WO2011156687A1

WO2011156687A1 - Apparatus for fluidic sample processing

Info

Publication number: WO2011156687A1
Application number: PCT/US2011/039940
Authority: WO
Inventors: Sergey E. Ilyin; Andrei Khodak
Original assignee: Serigene, Llc
Priority date: 2010-06-11
Filing date: 2011-06-10
Publication date: 2011-12-15

Abstract

The present invention provides a system and device for processing fluidic samples, utilizing the capillary effects of porous material and a capillary tube to draw fluid. When used in combination, differential capillary forces enable fluid to move in and out of the capillary tube. Fluid control is accomplished through fluid communication in a predetermined sequence between the capillary tube and chambers filled with porous material and solutions. The device can be configured for specific protocols and is particularly applicable in a resource limited setting or in a point of care environment.

Description

TITLE: APPARATUS FOR FLUIDIC SAMPLE PROCESSING

INVENTORS: Sergey E. Ilyin and Andrei Khodak

CROSS-REFERENCE:

The present application claims the benefit of priority of U.S. Provisional Application No.

61/353,744, filed June 11, 2010, which is incorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to a system for processing fluidic samples. Embodiments of the invention perform various fluid handling protocols including sample preparation, heat treatment and specific analyte detection.

BACKGROUND OF THE INVENTION

Analytical and preparative technologies require execution of fluid handling protocols. Fluid handling protocols could involve multiple steps. It is highly desirable to simplify and automate complex liquid handling protocols. Existing means to automate fluid management procedures rely on active fluidics control. Examples of active control include automated pipeting stations or use of externally controlled pumps and valves in integrating processing stations, such as Cepheid GeneExpert (disclosed in U.S. Pat. No. 6,374,684 BI entitled "Fluid control and processing system"). These approaches are difficult to adapt for use in the resource limited setting, Point of Care (POC) and field conditions. It would be highly desirable to simplify execution of fluid handling. Certain fluid management procedures may also require precise control of the temperature of the sample. Analysis of a specific analyte may also require changing temperature of the sample, for example, in PCR-based approaches. Low concentration of a specific analyte may require processing large sample volumes, but a large sample volume would in turn mean slow processing as it would take longer to heat up or cool down. Devices in the prior art, disclosed in US 2009/0129978 Al and U.S. Pat. No. 6,374,684 BI, rely on externally controlled valves and pumps to concentrate analytes prior to heat treatment. It would be highly

advantageous to address the need to process a large sample volume, and to achieve rapid changes in sample temperature. In order to perform a multi-step liquid handling protocols, control for transport, mixing and possibly detection should be executed. Fluidics control could be executed by different methods, for example, mechanical pumping, thermo pneumatic pumping, electrochemical actuation, and a capillary flow method by means of surface tension. The fluidic control devices using these principles could be classified into active fluidics control devices and passive control devices. Active fluidic controls employ a pump or a valve driven by external forces, while the passive fluidic control uses capillary force.

Examples of fluidic control devices employing active control are disclosed in U.S. Pat. No. 6,374,684 BI entitled "Fluid control and processing system" and in US 2009/0129978 Al "Reagent holder, and kits containing same". The U.S. Pat. No. 6,375,817 discloses an apparatus for transporting and separating a fine amount of fluid in a fast automated manner by using a mechanically generated pressure. Examples of fluidic control devices employing active control of fine droplets in the capillary tube are disclosed in U.S. Pat. No. 6,375,817 entitled "Apparatus and methods for sample analysis" and U.S. Pat. No. 6,193,471 entitled "Pneumatic control of formation and transport of small volume liquid samples".

Active and passive control can be used in combination. The U.S. Pat. No. 6,143,248 discloses a microvalve for transporting a fine amount of fluid from a micro storage chamber to a transfer chamber by controlling capillary and centrifugal forces, however, one disadvantage is the application of centrifugal force.

Typical examples of a fluidic control device employing capillary flow are disclosed in U.S. Pat. No. 6,271,040 Bl (Aug. 7, 2001) entitled "Diagnostic Devices Method and Apparatus for the Controlled Move. The U.S. Pat. No. 6,271,040 Bl discloses a structure for diagnosis, which transports the sample only using flow resulted from capillary force.. The device generates unidirectional flow of solution.

The U.S. Pat. No. 6,296,020 Bl discloses a structure, which allows fluid to be stopped using hydrophobic material or expansion of flow path. It describes a means for controlling the flow of samples through micro channels to allow mixing or diluting of the fluids and/or separation of the fluids or a fluid into several channels for multiprocessing.

