WO2011159537A2 - Method and device for analyte detection - Google Patents

Abstract

The present invention provides a method and a point-of-care device for detecting analytes by combining a complex fluid system with the lateral-flow immunoassay.

Description

METHOD AND DEVICE FOR ANALYTE DETECTION

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Patent Application No.

61/354,940, filed June 15, 2010, the teaching of which is incorporated herein by reference in its entirety.

FIELD OF THE INVENTION

The invention provides enhanced detection of analytes using complex fluids and methods for the rapid detection of proteins, viruses, allergens, bioterrorism agents, and the like.

BACKGROUND OF THE INVENTION

Detecting biomolecules is a crucial part of today's healthcare system. This includes, for example, viruses and proteins. The outbreak of swine-origin influenza A ("H1N1") virus infection in the past highlighted the need for a means to rapidly and accurately diagnose and detect infectious agents and pandemic pathogens at the point- of-care. Rapid and accurate diagnosis of such agents and pathogens at the point-of- care results in better patient management, such as timely use of appropriate antiviral treatments, isolation of confirmed cases, and prevention of outbreaks. One detection method that has gained much attention in recent years due to its ease of use, rapid time to result, and minimal power and laboratory equipment requirements is the lateral-flow immunoassay (LFA). LFA utilizes a test strip that collects a sample through lateral flow, and detects the presence of a target molecule through its specific antibody labeled with a colorimetric indicator. While LFA has been used to detect a wide range of biomolecules (see, e.g., Laderman, E.I., et al., Clinical and Vaccine

Immunology, 2008, 15(1): p. 159), its sensitivity in detecting viruses has been shown to be inferior to the gold standards, namely viral culture and real-time polymerase chain reaction (PCR) (see, e.g., Ginocchio, C.C., et al, Journal of Clinical Virology, 2009, 45(3): p. 191-195). Although LFA is superior to viral culture and real-time PCR in terms of ease of use and rapid time to result, its low sensitivity in detecting pathogens and viruses renders it ineffective as a point-of-care assay to prevent pandemic outbreaks. Therefore, the need to improve the sensitivity of LFA for detection of infectious agents exists. Furthermore, proteins are markers for a wide variety of applications, such as detecting food allergens and protein toxins. In the case of food allergens, ensuring the safety of manufactured goods is a vital component of public health. Therefore, food manufacturers have a duty to provide their customers with safe products, which includes protecting consumers from food allergens. In the US, under the Food

Allergen Labeling and Consumer Protection Act (FALCPA), food manufacturers are required to list the presence of any of the 8 pre-identified food allergens only if they are used by food manufacturers as ingredients (Taylor, S.L. et al, Current opinion in allergy and clinical immunology, 2006, 6(3): p. 186). However, this labeling does not account for food allergens present in products due to contamination caused by using shared transportation containers and production lines (Schappi, G.F., et al., Allergy, 2001, 56(12): p. 1216-1220). Therefore, a sensitive, yet rapid and inexpensive, assay, which can result in more frequent screening of food products by the manufacturers, could be beneficial to both food manufacturers and consumers. Although the recent development of rapid immunoassays based on the LFA technology precludes the need for sophisticated laboratory skills and caters to the manufacturers' preference for simplicity and speed, the traditional enzyme-linked immunosorbent assay (ELISA), which is currently the most widely used detection scheme to test for the presence of food allergens, is an order-of-magnitude more sensitive than its LFA counterpart (Schubert-Ullrich, P., et al., Analytical and Bioanalytical Chemistry, 2009, 395(1): p. 69-81). Therefore, the detection limit of LFA in detecting food allergens, which are typically proteins, needs to be improved before it could be used reliably.

In the case of protein toxins, bioterrorism agents (BAs), such as ricin toxin, present a great danger to the general public, since they are usually invisible to the naked eye, odorless, tasteless, and may not cause an immediate reaction (Broussard, L.A., Molecular Diagnosis, 2001, 6(4): p. 323-333; Ellison, D.H., Handbook of chemical and biological warfare agents. 2007: CRC). In order to minimize their spread and harmful impact to the civilian population, it is essential to provide the authorities with the means to rapidly detect BAs in-field and at the point-of-need. One approach that meets these requirements is the LFA, which has previously been used for the detection of BAs (see, e.g., Shyu, R.H., et al, Toxicon, 2002, 40(3): p. 255-258; King, 2003; and Chiao, D.J., J Chromatogr B Analyt Technol Biomed Life Sci, 2004, 809(1): p. 37-41). However, the detection limit of LFA is still inferior to lab-based assays, such as the ELISA, and needs to be improved (Peruski, A.H. et al., Clinical and Vaccine Immunology, 2003, 10(4): p. 506).

One approach to achieving a higher sensitivity for LFA is to improve the assay itself. Another approach is to concentrate the target biomolecule prior to the detection step. If the target biomolecules are viruses, the currently-accepted method for concentrating them is by using polyethylene glycol (PEG)-salt precipitation, followed by centrifugation of the viral particles. Millipore's centrifugal units, such as Amicon centrifugal filter units, are also used for concentrating viruses. The currently-accepted method for concentrating proteins, without denaturing them, is to employ centrifugal filter units from Millipore Corporation. These include Amicon, Microcon, and

Centriprep centrifugal filter units. However, centrifugation is needed in the above- mentioned methods, and as a result, they cannot be utilized at the point-of-care or point-of-need.

Therefore, this invention focuses on utilizing aqueous two-phase systems, such as aqueous two-phase micellar systems and aqueous two-phase PEG-salt systems, to concentrate the target biomolecules prior to their detection via LFA.

Aqueous two-phase systems typically exhibit a homogeneous, isotropic phase at low temperatures. Upon increasing the temperature, the solution undergoes a macroscopic phase separation to yield two phases, one more hydrophobic than the other. Biomolecules, such as viruses and proteins, partition unevenly between the two phases based on their physico-chemical characteristics, such as hydrophobicity and size. Once phase separation is established, the phase that contains the concentrated target biomolecules is sampled and applied to an LFA strip for the subsequent detection step. Therefore, this invention provides an enhanced detection of analytes via LFA by utilizing complex fluid systems for the rapid detection of proteins, viruses, allergens, bioterrorism agents, and the like.

As a reference, some methods and devices of analyte detection and separation are described in, e.g., United States Patents Nos. 5,798,273 (Direct read lateral flow assay for small analytes); 5,591,645 (Solid phase chromatographic immunoassay); 6,699,722 (Positive detection lateral-flow apparatus and method for small and large analytes); 5,772,888 (Separation and/or concentration of an analyte from a mixture using a two-phase aqueous micellar system); 6,437,101 (Methods for protein purification using aqueous two-phase extraction); and 4,579,661 (Process in the purification of biologically active substances); and European Patent Application EP- 0810436-A1 (Solid-phase analytical device).

SUMMARY OF THE INVENTION

In one aspect of the present invention, it provides a method for detecting an analyte in a sample. The method comprises:

(a) concentrating the analyte using a phase separation system to generate a sample of concentrated analyte,

(b) subjecting the sample of concentrated analyte to a lateral-flow

immunoassay, and

(c) detecting the analyte.

In some embodiments, the analyte is a target biomolecule. Such target biomolecule can be, e.g., a protein, a virus, a DNA, an RNA, a ribosome, a bacterium, a mammalian cell, a metabolite, or a combination thereof.

In some embodiments, the analyte can be an allergen, a bioterrorism agent, influenza, an infectious agent (e.g., sexually transmitted infectious agent), a biomarker for disease, or combinations thereof.

The phase separation system can be any phase separation system where a component, e.g., an analyte, can be concentrated or otherwise separated into a concentrated form. In some embodiments, the phase separation system is an aqueous two-phase system. In some embodiments, the aqueous two-phase system is an aqueous two-phase micellar system, an aqueous two-phase polyethylene glycol-salt system, an aqueous two-phase polymer 1 -polymer 2 system, or an aqueous two-phase polymer-surfactant system.

The lateral-flow immunoassay can take any form. In some embodiments, the lateral-flow immunoassay comprises an LFA strip, colloidal gold conjugates, and antibodies. In some embodiments, the LFA strip comprises a sample pad, a conjugate pad, a nitrocellulose membrane, an absorbent pad, and an adhesive vinyl backing. In some embodiments, the lateral-flow immunoassay is a sandwich assay or a competition assay.

