WO2017087834A1

WO2017087834A1 - Multiplex diagnostic assay cartridge for detection of a plurality of target molecules

Info

Publication number: WO2017087834A1
Application number: PCT/US2016/062832
Authority: WO
Inventors: David Erickson; Seoho LEE; Saurabh MEHTA; Dakota O'DELL
Original assignee: Cornell University
Priority date: 2015-11-18
Filing date: 2016-11-18
Publication date: 2017-05-26

Abstract

A multiplex diagnostic assay cartridge includes a plurality of diagnostic assay units within a chamber defined by walls of the cartridge. A pre-processing module is within the chamber and includes a sample input module positioned to receive material inserted through the inlet passage in the wall of the housing. One or more sample delivery channels are in fluid communication with the sample input module and at least one of the plurality of diagnostic assay units so that the sample inserted through the inlet passage in the wall of the housing and into the sample input module passes through the sample delivery channels to direct the sample to one or more of the diagnostic assay units. One or more sample processing units are in fluid communication with at least one of the sample delivery channels to selectively process the sample prior to entering at least one of the diagnostic assays. Methods of conducting diagnostic assays are also disclosed.

Description

MULTIPLEX DIAGNOSTIC ASSAY CARTRIDGE FOR DETECTION OF A

PLURALITY OF TARGET MOLECULES

[0001] This application claims the priority benefit of U.S. Provisional Patent Application

Serial No. 62/256,936, filed November 18, 2015, which is hereby incorporated by reference in its entirety.

GOVERNMENT FUNDING

[0002] This invention was made with Government support under Grant Number 1343058 awarded by NSF. The United States Government has certain rights in the invention.

FIELD OF THE INVENTION [0003] The present invention relates to a multiplex diagnostic assay cartridge for detection of a plurality of target molecules in a sample and methods of use thereof.

BACKGROUND OF THE INVENTION

[0004] Suboptimal nutrition is considered the biggest obstacle for human development and progress. More disability-adjusted life years are lost to malnutrition than any other medical condition worldwide; over 1,000,000 people die every year from vitamin A and zinc deficiencies alone. Domestically, nearly 30% of all cancers are estimated to be a result of poor diet.

Micronutrient deficiencies affect 75% of US adults and are responsible for a multitude of health conditions including anemia, rickets, scurvy, and cancer.

[0005] Many micronutrient deficiencies can be reversed through changes in diet or by taking supplements. However most people have little information about their micronutrient status and therefore are unaware of the severity of their deficiencies. Whether domestic or abroad, the key to solving this problem is the development of a simple, easy to deploy, tool that can inform people as to their micronutrient and broader nutritional status.

[0006] The present invention is directed to overcoming these and other deficiencies in the art. SUMMARY OF THE INVENTION

[0007] One aspect of the present invention relates to a multiplex diagnostic assay cartridge for detection of a plurality of target molecules in a sample. The multiplex diagnostic assay cartridge includes an elongate housing having walls defining a chamber. The cartridge extends between a first end, where the sample is inserted through an inlet passage in a wall of the housing and into the chamber, and a second end at which results of the assay can be assessed. A plurality of diagnostic assay units are within the chamber defined by the walls of the cartridge. A pre-processing module is within the chamber defined by the walls of the housing. The preprocessing module includes a sample input module positioned to receive material inserted through the inlet passage in the wall of the housing. One or more sample delivery channels are in fluid communication with the sample input module and at least one of the plurality of diagnostic assay units so that the sample inserted through the inlet passage in the wall of the housing and into the sample input module passes through the one or more sample delivery channels in the pre-processing module to direct the sample to one or more of the plurality of diagnostic assay units. One or more sample processing units are in fluid communication with at least one of the one or more sample delivery channels to selectively process the sample prior to entering at least one of the plurality of diagnostic assays.

[0008] Another aspect of the present invention relates to a method of conducting a multiplex diagnostic assay. The method includes providing a multiplex diagnostic cartridge according to the present invention. A sample is inserted through the inlet passage in the wall of the housing and into the chamber, whereby the inserting causes flow of the sample through the one or more sample delivery channels in the pre-processing module to direct the sample to one or more of the plurality of diagnostic assay units. The one or more sample processing units selectively process the sample prior to entering at least one of the plurality of diagnostic assays. The plurality of diagnostic assays are analyzed to determine whether a target material is present in the sample and/or what quantity of the target material is present.

[0009] Yet another aspect of the present invention relates to a method of conducting a multiplex diagnostic assay. The method includes providing a multiplex diagnostic assay cartridge according to the present invention. The sample is inserted through the inlet passage in the wall of the housing and into the chamber, whereby the inserting causes flow of the sample through the one or more sample delivery channels in said pre-processing module to direct the sample to one or more of the plurality of diagnostic assay units. The one or more sample processing units selectively process the sample prior to entering at least one of the plurality of diagnostic assays. A fluid is inserted through the second input module to the secondary delivery channel at substantially the same time as the sample is introduced into the sample input module, whereby the fluid is delayed in mixing with the sample. The plurality of diagnostic assays are analyzed to determine whether a target material is present in the sample and/or what quantity of the target material is present.

[0010] The present invention provides a multiplex test for different nutritional biomarkers including, but not limited to, vitamin Bi₂ vitamin D, vitamin A, and iron

deficiencies, all located on the same cartridge. The present invention may also include a test for C-Reactive Protein (CRP), which is an inflammation biomarker that determines the validity of the nutritional measurements. The present invention advantageously provides the speed and simplicity of traditional rapid diagnostic tests, while at the same time having the ability to: (1) multiplex for different targets, and (2) amplify the test strip signal without the need to perform extra procedures. The present invention relates to a diagnostic assay cartridge that provides a microscale fluid network design which allows for controlling the timing and location of the reagent delivery to and from the standard membrane components that have been widely accepted in rapid diagnostic tests, such as a blood filtration membrane.

