阅读说明：本技术 单步atps增强型lfa诊断设计 (Single step ATPS enhanced LFA diagnostic design ) 是由 D·T·卡梅 B·M·吴 G·L·莫斯利 Y·T·赵 D·Y·佩雷拉 C·M·吴 韩玥 于 2018-05-30 设计创作，主要内容包括：在各种实施方式中,提供了单步基于纸的ATPS的诊断测定法,其利用ATPS组分的依次再溶解的概念来引起纸内所需的相分离行为。在一个说明性实施方式中,提供了一种芯,其用于在纸中的双水相提取系统中浓缩分析物,其中,所述芯包括被构造成接收样品的纸,其中所述纸包括第一区域和第二区域,所述第一区域包含双水相系统(ATPS)的第一组分,其中所述第一组分为干燥状态,所述第二区域包含双水相系统(ATPS)的第二组分,其中所述第二组分为干燥状态；并且将所述第一区域和第二区域布置成使得当所述吸液芯与流体样品接触时,所述ATPS的第一组分在第二组分之前水化。在某些实施方式中,第一组分和第二组分布置为使得它们基本上同时水化。(In various embodiments, a single-step paper-based ATPS diagnostic assay is provided that utilizes the concept of sequential resolubilization of ATPS components to induce the desired phase separation behavior within the paper. In one illustrative embodiment, a cartridge is provided for concentrating an analyte in an aqueous two-phase extraction system in paper, wherein the cartridge comprises paper configured to receive a sample, wherein the paper comprises a first region comprising a first component of an aqueous two-phase system (ATPS), wherein the first component is in a dry state, and a second region comprising a second component of the aqueous two-phase system (ATPS), wherein the second component is in a dry state; and arranging the first and second regions such that when the wick is contacted with a fluid sample, the first component of the ATPS hydrates before the second component. In certain embodiments, the first component and the second component are disposed such that they are hydrated substantially simultaneously.)

1. A cartridge for concentrating an analyte in an aqueous two-phase extraction system of paper, the cartridge comprising:

a paper configured to receive the sample,

wherein the paper comprises:

a first zone containing a first component of an aqueous two-phase system (ATPS), wherein the first component is in a dry state; and

a second zone containing a second component of an Aqueous Two Phase System (ATPS), wherein the second component is in a dry state;

wherein the first and second regions are arranged such that the first component of the ATPS hydrates before the second component when the core is contacted with a fluid sample; or

Wherein the paper comprises: a region containing a first component of an aqueous two-phase system (ATPS) and a second component of the aqueous two-phase system, wherein the first component and the second component are in a dry state such that when the core is contacted with a fluid sample, the first component of the ATPS and the second component of the ATPS hydrate substantially simultaneously.

2. The core of claim 1, wherein the paper comprises:

a first zone containing a first component of an aqueous two-phase system (ATPS), wherein the first component is in a dry state; and

a second zone containing a second component of an Aqueous Two Phase System (ATPS), wherein the second component is in a dry state; and

wherein the first and second regions are arranged such that the first component of the ATPS hydrates before the second component when the core is contacted with a fluid sample.

3. The core of any of claims 1-2, wherein the core is configured such that a first component of the ATPS flows into the second component of the ATPS upon hydration, thereby segregating the second component to provide a mixed phase that separates into a first phase comprising the first component and a second phase comprising the second component as the ATPS moves through the core.

4. The core of any of claims 1-3, wherein the first component and the second component are components of a polymer/salt (ATPS), wherein the first component comprises a salt and the second component comprises a polymer.

5. The core of claim 4, wherein the salt comprises one or more salts selected from the group consisting of: potassium phosphate, sodium sulfate, magnesium sulfate, ammonium sulfate, sodium citrate, magnesium chloride, magnesium citrate, magnesium phosphate, sodium chloride, potassium citrate, and potassium carbonate.

