US20110312836A1 - Microfluidic device for electrochemiluminescent detection of target sequences - Google Patents

Microfluidic device for electrochemiluminescent detection of target sequences Download PDF

Info

Publication number
US20110312836A1
US20110312836A1 US13/150,267 US201113150267A US2011312836A1 US 20110312836 A1 US20110312836 A1 US 20110312836A1 US 201113150267 A US201113150267 A US 201113150267A US 2011312836 A1 US2011312836 A1 US 2011312836A1
Authority
US
United States
Prior art keywords
nucleic acid
ecl
microfluidic device
amplification
sample
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Abandoned
Application number
US13/150,267
Inventor
Mehdi Azimi
Kia Silverbrook
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Geneasys Pty Ltd
Original Assignee
Geneasys Pty Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority to US35601810P priority Critical
Priority to US201161437686P priority
Application filed by Geneasys Pty Ltd filed Critical Geneasys Pty Ltd
Priority to US13/150,267 priority patent/US20110312836A1/en
Assigned to Geneasys Pty Ltd reassignment Geneasys Pty Ltd ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: AZIMI, MEHDI, SILVERBROOK, KIA
Publication of US20110312836A1 publication Critical patent/US20110312836A1/en
Application status is Abandoned legal-status Critical

Links

Images

Classifications

    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12QMEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01LCHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5027Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01LCHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5027Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
    • B01L3/502707Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by the manufacture of the container or its components
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01LCHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5027Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
    • B01L3/502738Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by integrated valves
    • FMECHANICAL ENGINEERING; LIGHTING; HEATING; WEAPONS; BLASTING
    • F16ENGINEERING ELEMENTS AND UNITS; GENERAL MEASURES FOR PRODUCING AND MAINTAINING EFFECTIVE FUNCTIONING OF MACHINES OR INSTALLATIONS; THERMAL INSULATION IN GENERAL
    • F16KVALVES; TAPS; COCKS; ACTUATING-FLOATS; DEVICES FOR VENTING OR AERATING
    • F16K99/00Subject matter not provided for in other groups of this subclass
    • F16K99/0001Microvalves
    • F16K99/0003Constructional types of microvalves; Details of the cutting-off member
    • F16K99/003Valves for single use only
    • FMECHANICAL ENGINEERING; LIGHTING; HEATING; WEAPONS; BLASTING
    • F16ENGINEERING ELEMENTS AND UNITS; GENERAL MEASURES FOR PRODUCING AND MAINTAINING EFFECTIVE FUNCTIONING OF MACHINES OR INSTALLATIONS; THERMAL INSULATION IN GENERAL
    • F16KVALVES; TAPS; COCKS; ACTUATING-FLOATS; DEVICES FOR VENTING OR AERATING
    • F16K99/00Subject matter not provided for in other groups of this subclass
    • F16K99/0001Microvalves
    • F16K99/0034Operating means specially adapted for microvalves
    • F16K99/0036Operating means specially adapted for microvalves operated by temperature variations
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01LCHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2200/00Solutions for specific problems relating to chemical or physical laboratory apparatus
    • B01L2200/10Integrating sample preparation and analysis in single entity, e.g. lab-on-a-chip concept
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01LCHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00Additional constructional details
    • B01L2300/02Identification, exchange or storage of information
    • B01L2300/023Sending and receiving of information, e.g. using bluetooth
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01LCHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00Additional constructional details
    • B01L2300/02Identification, exchange or storage of information
    • B01L2300/024Storing results with means integrated into the container
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01LCHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00Additional constructional details
    • B01L2300/06Auxiliary integrated devices, integrated components
    • B01L2300/0627Sensor or part of a sensor is integrated
    • B01L2300/0636Integrated biosensor, microarrays
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01LCHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00Additional constructional details
    • B01L2300/06Auxiliary integrated devices, integrated components
    • B01L2300/0627Sensor or part of a sensor is integrated
    • B01L2300/0654Lenses; Optical fibres
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01LCHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00Additional constructional details
    • B01L2300/08Geometry, shape and general structure
    • B01L2300/0861Configuration of multiple channels and/or chambers in a single devices
    • B01L2300/0883Serpentine channels
