WO2009108224A1

WO2009108224A1 - Viral detection apparatus and method

Info

Publication number: WO2009108224A1
Application number: PCT/US2008/083184
Authority: WO
Inventors: Eugene Tu; Donald E. Ackley
Original assignee: Eugene Tu; Ackley Donald E
Priority date: 2007-11-16
Filing date: 2008-11-12
Publication date: 2009-09-03

Abstract

A method of fabricating a lateral flow test strip includes 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 liposome carriers with fluorescently labeled barcode oligos, drying down the liposome carriers with fluorescently labeled barcode oligos, and storing the dried down liposome carriers 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.

Description

VIRAL DETECTION APPARATUS AND METHOD

FIELD OF THE INVENTION

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 .

BACKGROUND OF THE INVENTION

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.

FDA approval of two drugs for influenza treatment stimulated development of rapid diagnostic methods because antivirals must be administered within 48 hours of symptoms. There are currently at least six different kits that identify influenza virus in clinical specimens in 30 minutes or less, and their use has become widespread in laboratories and in point-of-care (POC) testing venues. These tests are typically optical immunoassays and detect influenza A and B virus associated antigens in throat swabs, nasal swabs, or nasal lavages. They have an average sensitivity and specificity of 70% and 90%, respectively, but performance varies greatly with respect to viral types, sampling location, and patient age. None of these tests can subtype the virus so they are inadequate for community surveillance. The "gold" standard for typing and subtyping is viral culture but the method is tedious, requires trained personnel, and takes, on average, 2-14 days to isolate the virus. Molecular diagnostics, such as Reverse Transcriptase PCR (RT-PCR) are rapidly gaining favor because they are much faster to result, i.e. 6-24 hours. PCR (RT or Taqman) is also more accurate than culture methods because the technique is more sensitive and does not require replication competent viruses, making the method less sensitive to specimen quality. The 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. The authors show that RT-PCR using Taqman real time chemistry was more sensitive than culture and an immunofluorescence assay. However, the multiplex assay for influenza B was not as sensitive as the singleplex reactions. Templeton et al . reported multiplex real time PCR for 7 respiratory agents (influenza A and B and RSV were multiplexed in one reaction and Parainfluenza viruses 1, 2, 2, and 4 in another) . The sensitivity for PIV4 was 1 log less sensitive in the multiplex format. In spite of recent improvements, PCR assays are still technically demanding, expensive, difficult to multiplex, and not amenable to POC testing.

It is clear that current technology is either too complicated or lacks sensitivity to meet the challenges of rapid and direct viral analysis. In addition to identifying influenza, subtyping of the virus at POC would provide more information to public health officials and CDC in monitoring and controlling the spread of the virus. This information could be used to determine the composition of viral strains in vaccine development for the subsequent flu season.

It would be highly advantageous, therefore, to remedy the foregoing and other deficiencies inherent in the prior art. Accordingly, it is an object of the present invention to provide new and improved viral detection apparatus and methods of use.

It is another object of the present invention to provide new and improved viral detection apparatus including specifically engineered liposomes coupled to optical microcavity sensors .

It is another object of the present invention to provide a new and improved lateral flow triage strip test concept for influenza typing and subtyping.

SUMMARY OF THE INVENTION

Briefly, to achieve the desired objects of the present invention in accordance with a preferred embodiment thereof, apparatus is provided 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 . Also, to achieve the desired objects of the present invention, provided is a method of identifying viral genetic material. The method 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. 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. The objects and other aspects of the invention are further achieved by 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.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing and further and more specific objects and advantages of the instant invention will become readily apparent to those skilled in the art from the following detailed description of a preferred embodiment thereof taken in conjunction with the drawings; in which:

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' ; and

FIG. 9a) and b) are top plan and side views, respectively, of arrayed subtype encoded microcavities on the triage strip of FIG. 4.

DESCRIPTION OF THE PREFERRED EMBODIMENT

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. Using the ^λnanodroplet technology' (described in more detail below) or other synthesis means, 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. When 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. When liposome 10 has been "infected" by virus 16, the resulting combination will be referred to as a chimeric liposome generally designated 19.

