WO2009108224A1 - Appareil et méthode de détection virale - Google Patents

Appareil et méthode de détection virale Download PDF

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Publication number
WO2009108224A1
WO2009108224A1 PCT/US2008/083184 US2008083184W WO2009108224A1 WO 2009108224 A1 WO2009108224 A1 WO 2009108224A1 US 2008083184 W US2008083184 W US 2008083184W WO 2009108224 A1 WO2009108224 A1 WO 2009108224A1
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Prior art keywords
liposomes
area
viral
functionalized
reporter
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PCT/US2008/083184
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English (en)
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Eugene Tu
Donald E. Ackley
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Eugene Tu
Ackley Donald E
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Publication of WO2009108224A1 publication Critical patent/WO2009108224A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5027—Containers 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
    • B01L3/502761—Containers 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 specially adapted for handling suspended solids or molecules independently from the bulk fluid flow, e.g. for trapping or sorting beads, for physically stretching molecules
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
    • G01N33/5432—Liposomes or microcapsules
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01F—MIXING, e.g. DISSOLVING, EMULSIFYING OR DISPERSING
    • B01F23/00—Mixing according to the phases to be mixed, e.g. dispersing or emulsifying
    • B01F23/40—Mixing liquids with liquids; Emulsifying
    • B01F23/41—Emulsifying
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01F—MIXING, e.g. DISSOLVING, EMULSIFYING OR DISPERSING
    • B01F33/00—Other mixers; Mixing plants; Combinations of mixers
    • B01F33/30—Micromixers
    • B01F33/301—Micromixers using specific means for arranging the streams to be mixed, e.g. channel geometries or dispositions
    • B01F33/3011—Micromixers using specific means for arranging the streams to be mixed, e.g. channel geometries or dispositions using a sheathing stream of a fluid surrounding a central stream of a different fluid, e.g. for reducing the cross-section of the central stream or to produce droplets from the central stream
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/02—Identification, exchange or storage of information
    • B01L2300/021—Identification, e.g. bar codes
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/06—Auxiliary integrated devices, integrated components
    • B01L2300/0627—Sensor or part of a sensor is integrated
    • B01L2300/0654—Lenses; Optical fibres
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/08—Geometry, shape and general structure
    • B01L2300/0809—Geometry, shape and general structure rectangular shaped
    • B01L2300/0825—Test strips
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5027—Containers 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
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5027—Containers 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
    • B01L3/502769—Containers 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 multiphase flow arrangements
    • B01L3/502776—Containers 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 multiphase flow arrangements specially adapted for focusing or laminating flows
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5029—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures using swabs

Definitions

  • This invention relates to specifically engineered apparatus and methods of use for the detection of viral pathogens and the like including unknown, mutated, or engineered varieties. More specifically the invention relates to specifically engineered liposomes and microcavity sensors .
  • the detection and assaying of viral pathogens is very complicated and labor intensive. At a time when new and different viral pathogens appear regularly and with increasing frequency, the detection and identification as quickly and efficiently as possible is highly desirable. This of course is true for mutated and engineered varieties as well as any unknown varieties. At the present time, the only known method for detection and identification is by processing collected viral pathogens in a laboratory using well known testing or trial-and-error procedures such as cell cultures or PCR (polymerase chain reaction) .
  • Rapid detection of influenza virus is important because of the increased concern of a pandemic influenza caused by naturally occurring strains, such as avian H5N1, or a bio- terrorism threat from altered influenza virus or other agents with flu-like symptoms. Diagnosis on the basis of clinical presentation alone is not adequate because many infectious agents have similar symptoms. The ability to differentiate influenza from other respiratory pathogens and biological warfare agents is essential for public health and safety.
  • RT-PCR Reverse Transcriptase PCR
  • PCR RT or Taqman
  • PCR product is usually analyzed by agarose gel electrophoresis which is labor intensive, time consuming, and prone to contamination. Recently, a multiplex real time RT-PCR assay for differentiating influenza was reported.
  • apparatus for identifying viral pathogens.
  • the apparatus includes a receiving area for receiving a viral sample to be identified and a fusion area including functionalized liposomes with surface receptors complimentary to a desired viral target, and containing viral subtype specific oligo probes.
  • the fusion area receives the viral sample from the receiving area for binding the viral target to the functionalized liposomes and initiating fusion with the liposome, and subsequently internalizing and hybridizing the viral target genetic material with the subtype specific oligo probes.