Active control devices known in the art are capable to execute complex protocols, but are bulky for use in the field and POC settings. As mentioned above, additional mechanical devices used to control fluid in the related art, which cause its configuration to be complicated and production cost high. Passive control devices are generally adapted to assays amendable to execution by unidirectional fluid flow. Passive control devices known in the prior art are not able to execute complex fluid control protocol required for many types of assays.

SUMMARY OF THE INVENTION

The present invention overcomes at least some of the limitations with the systems and devices known in the prior art. A key feature of the invention is its reliance on the capillary effect of a porous material and a capillary tube to draw a fluid sample. Differential capillary force enables fluid to move in and out of the capillary tube. Porous material has a stronger capillary force and draws the fluid sample out of the capillary tube once it is in the contact with the fluid sample. In accordance with the invention, fluid control is achieved by establishing fluidic communication in a predetermined sequence between capillary tube and chambers filled with porous material and fluid samples to accommodate specific protocols. A variety of configurations can be used to establish fluidic communication. Fluidic communication can be established by moving one or more capillary tubes through the chambers filled with porous material and fluid samples. In this configuration, the capillary tube is emptied once in the contact with porous material and filled with fluid once in the communication with the fluid sample. Various chemical, biochemical, analytical and preparative procedures and protocols can be carried in the capillary tube as it is filled with different solutions. Alternatively, fluidic communication can be established utilizing a revolver type mechanism or other suitable means to establish fluidic communication. The present invention provides an apparatus and methods of manipulating fluid samples.

Embodiments of the invention process sample according to different protocols for analytical or preparative purposes. Methodology of the present invention enables processing of large and small volumes of samples in semi-automated or automated format. Principals of the present invention are ideally suited to simplify assays and techniques used in practicing molecular biology, for example, DNA, R A and protein purification, quantitation, ligation and multistep protocols involving a combination of methods. One embodiment of the present invention is the apparatus comprising: a cartridge having distal and proximal ends, wherein said cartridge comprises (a) test kit forming a proximal end and comprising of at least one reagent containing chamber and at least one chamber with porous materials; and (b) capillary tube located within the cartridge and contacting said test kit. In accordance with the invention, the capillary tube can function as a resistive heater. Another embodiment of the present invention provides an apparatus for analyte extraction, analyte amplification by means of thermocycling the reaction mixture followed by analyte detection. One embodiment of the present invention includes a disposable cartridge having distal and proximal ends and an instrument for receiving the disposable cartridge. The disposable cartridge includes (a) a sample fill port located at the distal end of the cartridge device; (b) a capillary tube or plurality of capillary tubes connecting the fill port with the proximal end, comprising a test kit of the cartridge; and (c) a test kit forming a proximal end and comprising one and more preferably several reagent containing chambers and one, and more preferably several, porous material containing chambers. In some embodiments of the present invention the test kit comprises one or more, preferably several, analyte detection strips. Analyte detection strips used in practicing this invention can be a lateral flow device. The capillary tube is located between and in contact with the fill port and the test kit, thus connecting the sample fill port and test kit. In some embodiments of the present invention, the instrument into which the cartridge is placed contains a plurality of electrical contacts coming in contact with the capillary tube. In certain embodiments of this invention, the capillary tube functions as a resistive heater. Miniature sensors may be used to monitor capillary tube temperature, alternatively, the temperature coefficient of resistance of the heater itself may be monitored to control the heat input. The instrument into which the cartridge is placed may contain one or more magnets. The magnets can be used to conveniently concentrate magnet bound analytes. In other embodiments of this invention, the instrument into which the cartridge is placed contains a plurality of mobile or stationary thermal control zones.

If desired, particular embodiments may optionally include means of real time sample

interrogation to provide quantitative data about analyte concentration. The invention may be used to a particular advantage in context of Point of Care (POC) testing. If desired, particular embodiments may optionally include means of antigen-based testing. BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a perspective view of the fluid control and processing system according to one embodiment of the present invention, comprising of fluid processing cartridge and apparatus into which the cartridge is placed.

FIG. 2 is a perspective view of the fluid processing apparatus with the cartridge removed. FIG. 3 is a drawing that represents the cartridge of FIG 1.

FIG. 4 is a drawing depicting a test kit according to one of the embodiments of the present invention.

