[0227] FIG. 118 is a schematic illustration of the architecture of
LOC variant XLIV;
[0228] FIG. 119 is a schematic illustration of the architecture of
LOC variant XLVII;
[0229] FIG. 120 is a diagram of a primer-linked, linear fluorescent
probe during the initial round of amplification;
[0230] FIG. 121 is a diagram of a primer-linked, linear fluorescent
probe during a subsequent amplification cycle;
[0231] FIGS. 122A to 122F diagrammatically illustrate thermal
cycling of a primer-linked fluorescent stem-and-loop probe;
[0232] FIG. 123 is a schematic illustration of the excitation LED
relative to the hybridization chamber array and the
photodiodes;
[0233] FIG. 124 is a schematic illustration of the excitation LED
and optical lens for directing light onto the hybridization chamber
array of the LOC device;
[0234] FIG. 125 is a schematic illustration of the excitation LED,
optical lens, and optical prisms for directing light onto the
hybridization chamber array of the LOC device;
[0235] FIG. 126 is a schematic illustration of the excitation LED,
optical lens and mirror arrangement for directing light onto the
hybridization chamber array of the LOC device;
[0236] FIG. 127 is a plan view showing all the features
superimposed on each other, and showing the location of Insets DA
to DK;
[0237] FIG. 128 is an enlarged view of Inset DG shown in FIG.
127;
[0238] FIG. 129 is an enlarged view of Inset DH shown in FIG.
127;
[0239] FIG. 130 is a diagrammatic representation of the
architecture of LOC variant XI;
[0240] FIG. 131 shows one embodiment of the shunt transistor for
the photodiodes;
[0241] FIG. 132 shows one embodiment of the shunt transistor for
the photodiodes;
[0242] FIG. 133 shows one embodiment of the shunt transistor for
the photodiodes;
[0243] FIG. 134 is a circuit diagram of the differential
imager;
[0244] FIG. 135 schematically illustrates a negative control
fluorescent probe in its stem-and-loop configuration;
[0245] FIG. 136 schematically illustrates the negative control
fluorescent probe of FIG. 135 in its open configuration;
[0246] FIG. 137 schematically illustrates a positive control
fluorescent probe in its stem-and-loop configuration;
[0247] FIG. 138 schematically illustrates the positive control
fluorescent probe of FIG. 137 in its open configuration;
[0248] FIG. 139 shows a test module and test module reader
configured for use with ECL detection;
[0249] FIG. 140 is a schematic overview of the electronic
components in the test module configured for use with ECL
detection;
[0250] FIG. 141 shows a test module and alternative test module
readers;
[0251] FIG. 142 shows a test module and test module reader along
with the hosting system housing various databases;
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
Overview
[0252] 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.
[0253] Referring to FIGS. 1, 2, 3, 139 and 140, the system has the
following top level components:
[0254] 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
139). Test module 10 schematically shown in FIG. 1 uses a
fluorescence-based detection technique to identify target
molecules, while test module 11 in FIG. 139 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.
[0255] 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.
[0256] 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 139 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.
[0257] 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. 140). 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.
[0258] 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.
[0259] FIGS. 1, 3 and 139 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. 141). 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.
[0260] Referring to FIG. 142, 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.
[0261] Referring back to FIGS. 1, 2, 139 and 140 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.
[0262] FIG. 71 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.
[0263] 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. 71 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
[0264] FIGS. 2 and 140 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. 139 and
140). 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.
[0265] 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.
[0266] Some examples of infectious diseases rapidly identified with
this system include:
[0267] Influenza--Influenza virus A, B, C, Isavirus,
Thogotovirus
[0268] Pneumonia--respiratory syncytial virus (RSV), adenovirus,
metapneumovirus, Streptococcus pneumoniae, Staphylococcus
aureus
[0269] Tuberculosis--Mycobacterium tuberculosis, bovis, africanum,
canetti, and microti
[0270] Plasmodium falciparum, Toxoplasma gondii and other protozoan
parasites
[0271] Typhoid--Salmonella enterica serovar typhi
[0272] Ebola virus
[0273] Human immunodeficiency virus (HIV)
[0274] Dengue Fever--Flavivirus
[0275] Hepatitis (A through E)
[0276] Hospital acquired infections--for example Clostridium
difficile, Vancomycin resistant Enterococcus, and Methicillin
resistant Staphylococcus aureus
[0277] Herpes simplex virus (HSV)
[0278] Cytomegalovirus (CMV)
[0279] Epstein-Barr virus (EBV)
[0280] Encephalitis--Japanese Encephalitis virus, Chandipura
virus
[0281] Whooping cough--Bordetella pertussis
[0282] Measles--paramyxovirus
[0283] Meningitis--Streptococcus pneumoniae and Neisseria
meningitidis
[0284] Anthrax--Bacillus anthracis
[0285] Some examples of genetic disorders identified with this
system include:
[0286] Cystic fibrosis
[0287] Haemophilia
[0288] Sickle cell disease
[0289] Tay-Sachs disease
[0290] Haemochromatosis
[0291] Cerebral arteriopathy
[0292] Crohn's disease
[0293] Polycistic kidney disease
[0294] Congential heart disease
[0295] Rett syndrome
[0296] A small selection of cancers identified by the diagnostic
system include:
[0297] Ovarian
[0298] Colon carcinoma
[0299] Multiple endocrine neoplasia
[0300] Retinoblastoma
[0301] Turcot syndrome
[0302] 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
[0303] 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.
[0304] 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.
[0305] 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.
[0306] 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.
[0307] 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 63,
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
[0308] 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.
[0309] 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.
[0310] 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.
[0311] 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.
[0312] 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.
[0313] A lower seal 64 encloses the cap channels 94 and the upper
seal layer 82 encloses the reservoirs 54, 56, 58, 60 and 62.
[0314] 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:
[0315] reservoir 54: anticoagulant with option to include
erythrocyte lysis buffer
[0316] reservoir 56: lysis reagent
[0317] reservoir 58: restriction enzymes, ligase and linkers (for
linker-primed PCR (see FIG. 70, extracted from T. Stachan et al.,
Human Molecular Genetics 2, Garland Science, NY and London,
1999))
[0318] reservoir 60: amplification mix (dNTPs, primers, buffer)
and
[0319] reservoir 62: DNA polymerase.
[0320] 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
[0321] 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.
[0322] 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.
[0323] 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.
[0324] 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.
[0325] 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.
[0326] 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.
[0327] 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).
[0328] 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.
[0329] 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.
[0330] 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.
[0331] When the boiling-initiated valve 126 opens, the lysed cells
flow into a mixing section 131 for pre-amplification restriction
digestion and linker ligation.
[0332] 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).
[0333] 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.
[0334] 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.
[0335] 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).
[0336] 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.
[0337] 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.
[0338] 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.
[0339] 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. 65). 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
[0340] 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.
[0341] 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.
[0342] 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.
[0343] 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.
[0344] 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.
[0345] 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
[0346] 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.
[0347] 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
[0348] 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
[0349] 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
[0350] 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).
[0351] 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.
[0352] The small constituents dialysis section 682 schematically
shown in FIG. 115 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 139) 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
[0353] 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.
[0354] 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
[0355] 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.
[0356] 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.
[0357] 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.
[0358] 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
[0359] 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.
[0360] 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.
[0361] 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
[0362] 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.
[0363] 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.
[0364] 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 Termal Cycle Times
[0365] 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.
[0366] 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
[0367] 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.
[0368] 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.
[0369] 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.
[0370] 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.
[0371] 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.
Humidifier and Humidity Sensor
[0372] 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 68, 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.
[0373] 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. 63, 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
[0374] 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.
[0375] 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.
[0376] 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.