Turning now to FIG. 2, a cross flow droplet generator generally designated 20 is illustrated schematically. Droplet generator 20 is a preferred apparatus for use in the ^λnanodroplet 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 ^λnanodroplet technology' , nano- and micro-droplets (e.g. 100 nm to 100 μm 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. The combination of flow focusing from cross channels 22 and viscous shear forces results in the formation of droplets 26 from the continuous flows of the two phases. 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. By varying the channel geometry (i.e. cross channels 22 and central channel 24) and relative flow rates of the fluid streams, 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.

Of particular interest are 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. In addition, unlike other immiscible oil and water solutions, it has a high vapor pressure, and may be readily separated from water by evaporation. Based on the solubility of ethyl acetate and water, this system is sufficiently immiscible to form droplets in the droplet generators while being sufficiently miscible to be exchanged with water.

In an example of the operation of generator 20, 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) carries strain specific oligos 12 and is injected through main channel 24. 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. A complete description of a preferred liposome formation can be found in copending United States Patent Application entitled "Droplet Generating Device and Method", filed 29 November 2006, bearing serial number 11/605,683, and incorporated herein by reference. After liposome formation, the 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. As an example, 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.

In some applications it may be desirable to form more stable bi-layer liposomes. 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. As an example, monodispersed 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 μm to 100 μm; (2) developing membrane compositions that have necessary stability and fusogenic potential; (3) producing asymmetrically functionalized membranes with specific viral receptors; and

(4) producing polymeric hollow shells that act as efficient microcavity lasers. Of particular interest is the use of the above droplet mechanism with multiple solvent inlet ports feeding the solvent cross channel, so as to enable the use of different membrane constituents that may be combined in a combinatorial fashion to optimize the aforementioned membrane compositions. As an example, one possible target of this viral based assay is the influenza virus. It is known that influenza viruses comprise a diverse mix of antigenic subtypes. Each subtype includes a specific hemagglutinin (HA) and a specific neuraminidase (NA) subtype, e.g., H5N1 or H3N2. 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) . Thus, even if the influenza virus were modified maliciously, the receptor would have to remain the same for host cell infectivity and would still be detectable with receptor decorated liposomes (e.g. liposome 10) because genetic sequences important for the viral life cycle are conserved.

For influenza, SA-2, 6 Gal decorated liposomes will capture virus particles, if present, and initiate membrane fusion, which introduces RNA genetic material into the liposomes. This protocol is especially useful for detecting emerging and unknown pathogens because genes coding for receptor binding proteins and polymerases are highly conserved. Thus a broad screen for infectious agents can be developed using all known surface receptors and strain specific oligos for capturing genetic material of viruses and the like.

In nature, the 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. It has been reported that sialic acid gangliosides, such as GDIa or GDIb, are sufficient for virus attachment and membrane fusion. In the disclosed process, 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. Thus, the proper insertion and orientation of membrane proteins is not a limiting factor for assay development. There are several methods to functionalize liposome surfaces with sialic acid: 1) SA residues can be conjugated to preformed liposomes with functional groups in batch mode; 2) SA-glycolipid conjugates can be exchanged into preformed liposomes in batch mode; or 3) SA-glycolipids can be incorporated into liposomes in the droplet generator in one pot real time mode. The latter method is preferred because it requires less purification steps. For these small carbohydrate receptors, random insertion into the inner or outer leaflet (first or second lipid layers of the liposome) will not materially affect virus binding and fusion.

For surface proteins that do not span the membrane bilayer, preferential incorporation of the receptor into the outer membrane leaf can be accomplished by methods 1 & 2 described above. For integral or transmembrane proteins, 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. Concurrently, 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. With reference to FIG. 6, to enhance the assay, 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. In this manner, 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.

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. 3, although it will be understood that these areas can be omitted if typing is not desired, an area 60 for storing dried down HA antibody conjugate and for rehydrating the conjugate and binding with the nucleoproteins, and an area 62 for visually typing the inputted sample virus.

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 70 in which the reporter liposome conjugates are lysed to release the barcode reporters, and an area 72 in which the released barcode reporters are hybridized to subtype encoded microcavities (also see FIG. 8) .

In a more specific example, 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) . This enables discrimination or typing of influenza A/B within 10-15 minutes. In addition, by utilizing the fusion step, 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. For subtyping, the lysis solution rehydrates a reagent zone containing reporter liposomes containing fluorescently labeled barcode oligos. Once these 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. Preferably 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.