  • the apparatus further includes an area containing antibody conjugate, an area containing reporter liposomes carrying fluorescently labeled barcode oligos, and an area containing subtype encoded microcavities .
  • a method of identifying viral genetic material includes providing functionalized liposomes containing subtype specific oligo probes, immobilized particles with functionalized antigens, liposome carriers with fluorescently labeled barcode oligos, and subtype encoded microcavities. In the preferred embodiment these are provided on a continuous, lateral flow test strip. A viral sample is received to be typed and subtyped.
  • An initial step includes binding the viral target to the receptors on the functionalized liposomes, initiating fusion of the viral target with the liposome membrane, and subsequent hybridization of the viral target genetic material with the subtype specific oligo probes contained within the vesicle to form a chimeric liposome. Lysis of the chimeric liposome releases viral proteins and genetic material for identification.
  • Typing of influenza A/B/avian can be accomplished using type specific antibodies to viral antigens, i.e. NP, MP, HA, NA, in an immunoassay format. Subtyping is accomplished by liposome based reporter amplification coupled to optical microcavity detection.
  • the subtype specific oligos contain linker sequences that are non-complementary to viral sequences but recognize complementary sequences on reporter liposomes. These liposomes contain either fluorescently labeled barcode oligos or barcode target sequences as payload and are present at equal concentrations. In the presence of the target analyte, a ternary complex is formed consisting of viral nucleic acid, subtype specific oligo with subtype encoded linker sequence, and reporter liposomes that carry either barcode oligos or target or both.
  • Lysis of the reporter liposomes allows a sandwich to be formed between the fluorescently labeled barcode oligo, barcode target and an encoded microcavity which has been functionalized with barcode sequences complimentary to the the barcode target but not overlapping the barcode oligo hybridization region.
  • this sandwich complex When excited with a pump laser source, this sandwich complex provides sufficient optical gain that lasing occurs and the sub-type encoding may be identified.
  • a method of fabricating a lateral flow test strip including the steps of providing a strip having a plurality of spaced apart areas, forming functionalized liposomes containing subtype specific oligo probes, drying down the functionalized liposomes, and storing the dried down functionalized liposomes in a first area on the strip, forming reporter liposomes with fluorescently labeled barcode oligos, drying down the reporter liposomes with fluorescently labeled barcode oligos, and storing the dried down reporter liposomes with fluorescently labeled barcode oligos in a second area on the strip, forming antibody conjugates, drying down the antibody conjugates, and storing the dried down antibody conjugates in a third area on the strip, and storing subtype encoded microcavities in a fourth area on the strip.
  • FIG. 1 is a schematic depiction of an embodiment of a viral detection liposome
  • FIG. 2 is a simplified schematic diagram of a liposome generator used in the formation and engineering of the viral detection liposomes
  • FIG. 3 illustrates a flow chart for an example of an assay in accordance with the present invention
  • FIG. 4 illustrates a lateral flow triage strip test concept for typing and subtyping
  • FIG. 5 illustrates hybridized viral RNA and proteins from lysed chimeric liposomes (step 34) of the process from FIG. 3;
  • FIG. 6 illustrates steps 38 and 39 of FIG. 3
  • FIG. 7 illustrates hybridizing subtype specific barcode liposome conjugates A and A' ;
  • FIG. 8 is a schematic illustration of microcavity with hybridized barcode markers A and A' ;
  • FIG. 9a) and b) are top plan and side views, respectively, of arrayed subtype encoded microcavities on the triage strip of FIG. 4.
  • the disclosed novel detection methods for a viral identification assay are based, in part, on mimicking the response mechanisms found in living cells.
  • By tailoring or engineering liposomes with the appropriate biomolecular and biochemical compositions these particles are comprised of the necessary biological elements to sense and identify potential biological threats.
  • the application of nanodroplet technology provides customized liposomes that are packaged with the suitable genetic substrates, biochemical reagents, and optical reporters. These liposomes are functionalized with pre-determined surface receptors that specifically target conserved characteristics of unknown or engineered pathogens of certain biological classes. Due to the flexible nature of the nanodroplet technology for providing various liposome compositions on demand, this approach has significant impact in the rapid development and deployment of reagents and receptor molecules for integration into existing detection systems. In addition to improvements in assay speed, accuracy, and sensitivity, the engineered liposomes also operate in complex backgrounds and provide the detection capability of multiple targets simultaneously.