FIG. 5 is a cross-sectional view of the fluid control and processing system according to one embodiment of the present invention, comprising a fluid processing cartridge and apparatus. This view represents an initial configuration of the fluid control and processing system according to one embodiment of the present invention

FIG. 6 is another cross-sectional view of the fluid control and processing system according to one embodiment of the present invention, comprising a fluid processing cartridge and apparatus. This view represents a sample introduction configuration of the fluid control and processing system according to one embodiment of the present invention

FIG. 7 is another cross-sectional view of the fluid control and processing system according to an embodiment of the present invention, comprising a fluid processing cartridge and apparatus. This view represents a sample amplification configuration of the fluid control and processing system according to one embodiment of the present invention

FIGS. 8A and 8B depict cross-sectional views of a fluid processing cartridge, according to the principles of the present invention.

FIG. 9 is another cross-sectional view of the fluid control and processing system according to one embodiment of the present invention, comprising a fluid processing cartridge and apparatus. This view represents a test result view configuration of the fluid control and processing system according to one embodiment of the present invention

FIG. 10 is another cross-sectional view of the fluid control and processing system according to an embodiment of the present invention, comprising a fiuid processing cartridge and apparatus with test kit removed for illustrative purposes.

FIG. 11 shows a cross-sectional view of the cartridge hold and release mechanism in the hold position.

FIG. 12 is cross-sectional view of the cartridge hold and release mechanism in the release position.

FIG. 13 is enlarged partial cross-sectional view of the fluid control and processing system according to an embodiment of the present invention, comprising of fluid processing cartridge and apparatus. This view represents initial configuration of the fluid control and processing system according to one embodiment of the present invention.

FIG. 14 is enlarged partial cross-sectional view of the fluid control and processing system according to one embodiment of the present invention, comprising a fluid processing cartridge and apparatus. This view represents a sample introduction configuration of the fluid control and processing system according to one embodiment of the present invention.

FIG. 15 is an enlarged partial cross-sectional view of the fluid control and processing system according to one embodiment of the present invention, comprising a fluid processing cartridge and apparatus. This view represents a sample amplification configuration of the fluid control and processing system according to one embodiment of the present invention.

FIG. 16 is an enlarged partial cross-sectional view of the fluid control and processing system according to one embodiment of the present invention, comprising a fluid processing cartridge and apparatus. This view represents a test result view configuration of the fluid control and processing system according to one embodiment of the present invention. FIG. 17 is an enlarged partial cross-sectional view of the fluid control and processing system according to one embodiment of the present invention, comprising a fluid processing cartridge and apparatus with the test kit removed for illustrative purposes.

FIG. 18 is an enlarged partial cross-sectional view of the fluid control and processing system according to one embodiment of the present invention, comprising a fluid processing cartridge and apparatus and illustrating a specific protocol. This view represents an initial configuration of the fluid control and processing system according to one embodiment of the present invention.

FIG. 19 is a view of the cartridge according to one embodiment of the fluid processing system, where electrical connection to the apparatus is achieved through plug-in contacts.

FIG. 20 is a view of the fluid processing system with the cartridge according to the embodiment presented on Fig. 19. Cartridge is shown in plugged-in position.

DESCRIPTION OF THE SPECIFIC EMBODIMENTS

The present invention provides a system for performing various operations with a fluid sample. One embodiment of the present invention consists of a cartridge and a device to receive said cartridge. The cartridges of the present invention allow for significantly improved processing of a fluid sample for the detection and/or analysis of chemical components in the sample, such as biological molecules. A pioneering improvement over the prior art is the ability to rapidly execute complex liquid handling protocols without use of valves and external pumps. Absence of external pumps significantly reduces risk of cross-contamination. Cartridge design allows processing a fluid sample that is larger in volume than the amplification region within the cartridge, thereby permitting increased sensitivity in the detection of low copy concentrations of analytes, such as nucleic acids.

A key feature of the invention is its reliance on the capillary effect of a porous material and a capillary tube to draw a f uid sample. Porous material has a stronger capillary force and draws a fluid sample out of the capillary tube once it is in contact with the f uid sample. Differential capillary force enables fluid to move in and out of the capillary tube. Positioning of the capillary tube in chambers filled with porous material and fluid samples enables the use of specific liquid handling protocols. FIG 1 shows a fluid control and processing system comprising fluid processing cartridge 1 and apparatus 2 for receiving said cartridge. The cartridge 1 is preferably used in combination with a portable, i.e. hand-held or desktop, external instrument 2 designed to accept one or more of the cartridges 1. As shown in FIG. 2 cartridge processing apparatus 2 of FIG 1 includes electrical contact left 21, electrical contact right 22, cooling nozzle lid 23, magnet 24, cartridge release handle 25, test stage dial 26, test stage gear 27. FIG. 3 show a fluid processing cartridge 1 comprising test kit 3, a sample fill port 11, test result window 12 and cooling air opening 13. FIG. 4 show test kit 3 comprising several test kit elements 38, analyte detection strip 33.

