US20060264779A1 - Fluidic medical devices and uses thereof - Google Patents

Fluidic medical devices and uses thereof Download PDF

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Publication number
US20060264779A1
US20060264779A1 US11/388,723 US38872306A US2006264779A1 US 20060264779 A1 US20060264779 A1 US 20060264779A1 US 38872306 A US38872306 A US 38872306A US 2006264779 A1 US2006264779 A1 US 2006264779A1
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US
United States
Prior art keywords
fluidic
reaction
reactants
fluidic device
analyte
Prior art date
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Abandoned
Application number
US11/388,723
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English (en)
Inventor
Timothy Kemp
Chris Todd
Ron Oral
Shulin Zeng
John Howard
Jeff Fenton
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Golden Diagnostics Corp
GOLDEN DIAGNOSTICS TOP CORP.
Theranos Inc
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Individual
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Family has litigation
First worldwide family litigation filed litigation Critical https://patents.darts-ip.com/?family=37397025&utm_source=google_patent&utm_medium=platform_link&utm_campaign=public_patent_search&patent=US20060264779(A1) "Global patent litigation dataset” by Darts-ip is licensed under a Creative Commons Attribution 4.0 International License.
Priority to US11/388,723 priority Critical patent/US20060264779A1/en
Application filed by Individual filed Critical Individual
Assigned to THERANOS, INC. reassignment THERANOS, INC. ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: ZENG, SHULIN, FENTON, JEFF, TODD, CHRIS, HOWARD, JOHN, KEMP, TIM, ORAL, RON
Priority to US11/554,509 priority patent/US8741230B2/en
Priority to PCT/US2006/042563 priority patent/WO2007111651A2/en
Publication of US20060264779A1 publication Critical patent/US20060264779A1/en
Priority to US12/625,430 priority patent/US9075046B2/en
Priority to US14/270,618 priority patent/US9176126B2/en
Priority to US14/727,547 priority patent/US9772291B2/en
Priority to US14/868,208 priority patent/US10533994B2/en
Priority to US15/689,905 priority patent/US11630069B2/en
Priority to US15/951,641 priority patent/US11287421B2/en
Priority to US17/706,102 priority patent/US20230035908A1/en
Priority to US18/301,835 priority patent/US20240085338A1/en
Assigned to GOLDEN DIAGNOSTICS CORP. reassignment GOLDEN DIAGNOSTICS CORP. ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: GOLDEN DIAGNOSTICS TOP CORP.
Assigned to GOLDEN DIAGNOSTICS TOP CORP. reassignment GOLDEN DIAGNOSTICS TOP CORP. ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: LABRADOR DIAGNOSTICS LLC
Abandoned legal-status Critical Current

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Definitions

  • Point-of-Care (POC) testing systems and fluidic devices or cartridges are becoming more common because of the advancement in microfabrication technology such as MEMS technology, which enables the fabrication of reliable and inexpensive fluidic based cartridges.
  • MEMS technology which enables the fabrication of reliable and inexpensive fluidic based cartridges.
  • Such systems use microvalves, micropumps, microneedles, etc. for moving the fluids through the fluidic system.
  • a common system contains a reagent reservoir, a mixing chamber, an analytical chamber and waste chamber. Fluids must therefore be moved from one chamber to another.
  • Some challenges in moving such fluids in a fluidic device include mixing the reagents with the sample, and washing unbound reagents from a detection site.
  • One of the common challenges is washing the unbound conjugates after the incubation period, particularly removing conjugates that remain stuck to the edges of the reaction site walls.
  • U.S. Pat. No. 5,600,993 provides a good summary of such exemplary problems.
  • U.S. Pat. No. 6,440,725 describes different fluid motive sources for moving liquids through the chambers.
  • One such example uses a fluid inside a sealed pouch wherein the fluid is converted to gas by an electrical current. This action pressurizes and expands the fluid pouch.