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01LCHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00Additional constructional details
    • B01L2300/10Means to control humidity and/or other gases
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01LCHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00Additional constructional details
    • B01L2300/18Means for temperature control
    • B01L2300/1805Conductive heating, heat from thermostatted solids is conducted to receptacles, e.g. heating plates, blocks
    • B01L2300/1827Conductive heating, heat from thermostatted solids is conducted to receptacles, e.g. heating plates, blocks using resistive heater
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01LCHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2400/00Moving or stopping fluids
    • B01L2400/04Moving fluids with specific forces or mechanical means
    • B01L2400/0403Moving fluids with specific forces or mechanical means specific forces
    • B01L2400/0406Moving fluids with specific forces or mechanical means specific forces capillary forces
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01LCHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2400/00Moving or stopping fluids
    • B01L2400/06Valves, specific forms thereof
    • B01L2400/0633Valves, specific forms thereof with moving parts
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01LCHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2400/00Moving or stopping fluids
    • B01L2400/06Valves, specific forms thereof
    • B01L2400/0677Valves, specific forms thereof phase change valves; Meltable, freezing, dissolvable plugs; Destructible barriers
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01LCHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2400/00Moving or stopping fluids
    • B01L2400/06Valves, specific forms thereof
    • B01L2400/0688Valves, specific forms thereof surface tension valves, capillary stop, capillary break
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01LCHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L7/00Heating or cooling apparatus; Heat insulating devices
    • B01L7/52Heating or cooling apparatus; Heat insulating devices with provision for submitting samples to a predetermined sequence of different temperatures, e.g. for treating nucleic acid samples
    • YGENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10TTECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION
    • Y10T137/00Fluid handling
    • Y10T137/0318Processes
    • Y10T137/0324With control of flow by a condition or characteristic of a fluid
    • Y10T137/0329Mixing of plural fluids of diverse characteristics or conditions
    • Y10T137/0352Controlled by pressure
    • YGENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10TTECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION
    • Y10T137/00Fluid handling
    • Y10T137/0318Processes
    • Y10T137/0391Affecting flow by the addition of material or energy
    • YGENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10TTECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION
    • Y10T137/00Fluid handling
    • Y10T137/0971Speed responsive valve control
    • Y10T137/1044With other condition responsive valve control
    • YGENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10TTECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION
    • Y10T137/00Fluid handling
    • Y10T137/206Flow affected by fluid contact, energy field or coanda effect [e.g., pure fluid device or system]
    • YGENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10TTECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION
    • Y10T137/00Fluid handling
    • Y10T137/206Flow affected by fluid contact, energy field or coanda effect [e.g., pure fluid device or system]
    • Y10T137/2076Utilizing diverse fluids
    • YGENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10TTECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION
    • Y10T137/00Fluid handling
    • Y10T137/206Flow affected by fluid contact, energy field or coanda effect [e.g., pure fluid device or system]
    • Y10T137/218Means to regulate or vary operation of device
    • Y10T137/2202By movable element
    • YGENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10TTECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION
    • Y10T436/00Chemistry: analytical and immunological testing
    • Y10T436/10Composition for standardization, calibration, simulation, stabilization, preparation or preservation; processes of use in preparation for chemical testing
    • Y10T436/107497Preparation composition [e.g., lysing or precipitation, etc.]
    • YGENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10TTECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION
    • Y10T436/00Chemistry: analytical and immunological testing
    • Y10T436/11Automated chemical analysis
    • YGENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10TTECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION
    • Y10T436/00Chemistry: analytical and immunological testing
    • Y10T436/14Heterocyclic carbon compound [i.e., O, S, N, Se, Te, as only ring hetero atom]
    • Y10T436/142222Hetero-O [e.g., ascorbic acid, etc.]