The colloidal gold conjugates comprise gold alone or a gold alloy. In some embodiments, the colloidal gold conjugates comprise gold. In some other

embodiments, the colloidal gold conjugates comprise a gold alloy. The colloidal gold conjugates can comprise gold nanoparticles, gold microparticles, or a mixture thereof. In another aspect of the present invention, it provides a point-of-care device for detecting an analyte in a source. The point-of-care device generally comprises (a) a concentrating component to concentrate the analyte using a phase separation system to generate a sample of concentrated analyte, and (b) a lateral-flow immunoassay component to subject the sample of concentrated analyte to a lateral-flow

immunoassay to detect the analyte.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is a schematic representation of the method for concentrating the target proteins in an aqueous two-phase system using colloidal gold particles that are decorated with target-specific antibodies.

Figure 2 is a schematic representation of an LFA strip.

Figure 3 is a schematic representation of (A) positive and (B) negative results for the LFA using the sandwich mechanism.

Figure 4 is a schematic representation of (A) negative and (B) positive results for the LFA using the competition mechanism.

Figure 5 is a schematic representation of the Triton X- l 14 aqueous micellar system phase separating upon increasing the temperature.

Figure 6 is a schematic presentation of the lateral-flow immunoassay for detecting bacteriophage Ml 3 using the sandwich mechanism. Figure 6A shows a positive result, whereas Figure 6B shows a negative result.

Figure 7 shows experimentally-measured partition coefficients for

bacteriophage Ml 3 at various temperatures. Error bars represent standard deviations from at least six measurements.

Figure 8 shows a comparison of experimentally measured (■) and theoretically predicted (— ) bacteriophage Ml 3 concentration factors for various volume ratios. Error bars represent standard deviations from triplicate measurements.

Figure 9 shows an LFA used to detect bacteriophage Ml 3 without a prior concentration step.

Figure 10 shows an LFA used to detect bacteriophage Ml 3 with the prior concentration step.

Figure 1 1 is a schematic representation of (a) positive and (b) negative results for detecting transferrin via the LFA using the competition mechanism.

Figure 12 shows LFA used to detect transferrin without a prior concentration step. Figure 13 shows LFA used to detect transferrin with the prior concentration step.

DETAILED DESCRIPTION OF THE INVENTION

In one aspect of the present invention, it provides a method for detecting an analyte in a sample. The method comprises:

(a) concentrating the analyte using a phase separation system to generate a sample of concentrated analyte,

(b) subjecting the sample of concentrated analyte to a lateral-flow

immunoassay, and

(c) detecting the analyte.

In some embodiments, the analyte is a target biomolecule. Such a target biomolecule can be, e.g., a protein, a virus, a DNA, an RNA, a ribosome, a bacterium, a mammalian cell, a metabolite, or a combination thereof.

In some embodiments, the analyte can be an allergen, a bioterrorism agent, influenza, an infectious agent (e.g., sexually transmitted infectious agent), a biomarker for disease, or combinations thereof.

The phase separation system can be any phase separation system where a component, e.g., an analyte, can be concentrated or otherwise separated into a concentrated form. In some embodiments, the phase separation system is an aqueous two-phase system. In some embodiments, the aqueous two-phase system is an aqueous two-phase micellar system, an aqueous two-phase polyethylene glycol-salt system, an aqueous two-phase polymer 1 -polymer 2 system, or an aqueous two-phase polymer-surfactant system.

The lateral-flow immunoassay can take any form. In some embodiments, the lateral-flow immunoassay comprises an LFA strip, colloidal gold conjugates, and antibodies. In some embodiments, the LFA strip comprises a sample pad, a conjugate pad, a nitrocellulose membrane, an absorbent pad, and an adhesive vinyl backing. In some embodiments, the lateral-flow immunoassay is a sandwich assay or a competition assay.

The colloidal gold conjugates comprise gold alone or a gold alloy. In some embodiments, the colloidal gold conjugates comprise gold. In some other

embodiments, the colloidal gold conjugates comprise a gold alloy. The colloidal gold conjugates can comprise gold nanoparticles, gold microparticles, or a mixture thereof. In another aspect of the present invention, it provides a point-of-care device for detecting an analyte in a source. The point-of-care device generally comprises (a) a concentrating component to concentrate the analyte using a phase separation system to generate a sample of concentrated analyte, and (b) a lateral-flow immunoassay component to subject the sample of concentrated analyte to a lateral-flow

immunoassay to detect the analyte.

As used herein, the term polymer can be any polymeric material. The polymer can be a synthetic polymer or natural polymer. The term "polymer 1 -polymer 2 system" refers to a phase separation system where the properties of polymer 1 and polymer 2 are sufficiently different so as to cause polymer 1 and polymer 2 to phase separate into polymer 1-rich and polymer 2-rich phases, respectively, and where the solubility of an analyte is different in the two respective phases.

As used herein, the term point-of-care device refers to any device suitable for use at the point-of-care. Such a device can be portable or non-portable. In some embodiments, the device is a portable device. In some further embodiments, the point-of-care device is a test kit.

As used herein, the term "gold alloy" shall mean an alloy comprising gold with another element, e.g., silver, copper, zinc, iron, platinum, etc. In some embodiments, a metallic or non-metallic element can be used in place of gold.

In some embodiments, the invention advantageously combines aqueous two- phase systems with the LFA technology. Specifically, the target of interest, such as a food allergen, is concentrated by utilizing aqueous two-phase systems, such as aqueous two-phase micellar systems and aqueous two-phase polyethylene glycol (PEG)-salt systems. Aqueous two-phase systems typically exhibit a homogeneous, isotropic phase at low temperatures. Upon increasing the temperature, the solution undergoes a macroscopic phase separation to yield two phases, one more hydrophobic than the other. In the case of aqueous two-phase micellar systems, the two phases are a micelle-poor phase and a micelle-rich phase. In the case of aqueous two-phase PEG-salt systems, the two phases are a polymer-rich phase and a polymer-poor phase.

Biomolecules, such as viruses and proteins, partition unevenly between the two phases based on their physico-chemical characteristics, such as hydrophobicity, and size. For example, viruses, which are large, hydrophilic macromolecules, partition extremely into the hydrophilic phase. However, water-soluble, hydrophilic proteins, for example, do not partition extremely into either phase of aqueous two- phase systems, which in turn would result in limited concentration of such proteins into one of the two phases. Therefore, a novel approach is used to concentrate the target proteins into the hydrophilic phase.

Figure 1 is a schematic representation of the method for concentrating a target protein in an aqueous two-phase system using colloidal gold particles that are decorated with target-specific antibodies. The y-shaped object, the red circle, and the orange circle represent the antibody, the colloidal gold particle, and the target molecule, respectively.

As set forth in Figure 1, the target proteins are "fished" into the hydrophilic phase by utilizing hydrophilic, colloidal gold particles, which are nanometers in diameter, and are coated with antibodies specific to a target protein. The colloidal gold-antibody-target protein complex would then partition extremely into the hydrophilic phase (micelle-poor or polymer-poor phase) based on repulsive, steric, excluded-volume interactions that operate between the colloidal gold particles and micelles or polymers that are present in the micelle-rich phase or polymer-rich phase, respectively.

Once phase separation is established, the hydrophilic phase, which typically contains the concentrated target biomolecules, is sampled and applied to an LFA strip. A traditional LFA test strip, shown schematically in Figure 2, consists of 4 main components: sample pad, nitrocellulose membrane, absorbent pad, and adhesive vinyl backing. Note that the adhesive vinyl backing is underneath the sample pad, the nitrocellulose membrane, and the absorbance pad, and therefore, is not shown in this schematic.

There are two different approaches for the lateral-flow immunoassay: the sandwich assay and the competition assay. Figure 3 is a schematic representation of an LFA using the sandwich mechanism. In the sandwich LFA, antibodies specific to the target of interest are immobilized on the nitrocellulose membrane in the form of a line. If the target molecules are present in the sample, they would first bind to their specific antibodies immobilized on colloidal gold particles. Subsequently, the target molecules present in the colloidal gold-antibody-target molecule complexes bind to their specific antibodies immobilized on the nitrocellulose membrane. Due to the "trapped" colloidal gold particles, which exhibit a purple red color, a visual band is formed that indicates a positive result, as depicted in Figure 3 A. Alternatively, if the target molecules are not present in the sample, the antibodies on the nitrocellulose membrane and colloidal gold cannot sandwich the target molecule, and therefore, a visual band is not formed. This indicates a negative result, as depicted in Figure 3B.

Figure 4 is a schematic representation of an LFA using the competition mechanism. In the competition LFA, the target of interest (or a fragment of the target of interest) is immobilized on the nitrocellulose membrane in the form of a line. If the sample does not have enough target molecules to saturate the antibodies on the colloidal gold particles, the antibodies on the gold particles can then bind to the immobilized target, and form a visual band indicating a negative result, as depicted in Figure 4A. Alternatively, if the sample does have enough target molecules to saturate the antibodies on the colloidal gold particles, the antibodies on the gold particles cannot then bind to the immobilized target, and hence do not form a visual band. This indicates a positive result, as depicted in Figure 4B.