6. The core of claim 5, wherein the salt comprises potassium phosphate.

7. The core of any of claims 4-6, wherein the salt ranges from about 0.1% w/w to about 40% w/w, or from about 1% w/w to at most about 30% w/w, or from about 5% w/w to at most about 25% w/w, or from about 10% w/w to at most about 20% w/w.

8. The core of claim 7, wherein the salt is present at about 15% (w/w).

9. The core of any of claims 4-8, wherein the polymer comprises a polymer selected from the group consisting of: polyethylene glycol (PEG), ethylene/propylene copolymers (e.g. UCON) ^TM50-HB), propylene glycol (PPG), methoxypolyethylene glycol and polyvinylpyrrolidone.

10. The core of claim 9, wherein the polymer comprises polyethylene glycol (PEG).

11. The core of claim 10, wherein the PEG has a molecular weight of about 1,000 to about 100,000, or about 4,000 to about 50,000, or about 5,000 to up to about 40,000, or up to about 30,000, or up to about 20,000.

12. The core of claim 11, wherein the polymer comprises polyethylene glycol (PEG)8000 MW.

13. The core of any of claims 4-12, wherein the polymer comprises from about 1% w/w to about 30% w/w, or from about 5% w/w to at most about 25% w/w, or from about 10% w/w to at most about 20% w/w of polymer.

14. The core of claim 13, wherein the polymer comprises about 10% (w/w).

15. The core of any of claims 1-14, wherein the paper comprises a material selected from the group consisting of: cellulose, glass fiber, nitrocellulose, polyvinylidene fluoride, nylon, charge modified nylon, polyethersulfone, Polytetrafluoroethylene (PTFE), and combinations thereof.

16. The core of claim 15 wherein the paper comprises glass fibers.

17. The core of any of claims 1-16, wherein the core comprises multiple layers of the paper.

18. The core of claim 17, wherein the core comprises at least 3, or at least 4, or at least 5, or at least 6, or at least 7, or at least 8, or at least 9, or at least 10, or at least 15 or at least 20 layers of the paper.

19. The core of claim 17 wherein the core comprises about 5 layers of the paper.

20. The core of any of claims 1-19, wherein a region free of ATPS component is disposed between the first region and the second region.

21. The core of any of claims 1-19, wherein the first region is disposed adjacent to the second region.

22. The core of any of claims 1-21, wherein the core comprises a sample application zone.

23. The cartridge of claim 22, wherein the sample application zone comprises a sample pad.

24. The cartridge of any one of claims 1-23, wherein the cartridge tapers in a region downstream of the second region and upstream of LFA when a Lateral Flow Assay (LFA) is in fluid communication with the cartridge.

25. The cartridge of any one of claims 1-24, wherein the cartridge is configured to couple to a Lateral Flow Assay (LFA) and provide fluid communication from the cartridge to the LFA.

26. The core of claim 25, wherein the core is configured to be coupled to an LFA such that a plane of the core is perpendicular to a plane of the LFA.

27. The core of claim 25, wherein the core is configured to be coupled to an LFA such that a plane of the core is parallel to a plane of the LFA.

28. The cartridge of claim 25, wherein the cartridge is coupled to a lateral flow immunoassay.

29. The core of claim 28 wherein the core is coupled to an LFA such that the plane of the core is parallel to the plane of the LFA.

30. The core of claim 28, wherein the core is connected to an LFA such that the plane of the core is perpendicular to the plane of the LFA.

31. The cartridge of any one of claims 28-30, wherein the lateral flow assay comprises:

an LFA paper comprising:

a conjugate zone comprising a conjugate comprising an indicator moiety linked to a binding moiety that binds to an analyte to be detected; or the conjugate region is configured to receive a nanoconjugate complexed with an analyte to be detected;

an absorption zone; and

a detection zone comprising a portion that captures the analyte/nanoconjugate complex.

32. The core of claim 31, wherein the detection zone comprises a detection line.

33. The core of any one of claims 31-32 wherein the LFA comprises a control zone comprising a portion that captures an analyte/nanoconjugate complex and the nanoconjugate in the absence of the analyte.