    • Y10T436/143333Saccharide [e.g., DNA, etc.]
    • YGENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10TTECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION
    • Y10T436/00Chemistry: analytical and immunological testing
    • Y10T436/14Heterocyclic carbon compound [i.e., O, S, N, Se, Te, as only ring hetero atom]
    • Y10T436/145555Hetero-N
    • YGENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10TTECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION
    • Y10T436/00Chemistry: analytical and immunological testing
    • Y10T436/17Nitrogen containing
    • Y10T436/173845Amine and quaternary ammonium
    • YGENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10TTECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION
    • Y10T436/00Chemistry: analytical and immunological testing
    • Y10T436/20Oxygen containing
    • Y10T436/203332Hydroxyl containing
    • YGENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10TTECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION
    • Y10T436/00Chemistry: analytical and immunological testing
    • Y10T436/25Chemistry: analytical and immunological testing including sample preparation
    • YGENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10TTECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION
    • Y10T436/00Chemistry: analytical and immunological testing
    • Y10T436/25Chemistry: analytical and immunological testing including sample preparation
    • Y10T436/25375Liberation or purification of sample or separation of material from a sample [e.g., filtering, centrifuging, etc.]

Abstract

A microfluidic device for detecting target nucleic acid sequences in a sample, the microfluidic device having reagent reservoirs for adding reagents to the sample prior to detection of the target nucleic acid sequences, probes with a nucleic acid sequence complementary to the target nucleic acid sequence for forming probe-target hybrids, and an electrochemiluminescent (ECL) luminophore, and, electrodes for generating an excited state in the ECL luminophore in which the ECL luminophore emits photons of light.

Description

    FIELD OF THE INVENTION
  • 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
  • The following applications have been filed by the Applicant which relate to the present application:
  • 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 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 GCF013US GCF014US GCF015US GCF016US GCF020US GCF021US GCF022US GCF023US GCF024US GCF025US GCF027US GCF028US GCF029US GCF030US GCF031US GCF032US GCF033US GCF034US GCF035US GCF036US GCF037US GSA001US GSA002US GSE001US GSE002US GSE003US GSE004US GDA001US GDA002US GDA003US GDA004US GDA005US GDA006US GDA007US GPK001US GMO001US GMV001US GMV002US GMV003US GMV004US GRD001US GRD002US GRD003US GRD004US GPD001US GPD003US GPD004US GPD005US GPD006US GPD007US GPD008US GPD009US GPD010US GPD011US GPD012US GPD013US GPD014US GPD015US GPD016US GPD017US GAL001US GPA001US GPA003US GPA004US GPA005US GSS001US GSL001US GCA001US GCA002US GCA003US
  • 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
  • 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:
      • Inherited disorders
      • Acquired disorders
      • Infectious diseases
      • Genetic predisposition to health-related conditions.
  • 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
  • A nucleic acid based test has four distinct steps:
  • 1. Sample preparation
  • 2. Nucleic acid extraction
  • 3. Nucleic acid amplification (optional)
  • 4. Detection
  • 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.
  • 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.
  • 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.
  • 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.
  • Other lysis methods include mechanical lysis via ultrasonic vibration and thermal lysis where the sample is heated to 94° C. to disrupt cell membranes.
  • 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.
  • 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.
  • 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.
  • PCR is an exponential process that proceeds as long as the conditions for sustaining the reaction are acceptable. The components of the reaction are:
  • 1. pair of primers—short single strands of DNA with around 10-30 nucleotides complementary to the regions flanking the target sequence
  • 2. DNA polymerase—a thermostable enzyme that synthesizes DNA
  • 3. deoxyribonucleoside triphosphates (dNTPs)—provide the nucleotides that are incorporated into the newly synthesized DNA strand
  • 4. buffer—to provide the optimal chemical environment for DNA synthesis
  • PCR typically involves placing these reactants in a small tube (˜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° C. to denature the DNA strands; in the annealing phase, the temperature is lowered to ˜50-60° 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° 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 108 fold amplification to 1010 fold amplification so that it is possible using this technique to amplify unique single copy molecules.