  • the structure of the engineered liposomes is relatively simple and is depicted schematically in FIG. 1.
  • strain specific oligos 12 (similar to strands of RNA or DNA) are encapsulated in the interior of a lipid shell 14 of liposome 10. While the illustrated droplet or particle is generically referred to as liposome 10 with lipid shell 14 for convenience of understanding in this disclosure, it should be understood that shell 14 can include lipids, proteins, carbohydrates, and polymers in substantially any desired ratios for any specific applications.
  • Strain specific oligos 12 will target the conserved sequences found across the wide range of influenza strains, for example.
  • Surface receptors 18 are attached to lipid shell 14 to form binding sites for viral target 16.
  • viral target 16 binds to surface receptors 18, within a short amount of time it will "inject" its genetic material, generally RNA but sometimes DNA, into the interior of liposome 10 by fusion with the lipid membrane, wherein the genetic material is able to hybridize to one or more strain specific oligos 12.
  • the entire process of viral fusion and hybridization generally occurs within approximately 360 seconds because of the enhanced binding rate due to the minute interior volume of the liposome, and further stabilizes the viral RNA or DNA.
  • liposome 10 has been "infected" by virus 16, the resulting combination will be referred to as a chimeric liposome generally designated 19.
  • Droplet generator 20 is a preferred apparatus for use in the ⁇ nanodroplet technology' described herein but other processes and apparatus can be employed for specific applications. Also, while the droplet formation process is generically referred to as the ⁇ nanodroplet technology' , nano- and micro-droplets (e.g. 100 nm to 100 ⁇ m in diameter) can be formed in generator 20 using immiscible fluids, such as oil, in cross channels 22 as focusing fluid and water in central channel 24 as a carrier fluid.
  • the carrier fluid can carry various materials such as drugs, proteins, etc. for encapsulation.
  • Particles or droplets formed using the droplet process can be polymeric or lipid based, or a combination of both, with surfaces that can be readily functionalized during the droplet formation process.
  • the droplet mechanism provides superior control of particle or liposome size distribution which is advantageous for sensitivity of the liposome based assay.
  • the size of droplets 26 can be precisely controlled and may be varied over a wide range, with diameters that can vary from hundreds of nanometers to hundreds of microns. Since the droplet size is determined by the flow rates, which can be made extremely consistent, the droplets are substantially monodispersed. However, for applications that demand size variations, the droplet size can be varied continuously in a controlled manner.
  • the nanodroplet technology adds an important new degree of freedom into the formation of complex nanoparticles in that the fluidic formation process can be used to control the size and structure of the particles, concurrently with the surface functionalization process but also somewhat independently of it, providing more flexibility to optimize the particle properties.
  • non-polar solvents which can dissolve lipids for the formation of liposomes in droplet generator 20.
  • specific non-polar solvents include ether, cyclohexane, butanol, ethyl acetate, benzyl alcohol, and the like.
  • Ethyl acetate is of particular interest for two reasons, first it is relatively nontoxic and second it is formed from ether and acetic acid and may be broken down into its constituents at relatively low concentrations. Overall, ethyl acetate was found to be about 8% miscible in water, which means that it can eventually be exchanged into a buffer solution.
  • this system is sufficiently immiscible to form droplets in the droplet generators while being sufficiently miscible to be exchanged with water.
  • lipids are carried in a partially miscible solvent (e.g., ethyl acetate) and used as the focusing fluid in droplet generator 20, injected through cross channels 22.
  • the carrier fluid water
  • the viscous shear forces between the focusing fluid and the carrier fluid generate droplets 26 of the carrier fluid, coated with a mono-layer of lipids (liposomes) .
  • the liposomes with a single lipid layer are carried in the focusing fluid in an outlet 28.
  • liposomes are flowed from outlet 28 and the focusing fluid is removed by diluting the focusing fluid in water, since the focusing fluid is partially miscible.
  • the focusing fluid with liposomes is directed into a large volume of water (50 to 100 times larger than the volume of the focusing fluid) .
  • the focusing fluid is then dissolved into the much greater volume of water significantly reducing the concentration of focusing fluid. By repeating the wash process several times, the focusing fluid concentration is reduced to negligible levels.