BRIEF DESCRIPTION OF THE DRAWINGS [0011] FIGS. 1 A and IB are a perspective view and a side cross-sectional view, respectively, of an embodiment of a multiplex diagnostic assay cartridge of the present invention.

[0012] FIGS. 2A-2C are perspective views of an embodiment of a test strip for use with the multiplex diagnostic assay cartridge of the present invention

[0013] FIG. 3 is a schematic view of another embodiment of a multiplex diagnostic assay cartridge of the present invention.

[0014] FIG. 4 is a schematic view of yet another embodiment of a multiplex diagnostic assay cartridge of the present invention.

[0015] FIG. 5 is a schematic view of a further embodiment of a multiplex diagnostic assay cartridge of the present invention. [0016] FIG. 6 is a schematic view of an additional multiplex diagnostic assay cartridge.

[0017] FIG. 7 is a schematic view of the layers of the experimental multiplex diagnostic assay cartridge shown in FIG. 6.

[0018] FIGS. 8A-8D illustrate preliminary results for a sandwich type lateral flow assay for CRP including strip images at CRP concentrations 0, 1, 3, 5, and lC^g/ml showing that T/C signal increases with increasing CRP concentrations (FIG. 8A); analysis of strip images revealing quantitative information about T/C ratios at different CRP concentrations and confirming that T/C increases with increasing CRP concentration (FIG. 8B); T/C ratio of CRP strips vs. CRP concentrations (FIG. 8C); and kinetics of T/C signal development to distinguish T/C signals of 10 vs. 25 μg/ml CRP (FIG. 8D).

DETAILED DESCRIPTION

[0019] The present invention relates to a multiplex diagnostic assay cartridge for detection of a plurality of target molecules in a sample and methods of use thereof.

[0020] One aspect of the present invention relates to a multiplex diagnostic assay cartridge for detection of a plurality of target molecules in a sample. The multiplex diagnostic assay cartridge includes an elongate housing having walls defining a chamber. The cartridge extends between a first end, where the sample is inserted through an inlet passage in a wall of the housing and into the chamber, and a second end at which results of the assay can be assessed. A plurality of diagnostic assay units are within the chamber defined by the walls of the cartridge. A pre-processing module is within the chamber defined by the walls of the housing. The preprocessing module includes a sample input module positioned to receive material inserted through the inlet passage in the wall of the housing. One or more sample delivery channels are in fluid communication with the sample input module and at least one of the plurality of diagnostic assay units so that the sample inserted through the inlet passage in the wall of the housing and into the sample input module passes through the one or more sample delivery channels in the pre-processing module to direct the sample to one or more of the plurality of diagnostic assay units. One or more sample processing units are in fluid communication with at least one of the one or more sample delivery channels to selectively process the sample prior to entering at least one of the plurality of diagnostic assays. [0021] FIGS. 1 A and IB are perspective and side views, respectively, of a first embodiment of multiplex diagnostic assay cartridge 10(1) of the present invention. Multiplex diagnostic assay cartridge 10(1) may be utilized for performing a multiplex test on a fluid sample for a number of different biomarkers, such as nutritional biomarkers on a single cartridge. In one example, the fluid sample is a blood sample from a finger prick, although other fluid samples such as a plasma sample, a serum sample, a urine sample, a saliva sample, a sweat sample, cerebral spinal fluid, or tears.

[0022] Multiplex diagnostic assay cartridge 10(1) includes elongate housing 12 extends between first end 14 and second end 16. In this example, elongate housing 12 is formed of a transparent plastic, although other suitable transparent materials may be utilized. In one example, housing 12 is formed of a plurality of laser etched layers. Exemplary materials for forming housing 12 include polycarbonate, polystyrene, polym ethyl methacrylate, polyamide, polysulfone, polyoxymethylene, polyetheretherketone, cyclic olefin polymer, polyethylene, polyethylene terephthalate, polyvinylchloride, perfluoroalkoxy, fluorinated etheylenepropylene, glass, cyclic olefin copolymer, silicon, quartz, and polydimethylsiloxane. The transparent nature of housing 12 allows for imaging the materials inside housing 12.

[0023] Housing 12 includes walls 18 defining a chamber 20 within housing 12 as shown in FIG. IB. Housing 12 includes inlet passage 22 located near first end 14 and extending into chamber 20. Inlet passage 22 is configured to receive a fluid sample into chamber 20 for processing and testing. In this example, housing 12 also includes optional inlet passages 23A and 23B for introducing additional fluids into chamber 20 as described in further detail below.

[0024] Chamber 20 in housing 12 is configured to house pre-processing module 24(1) and plurality of diagnostic assay units 26A-26C therein. Although three diagnostic assay units 26A-26C are illustrated and described, it is to be understood that chamber 20 may be configured to house any number of multiplexed diagnostic assay.