  • This sealed pumping pouch, or e-pump is positioned against a reagent pouch and forces the contents of the reagent pouch into the fluidic circuit as the pumping pouch expands.
  • the '725 patent also describes various other fluid motive sources such as pressure or vacuum source, or using a solenoid or stepper motor to provide a force to press against a reagent pouch.
  • US Patent Application No. 20050130292 describes using mechanical energy to move fluids within a fluidic device.
  • the inventors describe minimal or no external power to force the fluid through various chambers.
  • a sample is loaded on to a biochip and this biochip is inserted into a custom designed socket.
  • the work done in inserting the socket is converted to the energy required for the fluidic flow.
  • Subsequent steps of directing the sample to the desired chamber, mixing it, and assaying it are, according to the inventors, accomplished with minimal power consumption.
  • Such a device has several valves and pumps, even if the pumps are not driven by external electrical energy, which are difficult to include in a small disposable fluidic system.
  • reagents in a POC system are stored in a dry state to improve shelf-like. Buffers are generally stored separately until the assay is to be performed, at which time the reagents are hydrated. However, dry reagents may become wet or hydrated before they are intended to do so. Buffers may leak from their holding areas and mix with the dry reagents. It may thus be beneficial to keep the dry reagents in a dry state until the assay is initiated.
  • Cartridge or fluidic based POC systems may handle small volumes of fluids. Nanoliter or even picoliter amounts of fluids are sometimes forced to flow within fluidic channels. Either during the sample introduction or a venting process, there is a substantial likelihood that a bubble will be introduced into the microfluidics system. A bubble introduced into the system can cause an inaccurate measurement if the bubble is located in the detection site during the detection step.
  • the present invention provides an apparatus for detecting an analyte in a biological fluid of a subject.
  • the apparatus comprises a sample collection unit for introducing a biological fluid in fluid communication with a plurality of reaction sites, a plurality of reactant chambers carrying a plurality of reactants in fluid communication with said reaction sites wherein said plurality of reaction sites comprise a plurality of reactants bound thereto for detecting said analyte, and a system of fluidic channels to allow said biological fluid and said plurality of reactants to flow in said apparatus, wherein at least one channel located between said plurality of reaction sites comprises an optical barrier to reduce the amount of optical cross-talk between said plurality of said reaction sites during detection of said analyte.
  • the apparatus further comprising a plurality of waste chambers in fluid communication with at least one of said reaction sites.
  • each channel located between said plurality of reaction sites comprises an optical barrier.
  • the present invention further provides a method of manufacturing a fluidic device for detecting an analyte in a biological fluid of a subject.
  • the method comprises providing a plurality of layers of a fluidic device, and ultrasonically welding said layers together such that a fluidic network exists between a sample collection unit, at least one reactant chamber, at least one reaction site, and at least one waste chamber.
  • FIG. 1 illustrates exemplary multiple components of the present system.
  • FIG. 2 shows different layers of an exemplary fluidic device prior to assembly.
  • FIGS. 3 and 4 illustrate the fluidic network within an exemplary fluidic device.
  • FIG. 5 shows a top, side, and bottom view of exemplary reagent chambers of the present invention.
  • FIG. 6 illustrates an exemplary side view of a reagent chamber in fluidic communication with a fluidic device.
  • FIG. 7 illustrates exemplary reagent chambers being filled with reagents.
  • FIGS. 8 and 9 illustrate a side view of an exemplary fluidic device in combination with actuating elements of the reader assembly.
  • FIG. 10 illustrates a two-step assay and a competitive binding assay.
  • FIG. 11 shows an exemplary two-step chemiluminescence enzyme immunoassay.
  • FIG. 12 illustrates the increased sensitivity of the two-step chemiluminescence enzyme immunoassay.
  • FIGS. 13 A-C illustrate exemplary fluidic channels between reaction sites.
  • FIGS. 14A and 14B illustrate reaction sites to reduce the signal from unbound conjugates remaining in reaction sites.
  • FIG. 15 shows an exemplary bubble trapper or remover to prevent bubbles from entering the reaction sites.