  • 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.
  • 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.
  • 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.
  • 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 1012 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.
  • 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 109 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • More information regarding fluorescent probes can be found at: http://www.premierbiosoft.com/tech_notes/FRET_probe.html and http://www.invitrogen.com/site/us/en/home/References/Molecular-Probes-The-Handbook/Technical-Notes-and-Product-Highlights/Fluorescence-Resonance-Energy-Transfer-FRET.html
  • Point-of-Care Molecular Diagnostics
  • 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.
  • A point-of-care technology serving the physician's office, the hospital bedside or even consumer-based, at home, would offer many advantages including:
      • rapid availability of results enabling immediate facilitation of treatment and improved quality of care.
      • ability to obtain laboratory values from testing very small samples.
      • reduced clinical workload.
      • reduced laboratory workload and improved office efficiency by reducing administrative work.
      • 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.
      • facilitation of clinical management decisions such as infection control and antibiotic use.
    Lab-on-a-Chip (LOC) Based Molecular Diagnostics
  • 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.
  • 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
  • Accordingly, the present invention provides a microfluidic device for detecting target nucleic acid sequences in a sample, the microfluidic device comprising:
  • reagent reservoirs for adding reagents to the sample prior to detection of the target nucleic acid sequences;
  • probes with a nucleic acid sequence complementary to the target nucleic acid sequence for forming probe-target hybrids, and an electrochemiluminescent (ECL) luminophore; and,
  • electrodes for generating an excited state in the ECL luminophore in which the ECL luminophore emits photons of light.
  • Preferably, the probes each have a functional moiety for quenching photon emission from the ECL luminophore by resonant energy transfer.
  • Preferably, the probe is configured such that the functional moiety for quenching photon emission from the ECL luminophore is further from the ECL luminophore when the probe forms a probe-target hybrid.
  • Preferably, the microfluidic device also has CMOS circuitry configured to provide an electrical pulse to the electrodes.
  • Preferably, the electrical pulse has a duration less than 0.69 seconds.
  • Preferably, the electrical pulse has a current of 0.1 nanoamperes to 69.0 nanoamperes.
  • Preferably, the electrodes have an anode and a cathode each having fingers configured for mutual interdigitation.
  • Preferably, the anode and the cathode are separated by a dielectric gap between 0.4 microns and 2.0 microns wide.
  • Preferably, the microfluidic device also has a supporting substrate for the CMOS circuitry, and a cap in which the reagent reservoirs are defined, wherein the electrodes and the probes are between the cap and the CMOS circuitry.
  • Preferably, the reagent reservoirs each have an outlet valve for retaining liquid reagent in the reservoir until reagent addition to the sample is required.
  • Preferably, the CMOS circuitry incorporates a photosensor for sensing the photons emitted from the ECL luminophore.
  • Preferably, the microfluidic device also has an array of hybridization chambers wherein each of the hybridization chambers has a pair of the electrodes respectively and contains a plurality of the probes, the nucleic acid sequence in the probes in each of the hybridization chambers being different to the nucleic acid sequence in at least one other hybridization chamber in the array such that a plurality of target nucleic acid sequences are detectable.
  • Preferably, the microfluidic device also has a supporting substrate wherein the CMOS circuitry is positioned between the hybridization chambers and the supporting substrate such that the photosensor is adjacent the hybridization chambers.
  • Preferably, the photosensor is an array of photodiodes in registration with the array of hybridization chambers such that each of the hybridization chambers corresponds to one of the photodiodes respectively.