[0025] In this example, pre-processing module 24(1) includes sample input module 28 positioned to receive a liquid fluid sample inserted through inlet passage 22 of housing 12 for processing and testing. Pre-processing module 24(1) also includes sample delivery channels 30(1) in fluid communication with sample input module 28. In this embodiment, sample delivery channels 30(1) are also in fluid communication with diagnostic assay units 26A-26C. Sample delivery channels 30(1) provide a network of fluid delivery channels that are configured to receive the fluid sample from sample input module 28 and separately direct the fluid sample to one or more of diagnostic assay units 26A-26C. In this example, sample delivery channels 30(1) direct the sample fluid into each of diagnostic assay units 26A-26C for further processing by dividing the sample fluid among diagnostic assay units 26A-26C. [0026] In this example, pre-processing module 24(1) further includes sample processing units 32A and 32B in fluid communication with sample delivery channels 30(1), although preprocessing module 24(1) may have other numbers of sample processing units as described in the non-limiting examples set forth below that selectively process the fluid sample prior to entering at least one of diagnostic assays 26A-26C. According to the present invention, the sample processing units may be configured in a number of different arrangements along the sample delivery channels to provide selective processing of the fluid sample before it enters one or more of the diagnostic assay units as described with respect to the non-limiting examples below. By way of example only, the sample processing units may comprise one or more of a filtering unit, a dilution unit, a separating unit, or a mixing unit. [0027] Sample processing unit 32A is a filtering unit, such as a blood filtration membrane configured to filter blood added to sample input module 28 such that only plasma will be delivered through sample delivery channels 30(1). By way of example, sample processing unit 32A may be a Fusion 5 membrane produced by GE Whatman, St. Louis, Missouri, or an FR-1 membrane produced by MDI Membrane Technologies, India, although other filtering membranes may be utilized for other fluids. Sample processing unit 32B is a mixing unit for introducing a reagent through inlet passage 23B downstream of sample processing unit 32A. In this example, sample processing units 32A and 32B are located in series to allow both processing units to process the entire sample. However, other processing units may be located in parallel so that they each only process a portion of the sample as divided by sample delivery channels 30(1). The downstream location of sample processing unit 32B allows the additional reagent to be added only to the separated plasma emitted from sample processing unit 32A. By way of example, the additional reagent may be an extraction buffer or agent configured to separate an analyte, such as vitamins B_i2 or D, from their transport proteins. Other reagents can also be used to provide other pre-processing steps necessary prior to introducing the liquid sample to diagnostic assays 26A-26C. [0028] In this example, pre-processing module 24(1) also includes secondary input module 34 for receiving a fluid introduced into chamber 20 through inlet passage 23 A. Second input module 34 is in fluid communication with second delivery channel 36 to deliver a fluid introduced through inlet passage 23 A to diagnostic assay 26A. Additionally, second delivery channel 36 may be in fluid communication with other diagnostic assays in other embodiments. Secondary delivery channel 36 is configured such that a fluid delivered into inlet passage 23A at the same time as the sample is introduced in inlet passage 22 is delayed in interacting with the sample. In this example, secondary delivery channel 36 has a serpentine flow pattern that allows the timing of delivery of the fluid inserted through inlet passage 23 A to diagnostic assay 26A to be increased in a customized fashion depending on the requirements of the assay. In this example, second delivery channel 36 is separate from sample delivery channels 30(1). However, in other embodiments, second delivery channel 36 may be in fluid communication with a portion of sample delivery channels 30(1) to introduce the additional fluid downstream for delivery to one or more of diagnostic assays 26A-26C. [0029] Diagnostic assay units 26A-26C are test strips configured to receive and process a fluid sample to provide an indication of the quantity of a target molecule in the fluid sample. Diagnostic assay units 26A-26C, by way of example, may carry out a competitive assay, a sandwich assay, a colorimetric detection assay, a bead assay, or combinations thereof.

[0030] In a competitive assay, the target molecule in a sample competes with a labelled target molecule to bind a limited number of binding sites on an immobilized binding agent (see O'Kennedy et al., "A Review of Enzyme-Immunoassay and a Description of a Competitive Enzyme-Linked Immunosorbent Assay for the Detection of Immunoglobulin Concentrations," Biochem Edu. 18(3): 136-140 (1990), which is hereby incorporated by reference in its entirety). In one example, the amount of labeled, unbound target molecule detected at the detection region of the assay is inversely proportional to the concentration of target molecule in the sample (i.e., a greater amount of accumulated label indicates lower levels of analyte in the test sample).

[0031] In a sandwich assay or non-competitive assay, the target molecule in an unknown sample is bound to an immobilized binding agent and then labelled with a labeled binding agent (see O'Kennedy et al., "A Review of Enzyme-Immunoassay and a Description of a Competitive Enzyme-Linked Immunosorbent Assay for the Detection of Immunoglobulin Concentrations," Biochem Edu. 18(3): 136-140 (1990), which is hereby incorporated by reference in its entirety). The amount of labelled binding agent detected is proportional to the concentration of target molecule in the sample.

[0032] The concentration of a target molecule in a sample may be determined using various colorimetric methods, which are well known in the art (see Sapan et al., "Colorimetric Protein Assay Techniques," Biotechnol Appl Biochem. 29(Pt2):99-108 (1999) and Okutuku et al., "Comparison of Five Methods for Determination of Total Plasma Protein Concentration," J Biochem Biophys Methods 70(5):709-l 1 (2007), which are hereby incorporated by reference in their entirety).

[0033] In a bead assay, the immobilized binding agent may be bound to a bead. Bead assays are well known in the art (see Thompson et al., " Microfluidic, Bead-Based Assay:

Theory and Experiments," J Chromatogr B Analyt Technol Biomed Life Sci. 878(2):228 (2010), which is hereby incorporated by reference in its entirety).

[0034] As discussed below, the various assays may be located on paper test strips within chamber 20, although a bead assay may be incorporated directly into the plastic microchannel in chamber 20. Non-limiting examples of diagnostic assay unit combinations are discussed in detail below.