[0377] 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
[0378] 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
Leukocyte Target
[0379] The dialysis design described above in the LOC device 301
targets pathogens. FIG. 64 is a schematic section view of a
dialysis section 328 designed to concentrate leukocytes from a
blood sample for human DNA analysis. It will be appreciated that
the structure is essentially the same as that of the pathogen
target dialysis section 70 described above with the exception that
apertures in the form of 7.5 micron diameter holes 165 restrict
leukocytes from passing from the cap channel 94 to the dialysis MST
channels 204. In situations where the sample being analysed is a
blood sample, and the presence of haemoglobin from the erythrocytes
interferes with the subsequent reaction steps, addition of an
erythrocyte lysis buffer along with the anticoagulant in the
reservoir 54 (see FIG. 22), will ensure that the majority of the
lysed erythrocytes (and hence haemoglobin) will be removed from the
sample during this dialysis step. A commonly used erythrocyte lysis
buffer is 0.15M NH.sub.4CL, 10 mM KHCO.sub.3, 0.1 mM EDTA, pH
7.2-7.4, but a person skilled in the art will recognise that any
buffer which efficiently lyses erythrocytes can be used.
[0380] Downstream of the leukocyte dialysis section 328, the cap
channel 94 becomes the target channel 74 such that the leukocytes
continue as part of the assay. Furthermore, in this case, the
dialysis uptake holes 168 lead to a waste channel 72 so that all
smaller cells and components in the sample are removed. It should
be noted that this dialysis variant only reduces the concentration
of the unwanted specimens in the target channel 74.
[0381] FIG. 116 schematically illustrates a large constituents
dialysis section 686 which also separates any large target
constituents from a sample. The apertures in this dialysis section
are fabricated with a size and shape tailored to withhold the large
target constituents of interest in the target channel for further
analysis. As with the leukocyte dialysis section described above,
most (but not all) smaller sized cells, organisms or molecules flow
to a waste reservoir 768. Thus, other embodiments of the LOC device
are not limited to separating leukocytes that are larger than 7.5
.mu.m in size, but can be used to separate cells, organisms or
molecules of any size desired.
Dialysis Section with Flow Channel to Prevent Trapped Air
Bubbles
[0382] Described below is an embodiment of the LOC device referred
to as LOC variant VIII 518 and shown in FIGS. 74, 78, 79 and 103.
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.
[0383] Referring to FIG. 78, 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.
[0384] 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
[0385] Several variants of the LOC device have multiple
amplification sections operating in parallel. For example, LOC
variant VII 492 shown in FIG. 72 has parallel amplification
sections 112.1 to 112.4 which allow multiple nucleic acid
amplification assays to be performed simultaneously.
[0386] LOC variant XI 746 shown in FIG. 130 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. 105,
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.
[0387] 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.
Tandem PCR
[0388] FIGS. 106, 107, 108, 111 and 114 (amongst others)
schematically illustrate LOC devices in which the amplification
sections 112.1 and 112.2 operate in series. The first amplification
section 112.1 includes two reagent reservoirs, 60.1 for the
amplification mix and 62.1 for the polymerase. Each amplification
section added after the initial section also includes two reagent
reservoirs, reservoir 60.2 for the amplification mix and reservoir
62.2 for the polymerase.
[0389] Amplification sections in series allow tandem PCR assays
whereby the first amplification section 112.1 is for
pre-amplification to increase the sensitivity of the subsequent
nucleic acid amplification performed in section 112.2.
Amplification sections in series can also be used for nested PCR
reactions.
[0390] In tandem PCR used for pre-amplification, the first
amplification section 112.1 is used to amplify nucleic acid
sequences in the sample which include the target sequence. This
amplification need not be specific to the target sequence (for
example, whole genome amplification), but does increase the target
sequence concentration. After pre-amplification, the sample is
mixed with reagents from the reservoirs 60.2 and 62.2 and then
passes into the second amplification section 112.2. The reagents
stored in reservoir 60.2 include specific primers to amplify only
the target sequence in the pre-amplified sample mix. It should be
noted that a similar approach, where PCR is replaced by an
isothermal technique in the first or second amplification stages,
can also be employed to achieve the advantages of
preamplification.
[0391] Nested PCR is a particular form of tandem PCR, with the
additional advantage of high target specificity. In nested PCR, the
nucleic acid amplification step in the first amplification section
112.1 amplifies a sequence larger than the final target sequence by
using primers, forming part of the amplification mix reagents
stored in reservoir 60.1, which are complementary to regions
external to the target sequence. The reaction in the first
amplification section 112.1 results in amplicons which are the
target sequence plus the flanking sections. This amplified mixture
is mixed with reagents from reservoir 60.2 and polymerase from
62.2. The reagents stored in reservoir 60.2 include primers which
are complementary to sites at each end of the target sequence, i.e.
a subsection of the amplicon from the first amplification stage.
When nucleic acid amplification is performed in the second section
112.2, the chances of amplification arising from sequence locations
unrelated to the target are greatly diminished, as the amplicon
concentration from the first amplification stage is far greater
than the concentration of the original sample molecules. The
sensitivity and specificity advantages of nested PCR can also be
achieved when one or both of the PCR amplification stages are
replaced by a sequence-specific isothermal amplification
technique.
[0392] Storing polymerases separately and adding them independently
to the sample mix has the advantage that different polymerases can
be selected for the pre-amplification and final nucleic acid
amplification steps. For example, this allows selection of a
low-error-rate (e.g. proofreading) polymerase for the
pre-amplification step to avoid the creation of target sequences
containing errors, or false target sequences, while allowing the
use of a higher-speed, or more thermally tolerant, polymerase for
the final amplification.
Direct PCR
[0393] 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.
[0394] 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.
[0395] 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)
[0396] 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
[0397] 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.
[0398] 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.
[0399] 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.
[0400] 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).
[0401] 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. 117, 118
and 119, respectively. Direct isothermal amplification may also be
combined with one or more pre-amplification dialysis steps 70, 686
or 682 as shown in FIGS. 117 and 119 and/or a pre-hybridization
dialysis step 682 as indicated in FIG. 118 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.
[0402] Isothermal nucleic acid amplification can also be performed
in parallel amplification sections such as those schematically
represented in FIGS. 72, 104 and 105, multiplexed and some methods
of isothermal nucleic acid amplification, such as LAMP, are
compatible with an initial reverse transcription step to amplify
RNA.
Additional Details on the Fluorescence Detection System
[0403] FIGS. 58 and 59 show the hybridization-responsive FRET
probes 236. These are often referred to as molecular beacons and
are stem-and-loop probes, generated from a single strand of nucleic
acid, that fluoresce upon hybridization to complementary nucleic
acids. FIG. 58 shows a single FRET probe 236 prior to hybridization
with a target nucleic acid sequence 238. The probe has a loop 240,
stem 242, a fluorophore 246 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.
[0404] In the absence of a complementary target sequence, the probe
remains closed as shown in FIG. 58. The stem 242 keeps the
fluorophore-quencher pair in close proximity to each other, such
that significant resonant energy transfer can occur between them,
substantially eliminating the ability of the fluorophore to
fluoresce when illuminated with the excitation light 244.
[0405] FIG. 59 shows the FRET probe 236 in an open or hybridized
configuration. Upon hybridization to a complementary target nucleic
acid sequence 238, the stem-and-loop structure is disrupted, the
fluorophore and quencher are spatially separated, thus restoring
the ability of the fluorophore 246 to fluoresce. The fluorescence
emission 250 is optically detected as an indication that the probe
has hybridized.
[0406] 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 Probes
[0407] 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 could be 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 a separate amplification section on
the LOC device. Alternatively, the amplification section is left
unused or used for other reactions.
Primer-Linked Linear Probe
[0408] FIGS. 120 and 121 show a primer-linked linear probe 692
during the initial round of nucleic acid amplification and in its
hybridized configuration during subsequent rounds of nucleic acid
amplification, respectively. Referring to FIG. 120, the
primer-linked linear probe 692 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 fluorophore 246, 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 moiety 248. After an 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 (FIG. 120) 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
probe curls around and hybridizes to the amplified complementary
sequence 698 on the extended strand and fluorescence is detected
indicating the presence of the target DNA. Non-extended
primer-linked linear probes retain their double-stranded stem and
fluorescence remains quenched. This detection method is
particularly well suited for fast detection systems as it relies on
a single-molecule process.