  • Preferably, the photodiodes have a planar active surface area for receiving the light from the luminophore, each of the active surface areas being coplanar, and the electrodes are a layer of conductive material patterned to form the separate anodes and cathodes, the layer extending in a plane parallel to that of the active surface areas of the photodiodes.
  • Preferably, one of the electrodes in each of the electrode pairs is a working electrode which causes oxidation or reduction of the luminophore to generate an excited species that emits a photon, the working electrode being positioned such that the probes are between the photodiode and the working electrode.
  • Preferably, the photodiodes have a planar active surface area for receiving the light from the luminophore, and the working electrode has a surface area optically coupled to the active surface area of the photodiode, the working electrode being configured such that the optically coupled surface area is greater than 50% of the active surface area of the photodiode.
  • Preferably, the microfluidic device also has a polymerase chain reaction (PCR) section for amplifying the target nucleic acid sequences in the sample.
  • Preferably, the PCR section has a heater element for thermal cycling the target nucleic acid sequences with polymerase, the heater element being configured for operative control by the CMOS circuitry.
  • Preferably, the microfluidic device also has a plurality of sensors connected to the CMOS circuitry for feedback control of the electrodes and the heater element.
  • The electrochemiluminescence-based assay target detection obviates any need, of the assay system, for an excitation light source, excitation optics, and optical filter elements, in turn, providing for a more compact and more inexpensive assay system. The absence of the requirement for the rejection of any excitation light also simplifies the detector circuitry, making the assay system even more inexpensive.
  • BRIEF DESCRIPTION OF THE DRAWINGS
  • Preferred embodiments of the present invention will now be described by way of example only with reference to the accompanying drawings, in which:
  • FIG. 1 shows a test module and test module reader configured for fluorescence detection;
  • FIG. 2 is a schematic overview of the electronic components in the test module configured for fluorescence detection;
  • FIG. 3 is a schematic overview of the electronic components in the test module reader;
  • FIG. 4 is a schematic representation of the architecture of the LOC device;
  • FIG. 5 is a perspective of the LOC device;
  • FIG. 6 is a plan view of the LOC device with features and structures from all layers superimposed on each other;
  • FIG. 7 is a plan view of the LOC device with the structures of the cap shown in isolation;
  • FIG. 8 is a top perspective of the cap with internal channels and reservoirs shown in dotted line;
  • FIG. 9 is an exploded top perspective of the cap with internal channels and reservoirs shown in dotted line;
  • FIG. 10 is a bottom perspective of the cap showing the configuration of the top channels;
  • FIG. 11 is a plan view of the LOC device showing the structures of the CMOS+MST device in isolation;
  • FIG. 12 is a schematic section view of the LOC device at the sample inlet;
  • FIG. 13 is an enlarged view of Inset AA shown in FIG. 6;
  • FIG. 14 is an enlarged view of Inset AB shown in FIG. 6;
  • FIG. 15 is an enlarged view of Inset AE shown in FIG. 13;
  • FIG. 16 is a partial perspective illustrating the laminar structure of the LOC device within Inset AE;
  • FIG. 17 is a partial perspective illustrating the laminar structure of the LOC device within Inset AE;
  • FIG. 18 is a partial perspective illustrating the laminar structure of the LOC device within Inset AE;
  • FIG. 19 is a partial perspective illustrating the laminar structure of the LOC device within Inset AE;
  • FIG. 20 is a partial perspective illustrating the laminar structure of the LOC device within Inset AE;
  • FIG. 21 is a partial perspective illustrating the laminar structure of the LOC device within Inset AE;