  • One aspect of the present invention is a system for detecting an analyte in a sample of bodily fluid.
  • the subject system has one or more of the following components: a) a sample collection unit for introducing a biological fluid in fluid communication with a plurality of reaction sites, b) a plurality of reactant chambers carrying a plurality of reactants in fluid communication with said reaction sites wherein said plurality of reaction sites comprise a plurality of reactants bound thereto for detecting said analyte, and c) a system of fluidic channels to allow said biological fluid and said plurality of reactants to flow in said apparatus, wherein at least one channel located between said plurality of reaction sites comprises an optical barrier to reduce the amount of optical cross-talk between said plurality of said reaction sites during detection of said analyte.
  • the system may further comprise a reader assembly and a communication assembly.
  • the sample collection unit typically allows a sample of bodily fluid collected from a subject to react with reactants contained within the assay assembly for generating a signal indicative of the presence of the analyte of interest.
  • the reader assembly detects the signal, which is then transmitted via the communication assembly to an external device for further processing.
  • any bodily fluids suspected to contain an analyte of interest can be used in conjunction with the subject system or devices.
  • Commonly employed bodily fluids include but are not limited to blood, serum, saliva, urine, gastric and digestive fluid, tears, stool, semen, vaginal fluid, interstitial fluids derived from tumorous tissue, and cerebrospinal fluid.
  • the bodily fluids are used directly for detecting the analytes present therein with the subject fluidic device without further processing.
  • the bodily fluids can be pre-treated before performing the analysis with the subject fluidic devices. The choice of pre-treatments will depend on the type of bodily fluid used and/or the nature of the analyte under investigation.
  • the sample can be concentrated via any conventional means to enrich the analyte.
  • Methods of concentrating an analyte include but are not limited to drying, evaporation, centrifugation, sedimentation, precipitation, and amplification.
  • the analyte is a nucleic acid
  • it can be extracted using various lytic enzymes or chemical solutions according to the procedures set forth in Sambrook et al. (“Molecular Cloning: A Laboratory Manual”), or using nucleic acid binding resins following the accompanying instructions provided by manufactures.
  • analyte is a molecule present on or within a cell
  • extraction can be performed using lysing agents including but not limited to denaturing detergent such as SDS or non-denaturing detergent such as thesit, sodium deoxylate, triton X-100, and tween-20.
  • denaturing detergent such as SDS
  • non-denaturing detergent such as thesit, sodium deoxylate, triton X-100, and tween-20.
  • the volume of bodily fluid to be used with a fluidic device of the present invention is generally less than about 500 microliters, typically between about 1 to 100 microliters. Where desired, a sample of 1 to 50 microliters or 1 to 10 microliters can be used for detecting an analyte using the subject fluidic device.
  • a bodily fluid may be drawn from a patient and brought into the fluidic device in a variety of ways, including but not limited to, lancing, injection, or pipetting.
  • a lancet punctures the skin and draws the sample into the fluidic device using, for example, gravity, capillary action, aspiration, or vacuum force.
  • the lancet may be part of the fluidic device, or part of a reader assembly, or as a stand alone component. Where needed, the lancet may be activated by a variety of mechanical, electrical, electromechanical, or any other known activation mechanism or any combination of such methods.
  • a patient can simply provide a bodily fluid to the fluidic device, as for example, could occur with a saliva sample.
  • the collected fluid can be placed in the sample collection unit within the fluidic device.
  • the fluidic device comprises at least one microneedle which punctures the skin.
  • the microneedle can be used with a fluidic device alone, or can puncture the skin after the fluidic device is inserted into a reader assembly.
  • a microneedle is about the size of a human hair and has an integrated microreservoir or cuvette.
  • the microneedle may painlessly penetrate the skin and draw a small blood sample. More preferably, the microneedle collects about 0.01 to about 1 microliter, preferably about 0.05 to about 0.5 microliters and more preferably about 0.1 to about 0.3 microliters of capillary blood.