The LFA is widely used to detect various biomolecules in patient samples in the form of a point-of-care device. The invention is best practiced in such

applications where rapid detection of biomolecules is desired. In addition, the invention can be used by food manufacturers to detect the presence of food allergens, such as peanut proteins, in their products. Furthermore, the invention can be used by authorities to detect the presence of bioterrorism agents in various samples, such as water reservoirs.

The invention allows for lowering LFA's detection limit of an analyte by combining LFA with aqueous two-phase systems.

The invention is described in more detail in the following illustrative examples. Although the examples can represent only selected embodiments of the invention, it should be understood that the following examples are illustrative and not limiting.

EXAMPLES

Example 1. Enhancing the detection of viruses via the lateral-flow immunoassay using aqueous two-phase micellar systems

The studies in this example illustrate an embodiment of the present invention to enhance detection of infectious agents using LFA. The outbreak of swine-origin influenza A ("H1N1 ") virus infection in the past highlighted the need for a means to rapidly and accurately diagnose and detect infectious agents and pandemic pathogens at the point-of-care. Rapid and accurate diagnosis of such agents and pathogens at the point-of-care results in better patient management, such as timely use of appropriate antiviral treatments, isolation of confirmed cases, and prevention of outbreaks. One detection method that has gained much attention in recent years due to its ease of use, rapid time to result, and minimal power and laboratory equipment requirements is the lateral-flow immunoassay (LFA). LFA utilizes a test strip that collects a sample through lateral flow, and detects the presence of a target molecule through its specific antibody labeled with a colorimetric indicator. While LFA has been used to detect a wide range of biomolecules (see, e.g., Laderman, E.I., et al., Clinical and Vaccine Immunology, 2008, 15(1): p. 159), its sensitivity in detecting viruses has been shown to be inferior to the gold standards, namely viral culture and real-time polymerase chain reaction (PCR) (see, e.g., Ginocchio, C.C., et al, Journal of Clinical Virology, 2009, 45(3): p. 191 -195).

Although LFA is superior to viral culture and real-time PCR in terms of ease of use and rapid time to result, its low sensitivity in detecting pathogens and viruses renders it ineffective as a point-of-care assay to prevent pandemic outbreaks.

Therefore, the need to improve the sensitivity of LFA for detection of infectious agents exists. One approach to achieving a higher sensitivity for LFA is to improve the assay itself. Another approach is to concentrate the target biomolecule prior to the detection step.

The focus of this example is on the latter approach. The concentration of a model virus, namely bacteriophage Ml 3 (Ml 3), was investigated using an aqueous two-phase micellar system prior to the detection step. Since the goal is to combine the concentration step with LFA for a point-of-care device, the concentration method should also be easy to use, rapid, scalable (to require minimal sample volume), and not require any laboratory equipment. One concentration method that can be designed to meet the above criteria is the utilization of aqueous two-phase complex fluid systems. Proteins, bacteria, DNA fragments, and viruses have previously been separated in these systems (see, e.g., Mashayekhi, F., et al., Biotechnol Bioeng, 2009, 102(6): p. 1613-23). Furthermore, other advantages of these systems include low cost compared to chromatography (see, e.g., Champluvier, B. et al, Bioseparation, 1992, 2(6): p. 343-51) and the ability to concentrate a biomolecule by varying the relative volumes of the two phases (Mashayekhi, 2009; Johansson, G. et al., J Chromatogr B Biomed Sci Appl, 1998, 71 1(1-2): p. 161-72).

In this study, a solution comprised of the nonionic surfactant Triton X-l 14 and phosphate-buffered saline (PBS) was investigated for concentrating Ml 3. Surfactant molecules have a hydrophilic, or polar, "head" and a hydrophobic, or nonpolar, "tail." In an aqueous solution at concentrations above their critical micelle concentration ("CMC"), the surfactant molecules form aggregates known as micelles. In these micelles, the hydrophobic tails flock towards the interior to minimize their contact with water and maximize their contact with each other. On the other hand, the heads remain on the periphery of the micelles to maximize their contact with water

(Israelachvili, J.N., Intermolecular and surface forces, 2nd ed. 1992, San Diego, CA: Academic Press; Tanford, C, The hydrophobic effect: Formation of micelles and biological membranes, pi -10. 2nd ed. 1978, New York: John Wiley & Sons). The Triton X-l 14 micellar system exhibits a homogeneous, isotropic phase at low temperatures. Upon increasing the temperature, the solution undergoes a macroscopic phase separation to yield a top, micelle-poor phase and a bottom, micelle-rich phase as shown schematically in Figure 5. Biomolecules would then distribute, or partition, unevenly between the two phases based on their physico-chemical characteristics, such as hydrophobicity (Ginocchio, 2009; Bordier, C, J Biol Chem, 1981, 256(4): p. 1604-7) and size (Nikas, Y.J., et al, Macromolecules, 1992, 25(18): p. 4797-4806; and Lue, L. et al., Industrial & Engineering Chemistry Research, 1996, 35(9): p. 3032 -3043).

First, to gain an understanding of the main driving forces for Ml 3 partitioning in the Triton X-l 14 micellar system, the experimentally measured partitioning behavior of Ml 3 was compared with our theoretical predictions based on a model developed recently for DNA fragments partitioning in the micellar system

(Mashayekhi , 2009). Next, Ml 3 was concentrated in the top, micelle-poor phase by manipulating the volume ratio (the volume of the top, micelle-poor phase divided by that of the bottom, micelle-rich phase). After ensuring that Ml 3 was concentrated in the aqueous two-phase micellar system in a predictive manner, a lateral-flow immunoassay for detection of Ml 3 in a solution was conducted. Once the detection limit of the immunoassay was established, Ml 3 was concentrated by utilizing the aqueous two-phase micellar system prior to the detection step to investigate the effect of the concentration step on the immunoassay's detection limit.

MATERIALS AND METHODS

Bacteria and Bacteriophage Ml 3 Culture

Escherichia coli bacteria (American Type Culture Collection, ATCC, Manassas, VA) were incubated in 6 mL of lysogeny broth (LB, 10 g/L tryptone (BD, Franklin Lakes, NJ), 5 g/L yeast extract (BD), and 10 g/L NaC l) at 37°C and

240 RPM in a shaker incubator for 12 hours. This bacteria solution was then used in the plaque assay to quantify the concentration of Ml 3 (see Bacteriophage Ml 3 Quantification). In order to culture Ml 3 (ATCC), 10 of the stock Ml 3 solution was added to a bacteria solution as described above. The bacteria solution was then incubated in a shaker incubator at 37°C and 240 RPM for 5 hours. The solution was then centrifuged at 4°C and 8000g for 15 min to remove the bacteria. The supernatant containing Ml 3 was collected and filtered using a 0.22 μηι syringe filter (Millipore, Billerica, MA). All reagents and materials were purchased from Sigma-Aldrich (St. Louis, MO) unless otherwise noted.

Bacteriophage Ml 3 Quantification

The plaque assay was used to quantify the concentration of Ml 3 in a solution. In this assay, 100 μϋ of a diluted sample with an unknown concentration of Ml 3 was added to 200 μί of a bacteria solution and 3 raL of soft LB agar (LB, with 0.3% w/v agarose (Promega, Madison, WI)). This solution was then mixed and poured onto a petri dish covered with hard LB agar (LB, with 1.2% w/v agarose (Promega)). This assay relies on the fact that Ml 3 infects cells, replicates inside them, slows down their growth, and spreads to neighboring cells. Consequently, if a single Ml 3 viral particle is placed in an environment of growing bacteria cells, eventually, a plaque or hole will be visible in the opaque yellowish bacteria lawn due to the inhibited growth of bacterial cells that are infected by the replicating bacteriophage. Although many bacteriophages are responsible for one plaque, they all originated from a single infectious viral particle that was initially placed on the hard LB agar. Therefore, the concentration of Ml 3 is reported in the unit of plaque forming units (pfu) per mL. Partitioning Bacteriophage Ml 3 in Aqueous Micellar Solutions

For each Ml 3 partitioning experiment, four identical Triton X-l 14 solutions in Dulbecco's phosphate-buffered saline (PBS, Invitrogen, pH 7.4, containing 1.47 mM H₂P0₄, 8.10 mM Na₂HP0₄, 137.93 mM NaCl , 2.67 mM KC1, and 0.49 mM MgCl₂) were prepared. Ml 3 was added to three of the solutions at a concentration of 10⁸ bacteriophage particles/mL. The fourth solution served as the control which did not contain any bacteriophage. In order to ensure that each solution is in one phase prior to phase separation, the solutions were equilibrated at 4°C prior to the addition of Ml 3. Once Ml 3 was added, the solutions were mixed and placed in a water bath set at the appropriate temperature which yielded a volume ratio equal to 1. The operating conditions (i.e., temperature and initial surfactant concentration) are listed in Table I. After incubating the four solutions in the water bath for 18 hours, the two coexisting micellar phases were withdrawn carefully using syringe and needle sets, and the concentration of Ml 3 in each of the two phases was determined as described above. Each partitioning experiment, which involved triplicate solutions and the control, was repeated at least twice.