[0035] The target molecule may be selected from the group consisting of vitamins, such as vitamin Bi₂ or vitamin D, micronutrients, genetic biomarkers, such as DNA or RNA, carbohydrates, and proteins. Exemplary vitamin target molecules, include, but are not limited to, vitamin A, vitamin D, vitamin E, vitamin Bi (thiamine), vitamin B₂ (riboflavin), vitamin B₃ (niacin), vitamin B₆, vitamin B₇ (biotin), folate (folic acid and vitamin B₉), vitamin Bi₂

(cyanocobalamin), vitamin C, and pantothenic acid. Suitable micronutrients include, but are not limited to, iron, cobalt, zinc, manganese, copper, iodine, selenium, molybdenum, and chromium. Exemplary target nucleic acid molecules include, but are not limited to, viral, bacterial, and tumor-associated nucleic acid molecules. Carbohydrates may be selected from the group consisting of monosaccharides (such as glucose, galactose, fructose, xylose), disaccharides (such as sucrose, lactose, maltose, trehalose), oligosaccharides (such as malto-dextrin, raffinose, and stachyose), and polysaccharides (such as amylose, amylopectin, cellulose, hemicellulose, pectins, and hydrocolloids). Suitable target protein molecules include, but are not limited to, antibodies, antibody fragments, epitopes, hormones, neurotransmitters, cytokines, growth factors, cell recognition molecules, cell receptors, bacterial proteins, viral proteins, toxins, prions, disease-associated proteins, retinol binding proteins, and fragments thereof.

[0036] Different types of lateral flow assays may be utilized to accommodate different biomarker behaviors. For targets with significant molecular size (tens to hundreds of kg/mol) such as RBP, ferritin, soluble transferrin receptor (sTfR), and CRP, sandwich type lateral flow assays may be employed as the molecules allow multiple antibody binding at a time. On the other hand, smaller molecules such as vitamin Bi₂ (molecular size of 1.3kg/mol) and vitamin D (0.4kg/mol) do not allow more than one antibody binding at a time and thus, a competitive type lateral flow assay may be utilized. [0037] For sandwich type assays, the detectable target (RBP, ferritin, sTfR, and/or CRP) of the assay is determined by the choices of the detection and capture antibodies (anti-RBP, anti- ferritin, anti- sTfR, or anti-CRP) during the assay development. The main components of the test strip are a conjugate pad that stores the gold nanoparticle-labeled detection antibody, detection pad that immobilizes the capture antibody for the corresponding target, and the control pad that immobilizes the secondary antibody with an affinity for the common species (e.g., mouse, rabbit, goat, etc.) of the detection antibodies. In operation, the sample will flow downstream via capillary action and mix with the detection antibodies on the conjugate pad. The detection antibodies will interact with the target RBP, ferritin, sTfR, and/or CRP molecules if present in the sample and form a target-to-antibody complex. When transported over the detection pad, only the target-to-antibody complexes will be captured by the capture antibodies which have the affinity towards the corresponding targets but not the detection antibodies alone. The unreacted detection antibodies will flow further downstream and be captured by the secondary antibodies on the control pad. As a result, the detection band will turn increasingly redder for higher concentration of the target molecules in the sample as this will result in more target-to-antibody complexes that are captured. When the target concentration is low, the detection band will exhibit only a subtle change, while there will be a distinctly visible signal on the control pad.

[0038] For the competitive type assays, the detectable target, such as vitamin B_i2 or vitamin D, of the assay is determined by the choices of the detection antibodies (anti-vitamin Bi_2; anti-vitamin D) and the test line protein conjugate (B 12-BSA, vitamin D-BSA) during the assay development. As a competitive lateral flow test, the assay performance depends on the competitive interaction between the target (B_i2 and vitamin D molecules) in the sample and the respective protein conjugates (B12-BSA and vitamin D-BSA) on the test line for the limited binding regions on the gold nanoparticle-antibody conjugates. For samples with high target levels, most of the antibody conjugates are occupied with targets from the initial sample and thus become unavailable for binding to the associated protein conjugates on the test line.

Consequently, high target concentration results in only a subtle change in the colorimetric signal at the test line. For samples with low target levels, the test line develops an intense color that reflects the high number of antibody conjugates bound at the test line.

[0039] One or more of diagnostic assay units 26A-26C may include test strip 100 as shown in FIG. 2. Test strip 100 includes substrate 102, first layer 104, second layer 106, and third layer 108, although the test strip 100 may include other types or numbers of layers in other combinations, such as collection layer 110. In one example, test strip 100 is a competitive diagnostic assay strip, although test strip may be a sandwich assay test strip or a combination of both.

[0040] Substrate 102 has an elongate form extending between first end 130 at which the liquid sample inserted into inlet passage 22 in housing 12 is received and a second end 132. Substrate 112 is configured to support the various layers of test strip 100 as described below. In one example, substrate 112 is a Flow Plus 180 Membrane Card (EMD Millipore, Billerica, Massachusetts) with a 2mm clear polyester film backing to which the layers described below may be adhesively attached, by way of example, although other suitable substrates may be utilized.

[0041] First layer 114 is supported on substrate 112 proximate to first end 130 of test strip 100. First layer 114 provides a membrane for receiving, absorbing, and filtering a liquid sample, such as capillary blood from a finger stick. First layer 114 is made of a material selected from the group consisting of cellulose membranes, polyester matrix, glass fiber, and polysulfone membranes. First layer 114 is vertically stacked relative to substrate 112 to enhance receiving, absorbing, and filtering the liquid sample as described in U.S. Patent Application Publication No. 2016/0080548 and PCT Patent Application PCT/US 14/12263, the disclosures of which are hereby incorporated by reference in their entirety herein.

[0042] Second layer 116 provides a conjugate pad for storing antibody conjugates.