  • a microneedle may be constructed out of silicon and is about 10 to about 200 microns in diameter, preferably about 50 to about 150 microns in diameter, and most preferably about 100 microns in diameter, making their application to the skin virtually painless.
  • microneedles may be used for sample collection.
  • Such microneedles may be of the type marketed by Pelikan (Palo Alto, Calif.) and/or Kumetrix (Union City, Calif.).
  • U.S. Pat. No. 6,503,231 discloses microneedles which may be used with the present invention.
  • Microfabrication processes that may be used in making the microneedles disclosed herein include without limitation lithography; etching techniques such as wet chemical, dry, and photoresist removal; thermal oxidation of silicon; electroplating and electroless plating; diffusion processes such as boron, phosphorus, arsenic, and antimony diffusion; ion implantation; film deposition such as evaporation (filament, electron beam, flash, and shadowing and step coverage), sputtering, chemical vapor deposition (CVD), epitaxy (vapor phase, liquid phase, and molecular beam), electroplating, screen printing, and lamination. See generally Jaeger, Introduction to Microelectronic Fabrication (Addison-Wesley Publishing Co., Reading Mass.
  • microneedles may be molded in silicon wafers and then plated using conventional wire cutting techniques with nickel, gold, titanium or various other biocompatible metals.
  • microneedles can be fashioned from biopolymers.
  • microneedles may be fabricated and employed for the claimed devices according to the methods of Mukerjee et al., Sensors and Actuators A: Physical, Volume 114, Issues 2-3, 1 Sep. 2004, Pages 267-275.
  • a microneedle is only used once and then discarded.
  • a mechanical actuator can insert and withdraw the microneedle from the patient, discard the used needle, and reload a new microneedle.
  • the mechanical technologies developed and manufactured in very high volumes for very small disk drives have a similar set of motion and low cost requirements.
  • the actuator is a MEMS (micro machined electromechanical system) device fabricated using semiconductor-like batch processes. Such actuators include without limitation nickel titanium alloy, neumatic, or piezo electric devices.
  • the microneedles are about 1 micron to about 10 microns in thickness, preferably about 2 microns to about 6 microns in thickness, and most preferably about 4 microns in thickness.
  • the microneedles are about 10 microns to about 100 microns in height, preferably about 30 microns to about 60 microns in height, and most preferably about 40 microns in height.
  • FIG. 1 illustrates an exemplary system of the present invention.
  • a fluidic device provides a bodily fluid from a patient and can be inserted into a reader assembly.
  • the fluidic device may take a variety of configurations and in some embodiments the fluidic device may be in the form of a cartridge.
  • An identifier (ID) detector may detect an identifier on the fluidic device.
  • the identifier detector communicates with a communication assembly via a controller which transmits the identifier to an external device.
  • the external device sends a protocol stored on the external device to the communication assembly based on the identifier.
  • the protocol to be run on the fluidic device may comprise instructions to the controller of the reader assembly to perform the protocol on the fluidic device, including but not limited to a particular assay to be run and a detection method to be performed.
  • a signal indicative of an analyte in the bodily fluid sample is generated and detected by a detection assembly.
  • the detected signal may then be communicated to the communications assembly, where it can be transmitted to the external device for processing, including without limitation, calculation of the analyte concentration in the sample.
  • FIG. 2 illustrates exemplary layers of a fluidic device according to the present invention prior to assembly of the fluidic device which is disclosed in more detail below.
  • FIGS. 3 and 4 illustrate the fluidic network within an exemplary fluidic device. The different layers are designed and assembled to form a three dimensional fluidic channel network.
  • a sample collection unit 4 provides a sample of bodily fluid from a patient.
  • a reader assembly comprises actuating elements (not shown) can actuate the fluidic device to start and direct the flow of a bodily fluid sample and assay reagents in the fluidic device.
  • actuating elements first cause the flow of sample in the fluidic device 2 from sample collection unit 4 to reaction sites 6 , move the sample upward in the fluidic device from point G′ to point G, and then to waste chamber 8 .