Embodiments of the invention incorporate a plurality of porous materials for cleaning stage and plurality of reagent solution vials. FIG. 5 shows a cross-sectional view of a fluid control and processing station comprising cartridge 1, cartridge processing apparatus 2, air duct cover 20, electrical contact left 21, electrical contact right 22, magnet 24, cartridge release handle 25, test stage dial 26, test kit element containing porous material 32, test kit sealing tape slider 34, test kit element, reagent solution vial 35, test kit element lateral flow detection strip holder 36, test kit element spacer 37, spring fixing the capillary tube to temperature sensor 4, temperature sensor 5, capillary tube 6, controller block 7. The present invention considers all possible variations in the design of the capillary tube 6. One preferred embodiment of the design is with a hole or opening on the side or longitudinal surface of the capillary tube 6 adjacent to the sharp tip end of the tube which allows easy passage from the tube through the chambers without disturbing porous material. As shown in FIG. 5, electrical contacts 21 and 22 form a contact with capillary tube 6 when cartridge 1 is inserted for processing. Many connections can be made between cartridge and device. Suitable memory may be included on the cartridge, such as writable memory, electrically erasable programmable read-only memory and other types of memory. Test results, based on the sample introduced into the cartridge, can be written by the instrument into the cartridge's memory. Cartridge memory can also be used to store instruction on how to execute specific protocol and cartridge specific calibration data. In addition, lot manufacture

information, cartridge serial number and other information may be included. Engineers skilled in the art of data storage will recognize that many memory means can be used.

To demonstrate the fluid control mechanism, FIGS 6 - 7 illustrate fluid control. In FIG. 6, capillary tube 6 is placed in fluidic communication with porous material 32 of test kit 3. In accordance with the invention, communication between capillary tube 6 and porous material 32 results in a fluid sample going from the capillary tube 6 into porous material 32 by capillary force. In accordance with the invention (FIG. 7), capillary tube 6 is placed in fluidic

communication with reagent solution vial 35. This communication results in the solution moving from 35 into 6. FIG. 8B shows a cross-sectional view of a fluid control and processing cartridge 1. FIG. 8A is an enlarged view of test kit 3 portion of the cartridge comprising a test kit element porous material 32, analyte detection strip 33, test kit sealing tape slider 34, test kit element 35, reagent solution vial, test kit element lateral flow detection strip holder, test kit element spacer 37. In FIG. 9, capillary tube 6 is placed in fluidic communication with analyte detection strip 33 of test kit 3. FIG. 10 illustrates cartridge processing according to one of the embodiments of the present invention. Test stage gear 27 is rotated by motion communicated from test stage dial 26. This rotation advances compartments of the test kit and enables execution of specific protocols as illustrated in FIGS 5, 6, 7, 8 and 9. Test kit advancement can be automated by means of step motor and other mechanical and electro-mechanical motion producing systems known to those skilled in the art. FIG. 11 shows a cartridge hold and release mechanism in hold position according to one embodiment of the present invention. FIG 11 includes electrical contact right 22, cartridge release handle 25, electrical contact fixing spring 15, capillary tube 6. FIG. 12 shows a cartridge hold and release mechanism in release position according to one embodiment of the present invention. FIG. 13 depicts an initial configuration of the fluid control and processing system according to one embodiment of the present invention. FIG. 14 represents a sample introduction configuration of the fluid control and processing system according to one embodiment of the present invention and demonstrating a specific liquid handling protocol. Capillary tube 6 is in fluidic communication with test kit element porous material 32. FIG. 15 represents a fluid sample introduction configuration of the fluid control and processing system according to one embodiment of the present invention. Capillary tube 6 is in test kit element 35 and in fluidic communication with fluid sample 39. In certain

embodiments fluid sample 39 can be an amplification solution to perform PCR. FIG. 16

represents a test result view configuration of the fluid control and processing system according to one embodiment of the present invention. FIG. 17 is an enlarged partial cross-sectional view of the fluid control and processing system according to one embodiment of the present invention, comprising a fluid processing cartridge and apparatus with the test kit removed for illustrative purposes. FIG. 18 is an enlarged partial cross-sectional view of the fluid control and processing system according to one embodiment of the present invention, comprising a fluid processing cartridge and apparatus and illustrating a specific protocol. This view represents an initial configuration of the fluid control and processing system according to one embodiment of the present invention. FIGS. 19 and 20 show another embodiment of the present invention where electrical connection of the cartridge 1 to the apparatus 2 is achieved through a plurality of electrical contacts 61 positioned on the both sides of the active region of the capillary tube 6. Contacts 61 can be plugged in or otherwise electrically connected to the mating contacts 62 on the apparatus 2. FIG. 19 illustrates test kit positioning gear 31 according to one of the embodiments of the present invention.