Second layer 116 is supported on substrate 112, distal from both first end 130 and second end 132 of substrate 112 and downstream of first layer 114 such that the liquid sample may flow from first layer 114 to second layer 116. A portion of second layer 116 is overlapped by first layer 114 to enhance fluid flow between first layer 114 and second layer 116. In one example, second layer 116 is made of glass fibers, although other suitable materials may be utilized. Second layer 116 includes mobile labelled specific binding partner 134 located therein. [0043] Mobile labelled specific binding partner 134 is selected to be a binding partner of the target molecule of the assay, such as vitamin Bi₂ as illustrated in FIGS. 2A-2C, such that mobile labelled specific binding partner 134 will bind specifically to the target molecule when the target molecule passes from first layer 114 through second layer 116 toward second end 132 of test strip 100 and produce a complex of the target molecule bound to mobile labelled specific binding partner 134, as shown in FIGS. 2B and 2C. As shown in FIGS. 2A-2C, in one example, mobile labelled specific binding partner 134 is an Au P-anti-vitamin B_i2 conjugate that will specifically bind to vitamin B_i2 target molecules. Other binding partners may be utilized for other target molecules. Exemplary specific binding partners include, but are not limited to, nucleic acids (such as DNA or RNA aptamers, DNA or RNA ligands), and proteins (such as antibodies, antibody fragments, peptide aptamers, and ligands). The label for mobile labelled specific binding partner 34 is selected from the group consisting of carbon nano-particles, metallic nano-particles, fluorophores, quantum dots, and chemiluminescent particles. In this example, the label for mobile labelled specific binding partner 34 gold-nanoparticles (Au Ps).

[0044] Third layer 118 is supported on substrate 112 proximate to second end 132 of test strip 100 and downstream of second layer 116. Third layer 118 is made of a material selected from the group consisting of cellulose and nitrocellulose, although other suitable materials may be utilized. Third layer 1 18 includes test region 136 and control region 138 separated from test region 136.

[0045] Test region 136 has immobilized target molecules 140 located therein which will specifically bind to the complex and immobilize the complex in test region 136 as shown in FIGS. 2B and 2C. The immobilized target molecules may include vitamins, micronutrients, nucleic acids, carbohydrates, proteins, and peptides, as described above. In the example of Figures 2A-2C, the immobilized target molecules are vitamin B_i2 molecules. In other examples, test region 136 includes a plurality of different immobilized target molecules 140.

[0046] Control region 138 has an immobilized a moiety 142 located therein which will non-specifically bind to mobile labelled specific binding partner 134 of the target molecule and immobilize it in control region 138. Immobilized a moiety 142 may include a species specific anti-immunoglobulin reagent such as an anti-mouse, anti-horse, anti-bovine, anti-rat, anti-sheep, anti-goat, and anti-chicken antibody or various aptamers including, but not limited to, nonspecific protein and nucleic acid aptamers. Although a single test region 136 and a single control region 138 are described, a plurality of test regions and control regions, including different immobilized target molecules and immobilized moieties thereon, can be included on third layer 118 to provide a multiplexed assay for different target molecules as described in FIG. 5 below.

[0047] Collection layer 120 is supported on substrate 112 downstream of third layer 118 and proximate to second end 132 of test strip 100. Collection layer 120 provides an absorbent pad that is designed to receive materials passing through test strip 100 to collect the sample for test completion. Collection layer 120 is made of a material selected from the group consisting of cellulose membranes, polyester matrix, glass fiber, and polysulfone membranes.

[0048] FIG. 3 is a schematic view of the internal portion of another embodiment of multiplex diagnostic assay cartridge 10(2) of the present invention. Multiplex diagnostic assay cartridge 10(2) is the same as multiplex diagnostic assay cartridge 10(1) except as described below. In this example, diagnostic assay unit 26A is a vitamin Bi₂ immunoassay region, diagnostic assay unit 26B is a vitamin D (25-hydroxyvitamin D) immunoassay region, and diagnostic assay unit 26C is a retinol binding protein (RBP) immunoassay region. RBP has a 1 : 1 relationship with vitamin A and can thus be used as a proxy for vitamin A status. Multiplex diagnostic assay cartridge 10(2) includes pre-processing module 24(2) which allows the different assays to be performed with the same input on the same cartridge despite different processing requirements.

[0049] Pre-processing module 24(2) includes sample processing units 32A and 32B as described above, as well as sample delivery channels 30(2) which provide a network of channels for delivering the inserted sample to diagnostic assay units 26A-26C. Sample processing unit 32A filters blood added to sample input module 28 such that only plasma will be delivered through sample delivery channels 30(2). Sample processing unit 32B allows for introducing a reagent downstream of sample processing unit 32A. By way of example, the additional reagent may be an extraction buffer or agent configured to separate an analyte, such as vitamins Bi₂ or D, from their transport proteins. These reagents can interact with the sample in the downstream portion of sample delivery channels 30(2). The resulting separated samples are delivered to the vitamin Bi₂ diagnostic assay unit 26 A and the vitamin D diagnostic assay unit 26B, but not the RBP diagnostic assay unit 26C.

[0050] In this example, sample delivery channels 30(2) divert a portion of the filtered sample from sample processing unit 32A to a distinct channel for the RBP diagnostic assay unit 26C, such that the reagent inserted into sample processing unit 32B does not interact with the sample provided to RBP diagnostic assay unit 26C. Pre-processing module 24(2) also includes sample processing unit 32C, which is a diluting unit. Sample processing unit 32C is in parallel with sample processing units 32A and 32C, such that sample processing unit 32C allows for providing a diluting fluid only to the sample delivered RBP diagnostic assay unit 26C. Due to a much higher serum concentration of RBP, the sample needs to be diluted upstream of the RBP diagnostic assay unit.

[0051] Pre-processing module 24(2) also includes secondary input module 34 in fluid communication with second delivery channel 36 to deliver a fluid to vitamin Bi₂ diagnostic assay unit 26A. By way of example, a silver enhancement solution may be delivered through second delivery channel 36. The configuration of secondary delivery channel 36 allows the silver enhancement solution to be inserted at the same time as the sample, but arrive at vitamin B_i2 diagnostic assay unit 26A after a period of delay due to the longer path of secondary delivery channel 36. The period of delay allows for an incubation period on the vitamin Bi₂ diagnostic assay unit 26A prior to delivery of the enhancement solution. The flow pattern of secondary delivery channel 36 may be designed to provide a specific, optimized period of delay based on flow dynamics.