Table I. Operating conditions for the bacteriophage Ml 3 partitioning experiments with a 1:1 volume ratio.

Concentrating Bacteriophage Ml 3 in Aqueous Micellar Solutions

By altering the volume ratio of the partitioning experiments, Ml 3 can be concentrated in the top, micelle-poor phase. The same protocol described above for partitioning Ml 3 was used, except that the initial Triton X-l 14 surfactant

concentrations and operating temperatures were varied in order to achieve the desired volume ratios. The operating conditions and resulting volume ratios are listed in Table II.

Table II. Operating conditions for the bacteriophage Ml 3 concentration experiments with varying volume ratios.

Preparing Colloidal Gold Probes

The colloidal gold nanoparticles were prepared according to Frens (Frens, G., Colloid & Polymer Science, 1972, 250(7): p. 736-741 ). Briefly, 1 mL of a 10% gold (III) chloride hydrate was added to 1 L of 100°C deionized water. 1.5 mL of 12% sodium citrate was then added to reduce the gold chloride into gold atoms, which would subsequently nucleate to form gold colloids. Using this method, a one liter solution of 40 nm colloidal gold nanoparticles was obtained, which appeared as a clear, dark cherry-colored solution.

The colloidal gold-antibody probe was prepared as described by Horisberger and Clerc (Horisberger, M. and Clerc, M.F., Histochemistry, 1985, 82(3): p. 219-23 ). Briefly, the pH of a 2.5 mL colloidal gold nanoparticle solution was adjusted to pH 9 using 0.1 M NaOH. Subsequently, 40 μg of anti-M13 mouse monoclonal antibody (Abeam Inc., Cambridge, MA) at a concentration of 0.2 mg/mL was added to the colloidal gold solution and mixed for 10 min on a shaker. To prevent nonspecific binding of other proteins to the surfaces of the colloidal gold nanoparticles, 10% w/v bovine serum albumin was added to the mixture and mixed for 15 min on a shaker to block all excess surfaces on the colloidal gold nanoparticles. The mixture was then centrifuged for 30 min at 4°C and 9000g to remove free antibody and bovine serum albumin. The pellet, which contained the colloidal gold nanoparticles, was resuspended in 375 ih of 0.1 M sodium borate buffer, pH 9.

Preparing Lateral-Flow Immunoassay Test Strips

There are two different approaches for the lateral-flow immunoassay: the sandwich assay and the competition assay. In this study, we implemented the sandwich assay. As shown schematically in Figure 6, in the sandwich assay, antibodies specific to the target of interest are immobilized on a nitrocellulose membrane in the form of a line, called the test line. Secondary antibody specific to the primary antibody is also immobilized on the nitrocellulose membrane in the form of a line, called the control line. In LFA, a sample first comes into contact with the colloidal gold probes (see Bacteriophage Ml 3 Lateral-Flow Immunoassay). If the target molecules are present in the sample, they would first bind to their specific antibodies immobilized on the colloidal gold nanoparticles. Subsequently, as the colloidal gold nanoparticles move up the LFA strip, the target molecules present in the colloidal gold-antibody-target molecule complexes would bind to their specific antibodies immobilized on the test line. Due to the "trapped" colloidal gold particles, which exhibit a purple red color, a visual band is formed at the test line that indicates a positive result (Figure 6A). Alternatively, if the target molecule is not present in the solution, the colloidal gold-complexed antibodies would not get "trapped" by the immobilized antibodies at the test line. This indicates a negative result (Figure 6B). Furthermore, regardless of the presence of the target molecule in the sample, colloidal gold-complexed antibodies would bind and get "trapped" by the immobilized secondary antibodies on the control line, which indicates a valid test.

Using a similar approach to Schuurs and coworkers (Leuvering, J.H., et al, J. Immunoassay, 1980, 1(1): p. 77-91), the LFA test strips were prepared as follows: Goat polyclonal anti-mouse IgG (Abeam) and mouse monoclonal anti-M13 antibodies (Abeam) were first diluted to 0.5 mg/mL by using a 50% w/v sucrose solution. The antibody solutions were then sprayed near each end of a nitrocellulose membrane (Sartorius, Goettingen, Germany) using a syringe and PEEK™ tubings (Upchurch Scientific, Oak Harbor, WA). The nitrocellulose membrane was then dried under vacuum for two hours, and left overnight at room temperature and atmospheric pressure to allow the membrane to retain its relaxed form. Next, the treated nitrocellulose membrane, the sample pad (Whatman, Kent, UK) which is where the solution is applied, the absorbent pad (Whatman) which functions as a sink for excessive sample fluid, and adhesive vinyl backing (G&L, San Jose, CA) were assembled, and cut into test strips shown schematically in Figure 6.

Bacteriophage Ml 3 Lateral-Flow Immunoassay

The operating condition that was used for performing the concentration step prior to the detection step resulted in Ml 3 being concentrated in the top, micelle-poor phase that had a Triton X-l 14 concentration of 0.065% w/w in PBS. Therefore, to be consistent between the lateral-flow immunoassays performed with or without the concentration step, Ml 3 solutions in 0.065% w/w Triton X-l 14 in PBS were used to perform the immunoassay without the concentration step. The LFA was performed as follows: Solutions of M13 diluted in 0.065% w/w Triton X-l 14 in PBS were first prepared. 45 ih of the Ml 3 solutions were then added to 10 of the colloidal gold probes solution (see Preparing Colloidal Gold Probes) and 25 iL of test buffer

(0.2% bovine serum albumin, 0.3% Tween20, 0.2% sodium azide, 0.1% polyethylene glycol, 0.1 M Trizma base, pH 8), which was used to facilitate the flow of the samples through the test strips. The resulting solutions were mixed, and incubated for 5 minutes before a test strip (see Preparing Lateral-Flow Immunoassay Test Strips^') was dipped vertically into each solution. After 20 min, the test strips were taken out of the solution, and an image of each strip was immediately taken by a Canon EOS 1000D camera (Canon U.S.A., Inc., Lake Success, NY).

Combining Concentration of Ml 3 with the Lateral-Flow Immunoassay

To combine the concentration step with the detection step, Ml 3 was first concentrated following the same protocol as mentioned previously (see Concentrating Bacteriophage Ml 3 in Aqueous Micellar Solutions). Solutions of 9.50% w/w Triton X-1 14 in PBS were used, and the solutions were incubated at 26.1°C for 18 hours. In addition, various amounts of Ml 3 were added to each solution to obtain appropriate initial concentrations of Ml 3. After phase separation, the top phases were withdrawn carefully using syringe and needle sets. The LFA was performed as described in Bacteriophage Ml 3 Lateral-Flow Immunoassay, except instead of using 45 of the Ml 3 solutions diluted in 0.065% w/w Triton X-1 14 in PBS, 45 μΤ of the withdrawn top, micelle-poor phases were used.