In practicing one embodiment of the present invention, a sample is mixed with magnetic beads and introduced into cartridge 1 via fill port 11. Magnetic beads used in practicing this particular embodiment of the invention are coated with analyte-specific capturing reagents. A broad range of analyte capturing reagents can be used in practicing this invention. Analyte specific capturing reagents can be monoclonal or polyclonal antibodies against specific analytes, for example viruses and bacteria. Yet, in certain other embodiments analyte specific capture reagents can be nucleic acid binding reagents. A fluid sample containing a desired analyte and magnetic beads is added to the sample port 11 of the cartridge 1 and forced to flow by capillary force down a capillary tube 6 and into the porous material 32 of test kit 3. The fluid sample is absorbed in the porous material containing test kit element 32 and the magnetic beads are deposited in the capillary tube 6 by the action of a magnet 24. The Test kit moves by means of a test stage dial 26 and test kit gear 27, so that the capillary tube is positioned in a reagent solution vial 35 filled with reagent solution. In accordance with the invention, reagent solution fills the capillary tube. The temperature of the capillary tube is changed according to a specific protocol for a predetermined number of cycles to perform amplification. In one embodiment of the invention, the capillary tube 6 functions as a resistive heater. The temperature coefficient of resistance of the capillary tube itself may be monitored to control the heat input. A specific protocol may involve heat induced lysis to release nucleic acid into the reagent solution. Certain tests may require additional steps of washing magnetic beads prior to amplification. Test kit of FIG. 18 illustrates how principles of the present invention could be used to accommodate a liquid handling protocol with washing steps. A fluid sample containing a desired analyte and magnetic beads is added to the sample port 11 of the cartridge 1 and forced to flow by capillary force down a capillary tube 6 and into the porous material of test kit element 40. The fluid sample is absorbed in the porous material containing test kit element 40 and the magnetic beads are deposited in the capillary tube 6 by action of magnet 24. Test kit moves by the means of test stage dial 26 and test kit gear 27, so that capillary tube is positioned in the wash solution test kit element 41. In accordance with the invention, wash solution fills the capillary tube. Test kit moves by the means of test stage dial 26 and test kit gear 27, so that the capillary tube is positioned in fluidic communication with absorbent material of test kit element 42. The fluid sample is absorbed in the porous material containing test kit element 42. Principles of the invention allow designing effective sample handling protocols by adding elements to the test kit. The test kit moves by means of test stage dial 26 and test kit gear 27, so that the capillary tube is positioned in fluidic communication with reagent solution containing test kit element 43. In accordance with the invention, reagent solution fills the capillary tube. The temperature of the capillary tube is changed according to a specific protocol for a predetermined number of cycles to perform amplification. Once the amplification step is completed, the test kit moves by means of the test stage dial 26 and test kit gear 27, so that the capillary tube is positioned in fluidic communication with analyte detection strip 33. A person skilled in the art can appreciate how simple finger move enables execution of complex fluid management protocol. The fluid sample moves from the capillary tube 6 into analyte detection strip 33 by capillary force. The amplification step may result in the formation of PCR products or amplicons. These amplicons can be detected by analyte detection strip. In some embodiments of the present invention, analyte detection strip is a lateral flow device. Amplicons can be labeled directly by

incorporating suitable labels in the primers. Alternatively, amplicon-specific probe may be used for detection. A lateral flow detection strip comprises a test line zone. The test line zone displays a visual signal if the test is positive. In some embodiments of the present invention, the test line zone is conveniently observed through a test result window 12. Some embodiments of the present invention may incorporate a control line zone. In some embodiments of the present invention, detection is done via use of nanoparticles. Nanoparticles are coated with materials that bind to specific nucleic acids within a sample. This DNA-nanoparticle complex flows laterally through the membrane of the lateral flow detection strip until it is captured in the test line zone. A visual result can be achieved within a few minutes.

In practicing another embodiment of the present invention, a fluid sample containing a desired analyte is added to the sample port 11 of the cartridge 1 and forced to flow by capillary force down a capillary tube 6 and into the porous material test kit 40 of test kit 3. The fluid sample is absorbed in the porous material containing test kit element 40 and the analytes are captured by analyte capture reagents deposited in the capillary 6. The test kit moves by the means of a test stage dial 26 and test kit gear 27, so that the capillary tube is positioned in a wash solution vial 41 filled with reagent solution. In accordance with the invention, reagent solution fills the capillary tube. The temperature of the capillary tube is changed according to a specific protocol for a predetermined number of cycles to perform amplification. In one embodiment of the invention, the capillary tube 6 functions as a resistive heater. The temperature coefficient of resistance of the capillary tube itself may be monitored to control the heat input. A specific protocol may involve heat induced lysis to release nucleic acids into the reagent solution. The test kit moves by means of a test stage dial 26 and test kit gear 27, so that the capillary tube is positioned in fluidic communication with lateral flow detection strip 33.