34. The core of any of claims 31-33, wherein the control zone comprises a control line.

35. The cartridge of any one of claims 31-34, wherein the conjugate region comprises a conjugate pad.

36. The core of any of claims 31-35, wherein the absorbent region comprises an absorbent pad.

37. The core of any of claims 31-36 wherein the LFA paper material is the same as the paper comprising the core.

38. The core of any one of claims 31-37 wherein the LFA paper is a different material than the paper comprising the core.

39. The core of any one of claims 31-38, wherein the LFA paper comprises a material selected from the group consisting of: cellulose, glass fiber, nitrocellulose, polyvinylidene fluoride, nylon, charge modified nylon, polyethersulfone, Polytetrafluoroethylene (PTFE), polyester, and combinations thereof.

40. The core of claim 39 wherein the LFA paper comprises nitrocellulose.

41. The core of claim 39 wherein the LFA paper comprises glass fibers.

42. The cartridge of any one of claims 22-23 or 31-41, wherein the sample application zone of the cartridge or the conjugate zone of the LFA comprises a nanoconjugate comprising an indicator moiety linked to an analyte binding moiety that binds to an analyte to be detected.

43. The core of claim 42, wherein the analyte binding moiety is selected from the group consisting of: antibodies, lectins, proteins, glycoproteins, nucleic acids, monomeric nucleic acids, polymeric nucleic acids, aptamers, aptazymes, small molecules, polymers, lectins, carbohydrates, polysaccharides, sugars, and lipids.

44. The core of claim 43, wherein the analyte binding moiety comprises an antibody that binds to the analyte.

45. The core of any of claims 42-44, in which the indicator comprises a moiety selected from the group consisting of: a colorimetric indicator, a fluorescent indicator, and a moiety capable of being bound by a construct comprising a colorimetric or fluorescent indicator.

46. The core of any of claims 42-45, in which the indicator comprises a material selected from the group consisting of: synthetic polymers, metals, minerals, glass, quartz, ceramics, biopolymers, plastics, and combinations thereof.

47. The wick of any one of claims 42-46, wherein the indicator comprises a colorimetric indicator.

48. The core of claim 47, in which the indicator comprises gold nanoparticles.

49. A system for detecting an analyte, the system comprising:

a container comprising a dried nanoconjugate, the nanoconjugate comprising an indicator moiety linked to an analyte binding moiety that binds to the analyte; and

an apparatus comprising a first paper comprising components of an aqueous two-phase system, wherein the first paper is in fluid communication with a Lateral Flow Assay (LFA), and wherein the first paper comprises:

a first zone containing a first component of an aqueous two-phase system (ATPS), wherein the first component is in a dry state; and

a second zone comprising a second component of an Aqueous Two Phase System (ATPS), wherein the second component is in a dry state;

wherein:

the first and second regions being arranged such that the first component of the ATPS hydrates before the second component when the core is contacted with a fluid sample; or

The first region and the second region are the same region, and the first component and the second component are each distributed over substantially the same region.

50. The system of claim 49, wherein the first region and the second region are the same region, and the first component and second component are each distributed over substantially the same region.

51. The system of any one of claims 49-50, wherein the first component and the second component are components of a polymer/salt (ATPS), wherein the first component comprises a salt and the second component comprises a polymer.

52. The system of claim 51, wherein the salt comprises one or more salts selected from the group consisting of: potassium phosphate, sodium sulfate, magnesium sulfate, ammonium sulfate, sodium citrate, magnesium chloride, magnesium citrate, magnesium phosphate, sodium chloride, potassium citrate, and potassium carbonate.

53. The system of claim 52, wherein the salt comprises potassium phosphate.

54. The system of any one of claims 51-53, wherein the polymer comprises a polymer selected from the group consisting of: polyethylene glycol (PEG), ethylene/propylene copolymers (e.g. UCON) ^TM50-HB), propylene glycol (PPG), methoxypolyethylene glycol and polyvinylpyrrolidone.