RESULTS AND DISCUSSION

Partitioning Bacteriophage Ml 3 in the Triton X-114 Micellar System

The partitioning behavior of a biomolecule in an aqueous two-phase micellar system is quantified by evaluating the partition coefficient, K _m which is defined as follows: - bm.b where Cb_m and C_bm,_b are the concentrations of the biomolecule in the top and bottom phases, respectively. Based on its shape, Ml 3 can be modeled as a cylindrical biomolecule. We had previously extended a model, which was developed by

Blankschtein and coworkers (Nikas, 1992) for partitioning of globular, water-soluble proteins in aqueous two-phase nonionic micellar systems to cylindrical biomolecules (cb). This model incorporates the repulsive, steric, excluded-volume interactions that operate between the cylindrical biomolecule and the cylindrical micelles, and the following expression was obtained for the partition coefficient of a cylindrical biomolecule, such as Ml 3 (Mashayekhi, 2009):

where 0_t and 0 are the surfactant volume fractions in the top and bottom phases, respectively, R_cb and R_m are the cross-sectional radii of the cylindrical biomolecule and micelles, respectively, and l_cb and l_m are the lengths of the cylindrical biomolecule and micelles, respectively. The cross-sectional radius of Ml 3 was estimated to be 7 nm, and its length was estimated to be 900 nm (Day, L.A., et al, Annual review of biophysics and biophysical chemistry, 1988, 17(1): p. 509-539). The cross-sectional radius of Triton X-l 14 micelles was estimated to be 23.4 A (Mashayekhi, 2009), while their lengths were estimated to be between 10 nm and 1 μηι (see, e.g., Won, Y.Y., et al, Science, 1999, 283(5404): p. 960-3; Robson, R.J. et al, The Journal of Physical Chemistry, 1977, 81(1 1): p. 1075-1078; Lin, Z., et al, Langmuir, 1992, 8(9): p. 2200-2205; Acharya, D.P. et al, J Phys Chem B, 2003, 107(37): P. 10168-10175; Paradies, H. H., et al, Journal of Physical Chemistry, 1980, 84(6): p. 599-607; and Dalhaimer, P., et al., Macromolecules, 2003, 36(18): p. 6873 - 6877). Based on Eq. (2), the values for the dimensions of Ml 3 and Triton X-l 14 micelles, and the one-to- one correspondence between operating temperature and (0_t - ø_/,) found previously (Mashayekhi, 2009), extremely large (»1000) Ml 3 partition coefficients as a function of temperature were predicted. However, as shown previously for the partitioning behavior of spherical viruses and DNA fragments in aqueous two-phase nonionic micellar systems (Mashayekhi, 2009; amei, 2002), the entrainment of micelle-poor domains in the macroscopic micelle-rich phase has a significant impact on the partitioning behavior of such large hydrophilic macromolecules.

Due to the small density difference and interfacial tension between the micelle-rich and micelle-poor domains, even after waiting a long time, macroscopic phase separation equilibrium is not attained. As a result, some micelle-poor domains are entrained in the macroscopic micelle-rich phase, and similarly, some micelle-rich domains are entrained in the macroscopic micelle-poor phase. If Ml 3 partitions extremely into the micelle-poor domains, as predicted by the model, the concentration of Ml 3 in the micelle-poor domains would be orders of magnitude greater than that in the micelle-rich domains. Therefore, the effect of entrained micelle-poor, M13-rich domains on the measured concentration of Ml 3 in the macroscopic, micelle-rich, Ml 3 -poor phase would be drastic, while the effect of entrained micelle-rich, Ml 3- poor domains on the measured concentration of Ml 3 in the macroscopic, micelle- poor, M13-rich phase could be ignored. Defining x as the volume fraction of micelle- poor domains entrained in the bottom, macroscopic micelle-rich phase, the newly predicted partition coefficient could be written as follows (Mashayekhi, 2009;

(Kamei, D.T., et al., Biotechnol Bioeng, 2002, 78(2): p. 203-16):

_C EV

J EV +Em _ ^ M U.mp

M 1

where C^_{U mp} and C^'\_{3 mr} are the concentrations of Ml 3 in the micelle-poor and micelle-rich domains, respectively, as predicted by the excluded-volume theory. Dividing the numerator and denominator of the right-hand side of the above equation by _mr and rearranging the numerator results in the following expression for the Ml 3 partition coefficient:

where C^ is the partition coefficient of Ml 3 based only on excluded-volume interactions, and it is equal to the ratio of ^_{n mp} to C^ _mr . For large values of

K E_iV , Eq. (4) simplifies to:

Equation (5) indicates that the partition coefficient of Ml 3 is only dependent on x if (i) entrainment is present and (ii) is much, much greater than 1. It has also been shown by Blankschtein and coworkers that x is only a function of the volume ratio (Kamei, 2002). Therefore, if the volume ratio is maintained at 1 for all temperatures, the measured partition coefficients for Ml 3 should not change by varying the operating temperature. Figure 7 shows the partition coefficients of Ml 3 obtained experimentally at various operating temperatures, while the volume ratio was maintained at 1. As indicated in Figure 7, the partitioning behavior of Ml 3 in the aqueous two-phase Triton X-114 micellar system is approximately independent of the operating temperature, as expected, suggesting that Ml 3 partitioning is driven by the steric, excluded-volunie interactions between Ml 3 and micelles, but is limited by entrainment. Since the measured values of the Ml 3 partition coefficients are much greater than 1 , they can be exploited as described below.

Concentrating Bacteriophage Ml 3 by Manipulating the Volume Ratio

In an approach similar to that developed by others (Albertsson, P.A., Partition of Cell Particles and Macromolecules. 3^rd ed. 1986, New York: John Wiley & Sons), an expression for the concentration factor, that is, the concentration of virus in the top phase divided by the initial concentration, will now be derived. The starting point in the derivation is the mass balance of Ml 3 in the aqueous two-phase system: t M 13 ^' 'f ⁺ ί ~ *-- M J ^" ^r ⁺ /.?,* ^' where V, and V_b are the volumes of the top and bottom phases, respectively, C MI 3,0 is the initial concentration of Ml 3 in the homogeneous micellar solution prior to phase separation, and C_Mi3_,t and C n_,b are the concentrations of Ml 3 in the top and bottom phases, respectively. Dividin Eq. (6 by V_b and CMI3 yields the following equation:

where K^^' _n is the measured partition coefficient obtained from quantifying the concentration of Ml 3 in each phase. Therefore, for large values of K^' _u , the concentration factor could be approximated as follows:

V

concentration factor≡ = -rr- — « i +—

'M l 3 V,, Therefore, based on Eq. (8), and the large values of K™_n obtained experimentally

(Figure 7), the concentration factor can be manipulated by varying the volume ratio. Therefore, the volume ratio was varied from 1 to 1/8, and a 2- to 7-fold concentration of Ml 3 in the top phase was achieved (Figure 8). In addition, as indicated in Figure 8, there is reasonable agreement with the experimentally measured concentration factors and the model's predictions obtained from Eq. (8).

Detecting Bacteriophage Ml 3 via the Lateral-Flow Immunoassay

After demonstrating that Ml 3 could be concentrated via an aqueous two-phase micellar system, we prepared colloidal gold probes and LFA test strips by utilizing goat polyclonal anti-mouse IgG antibody and mouse monoclonal antibody to Ml 3 's coat protein pVIII (see Preparing Colloidal Gold Probes and Preparing Lateral-Flow Immunoassay Test Strips, respectively). The LFA without a prior concentration step was performed as described in Bacteriophage Ml 3 Lateral-Flow Immunoassay, and the results are shown in Figure 9. The negative control without any Ml 3 is shown in panel 9A. The remaining solutions contained Ml 3 at concentrations of 10¹⁰ pfu/mL (panel 9B), 5xl0⁹ pfu/mL (panel 9C), 10⁹ pfu/mL (panel 9D), 5xl0⁸ pfu/mL (panel 9E), and 10⁸ pfu/mL (panel 9F). As mentioned previously, the top line, which contains immobilized goat polyclonal anti-mouse IgG antibody, is the control line, indicating a valid test. The presence of the test line, which contains mouse monoclonal antibody to Ml 3 's coat protein pVIII, indicates the presence of Ml 3. As indicated in Figure 9, while no test line appeared for the negative control solution, which did not contain any Ml 3, the intensity of the test line decreased with the decreasing concentration of Ml 3 until no test line appeared for the solution containing 10 pfu/mL (panel (F) in Figure 9). This indicated a detection limit of 5x10 pfu/mL for the Ml 3 LFA performed without a prior concentration step.

Concentrating Bacteriophage Ml 3 Prior to the Lateral-Flow Immunoassay

After establishing the detection limit of the Ml 3 LFA, we investigated the possible improvement of the detection limit if Ml 3 were to be concentrated by utilizing an aqueous two-phase micellar system prior to the detection step. To do so, 9.50% w/w Triton X-l 14 in PBS solutions with different initial concentrations of Ml 3 were incubated at 26.1 °C for 18 hours. After phase separation, the top, micelle- poor, M13-rich phases were withdrawn using syringe, and needle sets, and were consequently used in the LFA as described previously (see Combining Concentration of Ml 3 with the Lateral-Flow Immunoassay). The results of the LFA with the prior concentration step are shown in Figure 10. The negative control without any Ml 3 is shown in panel 10A. The remaining solutions initially contained Ml 3 at

concentrations of 10¹⁰ pfu/mL (panel 10B), 5xl0⁹ pfu/mL (panel IOC), 10⁹ pfu/mL (panel 10D), 5xl0⁸ pfu/mL (panel 10E), 10⁸ pfu/mL (panel 10F), 5xl0⁷ pfu/mL (panel 10G), and 10⁷ pfu/mL (panel 10H). While no test line appeared for the negative control solution, which did not contain any Ml 3, the intensity of the test line decreased with the decreasing concentration of Ml 3 until no test line appeared for the solution containing 10⁷ pfu/mL (panel (H) in Figure 10). This indicated a detection 7

limit of 5x10 pfu/mL for the Ml 3 LFA when combined with the prior concentration step, which represented a 10-fold improvement of the detection limit of the assay. Furthermore, the intensity of the test line for all the detectable concentrations clearly increased when the concentration step was incorporated prior to the detection step. In the future, even lower volume ratios can be implemented to yield greater

concentration factors that can lead to even lower detection limits.