Primer-Linked Stem-and-Loop Probes
[0409] FIGS. 122A to 122F show the operation of a primer-linked
stem-and-loop probe 704. Referring to FIG. 122A, the primer-linked
stem-and-loop probe 704 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 fluorophore 246. 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. 122B), the strands of the target nucleic acid 238
separate, as does the stem 242 of the primer-linked, stem-and-loop
probe 704. When the temperature cools for the annealing phase (see
FIG. 122C), the oligonucleotide primer 700 on the primer-linked
stem-and-loop probe 704 hybridizes to the target nucleic acid
sequence 238. During extension (see FIG. 122D) the complement 706
to the target nucleic acid sequence 238 is synthesized forming a
DNA strand containing both the probe sequence 704 and the amplified
product. The amplification blocker 694 prevents the polymerase from
reading through and copying the probe region 704. When the probe
next anneals, following denaturation, the probe sequence of the
loop segment 240 of the primer-linked stem-and-loop probe (see FIG.
122F) anneals to the complementary sequence 706 on the extended
strand. This configuration leaves the fluorophore 246 relatively
remote from the quencher 248, resulting in a significant increase
in fluorescence emission.
Control Probes
[0410] The hybridization chamber array 110 includes some
hybridization chambers 180 with positive and negative control
probes used for assay quality control. FIGS. 135 and 136
schematically illustrate negative control probes without a
fluorophore 796, and FIGS. 137 and 138 are sketches of positive
control probes without a quencher 798. The positive and negative
control probes have a stem-and-loop structure like the FRET probes
described above. However, a fluorescence signal 250 will always be
emitted from positive control probes 798 and no fluorescence signal
250 is ever emitted from negative control probes 796, regardless of
whether the probes hybridize into an open configuration or remain
closed.
[0411] Referring to FIGS. 135 and 136, the negative control probe
796 has no fluorophore (and may or may not have a quencher 248).
Hence, whether the target nucleic acid sequence 238 hybridizes with
the probe (see FIG. 136), or the probe remains in its stem-and-loop
configuration (see FIG. 135), the response to the excitation light
244 is negligible. Alternatively, the negative control probe 796
could be designed so that it always remains quenched. For example,
by synthesizing the loop 240 to have a probe sequence 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 fluorophore and quencher will remain in close
proximity and no appreciable fluorescence signal will be emitted.
This negative control signal would correspond to low level
emissions from hybridization chambers 180 in which the probes has
not hybridized but the quencher does not quench all emissions from
the reporter.
[0412] Conversely, the positive control probe 798 is constructed
without a quencher as illustrated in FIGS. 137 and 138. Nothing
quenches the fluorescence emission 250 from the fluorophore 246 in
response to the excitation light 244 regardless of whether the
positive control probe 798 hybridizes with the target nucleic acid
sequence 238.
[0413] FIG. 52 shows a possible distribution of the positive and
negative control probes (378 and 380 respectively) throughout the
hybridization chamber array 110. The control probes 378 and 380 are
placed in hybridization chambers 180 positioned in a line 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).
Fluorophore Design
[0414] Fluorophores with long fluorescence lifetimes are required
in order to allow enough time for the excitation light to decay to
an intensity below that of the fluorescence emission at which time
the photosensor 44 is enabled, thereby providing a sufficient
signal to noise ratio. Also, longer fluorescence lifetime
translates into larger integrated fluorescence photon count.
[0415] The fluorophores 246 (see FIG. 59) have a fluorescence
lifetime greater than 100 nanoseconds, often greater than 200
nanoseconds, more commonly greater than 300 nanoseconds and in most
cases greater than 400 nanoseconds.
[0416] The metal-ligand complexes based on the transition metals or
lanthanides have long lifetimes (from hundreds of nanoseconds to
milliseconds), adequate quantum yields, and high thermal, chemical
and photochemical stability, which are all favourable properties
with respect to the fluorescence detection system requirements.
[0417] A particularly well-studied metal-ligand complex based on
the transition metal ion Ruthenium (Ru(II)) is
tris(2,2'-bipyridine)ruthenium(II) ([Ru(bpy).sub.3].sup.2+) which
has a lifetime of approximately 1 .mu.s. This complex is available
commercially from Biosearch Technologies under the brand name
Pulsar 650.
TABLE-US-00002 TABLE 1 Photophysical properties of Pulsar 650
(Ruthenium chelate) Parameter Symbol Value Unit Absorption
Wavelength .lamda..sub.abs 460 nm Emission Wavelength
.lamda..sub.em 650 nm Extinction Coefficient E 14800
M.sup.-1cm.sup.-1 Fluorescence Lifetime .tau..sub.f 1.0 .mu.s
Quantum Yield H 1 (deoxygenated) N/A
[0418] Terbium chelate, a lanthanide metal-ligand complex has been
successfully demonstrated as a fluorescent reporter in a FRET probe
system, and also has a long lifetime of 1600 .mu.s.
TABLE-US-00003 TABLE 2 Photophysical properties of terbium chelate
Parameter Symbol Value Unit Absorption Wavelength .lamda..sub.abs
330-350 nm Emission Wavelength .lamda..sub.em 548 nm Extinction
Coefficient E 13800 M.sup.-1cm.sup.-1 (.lamda..sub.abs and ligand
dependent, can be up to 30000 @ .lamda..sub.e=340 nm) Fluorescence
Lifetime .tau..sub.f 1600 .mu.s (hybridized probe) Quantum Yield H
1 N/A (ligand dependent)
[0419] The fluorescence detection system used by the LOC device 301
does not utilize filters to remove unwanted background
fluorescence. It is therefore advantageous if the quencher 248 has
no native emission in order to increase the signal-to-noise ratio.
With no native emission, there is no contribution to background
fluorescence from the quencher 248. High quenching efficiency is
also important so that fluorescence is prevented until a
hybridization event occurs. The Black Hole Quenchers (BHQ),
available from Biosearch Technologies, Inc. of Novato Calif., have
no native emission and high quenching efficiency, and are suitable
quenchers for the system. BHQ-1 has an absorption maximum at 534
nm, and a quenching range of 480-580 nm, making it a suitable
quencher for the Tb-chelate fluorophore. BHQ-2 has an absorption
maximum at 579 nm, and a quenching range of 560-670 nm, making it a
suitable quencher for Pulsar 650.
[0420] Iowa Black Quenchers (Iowa Black FQ and RQ), available from
Integrated DNA Technologies of Coralville, Iowa, are suitable
alternative quenchers with little or no background emission. Iowa
Black FQ has a quenching range from 420-620 nm, with an absorption
maximum at 531 nm and would therefore be a suitable quencher for
the Tb-chelate fluorophore. Iowa Black RQ has an absorption maximum
at 656 nm, and a quenching range of 500-700 nm, making it an ideal
quencher for Pulsar 650.
[0421] In the embodiments described here, the quencher 248 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.
Excitation Source
[0422] In the fluorescence detection based embodiments described
herein, a LED is chosen as the excitation source instead of a laser
diode, high power lamp or laser due to the low power consumption,
low cost and small size. Referring to FIG. 123, the LED 26 is
positioned directly above the hybridization chamber array 110 on an
external surface of the LOC device 301. On the opposing side of the
hybridization chamber array 110, is the photosensor 44, made up of
an array of photodiodes 184 (see FIGS. 53, 54 and 65) for detection
of fluorescence signals from each of the chambers.
[0423] FIGS. 124, 125 and 126 schematically illustrate other
embodiments for exposing the probes to excitation light. In the LOC
device 30 shown in FIG. 124, the excitation light 244 generated by
the excitation LED 26 is directed onto the hybridization chamber
array 110 by the lens 254. The excitation LED 26 is pulsed and the
fluorescence emissions are detected by the photosensor 44.
[0424] In the LOC device 30 shown in FIG. 125, the excitation light
244 generated by the excitation LED 26 is directed onto the
hybridization chamber array 110 by the lens 254, a first optical
prism 712 and second optical prism 714. The excitation LED 26 is
pulsed and the fluorescence emissions are detected by the
photosensor 44.