  • Bi-layer liposomes (or additional layers) can be formed in accordance with a procedure described in the above identified copending application including introducing the single lipid layer liposomes into a container of excess solution with excess lipids. As there are excess lipids in the container, in order for the vesicles to remain in the aqueous buffer it is energetically favorable for them to add a second lipid layer to the single lipid layer, thus protecting the hydrophobic tail groups and presenting hydrophilic head groups to the aqueous environment both inside and outside the now fully completed liposomes.
  • Several other methods are described in the above identified copending application for producing bi- layer liposomes or even tri-layer liposomes if desired.
  • a major advantage of the nanodroplet technology in forming liposomes (nanoparticles ) is the ability to encapsulate materials such as proteins and oligos without damage.
  • Many techniques currently being applied or investigated for drug encapsulation use high pressure or flow rates, or generate high shear forces, which can damage the structure of proteins or peptides. Since the droplet size is determined by the relative flow rates of solvent in cross channels 22 and water in main channel 24, adjusting these relative flow rates essentially varies the droplet- forming shear forces. Thus, varying the droplet size can ensure that the fragile proteins remain undamaged over a wide range of shear forces.
  • PCR microreactors containing Taq polymerase and ⁇ 1 kb DNA templates were fabricated using the droplet technology and amplified by PCR.
  • the amplification products and efficiency were determined by gel electrophoresis and showed no adverse effects when compared to control reactions.
  • the above described droplet mechanism represents a unified technology capable of: (1) producing liposomes that are monodispersed over a size range from 0.1 ⁇ m to 100 ⁇ m; (2) developing membrane compositions that have necessary stability and fusogenic potential; (3) producing asymmetrically functionalized membranes with specific viral receptors; and
  • influenza virus comprises a diverse mix of antigenic subtypes. Each subtype includes a specific hemagglutinin (HA) and a specific neuraminidase (NA) subtype, e.g., H5N1 or H3N2.
  • HA hemagglutinin
  • NA neuraminidase
  • the host range of influenza viruses is associated with differences in the specificity of HA for attachment to highly conserved sialic acid-containing receptors on susceptible cells.
  • HAs of human viruses have a preference for sialic acid alpha 2, 6-galactose beta 1,4-N-acetyl glucosamine (SA-2,6 Gal) .
  • SA-2,6 Gal sialic acid alpha 2, 6-galactose beta 1,4-N-acetyl glucosamine
  • influenza virus enters a target cell by binding to sialic acid residues on the cell surface, subsequent internalization by indocytosis, and finally delivery to endosomes .
  • Virus access to the cytosol occurs following fusion of the viral envelope and the endosomal membrane that is triggered by the envelope glycoprotein, hemagglutinin, conformational changes in acidic (pH 5-6) environment of the endosomes.
  • the proteolytic cleavage of HA produces a fusogenic protein with a hydrophobic peptide that can insert into the target membrane and induce fusion.
  • HA-mediated fusion process is necessary for viral infectivity but not for membrane fusion.
  • sialic acid gangliosides such as GDIa or GDIb
  • membrane fusion is all that is necessary to allow virus access to the liposome interior.
  • the pH of the interior can be made slightly acidic to dissociate the ribonucleoprotein (RNP) and RNA complex .
  • Fusion occurs within 5 minutes and can be influenced by the binding affinity of HA to the sialic acid derivatives presented on the liposome surface and the membrane lipid composition.
  • Liposome membrane composition can also be adjusted using cholesterol or nonlamellar phospholipids, such as phosphatidylethanolamine, that can induce membrane stress that is relieved by fusion events.
  • Physicochemical parameters, such as pH and divalent cations, can also affect the fusion rate.
  • Cell surface receptors are glycoproteins that are embedded or otherwise attached to the cell' s plasma membrane and have a binding site for specific ligands exposed to the extracellular environment.
  • Cell surface receptors are typically integral membrane proteins and have 3 basic domains: extracellular domain (ligand-binding domain); transmembrane domain; and cytoplasmic or intracellular domain.
  • Cell surface receptors may be purchased already purified or can be readily synthesized. In the case of influenza, the receptors can be much simpler, as sialic acid containing glycolipids can act as the receptors rather than an integral membrane protein. These lipids can be purchased through Avanti Polar Lipids or Sigma and used directly. Thus, there is not a need to implement the sometimes difficult and lengthy process of protein isolation, purification, and characterization in this effort. Other classes of viral targeted alternate receptors may be desired, and in those cases it is expected that commercial providers may be utilized to synthesize the receptors on either a standardized or custom basis.