CONCLUSION

Concentrating infectious agents, such as infectious viruses, prior to a detection step via LFA improves the detection limit of the immunoassay, which in turn significantly increases the effectiveness of using LFA for patient management at the point-of-care. In this study, the concentration of a model virus, namely bacteriophage Ml 3, in an aqueous two-phase micellar system was investigated. The micellar system was generated using the nonionic surfactant Triton X-l 14 and phosphate-buffered saline. We first compared experimentally measured partition coefficients with our theoretical predictions obtained from a model developed previously for cylindrical biomolecules partitioning in micellar systems. The agreement between theory and experiment indicated that the partitioning behavior of Ml 3 in the nonionic micellar system is primarily driven by repulsive, steric, excluded-volume interactions that operate between the micelles and Ml 3 particles, but is limited by the entrainment of micelle-poor, M13-rich domains in the macroscopic, micelle-rich phase. Next, the volume ratio was manipulated to concentrate Ml 3 particles in the top phase. By decreasing the volume ratio from 1 to 1/8, Ml 3 particles were concentrated in the top phase 2- to 7-fold in a predictive manner. After demonstrating that we could concentrate Ml 3 in the aqueous two-phase micellar system, we developed an LFA for the detection of Ml 3 in-solution. The detection limit of the Ml 3 lateral-flow immunoassay itself was found to be 5x10⁸ pfu/mL. Ml 3 was subsequently concentrated by utilizing the aqueous two-phase micellar system prior to the detection step. By combining the concentration step with the detection step, the detection limit of the Ml 3 LFA was improved 10-fold to 5x10⁷ pfu/mL. Therefore, we demonstrated that concentrating a target virus prior to the detection step enhances the detection limit of the viral LFA, thereby increasing the sensitivity of the immunoassay. Operating conditions can be further manipulated to obtain even lower volume ratios, which in turn will result in obtaining higher concentration factors that yield even lower detection limits. Furthermore, enhancing LFA by concentrating agents prior to the detection step via aqueous two-phase systems may be employed for in-field detection of biowarfare viral agents, such as the Ebola virus.

The disclosure of all publications, patent applications, and patents cited above and below are expressly incorporated herein by reference in their entireties for all purposes.

Example 2. Enhancing the detection of proteins via the lateral-flow

immunoassay using aqueous two-phase micellar systems

ABSTRACT

The studies in this example illustrate an embodiment of the present invention, which enhances detection of analytes using LFA. Proteins are markers for a wide variety of applications. These include detecting allergens in a food sample at the point-of-need and bioterrorism agents in-field. For such applications, a sensitive, yet rapid and inexpensive, detection assay that requires minimal training and power is desired. Due to its ease of use, rapid processing, and minimal power and laboratory equipment requirements, the lateral-flow immunoassay (LFA) represents a potential detection method for these applications. However, the detection limit of LFA is inferior to lab-based assays, such as the enzyme-linked immunosorbent assay (ELISA), and needs to be improved. A practical solution for improving the detection limit of LFA is to concentrate a target protein in a solution prior to the detection step. In this study, a novel approach was used along with an aqueous two-phase micellar system comprised of the nonionic surfactant Triton X-l 14 to concentrate a model protein, namely transferrin, prior to LFA. Proteins, however, have been shown to partition, or distribute, fairly evenly between the two phases of an aqueous two-phase system, which in turn results in their limited concentration in one of the two phases. Therefore, larger colloidal gold particles decorated with antibodies for transferrin was used in the concentration step to bind to transferrin and aid its partitioning into the top, micelle-poor phase. This use of the colloidal gold particles decorated with antibodies to alter the partitioning behavior of the protein led to its concentration in the top, micelle-poor phase. By manipulating the volume ratio of the two coexisting micellar phases, and combining the concentration step with LFA, the transferrin detection limit of LFA was improved by 10-fold from 0.5 μg/mL to 0.05 μg/mL.

INTRODUCTION

Proteins are markers for a wide variety of applications, such as detecting food allergens and protein toxins. In the case of food allergens, ensuring the safety of manufactured goods is a vital component of public health. Therefore, food

manufacturers have a duty to provide their customers with safe products, which includes protecting consumers from food allergens. In the US, under the Food Allergen Labeling and Consumer Protection Act (FALCPA), food manufacturers are required to list the presence of any of the 8 pre-identified food allergens only if they are used by food manufacturers as ingredients (Taylor, S.L. et al., Current opinion in allergy and clinical immunology, 2006, 6(3): p. 186). However, this labeling does not account for food allergens present in products due to contamination caused by using shared transportation containers and production lines (Schappi, G.F., et al., Allergy, 2001, 56(12): p. 1216-1220). Therefore, a sensitive, yet rapid and inexpensive, assay, which can result in more frequent screening of food products by the manufacturers, could be beneficial to both food manufactures and consumers. Although the recent development of rapid immunoassays based on the LFA technology precludes the need for sophisticated laboratory skills and caters to the manufacturers' preference for simplicity and speed, the traditional enzyme-linked immunosorbent assay (ELISA), which is currently the most widely used detection scheme to test for the presence of food allergens, is an order-of-magnitude more sensitive than its LFA counterpart (Schubert-Ullrich, P., et al., Analytical and Bioanalytical Chemistry, 2009, 395(1): p. 69-81). Therefore, the detection limit of LFA in detecting food allergens, which are typically proteins, needs to be improved before it could be used reliably. In the case of protein toxins, bioterrorism agents (BAs), such as ricin toxin, present a great danger to the general public, since they are usually invisible to the naked eye, odorless, tasteless, and may not cause an immediate reaction (Broussard, L.A., Molecular Diagnosis, 2001 , 6(4): p. 323-333; Ellison, D.H., Handbook of chemical and biological warfare agents. 2007: CRC). In order to minimize their spread and harmful impact to the civilian population, it is essential to provide the authorities with the means to rapidly detect BAs in-field and at the point-of-need. One approach that meets these requirements is the LFA, which has previously been used for the detection of BAs (see, e.g., Shyu, R.H., et al., Toxicon, 2002, 40(3): p. 255-258; King, 2003; and Chiao, D.J., J Chromatogr B Analyt Technol Biomed Life Sci, 2004,

809(1): p. 37-41). However, the detection limit of LFA is still inferior to lab-based assays, such as the ELISA, and needs to be improved (Peruski, A.H. et al., Clinical and Vaccine Immunology, 2003, 10(4): p. 506). One approach to achieve a higher sensitivity for LFA is to improve the assay itself. Another approach is to concentrate the target molecule prior to the detection step. However, for the detection assay to still be implementable at the point-of-need, it is essential for the concentration step to also be used at the point-of-need, meaning it must be simple to perform and require minimal training and power. One approach that meets these criteria is liquid-liquid extraction using aqueous two-phase systems, such as aqueous two-phase micellar systems and aqueous two-phase polyethylene glycol (PEG)-salt systems. Aqueous two-phase systems typically exhibit a homogeneous, isotropic phase at low temperatures. Upon increasing the temperature, the solution undergoes a macroscopic phase separation to yield two phases, one more hydrophobic than the other. In the case of aqueous two-phase micellar systems, the two phases are a micelle-poor phase and a micelle-rich phase. In the case of aqueous two-phase PEG-salt systems, the two phases are a polymer-rich phase and a polymer- poor phase.

In this study for protein markers, we investigated the concentration of human transferrin, our model protein, using the aqueous two-phase Triton X-l 14 micellar system prior to its detection via LFA. Transferrin (Tf) transports iron in serum, and has a molecular weight of about 78 kDa. It was chosen as our model protein due to its size being similar to that of the food allergen ovotransferrin from chicken egg white (78 kDa) and that of the BA ricin toxin (64 kDa).

Unlike large hydrophilic macromolecules, such as genomic DNA fragments and viruses, water-soluble proteins partition, or distribute, fairly evenly between the two phases of an aqueous two-phase system (see, e.g., Rangel-Yagui, et a.,

Biotechnology and Bioengineering, 2003, 82(4): p. 445-56), which in turn would result in limited concentration of such proteins into one of the two phases.