  • the actuating elements then initiate the flow of reagents from reagent chambers 10 to point B′, point C′, and point D′, then upward to points B, C, and D, respectively.
  • the reagents then move to point A, down to point A′, and then to waste chamber 8 in a manner similar to the sample.
  • a sample collection unit 4 in a fluidic device 2 may provide a bodily fluid sample from a patient by any of the methods described above. If necessary, the sample may first be processed by diluting the bodily fluid in a dilution chamber, and or may be filtered by separating the plasma from the red blood cells in a filtration chamber.
  • the sample collection unit, diluting chamber, and filtration chamber may be the same component, and in some embodiments they may be different components, or any two may be the same component and the other may be a separate component. In some embodiments there may be more than one sample collection unit in the fluidic device.
  • the fluidic device may include a lysing assembly to lyse cells present in the bodily fluid sample.
  • the lysing assembly may be incorporated with the sample collection unit, a dilution chamber, and/or a filtration chamber.
  • the sample collection unit, dilution chamber, and lysing component are within the same element in the fluidic device.
  • the lysing component may be incorporated with an assay reagent described below.
  • lysing agents may be impregnated and then dried into porous mats, glass fiber mats, sintered frits or particles such as Porex, paper, or other similar material. Lysing agents may be dried onto flat surfaces. Lysing agents may also be dissolved in liquid diluents or other liquid reagents.
  • porous materials are used to store the lysing agents because they can store a lysing agent in dry form likely to be very stable. They also facilitate the mixing of the bodily fluid sample with the lysing agent by providing a tortuous path for the sample as it moves through the porous material. In preferred embodiments such porous materials have a disc shape with a diameter greater than its thickness.
  • lysing agents may be dried onto porous materials using lyophilization, passive evaporation, exposure to warm dry flowing gas, or other known methods.
  • lysing agents are available in the art and are suitable for use in connection with the subject fluidic device.
  • Preferred lysing agents are non-denaturing, such as non-denaturing detergents.
  • Non-limiting examples of non-denaturing detergents include thesit, sodium deoxylate, triton X-100, and tween-20.
  • the agents are preferably non-volatile in embodiments where the agents are impregnated into a solid porous materials.
  • lysing agents are mixed together. Other materials may be mixed with the lysing agents to modify the lytic effects.
  • Such exemplary materials may be, without limitation, buffers, salts, and proteins.
  • lysing agents will be used in amounts that are in excess of the minimum amount required to lyse cells.
  • lysing agents will be used that can lyse both white and red cells.
  • any reagents necessary to perform an assay on a fluidic device according to the present invention are preferably on-board, or housed within the fluidic device before, during, and after the assay.
  • the only inlet or outlet from the fluidic device is preferably the bodily fluid sample initially provided by the fluidic device.
  • This design also helps create an easily disposable fluidic device where all fluids or liquids remain in the device.
  • the on-board design also prevents leakage from the fluidic device into the reader assembly which should remain free from contamination from the fluidic device.
  • a reagent chamber is preferably in fluid communication with at least one reaction site, and when the fluidic device is actuated as described herein, reagents contained in said reagent chambers are released into the fluidic channels within the fluidic device.
  • Reagents according to the present invention include without limitation wash buffers, enzyme substrates, dilution buffers, conjugates, enzyme-labeled conjugates, DNA amplifiers, sample diluents, wash solutions, sample pre-treatment reagents including additives such as detergents, polymers, chelating agents, albumin-binding reagents, enzyme inhibitors, enzymes, anticoagulants, red-cell agglutinating agents, antibodies, or other materials necessary to run an assay on a fluidic device.
  • An enzyme conjugate can be either a polyclonal antibody or monoclonal antibody labeled with an enzyme that can yield a detectable signal upon reaction with an appropriate substrate.
  • Non-limiting examples of such enzymes are alkaline phosphatase and horseradish peroxidase.
  • the reagents comprise immunoassay reagents.