  • FIG. 22 is schematic section view of the lysis reagent reservoir shown in FIG. 21;
  • FIG. 23 is a partial perspective illustrating the laminar structure of the LOC device within Inset AB;
  • FIG. 24 is a partial perspective illustrating the laminar structure of the LOC device within Inset AB;
  • FIG. 25 is a partial perspective illustrating the laminar structure of the LOC device within Inset AI;
  • FIG. 26 is a partial perspective illustrating the laminar structure of the LOC device within Inset AB;
  • FIG. 27 is a partial perspective illustrating the laminar structure of the LOC device within Inset AB;
  • FIG. 28 is a partial perspective illustrating the laminar structure of the LOC device within Inset AB;
  • FIG. 29 is a partial perspective illustrating the laminar structure of the LOC device within Inset AB;
  • FIG. 30 is a schematic section view of the amplification mix reservoir and the polymerase reservoir;
  • FIG. 31 show the features of a boiling-initiated valve in isolation;
  • FIG. 32 is a schematic section view of the boiling-initiated valve taken through line 33-33 shown in FIG. 31;
  • FIG. 33 is an enlarged view of Inset AF shown in FIG. 15;
  • FIG. 34 is a schematic section view of the upstream end of the dialysis section taken through line 35-35 shown in FIG. 33;
  • FIG. 35 is an enlarged view of Inset AC shown in FIG. 6;
  • FIG. 36 is a further enlarged view within Inset AC showing the amplification section;
  • FIG. 37 is a further enlarged view within Inset AC showing the amplification section;
  • FIG. 38 is a further enlarged view within Inset AC showing the amplification section;
  • FIG. 39 is a further enlarged view within Inset AK shown in FIG. 38;
  • FIG. 40 is a further enlarged view within Inset AC showing the amplification chamber;
  • FIG. 41 is a further enlarged view within Inset AC showing the amplification section;
  • FIG. 42 is a further enlarged view within Inset AC showing the amplification chamber;
  • FIG. 43 is a further enlarged view within Inset AL shown in FIG. 42;
  • FIG. 44 is a further enlarged view within Inset AC showing the amplification section;
  • FIG. 45 is a further enlarged view within Inset AM shown in FIG. 44;
  • FIG. 46 is a further enlarged view within Inset AC showing the amplification chamber;
  • FIG. 47 is a further enlarged view within Inset AN shown in FIG. 46;
  • FIG. 48 is a further enlarged view within Inset AC showing the amplification chamber;
  • FIG. 49 is a further enlarged view within Inset AC showing the amplification chamber;
  • FIG. 50 is a further enlarged view within Inset AC showing the amplification section;
  • FIG. 51 is a schematic section view of the amplification section;
  • FIG. 52 is an enlarged plan view of the hybridization section;
  • FIG. 53 is a further enlarged plan view of two hybridization chambers in isolation;
  • FIG. 54 is schematic section view of a single hybridization chamber;
  • FIG. 55 is an enlarged view of the humidifier illustrated in Inset AG shown in FIG. 6;
  • FIG. 56 is an enlarged view of Inset AD shown in FIG. 52;
  • FIG. 57 is an exploded perspective view of the LOC device within Inset AD;
  • FIG. 58 is an enlarged plan view of the humidity sensor shown in Inset AH of FIG. 6;
  • FIG. 59 is a schematic section view of a leukocyte target dialysis section;
  • FIG. 60 is a schematic showing part of the photodiode array of the photo sensor;
  • FIG. 61 is an enlarged view of the evaporator shown in Inset AP of FIG. 55;
  • FIG. 62 is a diagram of linker-primed PCR;
  • FIG. 63 is a schematic representation of a test module with a lancet;
  • FIG. 64 is a diagrammatic representation of the architecture of LOC variant VII;
  • FIG. 65 is a plan view of LOC variant VIII with features and structures from all layers superimposed on each other;
  • FIG. 66 is an enlarged view of Inset CA shown in FIG. 65;
  • FIG. 67 is a partial perspective illustrating the laminar structure of LOC variant VIII within Inset CA shown in FIG. 65;
  • FIG. 68 is an enlarged view of Inset CE shown in FIG. 66;