[0425] Similarly, the LOC device 30 shown in FIG. 126, the
excitation light 244 generated by the excitation LED 26 is directed
onto the hybridization chamber array 110 by the lens 254, a first
minor 716 and second minor 718. Again, the excitation LED 26 is
pulsed and the fluorescence emissions are detected by the
photosensor 44.
[0426] The excitation wavelength of the LED 26 is dependent on the
choice of fluorescent dye. The Philips LXK2-PR14-R00 is a suitable
excitation source for the Pulsar 650 dye. The SET UVTOP335TO39BL
LED is a suitable excitation source for the Tb-chelate label.
TABLE-US-00004 TABLE 3 Philips LXK2-PR14-R00 LED specifications
Parameter Symbol Value Unit Wavelength .lamda..sub.ex 460 nm
Emission Frequency .nu..sub.em 6.52(10).sup.14 Hz Output Power
p.sub.l 0.515 (min) @ 1 A W Radiation pattern Lambertian profile
N/A
TABLE-US-00005 TABLE 4 SET UVTOP334TO39BL LED Specifications
Parameter Symbol Value Unit Wavelength .lamda..sub.e 340 nm
Emission Frequency .nu..sub.e 8.82(10).sup.14 Hz Power p.sub.l
0.000240 (min) @ 20 mA W Pulse Forward Current I 200 mA Radiation
pattern Lambertian N/A
Ultra Violet Excitation Light
[0427] Silicon absorbs little light in the UV spectrum.
Accordingly, it is advantageous to use UV excitation light. A UV
LED excitation source can be used but the broad spectrum of the LED
26 reduces the effectiveness of this method. To address this, a
filtered UV LED can be used. Optionally, a UV laser can be the
excitation source unless the relatively high cost of the laser is
impractical for the particular test module market.
LED Driver
[0428] The LED driver 29 drives the LED 26 at a constant current
for the required duration. A lower power USB 2.0-certifiable device
can draw at most 1 unit load (100 mA), with a minimum operating
voltage of 4.4 V. A standard power conditioning circuit is used for
this purpose.
Photodiode
[0429] 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.
[0430] 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.
[0431] 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.
[0432] 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.
Calibration Chambers
[0433] The non-uniformity of the electrical characteristic of the
photodiode 184, autofluorescence, and residual excitation photon
flux that has not yet completely decayed, introduce background
noise and offset into the output signal. This background is removed
from each output signal using one or more calibration signals.
Calibration signals are generated by exposing one or more
calibration photodiodes 184 in the array to respective calibration
sources. A low calibration source is used for determining a
negative result in which a target has not reacted with a probe. A
high calibration source is indicative of a positive result from a
probe-target complex. In the embodiment described here, the low
calibration light source is provided by calibration chambers 382 in
the hybridization chamber array 110 which:
[0434] do not contain any probes;
[0435] contain probes that have no fluorescent reporter; or,
[0436] contain probes with a reporter and quencher configured such
that quenching is always expected to occur.
[0437] The output signal from such calibration chambers 382 closely
approximates the noise and offset in the output signal from all the
hybridization chambers in the LOC device. Subtracting the
calibration signal from the output signals generated by the other
hybridization chambers substantially removes the background and
leaves the signal generated by the fluorescence emission (if any).
Signals arising from ambient light in the region of the chamber
array are also subtracted.
[0438] It will be appreciated that the negative control probes
described above with reference to FIGS. 135 to 138 can be be used
in calibration chambers. However, as shown in FIGS. 128 and 129,
which are enlarged views of insets DG and DH of LOC variant X 728
shown in FIG. 127, another option is to fluidically isolate the
calibration chambers 382 from the amplicon. The background noise
and offset can be determined by leaving the fluidically isolated
chambers empty, or containing reporterless probes, or indeed any of
the `normal` probes with both reporter and quencher as
hybridization is precluded by fluidic isolation.
[0439] The calibration chambers 382 can provide a high calibration
source to generate a high signal in the corresponding photodiodes.
The high signal corresponds to all probes in a chamber having
hybridized. Spotting probes with reporters and no quenchers, or
just reporters will consistently provide a signal approximating
that of a hybridization chamber in which a predominant number of
the probes have hybridized. It will also be appreciated that
calibration chambers 382 can be used instead of control probes, or
in addition to control probes.
[0440] 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. 56, 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.
[0441] FIG. 134 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 excitation light, from the
fluorescence signal from the surrounding hybridization chambers
180. The differential imager circuit 788 samples the signal from
the pixel 790 and a "dummy" pixel 792. In one embodiment, the
"dummy" pixel 792 is shielded from light, so its output signal
provides a dark reference. Alternatively, the "dummy" pixel 792 can
be exposed to the excitation light along with the rest of the
array. In the embodiment where the "dummy" pixel 792 is open to
light, 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.
[0442] 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.
Suppression and Enablement of the Photodiode
[0443] The photodiode 184 needs to be suppressed during excitation
by the LED 26 and enabled during fluorescence. FIG. 66 is a circuit
diagram for a single photodiode 184 and FIG. 67 is a timing diagram
for the photodiode control signals. The circuit has photodiode 184
and six MOS transistors, M.sub.shunt 394, M.sub.tx 396, M.sub.reset
398, M.sub.sf 400, M.sub.read 402 and M.sub.bias 404. At the
beginning of the excitation cycle, t1, the transistors M.sub.shunt
394, and M.sub.reset 398 are turned on by pulling the M.sub.shunt
gate 384 and the reset gate 388 high. During this period, the
excitation photons generate carriers in the photodiode 184. These
carriers have to be removed, as the amount of generated carriers
can be sufficient to saturate the photodiode 184. During this
cycle, M.sub.shunt 394 directly removes the carriers generated in
photodiode 184, while M.sub.reset 398 resets any carriers that have
accumulated on node `NS` 406 due to leakage in transistors or due
to diffusion of excitation-produced carriers in the substrate.
After excitation, a capture cycle commences at t4. During this
cycle, the emitted response from the fluorophore is captured and
integrated in the circuit on node `NS` 406. This is achieved by
pulling tx gate 386 high, which turns on the transistor M.sub.tx
396 and transfers any accumulated carriers on the photodiode 184 to
node `NS` 406. The duration of the capture cycle can be as long as
the fluorophore emits. The outputs from all photodiodes 184 in the
hybridization chamber array 110 are captured simultaneously.
[0444] There is a delay between the end of the capture cycle t5 and
the start of the read cycle t6. This delay is due to the
requirement to read each photodiode 184 in the hybridization
chamber array 110 (see FIG. 52) separately following the capture
cycle. The first photodiode 184 to be read will have the shortest
delay before the read cycle, while the last photodiode 184 will
have the longest delay before the read cycle. During the read
cycle, transistor M.sub.read 402 is turned on by pulling the read
gate 393 high. The `NS` node 406 voltage is buffered and read out
using the source-follower transistor M.sub.sf 400.
[0445] There are additional, optional methods of enabling or
suppressing the photodiode as discussed below:
1. Suppression Methods
[0446] FIGS. 131, 132 and 133 show three possible configurations
778, 780, 782 for the M.sub.shunt transistor 394. The M.sub.shunt
transistor 394 has a very high off ratio at maximum |V.sub.GS|=5 V
which is enabled during excitation. As shown in FIG. 131, the
M.sub.shunt gate 384 is configured to be on the edge of the
photodiode 184. Optionally, as shown in FIG. 132, the M.sub.shunt
gate 384 may be configured to surround the photodiode 184. A third
option is to configure the M.sub.shunt gate 384 inside the
photodiode 184, as shown in FIG. 133. Under this third option there
would be less photodiode active area 185.
[0447] These three configurations 778, 780 and 782 reduce the
average path length from all locations in the photodiode 184 to the
M.sub.shunt gate 384. In FIG. 131, the M.sub.shunt gate 384 is on
one side of the photodiode 184. This configuration is simplest to
fabricate and impinges the least on the photodiode active area 185.