Accordingly, a novel approach was investigated to concentrate the target proteins into the more hydrophilic, micelle-poor phase. In this approach shown schematically in Figure 1 , the target proteins are "fished" into the more hydrophilic phase by utilizing hydrophilic, colloidal gold particles, which are nanometers in diameter, and are coated with antibodies specific for the target proteins. The colloidal gold-antibody-target protein complex is then expected to partition extremely into the top, micelle-poor phase based on the greater repulsive, steric, excluded-volume interactions that operate between the colloidal gold nanoparticles and micelles that are present in the bottom, micelle-rich phase. Therefore, similar to DNA and viral partitioning, if the target protein partitions extremely into the top, micelle-poor phase, it can be concentrated in that phase by reducing its volume relative to the bottom, micelle-rich phase.

Furthermore, another advantage of this approach is that the concentrated colloidal gold-antibody-target protein complexes could directly be utilized in the downstream LFA detection step.

Although partitioning of proteins in aqueous two-phase systems has been previously studied, the work reported here represents the first time the established technologies of LFA and aqueous two-phase systems have been combined to improve LFA's limit of protein detection. In this study, we developed an LFA for the detection of Tf in-solution. Once the detection limit of the immunoassay was established, the approach shown in Figure 1 was utilized to concentrate Tf prior to the detection step to investigate the effect of the concentration step on the detection limit of LFA.

MATERIALS AND METHODS

Preparing Gold Probes

The colloidal gold nanoparticles were prepared according to Frens (Frens, 1972). Briefly, 50 of a 10% gold (III) chloride hydrate was added to 50 mL of 100°C deionized water. 150 of 6% sodium citrate was then added to reduce the gold chloride into gold atoms, which would subsequently nucleate to form gold colloids. Using this method, a solution of colloidal gold nanoparticles with an average hydrodynamic radius of 19 nm was obtained, which appeared as a clear, dark cherry-colored solution. The size of the colloidal gold nanoparticles was obtained by using a Zetasizer Nano ZS particle analyzer (Malvern Instruments Inc, Westborough, Massachusetts) following the manufacturer's instructions.

The colloidal gold-anti-Tf antibody complexes, henceforth referred to as gold probes, were prepared as described by Horisberger and Clerc (Horisberger, 1985). Briefly, the pH of a 2.5 mL colloidal gold nanoparticle solution was adjusted to pH 9 using 0.1 M NaOH. Subsequently, 40 μg of anti-Tf antibody at a concentration of a 0.1 mg/mL was added to the colloidal gold solution and mixed for 1 hour on a shaker. To prevent nonspecific binding of other proteins to the surfaces of the colloidal gold nanoparticles, 250 iL of 10% w/v BSA was added to the mixture and mixed for 15 min on a shaker to block any remaining exposed surfaces on the colloidal gold nanoparticles. To remove free, unbound antibodies, the mixture was then centrifuged for 30 min at room temperature and 4000 g, followed by resuspending the pellet of colloidal gold nanoparticles in 1.3 niL of a 1% w/v BSA solution. The centrifugation and resuspension steps were repeated two more times, and after the third

centrifugation, the pellet of gold nanoparticles was resuspended in 375 μΐ, of 0.1 M sodium borate buffer at pH 9.0. The average hydrodynamic radius of the gold probes was also found by using the Zetasizer Nano ZS particle analyzer.

Preparing Transferrin Lateral-Flow Immunoassay Test Strips

There are two different approaches for the lateral-flow immunoassay: the sandwich assay and the competition assay. In this study, we implemented the competition assay. In the competition LFA assay, as shown schematically in Figure 1 1 , the entire target of interest, or a portion of the target (such as a nontoxic chain of a toxin molecule) is immobilized on a nitrocellulose membrane in the form of a line, called the test line. Secondary antibodies specific to the primary antibody (antibody specific to the target of interest) are also immobilized on the nitrocellulose membrane in the form of a line, called the control line. In LFA, a sample first comes into contact with the target-specific antibodies bound to a color indicator, such as colloidal gold nanoparticles. If the sample does have enough target molecules to saturate the target- specific antibodies immobilized on the colloidal gold nanoparticles, the target-specific antibodies in the colloidal gold probes cannot bind to the immobilized target molecules, and hence do not form a visual band. This indicates a positive result (Figure 1 1 a). Alternatively, if the sample does not contain the target molecules at a concentration that saturates the target-specific antibodies immobilized on the colloidal gold nanoparticles, the target-specific antibodies on the colloidal gold probes can bind to the immobilized target, and form a visual band indicating a negative result (Figure 1 1 b). Furthermore, regardless of the presence of the target molecule in the sample, colloidal gold probes will bind to the immobilized secondary antibodies on the control line, which indicates a valid test.

Using a similar approach to that of Schuurs and coworkers (Leuvering, 1980), the LFA test strips were prepared as follows: Rabbit polyclonal anti-goat IgG (Bethyl Laboratories) and Tf were first diluted to 0.05 mg/niL and 2 mg/niL, respectively, by using a 50% w/v sucrose solution. The antibody and Tf solutions were then sprayed at their corresponding locations on a nitrocellulose membrane (Sartorius, Goettingen, Germany) using a syringe and PEEK™ tubings (Upchurch Scientific, Oak Harbor, WA). The nitrocellulose membrane was then dried under vacuum for two hours, and left overnight at room temperature and atmospheric pressure to allow the membrane to retain its relaxed form. Next, the treated nitrocellulose membrane, the sample pad (Whatman, Kent, UK) which is where the solution is applied, the absorbent pad (Whatman) which functions as a sink for excessive sample fluid, and adhesive vinyl backing (G&L, San Jose, CA) were assembled, and cut into test strips, as shown schematically in Figure 11.

Transferrin Lateral-Flow Immunoassay

The operating condition that yielded a 1 :10 volume ratio (the volume of the top, micelle-poor phase divided by that of the bottom, micelle-rich phase) and was used for performing the concentration step prior to the detection step resulted in Tf and gold probes being concentrated in the top, micelle-poor phase that had a Triton X- 1 14 concentration of 0.065% w/w in PBS. Therefore, to be consistent between the lateral-flow immunoassays performed with or without the concentration step, Tf solutions in 0.065% w/w Triton X-1 14 in PBS were used to perform the immunoassay without the concentration step. For the LFA, solutions of Tf diluted in 0.065% w/w Triton X-1 14 in PBS were first prepared. 45 \iL of the Tf solutions were then added to 5 xL of the gold probes solution (see Preparing Gold Probes) and 10 of test buffer (0.2% BSA, 0.3% Tween20, 0.2% sodium azide, 0.1% polyethylene glycol, 0.1 M Trizma base, pH 8), which was used to facilitate the flow of the samples through the test strips. The resulting solutions were mixed and incubated for 18 hours. This incubation time was chosen to allow the same time for anti-Tf antibodies present on the gold probes to bind to Tf with or without the concentration step. After the incubation period, a test strip (see Preparing Transferrin Lateral-Flow Immunoassay Test Strips) was dipped vertically into each solution. After 15 minutes, the test strips were taken out of the solution, and an image of each strip was immediately taken by a Canon EOS 1000D camera (Canon U.S.A., Inc., Lake Success, NY).

Combining Concentration of Transferrin with the Lateral-Flow Immunoassay

To combine the concentration step with the detection step, Tf was first concentrated using the following protocol. First, 5 mL solutions of 9.50% w/w Triton X-114 in Dulbecco's phosphate-buffered saline (PBS, Invitrogen, pH 7.4, containing 1.47 mM KH₂P0₄, 8.10 mM Na₂HP0₄, 138 mM NaCl, 2.67 mM KC1, and 0.495 mM MgCl₂) were prepared. Next, 50 L of colloidal gold probes were added to the aqueous micellar solutions, followed by mixing the solutions well. Various amounts of Tf were then added to each solution to obtain appropriate initial concentrations of Tf. After mixing the solutions well, the solutions were incubated at 26.1°C for 18 hours, which yielded a 1 : 10 volume ratio. After phase separation, the top phases were withdrawn carefully using syringe and needle sets. The LFA was performed as described in Transferrin Lateral-Flow Immunoassay, except instead of using 45 ih of the Tf solutions diluted in 0.065% w/w Triton X-l 14 in PBS and 5 xL of colloidal gold probes, 50 ih of the withdrawn top, micelle-poor phases were added to 10 iL of test buffer before a test strip was immediately dipped vertically into each solution. Since the amount of gold probe used affects the detection limit of LFA, 50 μL· of the top, micelle-poor phase was used, since it contained approximately the same amount of gold probes utilized in the LFA without the concentration step.