[0052] FIG. 4 is a schematic view of the internal portion of another embodiment of multiplex diagnostic assay cartridge 10(3). Multiplex diagnostic assay cartridge 10(3) is the same as multiplex diagnostic assay cartridges 10(1) and 10(2) except as described below.

Multiplex diagnostic assay cartridge 10(3) is designed to provide a specific diagnostic of determining iron deficiency or iron anemia. However, the multiplex diagnostic assay cartridges of the present invention may be configured to provide other diagnostic functions. In this embodiment, diagnostic assay unit 26A is a ferritin immunoassay region, diagnostic assay unit 26B is a serum transferrin receptor (sTfR) immunoassay region, diagnostic assay unit 26C is a C-reactive protein immunoassay region, and additional diagnostic assay unit 26D is a hemoglobin colorimetric assay region. Multiplex diagnostic assay cartridge 10(3) includes pre- processing module 24(3) which allows the different assays to be performed with the same input on the same cartridge despite different processing requirements.

[0053] In this example, sample processing unit 32A filters blood added to sample input module 28 at an area downstream of the sample input module. Sample delivery channels 30(3) include a diverted path for the sample input through sample input module 28 to hemoglobin colorimetric diagnostic assay unit 26D, as this unit requires whole blood. Diagnostic assay units 26A-26C require plasma and are therefore located downstream from sample processing unit 32A.

[0054] In this example, sample processing unit 32A filters blood added to sample input module 28 at an area downstream of the sample input module. Sample delivery channels 30(3) include a diverted path for the sample input through sample input module 28 to hemoglobin colorimetric diagnostic assay unit 26D, as this unit requires whole blood. Diagnostic assay units 26A-26C require plasma and are therefore located downstream from sample processing unit 32A. [0055] FIG. 5 is a schematic view of the internal portion of another embodiment of multiplex diagnostic assay cartridge 10(4). Multiplex diagnostic assay cartridge 10(4) is the same as multiplex diagnostic assay cartridge 10(3) except as described below. In this example, diagnostic assay units 26A-26C are located on a single test strip including a plurality of test regions and control regions for the different tests performed. [0056] Another aspect of the present invention relates to a method of conducting a multiplex diagnostic assay. The method includes providing a multiplex diagnostic cartridge according to the present invention. A sample is inserted through the inlet passage in the wall of the housing and into the chamber, whereby the inserting causes flow of the sample through the one or more sample delivery channels in the pre-processing module to direct the sample to one or more of the plurality of diagnostic assay units. The one or more sample processing units selectively process the sample prior to entering at least one of the plurality of diagnostic assays. The plurality of diagnostic assays are analyzed to determine whether a target material is present in the sample and/or what quantity of the target material is present.

[0057] First, a multiplex diagnostic assay cartridge of the present invention is provided. An exemplary method of the present invention will now be described with respect to multiplex diagnostic assay cartridge 10(2), although the method may be applied with any of the multiplex diagnostic assays described herein. To operate multiplex diagnostic assay cartridge 10(2), as shown in FIG. 3, the user applies a drop of blood into chamber 20 through inlet passage 22. However, the method may be performed using other fluids. The blood is transferred through inlet passage 22 to sample input module 28 and onto sample processing unit 32, which in this example is a filtration membrane. Inserting the sample into inlet passage 22 causes a flow of fluid to be generated in the sample delivery channels 30(2) of pre-processing module 24(2). The plasma leaving sample processing unit 32A is separated by sample deliver channels 30(2) as shown in FIG. 3 such that a portion is delivered solely to diagnostic assay unit 26C. [0058] The sample passing through sample processing unit 32A to vitamin Bi₂ diagnostic assay unit 26A and vitamin D diagnostic assay unit 26B may be separately processed using sample processing unit 32B. By way of example, the user then applies an extracting agent onto sample processing module 32B which extracts the vitamins from their binding proteins prior to reaching their respective diagnostic assay units. The user may also apply an additional fluid, such as a silver enhancement solution through second delivery channel 36. Because second delivery channel 36 defines a longer, tortuous path, the fluid inside reaches the vitamin B_i2 diagnostic assay unit 26C a delayed period after the plasma sample. The second delivery channel 36 may be designed to provide a desired time delay to expose the test strips to the enhancement solutions. In this example, the enhancement solution is used for vitamin B_i2 but not the other biomarkers, because vitamin B_i2 is found in very low concentrations (pg/ml) blood. The various assays are then carried out. As set forth above, the assays may include competitive assays, sandwich assays, colorimetric detection assays, bead assays, or combinations thereof.

[0059] Next, the diagnostic assay units 26A-26C are analyzed. In one example, diagnostic assay units 26A-26C are analyzed using the methods described in U.S. Patent Application Publication No. 2016/0080548 and PCT Patent Application PCT/US 14/12263, the disclosures of which are hereby incorporated by reference in their entirety herein. However, other testing methods that employ image processing may be utilized. These include commercial lateral assay flow readers (e.g., ESEQuant Lateral Flow Reader produced by Qiagen, Germany). Alternatively, the signal can be recorded over time optically with a camera, photomultiplier, or similar optical sensor. The images are then processed to provide a quantitative analysis of the amount of various biomarkers in the sample. In one example, the cartridge is configured to be utilized in conjunction with a smartphone as described in U.S. Patent Application Publication No. 2016/0080548 and PCT Patent Application PCT/US 14/12263, the disclosures of which are hereby incorporated by reference in their entirety herein. Specifically, the cartridge may be inserted into a smartphone accessory that provides for analysis of diagnostic assay units 26A- 26C. The smartphone receives image data from the smartphone accessory to provide a quantification of the results of the diagnostic assay. The results of the method are then displayed, such as on the screen of a mobile computing device.