However, any carriers lingering on the remote side of the
photodiode 184 would take longer to propagate through to the
M.sub.shunt gate 384.
[0448] In FIG. 132, the M.sub.shunt gate 384 surrounds the
photodiode 184. This further reduces the average path length for
carriers in the photodiode 184 to the M.sub.shunt gate 384.
However, extending the M.sub.shunt gate 384 about the periphery of
the photodiode 184 imposes a greater reduction of the photodiode
active area 185. The configuration 782 in FIG. 133 positions the
M.sub.shunt gate 384 within the active area 185. This provides the
shortest average path length to the M.sub.shunt gate 384 and hence
the shortest transition time. However, the impingement on the
active area 185 is greatest. It also poses a wider leakage
path.
2. Enabling Methods
[0449] a. A trigger photodiode drives the shunt transistor with a
fixed delay. [0450] b. A trigger photodiode drives the shunt
transistor with programmable delay. [0451] c. The shunt transistor
is driven from the LED drive pulse with a fixed delay. [0452] d.
The shunt transistor is driven as in 2c but with programmable
delay.
[0453] FIG. 69 is a schematic section view through a hybridization
chamber 180 showing a photodiode 184 and trigger photodiode 187
embedded in the CMOS circuitry 86. A small area in the corner of
the photodiode 184 is replaced with the trigger photodiode 187. A
trigger photodiode 187 with a small area is sufficient as the
intensity of the excitation light will be high in comparison with
the fluorescence emission. The trigger photodiode 187 is sensitive
to the excitation light 244. The trigger photodiode 187 registers
that the excitation light 244 has extinguished and activates the
photodiode 184 after a short time delay .DELTA.t 300 (see FIG. 2).
This delay allows the fluorescence photodiode 184 to detect the
fluorescence emission from the FRET probes 186 in the absence of
the excitation light 244. This enables detection and improves the
signal to noise ratio.
[0454] Both photodiodes 184 and trigger photodiodes 187 are located
in the CMOS circuitry 86 under each hybridization chamber 180. The
array of photodiodes combines, along with appropriate electronics,
to form the photosensor 44 (see FIG. 65). The photodiodes 184 are
pn-junction fabricated during CMOS structure manufacturing without
additional masks or steps. During MST fabrication, the dielectric
layer (not shown) above the photodiodes 184 is optionally thinned
using the standard MST photolithography techniques to allow more
fluorescent light to illuminate the active area 185 of the
photodiode 184. The photodiode 184 has a field of view such that
the fluorescence signal from the probe-target hybrids within the
hybridization chamber 180 is incident on the sensor face. The
fluorescent light is converted into a photocurrent which can then
be measured using CMOS circuitry 86.
[0455] Alternatively, one or more hybridization chambers 180 can be
dedicated to a trigger photodiode 187 only. These options can be
used in these in combination with 2a and 2b above.
Delayed Detection of Fluorescence
[0456] The following derivations elucidate the delayed detection of
fluorescence using a long-lifetime fluorophore for the
LED/fluorophore combinations described above. The fluorescence
intensity is derived as a function of time after excitation by an
ideal pulse of constant intensity I.sub.e between time t.sub.1 and
t.sub.2 as shown in FIG. 60.
[0457] Let [S1](t) equal the density of excited states at time t,
then during and after excitation, the number of excited states per
unit time per unit volume is described by the following
differential equation:
[ S 1 ] t ( t ) + [ S 1 ] ( t ) .tau. F = I e c hv e ( 1 )
##EQU00001##
where c is the molar concentration of fluorophores, E is the molar
extinction coefficient, v.sub.e is the excitation frequency, and
h=6.62606896(10).sup.-34 Js is the Planck constant. [0458] This
differential equation has the general form:
[0458] y x + p ( x ) y = q ( x ) ##EQU00002##
which has the solution:
y ( x ) = .intg. .intg. p ( x ) x q ( x ) x + k .intg. p ( x ) x (
2 ) ##EQU00003##
[0459] Using this now to solve equation (1),
[ S 1 ] ( t ) = I e c .tau. f hv e + k - t / .tau. f ( 3 )
##EQU00004##
[0460] Now at time t.sub.1, [S1](t.sub.1)=0, and from (3):
k = - I e c .tau. f hv e t 1 / .tau. f ( 4 ) ##EQU00005##
[0461] Substituting (4) into (3):
[ S 1 ] ( t ) = I e c .tau. f hv e - I e c .tau. f hv e - ( t - t 1
) / .tau. f ##EQU00006##
[0462] At time t.sub.2:
[ S 1 ] ( t 2 ) = I e c .tau. f hv e - I e c .tau. f hv e - ( t 2 -
t 1 ) / .tau. f ( 5 ) ##EQU00007##
[0463] For t.gtoreq.t.sub.2, the excited states decay exponentially
and this is described by:
[S1](t)=[S1](t.sub.2)e.sup.-(t-t.sup.2.sup.)/.tau..sup.f (6)
[0464] Substituting (5) into (6):
[ S 1 ] ( t ) = I e c .tau. f hv e [ 1 - - ( t 2 - t 1 ) / .tau. f
] - ( t - t 2 ) / .tau. f ( 7 ) ##EQU00008##
[0465] The fluorescence intensity is given by the following
equation:
I f ( t ) = - [ S 1 ] ( t ) x hV f .eta. l ( 8 ) ##EQU00009##
where v.sub.f is the fluorescence frequency, .eta. is the quantum
yield and 1 is the optical path length.
[0466] Now from (7):
[ S 1 ] ( t ) t = - I e c hv e [ 1 - - ( t 2 - t 1 ) / .tau. f ] -
( t - t 2 ) / .tau. f ( 9 ) ##EQU00010##
[0467] Substituting (9) into (8):
I f ( t ) = I e cl .eta. v f v e [ 1 - - ( t 2 - t 1 ) / .tau. f ]
- ( t - t 2 ) / .tau. f For t 2 - t 1 .tau. f .fwdarw. .infin. , I
f ( t ) .fwdarw. I e cl .eta. v f v e - ( t - t 2 ) / .tau. f ( 10
) ##EQU00011##
[0468] Therefore, we can write the following approximate equation
which describes the fluorescence intensity decay after a
sufficiently long excitation pulse
(t.sub.2-t.sub.1>>.tau..sub.f):
I f ( t ) = I e cl .eta. v f v e - ( t - t 2 ) / .tau. f for t
.gtoreq. t 2 ( 11 ) ##EQU00012##
[0469] In the previous section, we concluded that for
t.sub.2-t.sub.1>>.tau..sub.f,
I f ( t ) = I e cl .eta. v f v e - ( t - t 2 ) / .tau. f for t
.gtoreq. t 2 . ##EQU00013##
[0470] From the above equation, we can derive the following:
n f ( t ) = n e cl .eta. - ( t - t 2 ) / .tau. f ( 12 )
##EQU00014##
where
n f ( t ) = I f ( t ) hv f ##EQU00015##
is the number of fluorescent photons per unit time per unit area
and
n e = I e hv e ##EQU00016##
is the number of excitation photons per unit time per unit
area.
[0471] Consequently,
n f ( t ) = .intg. t 3 .infin. n f ( t ) t ( 13 ) ##EQU00017##
where {umlaut over (n)}.sub.f is the number of fluorescent photons
per unit area and t.sub.3 is the instant of time at which the
photodiode is turned on. Substituting (12) into (13):
n f ( t ) = .intg. t 3 .infin. n e cl .eta. - ( t - t 2 ) / .tau. f
t ( 14 ) ##EQU00018##
[0472] Now, the number of fluorescent photons that reach the
photodiode per unit time per unit area, (t), is given by the
following:
n s ( t ) = n f ( t ) .phi. 0 ( 15 ) ##EQU00019##
where .phi..sub.0 is the light gathering efficiency of the optical
system.