55. The system of claim 54, wherein the polymer comprises an ethylene/propylene copolymer (e.g., UCON) ^TM50-HB)。

56. The system of any of claims 49-55, wherein the first paper comprises a material in the group consisting of: cellulose, glass fiber, nitrocellulose, polyvinylidene fluoride, nylon, charge modified nylon, polyethersulfone, Polytetrafluoroethylene (PTFE), polyester, and combinations thereof.

57. The system of claim 56, wherein the first paper comprises fiberglass.

58. The system of any one of claims 49-57, wherein the first paper comprises a single layer of the paper.

59. The system of any one of claims 49-57, wherein the first paper comprises a plurality of layers of the paper.

60. The system of claim 59, wherein the first paper comprises at least 3, or at least 4, or at least 5, or at least 6, or at least 7, or at least 8, or at least 9, or at least 10, or at least 15, or at least 20 layers of the paper.

61. The system of any one of claims 49-60, wherein a spacer is disposed between the first paper and the lateral flow assay, wherein the spacer provides fluid communication between the first paper and the lateral flow assay.

62. The system of claim 61, wherein the spacer is treated to reduce non-specific binding of analytes and/or nanoconjugates and/or nanoconjugate/analyte complexes.

63. The system of claim 62, wherein the spacers are treated with BSA.

64. The system of any one of claims 62-63, wherein the spacer comprises a material selected from the group consisting of: cellulose, glass fiber, nitrocellulose, polyvinylidene fluoride, nylon, charge modified nylon, polyethersulfone, Polytetrafluoroethylene (PTFE), polyester, and combinations thereof.

65. The system of claim 64, wherein the spacer paper comprises fiberglass.

66. The system of any one of claims 49-60, wherein the paper is disposed adjacent to a lateral flow assay.

67. The system of any one of claims 49-66, wherein the lateral flow assay comprises:

LFA paper, the LFA paper comprising:

an absorption zone; and

a detection zone comprising a portion that captures the analyte/nanoconjugate complex.

68. The system of claim 67, wherein the detection zone comprises a detection line.

69. The system of any one of claims 67-68, wherein the LFA comprises a control zone comprising a portion that captures an analyte/nanoconjugate complex and the nanoconjugate in the absence of the analyte.

70. The system of claim 69, wherein the control zone comprises a control line.

71. The system of any one of claims 67-70, wherein the absorbent region comprises an absorbent pad.

72. The system of any one of claims 67-71, wherein the LFA paper is the same material as the first paper.

73. The system of any one of claims 67-71, wherein the LFA paper is a different material than the first paper.

74. The system of any one of claims 67-73, wherein the LFA paper comprises a material selected from the group consisting of: cellulose, glass fiber, nitrocellulose, polyvinylidene fluoride, nylon, charge modified nylon, polyethersulfone, Polytetrafluoroethylene (PTFE), polyester, and combinations thereof.

75. The system of claim 74, wherein the LFA paper comprises nitrocellulose.

76. The system of any one of claims 49-75, wherein the analyte binding moiety is selected from the group consisting of: antibodies, lectins, proteins, glycoproteins, nucleic acids, monomeric nucleic acids, polymeric nucleic acids, aptamers, aptazymes, small molecules, polymers, lectins, carbohydrates, polysaccharides, sugars, and lipids.

77. The system of claim 76, wherein the analyte binding moiety comprises an antibody that binds to the analyte.

78. The system of any one of claims 76-77, wherein the indicator comprises a moiety selected from the group consisting of: a colorimetric indicator, a fluorescent indicator, and a moiety capable of being bound by a construct comprising a colorimetric or fluorescent indicator.

79. The system of any one of claims 76-78, wherein the indicator comprises a material selected from the group consisting of: synthetic polymers, metals, minerals, glass, quartz, ceramics, biopolymers, plastics, and combinations thereof.