RESULTS AND DISCUSSION

Detecting Transferrin via the Lateral-Flow Immunoassay

There are two different approaches for the lateral-flow immunoassay: the sandwich assay and the competition assay. In this study, we implemented the competition assay to detect Tf in-solution. To establish the limit of Tf detection via LFA, test strips were prepared by utilizing rabbit polyclonal anti-goat IgG antibody and Tf (see Preparing Transferrin Lateral-Flow Immunoassay Test Strips). The LFA without a prior concentration step was performed as described in Transferrin Lateral- Flow Immunoassay, and the results are shown in Figure 12. The negative control without any Tf is shown in panel (a). The remaining solutions contained Tf at concentrations of (b) 10, (c) 5, (d) 1 , (e) 0.5, (f) 0.1, and (g) 0.05 μg/mL. As mentioned previously, the top line, which contains immobilized rabbit polyclonal anti-goat IgG antibody, is the control line, indicating a valid test. In the competition LFA, as shown schematically in Figure 1 1, Tf was immobilized on the nitrocellulose membrane on the test line. If the solution being tested contained enough Tf molecules to saturate the anti-Tf antibodies bound to the colloidal gold nanoparticles, then these anti-Tf antibodies could not bind to the immobilized Tf molecules at the test line, and hence, would not form a visual band at the test line. This indicated a positive result, which was observed for solutions with Tf concentrations as low as 0.5 μg/mL (Figure 12 e). Alternatively, if the sample did not contain Tf at a concentration that saturated the anti-Tf antibodies immobilized on the colloidal gold nanoparticles, then some of these anti-Tf antibodies would bind to the immobilized Tf at the test line, and therefore, form a visual band indicating a negative result. This was observed for the negative control, which did not contain any Tf (Figure 12 a), as well as for solutions with Tf concentrations of 0.1 and 0.05 g/mL (Figures 12 f and g). This indicated a detection limit of approximately 0.5 iglmL for the Tf LFA performed without a prior concentration step.

Concentrating Transferrin Prior to the Lateral-Flow Immunoassay

After establishing the detection limit of the Tf LFA, we investigated the possibility of improving the detection limit of LFA if an aqueous two-phase micellar system was utilized to concentrate Tf molecules prior to the detection step. To combine the concentration step with LFA, 9.50% w/w Triton X-l 14 in PBS solutions that contained gold probes and had different initial concentrations of Tf were prepared. The solutions were mixed well, and subsequently incubated at 26.1 °C for 18 hours. After phase separation, which yielded a 1/10 volume ratio, the top, micelle- poor, gold probe-rich phases were withdrawn using syringe and needle sets, and were consequently used in the LFA as described previously (see Combining Concentration of Transferrin with the Lateral-Flow Immunoassay). The results of the LFA with the prior concentration step are shown in Figure 13. The negative control without any Tf is shown in panel (a). The remaining solutions contained Tf at concentrations of (b) 10, (c) 5, (d) 1 , (e) 0.5, (f) 0.1, (g) 0.05, (h) 0.01 , and (i) 0.005 μg/mL. While the test line appeared for the negative control solution, which did not contain any Tf, indicating a negative result, the test line did not appear for the decreasing

concentration of Tf until 0.01 and 0.005 μg/mL (Figures 13 h and i). This indicated a detection limit of approximately 0.05 μg/mL for the Tf LFA when combined with the aqueous two-phase micellar system, which represented a 10-fold improvement of the detection limit of the LFA assay. It should be noted that, in this study, a volume ratio of only 1/10 was utilized to demonstrate the improvement of the concentration step on LFA's detection limit. In the future, even lower volume ratios or extractions-in- series can be used to obtain even lower detection limits.

CONCLUSIONS

Concentrating proteins, such as food allergens and protein toxins, prior to a detection step via LFA can improve the detection limit of the immunoassay. In this study, the partitioning and concentration of a model protein, namely human transferrin, using a novel modification of the aqueous two-phase Triton X-l 14 micellar system was investigated. Specifically, anti-Tf antibodies bound to colloidal gold nanoparticles, or gold probes, were utilized to enhance the partitioning of Tf in the aqueous two-phase micellar system. By utilizing an aqueous two-phase system that yielded a volume ratio of 1/10 prior to LFA, gold probes bound to Tf molecules were concentrated in the top, micelle-poor phase 10-fold in a predictive manner, and the detection limit of LFA was improved by approximately 10-fold. In the future, the operating conditions could be manipulated to obtain even lower volume ratios or extractions-in-series could be performed, which in turn should result in even lower detection limits of LFA.

While particular embodiments of the present invention have been shown and described, it will be obvious to those skilled in the art that changes and modifications can be made without departing from this invention in its broader aspects. Therefore, the appended claims are to encompass within their scope all such changes and modifications as fall within the true spirit and scope of this invention.

Claims

We Claim:

1. A method for detecting an analyte in a source, comprising:

(a) concentrating the analyte using a phase separation system to generate a sample of concentrated analyte,

(b) subjecting the sample of concentrated analyte to a lateral-flow

immunoassay, and

(c) detecting the analyte.

2. The method according to Claim 1 , wherein the analyte is a target biomolecule.

3. The method according to Claim 1, wherein the analyte is a protein, a virus, a DNA, an RNA, a ribosome, a bacterium, a mammalian cell, a metabolite, or combinations thereof.

4. The method according to Claim 1, wherein the analyte is an allergen, a bioterrorism agent, influenza, an infectious agent, a biomarker for disease, or combinations thereof.

5. The method according to Claim 1 , wherein the phase separation system is an aqueous two-phase system.

6. The method according to Claim 5, wherein the aqueous two-phase system is an aqueous two-phase micellar system, an aqueous two-phase polyethylene glycol-salt system, an aqueous two-phase polymer 1 -polymer 2 system, or an aqueous two-phase polymer-surfactant system.

7. The method according to Claim 1, wherein the lateral-flow immunoassay comprises an LFA strip, colloidal gold conjugates, and antibodies.

8. The method according to Claim 7, wherein the LFA strip comprises a sample pad, a conjugate pad, a nitrocellulose membrane, an absorbent pad, and an adhesive vinyl backing.

9. The method according to Claim 1, wherein the lateral-flow immunoassay is a sandwich assay.

10. The method according to Claim 1, wherein the lateral-flow immunoassay is a competition assay.

1. The method according to Claim 7, where the colloidal gold conjugates comprise a gold alloy.

12. The method according to Claim 7, wherein the colloidal gold conjugates comprise gold nanoparticles, gold microparticles, or a mixture thereof.

13. A point-of-care device for detecting an analyte in a source, comprising:

(a) a concentrating component to concentrate the analyte using a phase separation system to generate a sample of concentrated analyte, and

(b) a lateral-flow immunoassay component to subject the sample of

concentrated analyte to a lateral-flow immunoassay to detect the analyte.

14. The point-of-care device according to Claim 13, wherein the analyte is a target biomolecule.

15. The point-of-care device according to Claim 13, wherein the analyte is a protein, a virus, a DNA, an RNA, a ribosome, a bacterium, a mammalian cell, a metabolite, or combinations thereof.

16. The point-of-care device according to Claim 13, wherein the analyte is an allergen, a bioterrorism agent, influenza, an infectious agent, a biomarker for disease, or combinations thereof.

17. The point-of-care device according to Claim 13, wherein the phase separation system is an aqueous two-phase system.

18. The point-of-care device according to Claim 17, wherein the aqueous two- phase system is an aqueous two-phase micellar system, an aqueous two-phase polyethylene glycol-salt system, an aqueous two-phase polymer 1 -polymer 2 system, or an aqueous two-phase polymer-surfactant system.

19. The point-of-care device according to Claim 13, wherein the lateral-flow immunoassay comprises an LFA strip, colloidal gold conjugates, and antibodies.

20. The point-of-care device according to Claim 19, wherein the LFA strip comprises a sample pad, a conjugate pad, a nitrocellulose membrane, an absorbent pad, and an adhesive vinyl backing.

21. The point-of-care device according to Claim 13, wherein the lateral-flow immunoassay is a sandwich assay.

22. The point-of-care device according to Claim 13, wherein the lateral-flow immunoassay is a competition assay.

23. The point-of-care device according to Claim 20, wherein the colloidal gold conjugates comprise a gold alloy.

24. The point-of-care device according to Claim 20, wherein the colloidal gold conjugates comprise gold nanoparticles, gold microparticles, or a mixture thereof.

25. The point-of-care device according to Claim 20, which is a test kit.