It may be highly advantageous to detect different types of analytes present in the sample. For example, a sample may contain specific pathogens and antibodies generated by a host against this pathogen. Alternatively, one pathogen can be detected by one method and another pathogen or pathogen related analyte by another method. Principles of present invention allow detecting different types of analytes. In practicing one embodiment of the present invention design for co- detection, a sample is introduced into cartridge 1 via fill port 11. The fluid sample is forced to flow by capillary force down a capillary tube 6 and into the porous material 32 of test kit 3. Porous material 32 used in practicing embodied in a lateral flow device configured to detect antigens or antibodies present in the sample. Nucleic acids potentially present in the sample are detected as described for other embodiments in the present invention.

One advantage of the cartridge according to principals of the present invention is that it allows the analyte from a relatively large volume of fluid sample, e.g. several milliliters or more, to be concentrated into a much smaller volume of amplification solution in the amplification zone. In certain embodiments of the present invention, concentrated analytes could be visualized on lateral flow devices without amplification. In contrast to devices in the prior art, the cartridge of the present invention permits concentration without use of pumps and valves.

As used herein, the term "capillary tube" refers to any means of fluidics communication by capillary action. Capillary tube could be manufactured from hydrophilic materials such as glass; alternatively capillary tube could be manufactured from hydrophobic material, for example steel, and coated or otherwise altered to render the surface to be hydrophilic. The capillary tube can be made from variety of materials, for example, glass, plastic, metal or ceramic with internal diameter ranging from 0.01 to about 3.00 mm. The capillary tube can be hollow or filled with porous material. In certain embodiments of this invention, the capillary tube functions as a resistive heater.

As used herein, the term "test kit" refers to a means to establish fluidic communication between the capillary tube and the test kit elements.

As used herein, the term "test kit element" refers to containers with porous materials or reagents within the test kit of the cartridge. Porous material can be incorporated into the test kit element using conventional pouching or packaging techniques. Reagents can be contained as liquids within the test kit elements of the cartridge, using conventional pouching or packaging techniques, the designs of which are optimized to allow integration into the cartridge. Reagents may be introduced into the cartridge before use or reagents may be placed in the cartridge during manufacture. Dried reagents can be employed as precursor materials for reconstitution.

Additives to stabilize reagents, such as sugars, methylcelluloses, proteins may be added to the reagent before or after drying.

The term "porous material" as used herein, refers to a length of material along which a reaction mixture can travel by capillary action.

The term "sample fill port," as used herein, is a receptacle for the application of a sample into the cartridge. For example, the sample fill port of the present invention may be a tube into which the distal end of the capillary tube is inserted to allow a sample or reaction mixture to enter the capillary tube. Alternatively, the sample fill port may be a pad located at the distal end of the capillary tube and in contact with the distal end of the capillary tube. A sample or reaction mixture may be applied to the pad and absorbed into the pad. The reaction mixture subsequently enters into the distal end of the capillary tube by means of capillary action. In some

embodiments of the invention, a syringe with or without a needle can be used as the sample fill port. A fluid sample may be introduced into the cartridge by a variety of means. For manual addition, a measured volume of material may be placed into a sample fill port of the cartridge. The cartridge itself may also serve as the actual specimen collection device, thereby facilitating handling.

As used herein, the term "analyte" refers to any molecular entity under investigation. Examples include pathogens such as bacteria and viruses, proteins and nucleic acids.

As used herein, the term "nucleic acid" refers to any nucleic acid, such as DNA or RNA in any possible configuration, i.e. in the form of double-stranded nucleic acid, single-stranded nucleic acid, or any combination thereof.

The fluid sample may be an aqueous solution containing microorganisms, cells, bodily fluids. The fluid sample may be introduced directly into the sample fill port. The fluid sample may also be pretreated, for example, mixed with chemicals, sonicated or centrifuged prior to introduction into the cartridge.

The term "temperature controller," as used herein, refers to a device that regulates temperature.

The term "amplification zone," as used herein, refers to the region of the capillary tube within which the thermal cycles necessary for amplification occur.

The term "instrument" is the apparatus for receiving the cartridge.