80. The system of any one of claims 76-79, wherein the indicator comprises a colorimetric indicator.

81. The system of claim 80, wherein the indicator comprises gold nanoparticles.

82. A method of detecting and/or quantifying an analyte in a sample, the method comprising:

providing an aqueous solution or suspension comprising the sample; and

applying the solution to the core of any one of claims 1-48, wherein the solution sequentially hydrates the first component and the second component as the solution moves through the core and causes the analyte to partition into the phases of the ATPS;

delivering the ATPS into the lateral flow assay; and

detecting and/or quantifying the analyte in the lateral flow assay if the analyte is present.

83. The method of claim 82, wherein the delivering comprises contacting the wick of any one of claims 1-30 with a sample-receiving region of the lateral flow assay.

84. The method of claim 82, wherein the wick is in fluid communication with the wick and the ATPS flows into the LFA.

85. The method of claim 84, wherein the core is the core of any one of claims 28-48.

86. A method of detecting and/or quantifying an analyte in a sample, the method comprising:

providing the system of any one of claims 49-81;

introducing the sample into the container containing dried nanoconjugates to hydrate the nanoconjugates and contact the nanoconjugates with the sample, wherein the nanoconjugates form nanoconjugate/analyte complexes when the analyte is present in the sample;

contacting a region of the device containing the components of an aqueous two-phase system and hydrating the components, wherein the hydrated components flow through the lateral flow assay; and

detecting and/or quantifying the analyte in the lateral flow assay if the analyte is present.

87. The method of any one of claims 82-86, wherein the sample is untreated prior to application to the device.

88. The method of any one of claims 82-86, wherein the sample is diluted prior to application to the device.

89. The method of claim 88, wherein the sample is diluted with Phosphate Buffered Saline (PBS).

90. The method of any one of claims 82-89, wherein the subject is a human.

91. The method of any one of claims 82-89, wherein the subject is a non-human mammal.

92. The method of any one of claims 82-91, wherein the sample is selected from the group consisting of: biological samples (e.g., oral fluid or tissue samples, nasal fluid, urine, blood or blood fractions, cerebrospinal fluid, lymph, tissue biopsies, vaginal samples, etc.), food samples, and environmental samples.

93. The method of any one of claims 82-92, wherein the analyte comprises a bacterium, fungus, protozoan, virus, or component thereof.

94. The method of any one of claims 82-92, wherein the analyte comprises a marker of infection.

95. The method of claim 94, wherein the marker comprises an antibody against an infectious pathogen (e.g., an anti-HIV antibody).

96. A kit, comprising:

a container containing the wick of any one of claims 1-48; and/or

A container housing the container and/or apparatus of the system of any one of claims 49-82.

Technical Field

This application claims the benefits and priority of USSN62/513,347 filed on 31/5/2017, which is incorporated herein by reference in its entirety for all purposes.

Statement of government support

The invention was made with government support of the national science foundation of the united states, grant number 1549003. The government has certain rights in the invention.

Background

Infectious diseases such as chlamydia and HIV greatly affect developed and developing countries. Chlamydia infection is a Sexually Transmitted Infection (STI) caused by Chlamydia trachomatis (Chlamydia trachomatis) bacteria, and if left untreated, can lead to pelvic inflammatory disease in women and cause permanent damage to the reproductive system (Hafner (2015) content, 92: 108-115). Since 1993, the prevalence of chlamydia has steadily increased in the United states, and more than 140 million new chlamydia infections have been reported in 2014 (Centers for Disease Control and preservation (2014) Sexually Transmitted Disease Surveillance 2014: 1-176). While chlamydia is relatively straightforward to treat, it shows no evidence of resistance to the primary drug treatment options (Krupp & Madhivanan (2015) Indian JSex trans Dis 36: 3-8), it is still one of the most common STIs in the united states (Centers for Disease control and preservation (2014) Sexually Transmitted Disease 2014: 1-176). On the other hand, HIV is caused by the human immunodeficiency virus, which attacks the human immune system, in particular CD4 cells. Only in 2015 there were approximately 210 new HIV cases worldwide, and approximately 39,513 people were diagnosed with HIV in the united states (CDC (2015) HIV surfecil. rep.27: 1-82). One approach to address the increasing incidence of chlamydial infection and aids is through Low-cost point-of-care (POC) screening of at-risk populations, with encouraging results shown by theoretical models (Huang et al (2013) Sex trans. infection.89: 108-114.doi: 10.1136/textrans-2011-.