  • the influenza surface receptor can be incorporated into the lipid membrane forming the liposomes via attachment to phospholipids or to membrane proteins.
  • the proper insertion and orientation of membrane proteins is not a limiting factor for assay development.
  • insertion and orientation is evaluated by investigating signal transduction events across the lipid bilayer, such as GPCR signaling for chemical or drug interactions. For proper insertion and orientation, optimizing the lipid composition, membrane asymmetry, and protein-lipid ratios enables the proteins to self-assemble into the correct configuration .
  • a flow chart for a general typing and subtyping assay procedure is depicted in FIG. 3, and includes the following steps. A sample of a virus to be assayed is supplied to a test area (30) .
  • the virus is captured by fusion with a liposome reagent, functionalized liposomes 10, for rapid hybridization of its RNA to capture oligos (i.e. strain specific oligos 12) contained within the functionalized liposomes 10 (32) .
  • the fused virus and functionalized liposome form an infected liposome, also referred to as a chimeric liposome 19 as illustrated in FIG. 1.
  • viral antigens expressed on the chimeric liposomes enable antibody capture and purification of "infected" liposomes.
  • Capture antibodies which can be fixed (immobilized) to a substrate, bind with the viral antigens and immobilize the chimeric liposomes.
  • the chimeric liposomes are then purified by washing away those materials not fixed to the substrate or test area (33) . Lysis (34) of chimeric liposomes releases the hybridized viral RNA 36 and nucleoproteins 37 as illustrated in FIG. 5. At this point, typing and/or subtyping can be performed.
  • Typing and subtyping can follow parallel or serial pathways .
  • typing is achieved by providing immobilized antibodies which are conjugates of the nucleoproteins present.
  • the antibody conjugates bind (38) with the proteins and can be assayed using colorimetric typing (39) also known as immunochromatographic analysis.
  • colorimetric typing 319 also known as immunochromatographic analysis.
  • immobilized antibody conjugates 40 can be employed to attach nucleoproteins 37 to the surface, which subsequently bind reporter antibodies carrying gold particles 42 (for light dispersion)
  • Subtyping is achieved by providing barcode reporter liposome conjugates, A & A' which are hybridized (43) to appropriate subtype targets (hybridized viral RNA) released from the lysed chimeric liposomes .
  • the complexes formed thereby are captured by immobilized universal capture oligos.
  • the barcode reporter liposome conjugates, A & A' hybridized with the hybridized viral RNA are immobilized.
  • the immobilized barcode reporter liposome conjugates, A & A' are then purified (44) by washing away those materials not fixed to the substrate or test area.
  • a simplified diagram illustrates this process in FIG. 7.
  • Barcode reporter liposomes (A and A' ) functionalized to be conjugates of the hybridized viral RNA 36 hybridize therewith.
  • Universal capture oligos 45 bind to and immobilized hybridized viral RNA 36.
  • the viral RNA complex can also be immobilized by an affinity interaction, such as antibody-antigen or avidin-biotin . In the latter example the subtype specific capture oligo would be functionalized with biotin.
  • Barcode reporters are then released by lysis (46) of the reporter liposomes .
  • the released barcode reporters are then hybridized (48) to subtype encoded microcavities .
  • Optical detection of spatially arrayed optical microcavity lasers reports subtype. Sandwich complex between A, A', and microcavity must be formed for lasing to occur which provides high specificity and sensitivity. This process is described in greater detail later.
  • the capture antibodies In the case of influenza typing, for example, the capture antibodies would be chosen to be hemagglutinin, neuraminidase, matrix protein, or combination thereof. Use of several capture antibodies can increase binding efficiency and overcome patient sample issues such as influenza antibodies from previous vaccinations. Current influenza vaccines generally immunize against HA. Preliminary typing of influenza A/B/avian can be accomplished by applying reagents used in current immunochromatographic strip tests. Turning now to FIG. 4, a lateral flow membrane test strip 50 that might be utilized in influenza identification, is illustrated. Test strip 50 is specifically designed for a step-by-step typing/subtyping assay, although a strip which will only subtype can be provided.