[0473] Substituting (12) into (15) we find
n s ( t ) = .phi. 0 n e cl .eta. - ( t - t 2 ) / .tau. f ( 16 )
##EQU00020##
[0474] Similarly, the number of fluorescence photons that reach the
photodiode per unit fluorescent area {umlaut over (n)}.sub.s, will
be as follows:
n s = .intg. t 3 .infin. n s ( t ) t ##EQU00021##
and substituting in (16) and integrating:
n s = .phi. 0 n e cl .eta. .tau. f - ( t 3 - t 2 ) / .tau. f
##EQU00022## Therefore,
n.sub.s=.phi..sub.0{dot over
(n)}.sub.e.epsilon.cl.eta..tau..sub.fe.sup.-.DELTA.t/.tau..sup.f
(17)
[0475] The optimal value of t.sub.3 is when the rate of electrons
generated in the photodiode 184 due to fluorescence photons becomes
equal to the rate of electrons generated in the photodiode 184 by
the excitation photons, as the flux of the excitation photons
decays much faster than that of the fluorescence photons.
[0476] The rate of sensor output electrons per unit fluorescent
area due to fluorescence is:
e f ( t ) = .phi. f n s ( t ) ##EQU00023##
where .phi..sub.f is the quantum efficiency of the sensor at the
fluorescence wavelength.
[0477] Substituting in (17) we have:
e f ( t ) = .phi. f .phi. 0 n e cl .eta. - ( t - t 2 ) / .tau. f (
18 ) ##EQU00024##
[0478] Similarly, the rate of sensor output electrons per unit
fluorescent area due to the excitation photons is:
e e ( t ) = .phi. e n e - ( t - t 2 ) / .tau. e ( 19 )
##EQU00025##
where .phi..sub.e is the quantum efficiency of the sensor at the
excitation wavelength, and .tau..sub.e is the time-constant
corresponding to the "off" characteristics of the excitation LED.
After time t.sub.2, the LED's decaying photon flux would increase
the intensity of the fluorescence signal and extend its decay time,
but we are assuming that this has a negligible effect on
I.sub.f(t), thus we are taking a conservative approach.
[0479] Now, as mentioned earlier, the optimal value of t.sub.3 is
when:
e f ( t 3 ) = e e ( t 3 ) ##EQU00026##
[0480] Therefore, from (18) and (19) we have:
.phi. f .phi. 0 n e cl .eta. - ( t 3 - t 2 ) / .tau. f = .phi. e n
e - ( t 3 - t 2 ) / .tau. e ##EQU00027##
and rearranging we find:
t 3 - t 2 = ln ( cl .eta. .phi. f .phi. 0 .phi. e ) 1 .tau. f - 1
.tau. e ( 20 ) ##EQU00028##
[0481] From the previous two sections, we have the following two
working equations:
n s = .phi. 0 n . e F .tau. f - .DELTA. t / .tau. f ( 21 ) .DELTA.
t = ln ( F .phi. f .phi. 0 .phi. e ) 1 .tau. f - 1 .tau. e ( 22 )
##EQU00029##
where F=.epsilon.cl.eta. and .DELTA.t=t.sub.3-t.sub.2. We also know
that, in practice, t.sub.2-t.sub.1>>.tau..sub.f.
[0482] The optimal time for fluorescence detection and the number
of fluorescence photons detected using the Philips LXK2-PR14-R00
LED and Pulsar 650 dye are determined as follows. The optimum
detection time is determined using equation (22):
[0483] Recalling the concentration of amplicon, and assuming that
all amplicons hybridize, then the concentration of fluorescent
fluorophores is: c=2.89(10).sup.-6 mol/L
[0484] The height of the chamber is the optical path length
l=8(10).sup.-6 m.
[0485] We have taken the fluorescence area to be equal to our
photodiode area, yet our actual fluorescence area is substantially
larger than our photodiode area; consequently we can approximately
assume .phi..sub.0=0.5 for the light gathering efficiency of our
optical system. From the photodiode characteristics,
.phi. f .phi. e = 10 ##EQU00030##
is a very conservative value for the ratio of the photodiode
quantum efficiency at the fluorescence wavelength to its quantum
efficiency at the excitation wavelength.
[0486] With a typical LED decay lifetime of .tau..sub.e=0.5 ns and
using Pulsar 650 specifications, .DELTA.t can be determined:
F = [ 1.48 ( 10 ) 6 ] [ 2.89 ( 10 ) - 6 ] [ 8 ( 10 ) - 6 ] ( 1 ) =
3.42 ( 10 ) - 5 ##EQU00031## .DELTA. t = ln ( 3.42 ( 10 ) - 5 ] (
10 ) ( 0.5 ) 1 1 ( 10 ) - 6 - 1 0.5 ( 10 ) - 9 = 4.34 ( 10 ) - 9 s
##EQU00031.2##
[0487] The number of photons detected is determined using equation
(21). First, the number of excitation photons emitted per unit time
{dot over (n)}.sub.e is determined by examining the illumination
geometry.
[0488] The Philips LXK2-PR14-R00 LED has a Lambertian radiation
pattern, therefore:
n l = n l 0 cos ( .theta. ) ( 23 ) ##EQU00032##
where is the number of photons emitted per unit time per unit solid
angle at an angle of .theta. off the LED's forward axial direction,
and is the valve of in the forward axial direction.
[0489] The total number of photons emitted by the LED per unit time
is:
n . l = .intg. .OMEGA. n l .OMEGA. = .intg. .OMEGA. n l 0 cos (
.theta. ) .OMEGA. ( 24 ) ##EQU00033##
[0490] Now,
.DELTA..OMEGA. = 2 .pi. [ 1 - cos ( .theta. + .DELTA..theta. ) ] -
2 .pi. [ 1 - cos ( .theta. ) ] ##EQU00034## .DELTA..OMEGA. = 2 .pi.
[ cos ( .theta. ) - cos ( .theta. + .DELTA..theta. ) ] = 4 .pi. sin
( .theta. ) cos ( .DELTA..theta. 2 ) sin ( .DELTA..theta. 2 ) + 4
.pi. cos ( .theta. ) sin 2 ( .DELTA..theta. 2 ) ##EQU00034.2##
.OMEGA. = 2 .pi. sin ( .theta. ) .theta. ##EQU00034.3##
[0491] Substituting this into (24):
n . l = .intg. 0 .pi. 2 2 .pi. n l 0 cos ( .theta. ) sin ( .theta.
) .theta. = .pi. n l 0 ##EQU00035##
[0492] Rearranging, we have:
n l 0 = n . l .pi. ( 26 ) ##EQU00036##
[0493] The LED's output power is 0.515 W and
v.sub.e=6.52(10).sup.14 Hz, therefore:
n . l = p 1 hv e = 0.515 [ 6.63 ( 10 ) - 34 ] [ 6.52 ( 10 ) 14 ] =
1.19 ( 10 ) 18 photons / s ( 27 ) ##EQU00037##
[0494] Substituting this value into (26) we have:
n l 0 = 1.19 ( 10 ) 18 .pi. = 3.79 ( 10 ) 17 photons / s / sr
##EQU00038##
[0495] Referring to FIG. 61, the optical centre 252 and the lens
254 of the LED 26 are schematically shown. The photodiodes are 16
.mu.m.times.16 .mu.m, and for the photodiode in the middle of the
array, the solid angle (.OMEGA.) of the cone of light emitted from
the LED 26 to the photodiode 184 is approximately:
.OMEGA. = area of sensor / r 2 = [ 16 ( 10 ) - 6 ] [ 16 ( 10 ) - 6
] [ 2.825 ( 10 ) - 3 ] 2 = 3.21 ( 10 ) - 5 sr ##EQU00039##
[0496] It will be appreciated that the central photodiode 184 of
the photodiode array 44 is used for the purpose of these
calculations. A sensor located at the edge of the array would only
receive 2% less photons upon a hybridization event for a Lambertian
excitation source intensity distribution.