Unfortunately, none of the current gold-based standard laboratory diagnostic methods (e.g., ELISA tests, Nucleic Acid Amplification Tests (NAAT), or cell culture methods) are suitable for POC screening. This is due to the high cost of the equipment, the need for trained personnel and the long time required to obtain the results. In contrast, paper-based diagnostics is a more suitable technique, with two components required to efficiently conduct large-scale screening: on-site diagnosis and treatment at the same visit, and management of untrained or minimally trained personnel. The most commonly used paper diagnostic method is Lateral flow assay technology (LFA), an antibody-based diagnostic method that is illustrated visually and is recognized for its wide application in pregnancy tests (Wong & Tse (2009) terrestrial flow immunoassay,1st ed. springer, New York). Unfortunately, chlamydia LFA detection is currently not sensitive enough to be effectively diagnosed (Land et al (2009) hum. reprod. update,16: 189-. Although the HIVLFA assay is more mature in the consumer market than the chlamydia LFA assay, there is still room to improve its sensitivity to further reduce the risk of false negatives and potential spread of the virus.

In recent years, great efforts have been made to improve the sensitivity of paper-based assays. Some key innovations include the work of Yager laboratories using two-dimensional paper networks (Fu et al (2010) transmitters, BChem.149: 325-328; Fu et al (2010) Lab Chip,10: 918-920; Osborn et al (2010) Labchip,10: 2659-2565; Fu et al (2011) Microfluid nanofluid diodes, 10: 29-35; Kauffman et al (2010) Lab Chip,10: 2614-2617; Fridley et al (2012) Lab Chip,12: 4321; Fu et al (2012) Anle.84: 4574-4579; Lutz et al (2013) Lab Chip,13: 2840) and Baeid et al 4248; Baeid et al 4253: 4248; Taber et al 4248: 2014-42), 15:655-659). Previously, our laboratory developed a device-free method for thermodynamic preconcentration of target analytes prior to their application to LFA testing. Briefly, this is accomplished by using an aqueous two-phase system (ATPS) that separates into two distinct liquid phases, wherein the target analyte partitions thoroughly into one of the two phases, effectively concentrating the target. In the first approach, our three-step diagnostic process involves (i) mixing a large amount of target solution with the ATPS component, (ii) waiting for macroscopic phase separation, and (iii) extracting the concentrated target phase and applying it to the LFA test. Using this method, we demonstrated improved detection limits for both large viruses (Jue et al (2014) Biotechnol. Bioeng.111: 2499-2507; Mashayekhi et al (2010) Anal. Bioanal. chem.398:2955-2961) and small protein targets (Mashayekhi et al (2012) Anal. Bional. chem.404: 2057-2066; Chiu et al (2014) Ann. biomed. Eng.42(11): 2322-2332). Recently, we have found that the phase separation process is accelerated as the ATPS flows through the paper, reducing the total diagnostic time from hours to minutes by omitting the waiting and extraction steps. Using this phenomenon, our laboratory demonstrated the ability to simultaneously concentrate and detect protein biomarkers in paper (Chiu et al (2014) LabChip,14: 3021-. The diagnostic process still requires an ATPS component mixing step prior to applying the solution to the LFA test strip, which may be suitable for applications that otherwise require mixing into a predetermined buffer (e.g., swab-based diagnostics).

Disclosure of Invention

In various embodiments, described herein is a single-step ATPS paper-based diagnostic assay based on a novel concept of sequentially redissolving ATPS components to produce the desired phase separation behavior within the paper.

Various embodiments contemplated herein may include, but are not necessarily limited to, one or more of the following:

Drawings

FIG. 1 shows a schematic of a typical lateral flow immunoassay test strip (top panel) and a lateral flow immunoassay in sandwich format (bottom panel).