  • a reagent chamber contains approximately about 50 ⁇ l to about 1 ml of fluid. In some embodiments the chamber may contain about 100 ⁇ l of fluid.
  • the volume of liquid in a reagent chamber may vary depending on the type of assay being run or the sample of bodily fluid provided. In some embodiments the reagents are initially stored dry and liquified upon initiation of the assay being run on the fluidic device.
  • a reagent chamber is preferably in fluid communication with at least one reaction site, and when the fluidic device is actuated as described herein, reagents contained in said reagent chambers are released into the fluidic channels within the fluidic device.
  • Reagents according to the present invention include without limitation wash buffers, substrates, dilution buffers, conjugates, enzyme-labeled conjugates, DNA amplifiers, sample diluents, wash solutions, sample pre-treatment reagents including additives such as detergents, polymers, chelating agents, albumin-binding reagents, enzyme inhibitors, enzymes, anticoagulants, red-cell agglutinating agents, antibodies or other materials necessary to run an assay on a fluidic device.
  • An enzyme conjugate can be either a polyclonal antibody or monoclonal antibody labeled with an enzyme, such as alkaline phosphatase or horseradish peroxidase.
  • the reagents are immunoassay reagents.
  • a reagent chamber contains approximately about 50 ⁇ l to about 1 ml of fluid. In some embodiments the chamber may contain about 100 ⁇ l of fluid.
  • the volume of liquid in a reagent chamber may vary depending on the type of assay being run or the sample of bodily fluid provided. In some embodiments the reagents are initially stored dry and liquified upon initiation of the assay being run on the fluidic device.
  • FIG. 5 illustrate a different embodiment of a sealed reagent chamber.
  • FIG. 5 shows a top, side, and bottom view of a reagent chamber.
  • a top layer 11 contains a plurality of bubbles or pouches 13 .
  • a bottom layer 15 has a bottom surface that is bonded to the fluidic device base 17 as shown in FIG. 6 .
  • the bottom layer 15 has a plurality of fluidic channels 19 dispersed through the entire surface, where each channel traverses the bottom layer 15 .
  • the fluid in the reagent chamber is contained within the chamber by pressure burstable seal 21 between the fluidic channel 19 and the chamber 13 .
  • the burstable seal 21 is designed such that at a pre-determined pressure the seal bursts allowing the fluid in the chamber 13 to flow out into a fluidic channel 19 .
  • FIG. 7 shows an exemplary process of filling the reagent chambers 13 with, for example, reagents.
  • Reagent chambers 13 may be filled with fluid using a fill channel and a vacuum draw channel.
  • the process of filling the reagents involves first removing all the air from the chamber. This is done by drawing a vacuum through the vacuum draw channel. Once the vacuum is drawn, a permanent seal is placed between the fill channel and the vacuum draw channel. Next, required reagents are dispensed into the chamber through the fill channel. Then, a permanent seal is placed between the chamber and the fill channel. This ensures that when the chamber is compressed, the fluid can flow in only one direction, towards the burstable seal. If the compression imparts a pressure larger than the burst pressure of seal, the seal bursts and the fluid flows into the fluidic channel.
  • FIGS. 8 and 9 illustrate an embodiment of a fluidic device in operation with actuating elements as described herein.
  • Fluidic device 2 contains a reagent chamber 10 and a layer of burstable foil 12 enclosing the reagent chamber. Above the burstable foil 12 is a portion of the microfluidic circuit 14 .
  • a tough, but elastomeric top cover 16 acts as the top layer of the fluidic device 2 .
  • the reader assembly includes a valve actuation plate 18 . Securely attached to the plate 18 is a non-coring needle 20 such that when the plate is lowered, the sharp edge of the needle contacts the elastomeric cover 16 .
  • the top cover could also be made of flexible silicone material that would act as a moisture impermeable seal.
  • This embodiment also provides a solution to liquid evaporation and leakage from a fluidic device by isolating any liquid reagents in the fluidic device from any dry reagents until the assay is initiated.