  • FIG. 69 is a diagrammatic representation of the architecture of LOC variant VIII;
  • FIG. 70 is a schematic illustration of the architecture of LOC variant XIV;
  • FIG. 71 is a schematic illustration of the architecture of LOC variant XLI;
  • FIG. 72 is a schematic illustration of the architecture of LOC variant XLII;
  • FIG. 73 is a schematic illustration of the architecture of LOC variant XLIII;
  • FIG. 74 is a schematic illustration of the architecture of LOC variant XLIV;
  • FIG. 75 is a schematic illustration of the architecture of LOC variant XLVII;
  • FIG. 76 is a diagrammatic representation of the architecture of LOC variant X;
  • FIG. 77 is a perspective view of LOC variant X;
  • FIG. 78 is a plan view of LOC variant X showing the structures of the CMOS+MST device in isolation;
  • FIG. 79 is a perspective view of the underside of the cap with the reagent reservoirs shown in dotted line;
  • FIG. 80 is a plan view showing only the features of the cap in isolation;
  • FIG. 81 is a plan view showing all the features superimposed on each other, and showing the location of Insets DA to DK;
  • FIG. 82 is an enlarged view of Inset DA shown in FIG. 81;
  • FIG. 83 is an enlarged view of Inset DB shown in FIG. 81;
  • FIG. 84 is an enlarged view of Inset DC shown in FIG. 81;
  • FIG. 85 is an enlarged view of Inset DD shown in FIG. 81;
  • FIG. 86 is an enlarged view of Inset DE shown in FIG. 81;
  • FIG. 87 is an enlarged view of Inset DF shown in FIG. 81;
  • FIG. 88 is an enlarged view of Inset DG shown in FIG. 81;
  • FIG. 89 is an enlarged view of Inset DH shown in FIG. 81;
  • FIG. 90 is an enlarged view of Inset DJ shown in FIG. 81;
  • FIG. 91 is an enlarged view of Inset DK shown in FIG. 81;
  • FIG. 92 is an enlarged view of Inset DL shown in FIG. 81;
  • FIG. 93 is a circuit diagram of the differential imager;
  • FIG. 94 schematically illustrates a CMOS-controlled flow rate sensor;
  • FIG. 95 illustrates the reactions occurring during an electrochemiluminescence (ECL) process;
  • FIG. 96 schematically illustrates three different anode configurations;
  • FIG. 97 is a schematic partial cross-section of the anode and cathode in the hybridization chamber;
  • FIG. 98 schematically illustrates an anode in a ring geometry around the peripheral edge of a photodiode;
  • FIG. 99 schematically illustrates an anode in a ring geometry within the peripheral edge of a photodiode;
  • FIG. 100 schematically illustrates an anode with a series of fingers to increase the length of its lateral edges;
  • FIG. 101 schematically illustrates the use of a transparent anode to maximise surface area coupling and ECL signal detection;
  • FIG. 102 schematically illustrates the use of an anode affixed to the roof of the hybridization chamber to maximise surface area coupling and ECL signal detection;
  • FIG. 103 schematically illustrates an anode interdigitated with a cathode;
  • FIG. 104 shows a test module and test module reader configured for use with ECL detection;
  • FIG. 105 is a schematic overview of the electronic components in the test module configured for use with ECL detection;
  • FIG. 106 shows a test module and alternative test module readers;
  • FIG. 107 shows a test module and test module reader along with the hosting system housing various databases;
  • FIGS. 108A and 108B is a diagram illustrating binding of an aptamer to a protein to produce a detectable signal;
  • FIGS. 109A and 109B are diagrams illustrating binding of two aptamers to a protein to produce a detectable signal;
  • FIGS. 110A and 110B are diagrams illustrating binding of two antibodies to a protein to produce a detectable signal;
  • FIG. 111 is a diagrammatic representation of the architecture of LOC variant L with ECL detection;
  • FIG. 112 is a perspective view of LOC variant L;
  • FIG. 113 is a plan view of LOC variant L showing the structures of the CMOS+MST device in isolation;
  • FIG. 114 is a perspective view of the underside of the cap of LOC variant L with the reagent reservoirs shown in dotted lines;
  • FIG. 115 is a plan view of