In order to achieve low lasing thresholds and enhance assay sensitivity, 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 10⁹ for spherical geometries with a large index refractive change to their environment. To efficiently couple the input excitation into the spheres, 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. By 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. While 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. The choice of a triangular waveguide array enables use of a very low cost plastic embossed waveguide such as can be seen in children' s toys . It also enables effective binding of the microsphere lasers to the waveguide at exactly two points. In that configuration, 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. It is important to realize that this optical arrangement is extremely efficient in suppressing optical background, for a number of reasons. First, microspheres that are not bound in close contact to the waveguide are not efficiently coupled to the waveguide modes, either for excitation or collection. Second, only waveguide modes propagating at high angles within the microspheres are collected in the waveguides. This ensures that spontaneous emission from the dyes or quantum dots are not efficiently connected. The same is true for any background fluorescence excited in the waveguide, as only a small fraction of the background emitted at the appropriate angle will be coupled to the waveguide. Third, since the coupled microcavity emission will exit the waveguide at discrete angles, the appropriate output angle can be selected to form a spatial filter. Also, since the microsphere laser emission has quite a narrow spectral width, spectral filter techniques can be applied to tightly select that emission. In an alternative embodiment to inducing lasing of the microcavities, 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. Alternatively, 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.

In an alternative embodiment to the use of a test strip, 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 (chimeric liposomes) 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.

In an alternative embodiment to the paramagnetic bead based assay, 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 .

Thus, a new and improved viral detection apparatus and method has been disclosed. At several steps in the process, liposomes with receptors and subtype specific oligo probes (functionalized liposomes), liposomes with barcode reporters (reporter liposomes), and 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 μm to 30 μm; (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 .

The described 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.

Various changes and modifications to the embodiments herein chosen for purposes of illustration will readily occur to those skilled in the art . To the extent that such modifications and variations do not depart from the spirit of the invention, they are intended to be included within the scope thereof, which is assessed only by a fair interpretation of the following claims.

Having fully described the invention in such clear and concise terms as to enable those skilled in the art to understand and practice the same, the invention claimed is:

Claims

1. Apparatus for identifying viral pathogens comprising : a receiving area for receiving a viral sample to be identified; a fusion area including functionalized liposomes containing subtype specific oligo probes, the fusion area receiving the viral sample from the receiving area for permitting binding of the virus in the viral sample to the functionalized liposomes and initiating fusion and subsequent hybridization the viral sample with the subtype specific oligo probes to form a chimeric liposome; an area containing immobilized particles with functionalized antibodies for receiving and binding the chimeric liposomes; an area containing functionalized reporter liposomes containing fluorescently labeled barcode oligos; and an area containing subtype encoded optical microcavities .

2. Apparatus for identifying viral pathogens as claimed in claim 1 further including: an area containing antibody conjugate to bind antigens for typing; and an area containing immobilized secondary capture antibody to bind antibody-antigen complexes for typing.

3. Apparatus for identifying viral pathogens as claimed in claim 1 wherein the fusion area including functionalized liposomes containing subtype specific oligo probes includes dried down functionalized liposomes containing subtype specific oligo probes.

4. Apparatus for identifying viral pathogens as claimed in claim 2 wherein the area containing antibody conjugate contains dried down antibody conjugates.

5. Apparatus for identifying viral pathogens as claimed in claim 1 wherein the areas containing immobilized particles with functionalized antibodies contain immobilized and dried down particles with functionalized antibodies.

6. Apparatus for identifying viral pathogens as claimed in claim 1 wherein the area containing reporter liposomes with fluorescently labeled barcode oligos includes dried down reporter liposomes with fluorescently labeled barcode oligos .

7. Apparatus for identifying viral pathogens as claimed in claim 1 wherein the receiving area, the fusion area, the area containing immobilized particles with functionalized antibodies, the area containing reporter liposomes with fluorescently labeled barcode oligos, and the area containing subtype encoded microcavities are all included on a continuous test strip.

8. Apparatus for identifying viral pathogens as claimed in claim 7 wherein the functionalized liposomes containing subtype specific oligo probes in the fusion area are dried down and stored on the test strip, and the reporter liposomes with fluorescently labeled barcode oligos are dried down and stored on the test strip.

9. Apparatus for identifying viral pathogens as claimed in claim 2 wherein the receiving area, the fusion area, the area containing immobilized particles with functionalized antibodies, the area containing the antibody conjugate, the area containing secondary capture antibody, the area containing reporter liposomes with fluorescently labeled barcode oligos, and the area containing subtype encoded microcavities are all included on a continuous test strip .