EXAMPLES Example 1 - Fabrication of Plastic Cartridge [0060] A cartridge with mm-scale channels, multiple layers with different features was laser-cut and assembled to define the fluid network. The preliminary cartridge was a 4-layer plastic device as shown in Figures 6 and 7. As illustrated in Figures 6 and 7, the multiplexed diagnostic assay cartridge includes diagnostic assay units for vitamin Bi₂, vitamin A, and C- reactive protein (CRP). The cartridge includes separate inputs for a sample, (e.g., blood) and a running buffer along a multiplexed delivery channel that delivers the sample to each of the diagnostic assay units. The cartridge also includes a second path for delivering a fluid only to the vitamin Bi₂ assay unit. The path intersects a pair of membranes that may include, for example, silver enhancer pads. As illustrated in FIG. 7, on the base plastic layer are indents where a fusion 5 sample pad and lateral flow assays for different targets could be aligned and placed. The subsequent layer defines the fluid network for the sample to travel from the fusion 5 membrane to the conjugate pad inlets of different lateral flow assays. The layer on top defines the longer, tortuous fluid network for the silver enhancement solution to be delivered to the lateral flow assays -10 min after their sample interaction. The upper-most layer is for enclosure and leaves only the application areas for the sample and running buffers. Example 2 - Sandwich Lateral Flow Assay for CRP

[0061] A sandwich lateral flow assay for CRP was prepared using Flow Plus 180

Membrane Cards (EMD Millipore, Billerica, Massachusetts) with a 2mm clear polyester film backing as the assay platform. The membrane card housed the nitrocellulose membrane and the adhesive parts where the conjugate, sample and absorbent pads could be attached. Before the assembly, the test and control lines were prepared on the nitrocellulose membrane using the Lateral Flow Reagent Dispenser (Claremont Biosolutions, Claremont, California) to dispense l .Omg/ml polyclonal anti-CRP (CalBioreagents Inc., San Mateo, California) and l.Omg/ml anti- mouse IgG produced in goat (Sigma-Aldrich Co. LLC, St. Louis, Missouri), respectively.

[0062] The two lines were separated by 3mm and uniform line widths of 1mm could be obtained by operating the Legato 200 Dual Syringe Pump (Claremont Biosolutions, Claremont, California) at 6.4μ1/ιηίη. The membrane cards were subsequently dried for 2h at 37°, then at room temperature overnight. The monoclonal anti-CRP IgG produced in mouse (antibodies- online) came in >95% purity and was conjugated with 40nm AuNPs using the InnovaCoat Gold Conjugation Kit (Innova Biosciences Ltd., United Kingdom).

[0063] To prepare the conjugate pads for the CRP assay, the AuNP-anti-CRP conjugates were first diluted to 1.0 O.D. in the conjugate buffer (2mM borate buffer with 5% sucrose). The Glass Fiber Conjugate Pads (EMD Millipore, Billerica, Massachusetts) with 30cm x 5mm dimensions were soaked in the diluted conjugate solution for lmin, followed by drying at 37°C for lOh. The CRP assay was assembled into its final form by first attaching the AuNP-anti-CRP treated conjugate pad to the adhesive region of the assay platform below the nitrocellulose membrane with an overlap of 2mm. The Fusion 5 Membrane (GE Whatman, St. Louis, Missouri) was then attached below the conjugate pad with the overlap to serve as the sample pad of the assay. The Cellulose Fiber Sample Pad (EMD Millipore, Billerica, Massachusetts) was then attached above the nitrocellulose membrane with the 2mm overlap to serve as the absorbant pad of the assay. The assembled assay was cut into individual strips of 4mm width using a rotary paper trimmer (Dahle North America, Inc., Peterborough, New Hampshire).

Example 3 - Preliminary Results

[0064] The CRP strips were tested in standard CRP calibrators in the 0-10 range. To test, 35 μΐ of CRP standard was applied to the sample pad of the CRP strips, immediately followed by 45 μΐ of running buffer (lx TBS with 1% BSA and 1% Tween 20). The test and control line signals fully developed in -lOmin as shown in Figure 8A. As is expected from sandwich type assays, the test line intensity increased with increasing CRP concentrations, T/C being the smallest at low CRP and the highest at high CRP. When analyzed with a computing system, the T/C ratio could be quantified at each CRP concentration and T/C ratio across the 0- 10 μg/ml CRP range was obtained (Figure 8C). [0065] Although the test line intensity saturated at 10 μ/ml resulting in the same T/C ratio for strips tested at >10 μg/ml CRP concentrations, the kinetics and test and control line development could be investigated to distinguish the CRP in >10 μg/ml range. In Figure 8D, 25 μg/ml causes the T/C signals on the CRP strips develop faster than at 10 μg/ml, although the T/C ratio reached approximately the same values after -lOmin.

[0066] Although preferred embodiments have been depicted and described in detail herein, it will be apparent to those skilled in the relevant art that various modifications, additions, substitutions, and the like can be made without departing from the spirit of the invention and these are therefore considered to be within the scope of the invention as defined in the claims that follow.

Claims

WHAT IS CLAIMED:

1. A multiplex diagnostic assay cartridge for detection of a plurality of target molecules in a sample, said cartridge comprising:

an elongate housing having walls defining a chamber, said cartridge extending between a first end, where the sample is inserted through an inlet passage in a wall of the housing and into the chamber, and a second end at which results of the assay can be assessed;

a plurality of diagnostic assay units within the chamber defined by the walls of the cartridge; and

a pre-processing module within the chamber defined by the walls of the housing and comprising:

a sample input module positioned to receive material inserted through the inlet passage in the wall of the housing;

one or more sample delivery channels in fluid communication with the sample input module and at least one of the plurality of diagnostic assay units so that the sample inserted through the inlet passage in the wall of the housing and into the sample input module passes through the one or more sample delivery channels in said pre-processing module to direct the sample to one or more of the plurality of diagnostic assay units; and

one or more sample processing units in fluid communication with at least one of the one or more sample delivery channels to selectively process the sample prior to entering at least one of the plurality of diagnostic assays.