  • Strip 50 in this preferred embodiment, includes areas for performing the process steps illustrated as boxes 30, 32, 33 and 34 in FIG. 3. These areas include a sample input area 52, an area 54 containing a dried down liposome fusion reagent (functionalized liposomes containing strain specific oligos as described above) , an area 56 for rehydration of the functionalized liposomes and fusion with the sample virus to create chimeric liposomes, this area can also be employed for purification as described above, and an area 58 for lysis of the chimeric liposomes. Strip 50 also includes the following areas for typing illustrated as boxes 38 and 39 in FIG.
  • test strip 50 Also included on test strip 50 are areas for subtyping the inputted sample virus once it is determined from the typing steps that the sample virus includes one of influenza A, B, or avian if the typing steps are not omitted.
  • the subtyping steps are illustrated as boxes 43, 44, 46 and 48 in FIG. 3.
  • These subtyping areas include an area 64 for storing dried down barcode reporter liposome conjugates A and A' , an area 66 for rehydrating the barcode reporter liposome conjugates, an area 68 for hybridizing the barcode reporter liposome conjugates and the viral targets
  • hybridized viral RNA to be subtyped
  • an area 72 in which the released barcode reporters are hybridized to subtype encoded microcavities also see FIG. 8) .
  • a sample of a virus or suspected influenza virus is collected using commercially available swab or lavage kits. Liquified aliquot is applied to a lateral flow membrane test strip 50 (see FIG. 4, step 30) and rehydrates sialic acid functionalized liposomes containing subtype specific oligo probes. Influenza viruses will recognize the receptor, bind to the functionalized liposomes, and initiate fusion under appropriate conditions (step 32) . Fusion is rapid and virus genetic material is sequestered in an optimal environment. Strain specific hybridization with oligos within the functionalized liposomes occurs rapidly because of enhanced concentration and further stabilizes the viral RNA.
  • Oligos are designed with linker sequences that provide unique barcode sequences such that the downstream assay steps are analyte agnostic. Since the viral membrane envelope is fused to the engineered liposome (FIG. 1), viral antigens are expressed on "infected" chimeric liposomes and these can be captured using capture antibodies. The capture antibodies fix the chimeric liposomes in place, allowing removal of uninfected liposomes and other sample matrix contaminants (step 33) . The chimeric liposomes are subsequently ruptured (step 34) to release viral nucleic acids and proteins.
  • Virus typing can be accomplished by immunoassays for viral antigens, such as nucleoprotein (NP), and detected visually, similar to conventional rapid immunochromatographic tests on the market today (step 39) .
  • NP nucleoprotein
  • This enables discrimination or typing of influenza A/B within 10-15 minutes.
  • complexity of the sample matrix and excess reagents are significantly reduced for subsequent subtyping. Removal of sample contaminants and interfering substances can improve assay sensitivity and specificity.
  • the lysis solution rehydrates a reagent zone containing reporter liposomes containing fluorescently labeled barcode oligos.
  • oligos are released by lysis (step 46) of the reporter liposomes, they will hybridize (step 48) to the appropriate subtype microcavity bead in a spatially separated array (FIG. 9) on test strip 50 and enable lasing.
  • Laser emission is orders of magnitude more detectable than fluorescence due to the spectral and spatial narrowing of the lasing mode.
  • this test is run in an orthogonal membrane (test strip) to avoid previous contaminants (e.g. in a manner similar to the ChemBio dual path lateral flow assay) .
  • the subtyping assay can be performed without executing the preliminary typing assay. Specificity can be increased by requiring a sandwich to be formed, essentially providing a microcavity trigger, at the optical detect step.
  • a simple means to accomplish this is to have 2 different reporter liposomes, one with dye labeled oligos and another with a synthetic target that has complementary sequences for the microcavity barcode (A) and for the dye labeled reporter barcode (A' ) . If they are present in equal concentrations then at the target hybridization step, the target will capture either A or A' in equal proportions. Although this reduces by 50% the amount of dye reporters, it significantly increases the downstream specificity because binding of the reporter barcode A' to the microcavity requires the intermediate barcode A. In this process the microcavity (see FIG. 8) will not lase unless both hybridization events occurs, substantially increasing the specificity.
  • An alternative embodiment is to replace the reporter liposome with a microcavity bead functionalized with a universal capture barcode sequence.
  • the bead-RNA complex flows across a test strip grating which has been functionalized with subtype specific barcode sequences in an array for spatial discrimination and is captured by the appropriate barcode sequence.
  • the test strip provides efficient coupling of the excitation source and collects the laser emission from the bound microcavities, which are spatially located according to the detected subtype.