The term "analyte detection strip" as used herein, typically refers to a means to detect specific analytes. In some embodiments of the present invention, a lateral flow device is used as an analyte detection strip.

The lateral flow device of this invention is comprised from one or more, preferably several, porous membranes. Lateral flow devices used in practicing some embodiments of the present invention can be configured to detect various types of analytes. A lateral flow device configured to detect specific nucleic acids would typically include test line zone, control zone and labeling zone.

The term "porous membrane" or "membrane," as used herein refers to a length of absorbent material along which a reaction mixture can travel by capillary action.

The term "test line zone," as used herein, refers to a region of the porous membrane, which includes an amplicon-capturing agent. The test line zone provides a means for detecting successful amplification by capturing the amplicon product within a narrow region of the porous membrane.

The term "labeling zone," as used herein, refers to a region that includes an amplicon-specific probe. Preferably, the labeling zone is located proximal to the amplification zone and distal to the test line zone. According to the invention, as the reaction mixture leaves the amplification zone and enters the labeling zone, the amplified product, or "amplicon," is bound by, and thereby labeled by, a probe that specifically recognizes the amplicon product.

The amplicons become conveniently associated with a readily detectable label upstream of the detection zone. The label may be any readily detectable suitable substance. A direct visible label, which is apparent to an observer without any prior processing is greatly preferred.

It is desirable that the labeling is amplicon-specific. One of the simplest ways of achieving this is to ensure that the amplicon has a sequence which is essentially unique amongst the nucleic acids entering a labeling zone and to provide a labeling reagent which comprises a base sequence complementary to that of the amplicon.

The term "amplicon-capturing agent," as used herein, refers to a means for binding to an amplified nucleic acid product. The term "amplicon-specific probe" or "probe," as used herein comprises a detectable label conjugated to a molecule capable of binding an amplicon.

It may be desirable to place certain samples into another device or accessory and then perform sonication or other type of mechanical treatment prior to introduction into the cartridge. The overall geometry of the cartridge may take a number of forms. For example, the cartridge may incorporate a plurality of sample fill ports, a plurality of capillaries, a plurality of test kits.

The cartridge may be fabricated using a variety of methods from a variety of polymeric materials, or may be silicon, glass, or the like.

The cartridge may also incorporate one or more filters for capturing sample components, for example cells, spores, or microorganisms to be analyzed. The filters may also be used for removing undesired contamination from the sample. The filters may be within any region of the cartridge. A variety of filter media may be used, including, e.g., cellulose, nitrocellulose, polysulfone, nylon, vinyl copolymers, glass fiber, microfabricated structures. Similarly, separation media, such as ion exchange resins, affinity resins or the like, may be included within the cartridge.

In some embodiments, enzymes, such as a polymerase enzyme, may be present within an amplification region, coupled to the walls and surfaces of the region.

Temperature control is generally supplied by resistive heaters which are prepared using methods that are well known in the art. For example, these heaters may be fabricated from thin metal films applied within or adjacent to the capillary tube. The heater is electrically connected to a power source which delivers a current across the heater.

In one embodiment, a controllable heater is the capillary tube itself. In another embodiment, the heating element is disposed within or adjacent to an amplification zone for thermal control of the sample. Thermal control may be carried out by varying the electric power supplied to the heater to achieve the desired temperature for the particular stage of the reaction. Alternatively, several heaters each at a different constant temperature can be positioned one at a time closely to a region changing the temperature of the region according to a specific protocol. Heating may alternatively be supplied by exposing the region to a laser. Resistive heater elements may also be incorporated into the walls of the amplification regions by introducing resistive heaters into the wall material using over molding or thin film deposition. Alternatively the walls of the capillary tube can be used as resistive heaters. Varieties of materials are suitable to fabricate such tubes. Controlled heating provides additional functional capabilities, such as mixing, dissolution of solid reagents, lysing, thermal denaturation of proteins and nucleic acids and lysis of cells, elution of bound molecules. Controlled heating provides means to conduct PCR, NASBA, TMA and ligase chain reactions. Cooling features may also be exploited in high surface area regions, for example, with external cooling fins.

The heaters may be incorporated within the cartridge by including a resistive heater in the cartridge, or alternatively, the heaters may be provided externally, in the instrument, and applied to the exterior of the cartridge, adjacent to a particular region, so that heat is conducted into the region.

An amplification zone may also include temperature sensors for monitoring temperatures and thereby controlling the application of current across the heater. A wide variety of sensors are available for determining temperatures, including, e.g., thermocouples, resistance thermometers, thermistors, IC temperature sensors, quartz thermometers and the like. Alternatively, the temperature coefficient of resistance of the heater itself may be monitored to control the heat input.