[0497] The number of excitation photons emitted per unit time
is:
n . e = n l .OMEGA. = [ 3.79 ( 10 ) 17 ] [ 3.21 ( 10 ) - 5 ] = 1.22
( 10 ) 13 photons / s ( 28 ) ##EQU00040##
[0498] Now referring to equation (29):
n s = .phi. 0 n . e F .tau. f - .DELTA. t / .tau. f ##EQU00041## n
s = ( 0.5 ) [ 1.22 ( 10 ) 13 ] [ 3.42 ( 10 ) - 5 ] [ 1 ( 10 ) - 6 ]
- 4.34 ( 10 ) - 9 / 1 ( 10 ) - 6 = 208 photons per sensor .
##EQU00041.2##
[0499] Therefore, using the Philips LXK2-PR14-R00 LED and Pulsar
650 fluorophore, we can easily detect any hybridization events
which results in this number of photons being emitted.
[0500] The SET LED illumination geometry is shown in FIG. 62. At
I.sub.D=20 mA, the LED has a minimum optical power output of
p.sub.l=240 .mu.W centred at .lamda..sub.e=340 nm (the absorption
wavelength of the terbium chelate). Driving the LED at I.sub.D=200
mA would increase the output power linearly to p.sub.l=2.4 mW. By
placing the LED's optical centre 252, 17.5 mm away from the
hybridization chamber array 110, we would approximately concentrate
this output flux in a circular spot size which has a maximum
diameter of 2 mm.
[0501] The photon flux in the 2 mm-diameter spot at the
hybridization away plane is given by equation 27.
n . l = p l hv e = 2.4 ( 10 ) - 3 [ 6.63 ( 10 ) - 34 ] [ 8.82 ( 10
) 14 ] = 4.10 ( 10 ) 15 photons / s ##EQU00042##
[0502] Using equation 28, we have:
n . e = n l .OMEGA. = 4.10 ( 10 ) 15 [ 16 ( 10 ) - 6 ] 2 .pi. [ 1 (
10 ) - 3 ] 2 = 3.34 ( 10 ) 11 photons / s ##EQU00043##
[0503] Now, recalling equation 22 and using the Tb chelate
properties listed previously,
.DELTA. t = ln [ ( 6.94 ( 10 ) - 5 ) ( 10 ) ( 0.5 ) ] 1 1 ( 10 ) -
3 - 1 0.5 ( 10 ) - 9 = 3.98 ( 10 ) - 9 s ##EQU00044##
[0504] Now from equation 21:
n s = ( 0.5 ) [ 3.34 ( 10 ) 11 ] [ 6.94 ( 10 ) - 5 ] [ 1 ( 10 ) - 3
] - 3.98 ( 10 ) - 9 / 1 ( 10 ) - 3 = 11 , 600 photons per sensor .
##EQU00045##
[0505] The theoretical number of photons emitted by hybridization
events using the SET LED and terbium chelate system are easily
detectable and well over the minimum of 30 photons required for
reliable detection by the photosensor as indicated above.
Maximum Spacing Between Probes and Photodiode
[0506] 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 of the order of 1 micron, the angle of collection of
emission light is up to 173.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 area 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 29 micron.times.19.75
micron hybridization chamber, the vertex angle is 173.degree.. A
separation between the chamber face and the photodiode active
surface of 1 micron or less is readily achievable.
[0507] 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 with reference to FIG. 54.
[0508] Utilizing a terbium chelate fluorophore and a SET
UVTOP335TO39BL LED, we calculated 11600 photons reaching our 16
micron.times.16 micron photodiode 184 from the respective
hybridization chamber 180. In performing this calculation we
assumed that the light-collecting region of our hybridization
chamber 180 has a base area which is the same as our photodiode
active area 185, and half of the total number of the hybridization
photons reaches the photodiode 184. That is, the light gathering
efficiency of the optical system is .phi..sub.0=0.5.
[0509] More accurately we can write .phi..sub.0=[(base area of the
light-collecting region of the hybridization chamber)/(photodiode
area)][.OMEGA./4.pi.], where .OMEGA.=solid angle subtended by the
photodiode 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.
[0510] Each hybridization chamber releases 23200 photons. The
selected photodiode has a detection threshold of 17 photons;
therefore, the minimum optical efficiency required is:
.phi..sub.0=17/23200=7.33.times.10.sup.-4
[0511] The base area of the light-collecting region of the
hybridization chamber 180 is 29 micron.times.19.75 micron.
[0512] Solving for d.sub.0, we will get the maximum limiting
distance between the bottom of our hybridization chamber and our
photodiode 184 to be d.sub.0=249 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
[0513] 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.
LOC Variant VIII
[0514] FIGS. 73 to 77 and 78 to 104 show LOC variant VIII 518.
Features and structures that correspond to equivalent features or
structures shown in the LOC device 301 are indicated by the same
reference numeral. Features that do not correspond to previously
described features are indicated with new reference numerals.
[0515] As diagrammatically shown in FIG. 104, this variant of the
LOC 518 extracts 290, incubates 291, amplifies 292 and detects 294
human DNA using twelve separate amplification sections (112.1 to
112.12). LOC variant VIII 518 uses multiple amplification sections
to increase assay sensitivity and improve signal to noise ratio of
the detected fluorescence.
[0516] Referring to FIGS. 73, 74 and 75, the blood sample enters
the sample inlet 68 and capillary action draws it along the cap
channel 94 to the anticoagulant surface tension valve 118. The cap
46 is fabricated with an alternative layer to the lower seal 64. In
this design, an 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. 75
superimposes the reservoirs, the top channels and the interface
channels to illustrate the more sophisticated plumbing achieved
with the interface layer 594.
[0517] As best shown in FIG. 103, the interface layer 594 requires
the anticoagulant surface tension valve 118 to have two interface
channels 596 and 598. 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 to
pin a meniscus 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 leukocyte dialysis section
328.
[0518] Referring to FIGS. 78 and 103, the leukocyte dialysis
section 328 incorporates a bypass channel 600 for filling the flow
channel structures without trapped air bubbles. The blood sample
flows through the cap channel 94 to the upstream end of the
interface target channel 602. The interface target channel 602 is
in fluid communication with dialysis MST channels 204 via apertures
in the form of 7.5 micron diameter holes 165. Each of the dialysis
MST channels 204 lead from the 7.5 micron diameter holes 165 to
respective dialysis uptakes 168. The dialysis uptakes 168 are open
to the interface waste channel 604. However the uptakes are
configured to pin a meniscus rather than allow capillary driven
flow to continue.
[0519] Conversely, the bypass channel 600 at the very upstream end
of the leukocyte dialysis section 328, has a CIF (capillary
initiation feature) 202 to promote capillary driven flow from the
bypass channel 600 into the interface waste channel 604 (see FIGS.
78 and 103). The bypass channel also has a wide meander to lengthen
the flow-path from the interface target channel 602 to the
interface waste channel 604. The longer flow-path delays the sample
flow such that it fills the interface waste channel 604 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 604. This ensures all the
dialysis MST channels fill with sample flow as the dialysis section
fills.
[0520] 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 waste
channel 604. In either case, flow through the dialysis section can
be substantially throttled.
[0521] Referring back to FIGS. 74 and 75, the interface waste
channel 604 feeds into the waste channel 72 which flows to the
waste reservoir 76. The interface target channel 602 feeds into the
target channel 74. The sample flow with the target cells is drawn
along the target channel 74 to the lysis surface tension valve
128.
[0522] 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. 75). 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. 74). 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.
[0523] In the chemical lysis 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. As best shown in FIGS. 78 and 103,
the mixing section exit valve is a boiling-initiated valve 206. The
lysed sample flows into a mixing section exit downtake 612 via an
interface duct 610. The sample continues along the MST channel 90
to the boiling-initiated valve 206 where it stops when a meniscus
pins at the valve uptake 151 in the roof layer 66 (see in
particular FIGS. 80A and 81A). The liquid sensor 174 upstream of
the valve provides feedback that the sample flow is about to reach
the valve uptake 151. 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.
[0524] After any delay period, the annular heater 152 is activated
via the heater contacts 156 (see FIGS. 81A and 81B). The sample
liquid at the valve uptake 151 boils and the meniscus is unpinned.