  • the reagent chamber and sample collection unit are fluidly connected to reaction sites where bound reactant can detect an analyte of interest in the bodily fluid sample using the assay.
  • a reaction site could then provide a signal indicative of the presence of the analyte of interest, which can then be detected by a detection device described in detail herein below.
  • the reactions sites are flat but they may take on a variety of alternative surface configurations.
  • the reaction site preferably forms a rigid support on which a reactant can be immobilized.
  • the reaction site surface is also chosen to provide appropriate light-absorbing characteristics.
  • the reaction site may be functionalized glass, Si, Ge, GaAs, GaP, SiO 2 , SiN 4 , modified silicon, or any one of a wide variety of gels or polymers such as (poly)tetrafluoroethylene, (poly)vinylidenedifluoride, polystyrene, polycarbonate, polypropylene, or combinations thereof. Other appropriate materials may be used in accordance with the present invention.
  • a reactant immobilized at a reaction site can be anything useful for detecting an analyte of interest in a sample of bodily fluid.
  • reactants include without limitation nucleic acid probes, antibodies, cell membrane receptors, monoclonal antibodies and antisera reactive with a specific analyte.
  • Various commercially available reactants such as a host of polyclonal and monoclonal antibodies specifically developed for specific analytes can be used.
  • the immobilization may be covalent or noncovalent, via a linker moiety, or tethering them to an immobilized moiety. These methods are well known in the field of solid phase synthesis and micro-arrays (Beier et al., Nucleic Acids Res. 27:1970-1-977 (1999).
  • Non-limiting exemplary binding moieties for attaching either nucleic acids or proteinaceous molecules such as antibodies to a solid support include streptavidin or avidin/biotin linkages, carbamate linkages, ester linkages, amide, thiolester, (N)-functionalized thiourea, functionalized maleimide, amino, disulfide, amide, hydrazone linkages, and among others.
  • a silyl moiety can be attached to a nucleic acid directly to a substrate such as glass using methods known in the art.
  • reaction sites there are more than one reaction sites which can allow for detection of multiple analytes of interest from the same sample of bodily fluid. In some embodiments there are two, three, four, five, six, or more reaction sites, or any other number of reaction sites as may be necessary to carry out the intent of the invention.
  • each reaction site may contain a probe different from a probe on a different reaction site.
  • there may be three different probes each bound to a different reaction site to bind to three different analytes of interest in the sample.
  • there may be different probes bound to a single reaction site if, for example, a CCD with multiple detection areas were used as the detection device, such that multiple different analytes could be detected in a single reaction site.
  • the capability to use multiple reaction sites in addition to multiple different probes on each reaction site enables the high-throughput characteristics of the present invention.
  • the present invention allows for the detection of multiple analytes on the same fluidic device. If assays with different luminescent intensities are run in adjacent reaction sites, photons (signals that emanate from the reactions) may travel from one reaction site to an adjacent reaction site, as reaction sites may be constructed of materials that allow photons to travel through the fluidic channels that connect the sites. This optical cross talk may compromise the accuracy of the detected photons.
  • FIGS. 13B and 13C illustrate different embodiments of this invention that can eliminate or reduce the amount of optical cross-talk. Non-linear channels 22 will not allow photons (light) to pass through. Hence, embodiments such as those shown in FIGS.
  • reaction sites 13B and 13C would not allow signals from a reaction site to contaminate a signal produced from an adjacent site from which a detection device may be detecting. Additionally, the edges or walls of a reaction site may be constructed using optically opaque materials so that light will not escape the wells. In some embodiments the reaction sites are white or opaque.
  • the bound reactants in the at least one reaction site are localized around the center of said reaction site.
  • an outer edge of the at least one reaction site is at a distance sufficiently far from said bound reactants to reduce signals unrelated to the presence of said analyte. Distancing the edge of the reaction site from the center area where bound reactants are concentrated allows reduction of interfering signals from the background that does not relate to the presence of the analyte of interest.