10. Apparatus for identifying viral pathogens as claimed in claim 1 wherein the area containing subtype encoded microcavities includes an array of optical waveguides positioned to excite and collect optical emission from the microcavities.

11. Apparatus for identifying viral pathogens as claimed in claim 10 wherein the array of optical waveguides comprises multimode optical waveguides.

12. Apparatus for identifying viral pathogens as claimed in claim 10 wherein microcavities with different subtyping oligos are located in different positions in the area containing subtype encoded microcavities.

13. Apparatus for identifying viral pathogens as claimed in claim 12 wherein the microcavities are positioned using one of hybridization, ligand-receptor binding, contact and noncontact dispensing, screen printing, and ink-jet printing .

14. A method of identifying viral pathogens comprising the steps of: providing functionalized liposomes containing subtype specific oligo probes, immobilized particles with functionalized antibodies, liposome carriers with fluorescently labeled barcode oligos, and subtype encoded microcavities; receiving a viral sample to be typed and subtyped; binding the viral sample to the functionalized liposomes initiating fusion and hybridization of the viral sample genetic material with the subtype specific oligo probes, creating chimeric liposomes; capturing chimeric liposomes by binding viral antigens using the immobilized particles with functionalized antibodies and discriminating the chimeric liposomes; lysing discriminated chimeric liposomes to release genetic material and proteins of the infected liposomes; hybridizing the released genetic material and the reporter liposomes containing fluorescently labeled barcode oligos; lysing the hybridized reporter liposomes to release the fluorescently labeled barcode oligos; hybridizing the fluorescently labeled barcode oligos with the subtype encoded microcavities; and exciting optical emission from the subtype encoded microcavities .

15. A method as claimed in claim 14 wherein the step of providing functionalized liposomes includes functionalizing the liposomes with one of sialic acid, glycolipids, glycoproteins, peripheral and integral membrane proteins, and transmembrane proteins.

16. A method as claimed in claim 14 further including the steps of: providing antibody conjugates and secondary capture antibodies; binding viral antigens with the antibody conjugates; and capturing viral antigens of the viral sample using secondary capture antibodies and discriminating the viral type.

17. A method as claimed in claim 14 wherein the step of providing the immobilized particles with functionalized antigens includes providing immobilized capture antibodies.

18. A method of identifying viral pathogens comprising the steps of: providing a lateral flow test strip having an area including dried down functionalized liposomes containing subtype specific oligo probes, an area containing immobilized capture antibody, an area including dried down antibody conjugate, an area containing immobilized secondary capture antibody, an area including dried down reporter liposomes containing fluorescently labeled barcode oligos, and an area including subtype encoded optical microcavities ; introducing a viral sample to be identified to the test strip; rehydrating the functionalized liposomes containing subtype specific oligo probes; binding the viral sample to the rehydrated functionalized liposomes initiating fusion and hybridization of the viral sample with the subtype specific oligo probes to form chimeric liposomes; capturing viral antigens of the viral sample using the immobilized capture antibody and discriminating chimeric liposomes by binding to a target viral type in the viral sample; lysing discriminated chimeric liposomes to release genetic material and proteins of the chimeric liposomes; rehydrating the dried down antibody conjugate and binding the released proteins with the antibody conjugate capturing antibody conjugate-protein complexes using immobilized secondary capture antibody and discriminating viral type; rehydrating the dried down reporter liposomes containing fluorescently labeled barcode oligos; hybridizing the released genetic material and the rehydrated reporter liposomes; discriminating the hybridized reporter liposomes; lysing the hybridized reporter liposomes hybridizing the fluorescently labeled barcode oligos with the subtype encoded microcavities; and optically exciting the subtype encoded microcavities so that the microcavities lase.

19. A method as claimed in claim 18 wherein the step of discriminating chimeric liposomes includes using immunochromatographic tests to visually determine whether chimeric liposomes are present in the viral sample.

20. A method of fabricating a lateral flow test strip comprising the steps of: providing a strip having a plurality of discrete reaction 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 containing fluorescently labeled barcode oligos, drying down the reporter liposomes containing 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.

21. A method as claimed in claim 20 wherein the step of forming functionalized liposomes containing subtype specific oligo probes includes using nanodroplet technology.

22. A method as claimed in claim 20 wherein the step of forming reporter liposomes containing fluorescently labeled barcode oligos includes using nanodroplet technology.