  • the microcavities are attached to a waveguide excitation and detection system using specific hybridization probes.
  • the low threshold power requirements are predominantly the result of the high cavity Q of the whispering gallery modes of the microcavity, which can approach 10 9 for spherical geometries with a large index refractive change to their environment.
  • tapered fiber couplers have been used to achieve highly efficient excitation of the microsphere cavity modes. To achieve these efficiencies, careful tuning of the excitation frequency and an extremely narrow tapered fiber region were required. To relax those restrictions, multimode waveguide excitation of the resonant modes of the microcavities is used.
  • a multimode linear waveguide has sufficient width and index change that a large number of propagating waveguide modes is allowed.
  • exciting all the modes allowed in the waveguide i.e. by uniformly illuminating the end of the guide
  • we can ensure that one of the oscillating modes of the microsphere laser is phase matched to a waveguide mode, effectively pumping that particular mode of the microsphere.
  • the fraction of power propagating in the waveguide that is coupled into the sphere is quite small, since we only couple one mode of many, the advantages in terms of the ability to build larger waveguides with wide alignment tolerances is significant.
  • Use of the multimode guide will allow the use of inexpensive plastic embossed structures on the test strip.
  • each waveguide can efficiently pump a mode of the microsphere. More importantly, the microsphere lasing mode can be effectively coupled back out of the microsphere into the waveguide, without introducing excess coupling loss and spoiling the cavity Q of the microsphere. Since the microsphere laser output is also coupled to one of the propagating modes of the waveguide, the same optical system that couples blue excitation light into the microsphere can be used to effectively collect the coupled microsphere emission, using a standard fluorescence cube arrangement.
  • spectral filter techniques can be applied to tightly select that emission.
  • binding of the microspheres to the waveguide array scatters source laser emission out of the waveguide. This scattered emission may be imaged using a camera oriented to observe the surface of the waveguide array.
  • waveguide loss within the waveguide array is measured to determine the number of microspheres bound to each waveguide in the waveguide array. Spectral discrimination may be used to increase the sensitivity of the scattered light measurement.
  • the assay may be performed in a format that utilizes magnetic beads.
  • the sample to be typed/subtyped is combined with a solution of receptor labeled liposomes (functionalized liposomes) and target virus binds to the receptors, initiating fusion and hybridization of viral nucleic acid with subtype specific oligo probes contained therein.
  • the functionalized liposomes with bound virus are then captured with paramagnetic particles functionalized with antibodies.
  • a magnetic field is introduced to immobilize the bead bound chimeric liposomes, and unbound liposomes and other matrix contaminants are washed away.
  • the remaining bound chimeric liposomes are then lysed to release functional viral nucleic acids and proteins into buffer solution.
  • the released nucleic acids are subsequently mixed with and bound to a second solution of paramagnetic particles functionalized with oligo capture sequences complementary to the viral sequences, with universal capture sequences being preferred.
  • a portion of the lysed solution can be used for viral typing performed by standard immunoassay techniques for viral antigens, such as ELISA, etc.
  • a solution of reporter liposomes is added to the paramagnetic particles with captured viral nucleic acids and subtype specific oligo probes.
  • the reporter liposomes (2 versions for each subtype) are functionalized with oligo sequences that are complementary to the unhybridized linker of the subtype specific oligos .
  • the linker sequence is unique for each subtype.
  • the reporter liposomes carry a payload of either fluorescently labeled barcode oligos or a barcode target.
  • the barcode target has two hybridization domains which are sequences complementary to both the fluorescently labeled barcode oligo and to a barcode sequence attached to microcavity microsphere.
  • a magnetic field is subsequently applied to collect the bead bound reporter liposomes, and unhybridized liposomes are washed away.
  • the reporter liposomes are lysed and the solution is applied to a microcavity array as previously described which is spatially encoded for each subtype.
  • Hybridization forms a sandwich between a microcavity and the fluorescently labeled barcode oligo via the barcode target. Illumination of the microcavity array causes lasing of fluor hybridized microcavities, and position in the array corrresponds to subtype.
  • Use of barcode sequences allows reagents (reporter liposomes + payload, microcavities, and microcavity array) to be conveniently formatted for different viral targets.
  • the assay could be performed in a microplate format, with the binding steps to the paramagnetic beads being replaced by binding to functionalized wells in the plate .