The sample flows into the boiling-initiated valve interface cavity
616 (see FIG. 82) and out of the valve downtake 150 (see FIG. 81B).
The downstream liquid sensor 174 registers that the flow has
resumed along the MST channel 90.
[0525] The lysed sample flow continues to the uptakes 96 of the
restriction enzyme, ligase and linker surface tension valve 132
(see FIG. 78). Referring to FIGS. 80A, 81A, 82, 83 and 84,
restriction enzymes, ligase and linker primers in the reservoir 58
flow into the cap channel 94 leading to the restriction enzyme,
ligase and linker valve interface channel 614. The restriction
enzyme, ligase and linker valve interface channel 614 opens to
three uptakes 96 where the enzymes and linker primers are retained
by a meniscus. The lysed sample flow in the MST channel 90 passes
the uptakes 96 and removes the menisci such that the restriction
enzymes, ligase and linker primers mix with the sample flow.
[0526] Referring to FIG. 78, the sample, restriction enzymes,
ligase and linker primers flow through a MST channel mixing section
131 for diffusion mixing prior to entering the heated microchannels
of the incubation section 114. The incubation section 114 is
composed of a serpentine microchannel 210 (see FIG. 79) heated by
respective heaters 154 (see FIG. 81A) supported on the roof layer
66 above. The heaters 154 extend between respective pairs of heater
contacts 156 connected to the CMOS circuitry 86.
[0527] Referring to FIG. 85, the sample flow is stopped at the
incubator exit valve 207. The incubator exit valve 207 is a
boiling-initiated valve similar to the mixing section exit valve
206. The liquid sensor 174 immediately upstream of the incubator
exit valve 207 indicates when the sample flow is about to stop at
the valve uptake 151 (see FIGS. 85, 87 and 88B). The CMOS circuitry
86 initiates an incubation time delay (if required) in response to
the liquid sensor.
[0528] After sufficient incubation, the annular heater 152 boils
liquid at the valve uptake 151 to unpin the meniscus. Flow resumes
into the boiling-initiated valve interface cavity 616 (see FIG. 89)
and out of the valve downtake 150 (see FIG. 87). From the valve
downtake 150, the sample flows along the MST incubation exit
channel 630 (see FIG. 85) to the polymerase surface tension valve
140 (see FIGS. 74 and 75). Polymerase from the reservoir 62
combines with the sample flow as it travels the serpentine path of
the amplification input channel 632.
[0529] Referring back to FIG. 85, 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 to 60.12 (see FIG. 75) flows
through respective cap channels 94 (see FIGS. 90 and 91) and
respective amplification interface ducts 618-629 (see FIG. 89) to
pin menisci at the amplification mix surface tension valves 138
(see FIG. 95). The sample flow opens each of the surface tension
valves in turn, and the amplification mix from the respective
amplification mix reservoirs 60.1 to 60.12 (see FIG. 75) entrains
with the sample flow into the respective heated microchannel 158 of
each of the twelve amplification sections 112.1 to 112.12.
[0530] Referring to FIG. 92, each of the twelve amplification
sections 112.1 to 112.12 has one of the amplification outlet valves
108 respectively. The sample flow stops at the valve uptakes 151 of
each amplification outlet valve 108. After thermal cycling, the
valve heater 152 boils liquid at the valve uptake 151 (best shown
in FIG. 96B) and sample flows to the valve interface cavity 616
(see FIG. 97) and out through the valve downtake 150.
[0531] Downstream of the amplification outlet valves 108 are the
separate arrays 110.1 to 110.12 of hybridization chambers 180 for
each of the twelve separate amplicons (see FIGS. 92 and 100). The
sample is drawn along the flow-path 176 through each of the
separate arrays 110.1 to 110.12 and into the individual
hybridization chambers 180 via respective diffusion barrier inlets
175. Referring to FIG. 100, when the sample flow reaches the
end-point liquid sensor 178, the hybridization heaters 182 are
energized for optimum probe-target hybridization.
[0532] Referring to FIG. 77 and FIGS. 100 to 102, the humidity
sensor 232 and hybridization and detection section 52 are
surrounded by a strip of titanium nitride deposited on the roof
layer 66. The strip of TiN provides a LED chip support surface 634
for the excitation LED 26 (see FIG. 3). The excitation LED is
sealed to the LED chip support surface 634 and the air pressure in
the hybridization chambers 180 is equalized with the atmosphere
through the vent holes 122 and the vent channel 636 in the MST
layer 87 (see FIGS. 100 and 102).
[0533] Referring to FIG. 104, the hybridization chamber arrays
110.1 to 110.12 for each of the twelve amplicons have a single
photosensor 44 in the underlying CMOS circuitry 86 (see FIG. 12).
Probe-target hybrids in any of the hybridization chambers 180 emit
a fluorescence signal that is detected by the corresponding
photodiode 184. Each of the hybridization chamber arrays 110.1 to
110.12 has at least one calibration chamber 382 which is isolated
from the sample flow such that no amplicon enters the calibration
chamber 382. The calibration chambers 382 are used to calibrate the
photodiode outputs to adjust for system noise, in the sense of
readout error, as described elsewhere in this specification.
LOC Variant XXIII
[0534] The LOC variant XXIII 650 of FIG. 109 is for genetic
analysis and uses a leukocyte dialysis section 328 to substantially
reduce the erythrocyte concentration in the sample. In the chemical
lysis section 130, lysis reagent from reservoir 56 releases the
genetic material in the leukocytes. Downstream of the chemical
lysis section 130, the sample combines with restriction enzymes,
ligase and linker primers from reservoir 58 and fills the
incubation section 114. After incubation, the boiling-initiated
valve 108 immediately downstream of the incubation section 114
opens for the sample to flow into the amplification section 112 and
ultimately the hybridization chamber array 110.
LOC Variant XXIV
[0535] LOC variant XXIV 651 is a genetic analysis LOC device (see
FIG. 110) with a leukocyte dialysis section 328, chemical lysis
section 130 and restriction enzyme, ligase and linker incubation
section 114. LOC variant XXIV 651 uses parallel amplification
sections 112.1, 112.2, . . . 112.X and respective hybridization
chamber arrays 110.1, 110.2, . . . 110.X.
LOC Variant XXV
[0536] LOC variant XXV 652 (see FIG. 111) is an embodiment of a
genetic analysis LOC device. LOC variant XXV 652 uses a leukocyte
dialysis section 328, chemical lysis section 130 and restriction
enzyme, ligase and linker incubation section 114. The sample is
then fed to tandem amplification sections 112.1 and 112.2 before
detection in a single hybridization chamber array 110.
LOC Variant XXVI
[0537] LOC variant XXVI 653 shown in FIG. 112 is for genetic
analysis and uses a leukocyte dialysis section 328 to substantially
reduce the erythrocyte concentration in the sample. In the chemical
lysis section 130, lysis reagent from reservoir 56 releases the
genetic material in the leukocytes. After chemical lysis, the
boiling-initiated valve 108 immediately downstream of the chemical
lysis section 114 opens for the sample to flow into the
amplification section 112 and ultimately the hybridization chamber
array 110.
LOC Variant XXVII
[0538] LOC variant XXVII 654 shown in FIG. 113 is a genetic
analysis LOC device with a leukocyte dialysis section 328 and
chemical lysis section 130. LOC variant XXVII 654 then uses
parallel amplification sections 112.1, 112.2, . . . 112.X and
respective hybridization chamber arrays 110.1, 110.2, . . .
110.X.
LOC Variant XXVIII
[0539] LOC variant XXVIII 655 shown in FIG. 114 is an embodiment of
a genetic analysis LOC device. LOC variant XXVIII 655 uses a
leukocyte dialysis section 328 and chemical lysis section 130. The
sample is then fed to tandem amplification sections 112.1 and 112.2
before detection in a single hybridization chamber array 110.
Conclusion
[0540] The devices, systems and methods described here facilitate
molecular diagnostic tests at low cost with high speed and at the
point-of-care.
[0541] 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