2. The multiplex diagnostic assay cartridge of claim 1, wherein at least one of plurality of diagnostic assay units comprises a test strip comprising:

an elongate substrate extending between a first end, proximate to the first end of said cartridge and a second end proximate to the second end of said cartridge;

a first layer supported on said elongate substrate proximate to the first end of the strip for receiving, absorbing, and filtering a liquid sample inserted through the inlet passage in the walls of the housing;

a second layer supported on said elongate substrate downstream of said first layer and comprising a mobile labelled specific binding partner of said target molecule, wherein the labelled specific binding partner will bind specifically to said target molecule passing through said first layer toward the second end of the strip and produce a complex of said target molecule bound to the labelled specific binding partner; and

a third layer supported on said elongate substrate proximate to the second end of the strip and downstream of said second layer, said third layer comprising a test region and a control region separated from the test region.

3. The multiplex diagnostic assay cartridge of claim 2, wherein the test strip carries out a competitive assay.

4. The multiplex diagnostic assay cartridge of claim 2, wherein said test strip carries out a sandwich assay.

5. The multiplex diagnostic assay cartridge of claim 2, wherein said test strip carries out both a sandwich assay and a competitive assay.

6. The multiplex diagnostic assay cartridge of claim 2, wherein the target molecule is selected from the group consisting of micronutrients, peptides, proteins, and carbohydrates and combinations thereof.

7. The multiplex diagnostic assay cartridge of claim 2, wherein the target molecule is vitamin D.

8. The multiplex diagnostic assay cartridge of claim 2, wherein the target molecule is vitamin Bi₂.

9. The multiplex diagnostic assay cartridge of claim 2, wherein the third layer comprises at least two test regions and at least two control regions separated from the at least two test regions.

10. The multiplex diagnostic assay cartridge of claim 1, wherein at least one of the plurality of diagnostic assay units comprises a colorimetric detection assay.

11. The multiplex diagnostic assay cartridge of claim 1, wherein at least one of the plurality of diagnostic assay units comprises a bead assay.

12. The multiplex diagnostic assay cartridge of claim 1, wherein the one or more sample processing units comprise a filter unit, a dilution unit, a separating unit, or a mixing unit.

13. The multiplex diagnostic assay cartridge of claim 1, wherein cartridge is formed from a material selected from the group consisting of polycarbonate, polystyrene, polymethyl methacrylate, polyamide, polysulfone, polyoxymethylene, polyetheretherketone, cyclic olefin polymer, polyethylene, polyethylene terephthalate, polyvinylchloride,

perfluoroalkoxy, fluorinated ethylenepropylene, glass, topascyclic olefin copolymer, silicon, quartz, and polydimethylsiloxane.

14. The multiplex diagnostic assay cartridge of claim 1, wherein the preprocessing module further comprises:

a secondary input module; and

a secondary delivery channel in fluid communication with the secondary input module and at least one of the plurality of diagnostic assays so that a fluid delivered though the secondary input module at substantially the same time as the sample is introduced into the sample input module is delayed in mixing with the sample.

15. The multiplex diagnostic assay cartridge of claim 14, wherein the second delivery channel is in fluid communication with at least one of the one or more sample delivery channels.

16. The multiplex diagnostic assay cartridge of claim 14, wherein the second delivery channel is separate from the one or more sample delivery channels.

17. The multiplex diagnostic assay cartridge of claim 14, wherein the second delivery channel has a serpentine flow pattern.

18. The multiplex diagnostic assay cartridge of claim 1 comprising at least two pre-processing sample processing units arranged in parallel.

19. The multiplex diagnostic assay cartridge of claim 1 comprising at least two pre-processing sample processing units arranged in series.

20. A method of conducting a multiplex diagnostic assay, said method comprising:

providing the multiplex diagnostic assay cartridge of claim 1;

inserting a sample through the inlet passage in the wall of the housing and into the chamber, whereby said inserting causes flow of the sample through the one or more sample delivery channels in said pre-processing module to direct the sample to one or more of the plurality of diagnostic assay units, whereby the one or more sample processing units selectively process the sample prior to entering at least one of the plurality of diagnostic assays; and

analyzing the plurality of diagnostic assays to determine whether a target material is present in the sample and/or what quantity of the target material is present.

21. The method of claim 21 further comprising:

displaying the results of said method.

22. The method of claim 21, wherein at least one of the plurality of diagnostic assays carries out a competitive assay.

23. The method of claim 21, wherein at least one of the plurality of diagnostic assays carries out a sandwich assay.

24. The method of claim 21, wherein at least one of the plurality of diagnostic assays carries out both a sandwich and a competitive assay.

25. The method of claim 21, wherein at least one of the plurality of diagnostic assay units carries out a colorimetric detection assay.

26. The method of claim 21, wherein at least one of the plurality of diagnostic assay units carries out a bead assay.

27. The method of claim 21, wherein the one or more sample processing units carry out one or more of filtering, diluting, separating, or mixing of the sample.

28. A method of conducting a multiplex diagnostic assay, said method comprising:

providing the multiplex diagnostic assay cartridge of claim 14;

inserting the sample through the inlet passage in the wall of the housing and into the chamber, whereby said inserting causes flow of the sample through the one or more sample delivery channels in said pre-processing module to direct the sample to one or more of the plurality of diagnostic assay units, whereby the one or more sample processing units selectively process the sample prior to entering at least one of the plurality of diagnostic assays;

inserting a fluid through the second input module to the secondary delivery channel at substantially the same time as the sample is introduced into the sample input module, whereby the fluid is delayed in mixing with the sample; and