  • liposomes with receptors and subtype specific oligo probes functionalized liposomes
  • liposomes with barcode reporters reporter liposomes
  • microcavities are used that are generated in droplet generators.
  • the nanodroplet technology represents the only technology capable of: (1) producing liposomes that are monodispersed from 0.3 ⁇ m to 30 ⁇ m; (2) developing membrane compositions that have necessary stability and fusogenic potential; (3) producing asymmetrically functionalized membranes with specific viral receptors; and (4) producing organic or inorganic polymeric hollow shells that act as efficient microcavity lasers .
  • test strip is unique in that viral typing and/or subtyping can be performed in a matter of minutes on- site without the necessity of taking a sample to a laboratory and waiting days or weeks to grow a culture. Also, the test strips can be produced relatively inexpensively and can be used by virtually any practitioners at the site.
  • the steps of dehydrating and the ability to rehydrate the various liposomes is a substantial step in the production of the test strips, as is the printing of spatially distinct optical microcavities using screen printing, hybridization, dispensing, or ink-jet techniques.

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Abstract

Cette invention concerne une méthode de fabrication d’une bandelette de test d’immunochromatographie sur membrane comprenant les étapes consistant à : produire une bandelette à plusieurs zones espacées les unes des autres, à former des liposomes fonctionnalisés contenant des oligo-sondes spécifiques d’un sous-type, à sécher les liposomes fonctionnalisés, puis à stocker les liposomes fonctionnalisés séchés au niveau d’une première zone de la bandelette; à former des vecteurs de liposome avec des oligos codes barres marqués par fluorescence, à sécher les vecteurs de liposomes avec oligos codes barres marqués par fluorescence, puis à stocker ces vecteurs de liposomes séchés avec oligos codes barres marqués par fluorescence au niveau d’une seconde zone de la bandelette; à former des conjugués d’anticorps, à sécher les conjugués d’anticorps, puis à stocker les conjugués d’anticorps au niveau d’une troisième zone de la bandelette; et à stocker les microcavités codées par le sous-type au niveau d’une quatrième zone de la bandelette.
PCT/US2008/083184 2007-11-16 2008-11-12 Appareil et méthode de détection virale WO2009108224A1 (fr)

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US10808287B2 (en) 2015-10-23 2020-10-20 Rapid Pathogen Screening, Inc. Methods and devices for accurate diagnosis of infections
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US10379121B2 (en) 2008-05-20 2019-08-13 Rapid Pathogen Screening, Inc. Method and device for combined detection of viral and bacterial infections
US9933423B2 (en) 2008-05-20 2018-04-03 Rapid Pathogen Screening, Inc. Method and device for combined detection of viral and bacterial infections
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KR20150125002A (ko) * 2013-03-07 2015-11-06 레피드 페써겐 스크리닝, 아이엔씨. 바이러스 및 박테리아 감염의 복합 검출을 위한 방법 및 장치
KR102489679B1 (ko) 2013-03-07 2023-01-18 레피드 페써겐 스크리닝, 아이엔씨. 바이러스 및 박테리아 감염의 복합 검출을 위한 방법 및 장치
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KR102322094B1 (ko) 2013-03-07 2021-11-08 레피드 페써겐 스크리닝, 아이엔씨. 바이러스 및 박테리아 감염의 복합 검출을 위한 방법 및 장치
KR102209489B1 (ko) 2013-03-07 2021-02-02 레피드 페써겐 스크리닝, 아이엔씨. 바이러스 및 박테리아 감염의 복합 검출을 위한 방법 및 장치
KR20210013646A (ko) * 2013-03-07 2021-02-04 레피드 페써겐 스크리닝, 아이엔씨. 바이러스 및 박테리아 감염의 복합 검출을 위한 방법 및 장치
KR20210134994A (ko) * 2013-03-07 2021-11-11 레피드 페써겐 스크리닝, 아이엔씨. 바이러스 및 박테리아 감염의 복합 검출을 위한 방법 및 장치
US20160298187A1 (en) * 2015-04-08 2016-10-13 Verily Life Sciences Llc Methods of tagging particles for multiplexed functional screening
US10808287B2 (en) 2015-10-23 2020-10-20 Rapid Pathogen Screening, Inc. Methods and devices for accurate diagnosis of infections
WO2021212104A1 (fr) * 2020-04-18 2021-10-21 Apollos Diagnostics, Llc Suivi et test d'une infection virale ou d'une autre infection

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