The temperature measured by the temperature sensor will typically be input to a processor in the external instrument, which is programmed to receive and record this data. The same processor will typically include programming for instructing the delivery of appropriate electric power for raising and lowering the temperature of the amplification zone. For example, the processor may be programmed to take the amplification zone through any number of predetermined

time/temperature profiles, e.g., thermal cycling for PCR, NASBA, TMA and ligase chain reactions, and the like. Given the small size of the cartridges of the invention, cooling of an interactive region will typically occur through exposure to ambient temperature. However, additional cooling elements may be included if desired, for example, Peltier coolers may be included. In addition to sensors for monitoring temperature, the cartridge may contain sensors to monitor the progress of one or more of the operations of the device. For example, optical sensors may be incorporated into one or more regions to monitor the progress of the various reactions. An electrical circuit can be used to sense the presence or absence of the fluid. By placing several such circuits along the length of fluid channel, the presence of the fluid in various regions can be observed.

The exact dimensions and structure of the cartridge elements are manufactured to suit the cartridge to a particular application.

The device may also include an ultrasonic transducer, that is coupled to the cartridge for transferring ultrasonic energy to the components captured on the magnetic beads.

Although the above description contains many specificities, these should not be construed as limitations on the scope of the invention, but merely as illustrations of some of the presently preferred embodiments. Many possible variations and modifications to the invention will be apparent to one skilled in the art upon consideration of this disclosure. Therefore, the scope of the invention should be determined by the following claims and their legal equivalents.

Claims

Claims What is claimed is:

1. A fluidic control device for assessing target analytes within a fluid sample, said device

comprising:

a. a capillary tube ;

b. at least one chamber filled with a porous material;

c. at least one chamber filled with reagent;

d. means for establishing fluidic communication between said sample when contained within said capillary tube and elements (b) and (c) wherein fluidic communication is established by positioning the capillary tube, said chambers, or both in a predetermined configuration; and

e. means for detecting said target analytes.

2. The device of claim 1 wherein said analytes are from a group consisting of bacteria, viruses, nucleic acids, proteins, and combinations thereof.

3. The device of claim 1 wherein said capillary tube is coated on its inner surface with an

analyte-specific capturing reagent.

4. The device of claim 3 wherein said analyte-specific capturing reagent is from a group

consisting of monoclonal antibodies, polyclonal antibodies, nucleic acid binding agents, and combinations thereof.

5. The device of claim 1 further includes a magnetic means for capturing a target analyte

complex wherein said complex comprises a target analyte linked to a magnetically responsive particle.

6. The device of claim 5 wherein said magnetically responsive particle is a magnetic

nanoparticle.

7. The device of claim 1 wherein said predetermined configuration is obtained with revolver mechanism.

8. The device of claim 1 where said means for detecting target analytes comprises:

a. means for thermocycling; and

b. means for confirming amplicons from said thermocycling.

9. The device of claim 8 wherein said thermocycling means is an amplification zone containing a heating element along the external surface of the capillary tube.

10. The device of claim 1 wherein said means for assessing target analytes includes an analyte detection strip in fluidic communication with said capillary tube.

11. The device of claim 10 wherein said analyte detection strip is a lateral flow device.

12. The device of claim 1 wherein said capillary tube further includes a controlled heating means.

13. The device of claim 12 wherein said controlled heating means is a resistive heater.

14. A cartridge for analysis of target analytes in a fluid sample, said cartridge comprising:

a. a sample fill port;

b. at least one capillary tube connected to said sample fill port; and

c. a test kit having multiple chambers wherein at least one chamber contains porous

material and a second chamber containing reagent, said test kit in fluidic communication with said capillary tube.

15. The cartridge of claim 14 wherein the capillary tube is coated on its inner surface with an analyte specific capturing reagent.

16. The cartridge of claim 15 wherein the analyte-specific capturing reagent is from a group

consisting of monoclonal antibodies, polyclonal antibodies, nucleic acid binding agents and combinations thereof.

17. The cartridge of claim 14 wherein the capillary tube includes a controlled heating means.

18. The cartridge of claim 14 further includes at least one analyte detection strip.

19. The cartridge of claim 14 further includes at least one filter for separating sample

components.

20. A method for the control of fluid movement comprising:

a. depositing a fluid sample in a capillary tube;

b. placing said capillary tube in fluidic communication with a porous material;

c. drawing said sample from the capillary tube;

d. placing said capillary tube in second fluidic communication with a second fluid reservoir; and

e. filling said capillary tube with second fluid.