Figure 2 illustrates the rehydration sequence for the PEG/salt ATPS component. The phase separation in the single sheet of the ARROW design was visualized over time when PEG and potassium phosphate were rehydrated in separate regions and they were rehydrated as a mixture. A close-up image of the downstream region where phase separation occurred is shown, so the first image is located at t 6 seconds instead of t 0. Visualization and identification of the PEG-rich, PEG-poor and macro-mixed domain regions was achieved by flowing suspensions of BSA-DGNP and bright blue dye.

FIG. 3 illustrates the rehydration sequence for the UCON/SALT ATPS component. When UCON ^TMThe phase separation within a single glass fiber rod was visualized over time upon rehydration of-50-HB-5100 and potassium phosphate in separate regions and their rehydration as a mixture. The image was cut to the same area containing the fiberglass strip to observe the relative flow rate. Visualization and identification of the UCON-rich, UCON-poor and macro-mixed domain regions was achieved by flowing a suspension of BSA-GNPs and brilliant blue dye.

FIG. 4(a-b diagram) shows the kinetics of phase separation. Graph a) captures an image over time of ARROW with separate two-phase components during fluid flow. The liquid consisted of a suspension of BSA-DGNP and bright blue dye, which allowed phase separation to be observed. Panel b) takes a time image of the mixed UCON/salt design during rehydration by suspension of BSA-GNP and brilliant blue dye.

FIGS. 5A and 5B illustrate one embodiment of an ARROW and LFA integrated diagnostic design layout. Fig. 5A shows the ARROW and LFA integrated diagnostics (note that in certain embodiments, the glass fibers may be replaced with other materials). Fig. 5B is an ARROW and SEM image of the ARROW and LFA integrated diagnostic design layout and includes dehydrated PEG on glass fibers, blank glass fibers, and dehydrated potassium phosphate on glass fibers. In the embodiment shown, the top and bottom tips of the fiberglass paper are also blank fiberglass.

Figure 6 shows one embodiment of the TUBE and LFA integrated design, which includes a sample TUBE containing dried GNP conjugate and a test strip containing UCON/SALT ATPS dehydrated in a glass fiber pad. SEM images of the UCON/salt pad, BSA treated spacer and nitrocellulose membrane are also shown.

Figure 7 illustrates that by introducing ARROW, the detection limit of chlamydia trachomatis LFA can be improved. A comparison of LFA results at different concentrations of chlamydia trachomatis with and without ARROWs is presented. The test line is located at the bottom of the LFA strip and the control line is located at the top of the LFA strip. For 0 ng/. mu.L Chlamydia trachomatis, the results of the negative control are shown in the left-most panel.

Figure 8 illustrates the improvement in the detection limit of human IgM LFA by introduction of TUBE. Comparison of LFA results at different human IgM concentrations with and without TUBE was compared. The test line is located at the bottom of the LFA strip and the control line is located at the top of the LFA strip. The negative control results are shown in the leftmost panel.

FIG. 9 is a-b graph showing quantitative LFA test line intensity profiles for ARROW/LFA system and LFA only system (Panel a), TUBE/LFA system and LFA only system (Panel b).

embodiment 1: a cartridge for concentrating an analyte in an aqueous two-phase extraction system of paper, the cartridge comprising:

a paper configured to receive a sample, wherein the paper comprises:

a first zone containing a first component of an aqueous two-phase system (ATPS), wherein the first component is in a dry state; and

a second zone containing a second component of an Aqueous Two Phase System (ATPS), wherein the second component is in a dry state;

wherein the first and second regions are arranged such that the first component of the ATPS hydrates before the second component when the core is contacted with a fluid sample; or wherein the paper comprises:

a region comprising both a first component of an aqueous two-phase system (ATPS) and a second component of an aqueous two-phase system, wherein the first component and the second component are in a dry state such that when the core is contacted with a fluid sample, the first component of the ATPS and the second component of the ATPS hydrate substantially simultaneously.