  • At least one of these channels will typically have small cross sectional dimensions. In some embodiments the dimensions are from about 0.01 mm to about 5 mm, preferably from about 0.03 mm to about 3 mm, and more preferably from about 0.05 mm to about 2 mm.
  • Fluidic channels in the fluidic device may be created by, for example without limitation, precision injection molding, laser etching, or any other technique known in the art to carry out the intent of the invention.
  • FIG. 15 illustrates an embodiment where a bubble could be trapped in a filter 28 before it reaches a reaction site 6 .
  • a bubble trapper 28 can be positioned between a sample collection unit 4 and reaction site 6 . The bubble trapper can have such a geometry that the bubbles tend to migrate towards the edges of this surface and remain stuck at that service, thereby not entering into the reaction sites.
  • Manufacturing of the fluidic channels may generally be carried out by any number of microfabrication techniques that are well known in the art.
  • lithographic techniques are optionally employed in fabricating, for example, glass, quartz or silicon substrates, using methods well known in the semiconductor manufacturing industries such as photolithographic etching, plasma etching or wet chemical etching.
  • micromachining methods such as laser drilling, micromilling and the like are optionally employed.
  • polymeric substrates well known manufacturing techniques may also be used. These techniques include injection molding or stamp molding methods where large numbers of substrates are optionally produced using, for example, rolling stamps to produce large sheets of microscale substrates or polymer microcasting techniques where the substrate is polymerized within a micromachined mold.
  • At least one of the different layers of the fluidic device may be constructed of polymeric substrates.
  • polymeric materials include polystyrene, polycarbonate, polypropylene, polydimethysiloxanes (PDMS), polyurethane, polyvinylchloride (PVC), and polysulfone.
  • the fluidic device may be manufactured by stamping, thermal bonding, adhesives or, in the case of certain substrates, for example, glass, or semi-rigid and non-rigid polymeric substrates, a natural adhesion between the two components.
  • the fluidic device is manufactured by ultrasonic or acoustic welding.
  • FIG. 2 shows one embodiment of the invention in which fluidic device 2 is comprised of seven layers. Features as shown are, for example, cut in the polymeric substrate such that when the layers are properly positioned when assembly will form a fluidic network. In some embodiments more or fewer layers may be used to construct a fluidic device to carry out the purpose of the invention.
  • One goal of the present invention is to prevent fluid inside a fluidic device from contacting the components of a reader assembly which may need to remain dry and or uncontaminated, and also to prevent contamination to a detection device within the reader assembly.
  • a leak in the fluidic device could result in liquids, for example reagents or waste, escaping from the fluidic device and contaminating the reader.
  • a liquid absorbing material such as polymeric materials found in diapers, could be placed within a portion of the fluidic channel or waste chamber to absorb the waste liquid.
  • a non-limiting example of such a polymer is sodium polyacrylate. Such polymers can absorb fluids hundreds of times their weight. Hence, only minute quantities of such polymeric materials may be required to accomplish the goal of absorbing leaked fluids.
  • a waste chamber is filled with a superabsorbent material.
  • leaked liquid may be converted into a gel or other solid or semi-solid form.
  • FIGS. 8 and 9 illustrate an exemplary sequence to initiate the flow of a reagent within the fluidic device.
  • An actuation plate 18 in the reader assembly comprises a non-coring needle or pin 20 which when lowered flexes the top cover 16 , as it is preferably made of strong, flexible elastomeric material. However, the easily rupturable foil 12 then ruptures due to the stress induced by the flexing of top cover 16 . Valves located downstream to the reagent chamber puncture different areas of foil in the fluidic device and can then work in tandem with a pump within the reader assembly to create a vacuum force to pull the reagent out of the reagent chamber 6 into a fluidic channel and then direct the flow of the reagent to a reaction site.
  • At least one valve is preferably fluidically connected to a pump housed within the reader assembly.
  • the non-coring needle or pin 20 is removed from the fluidic device when the device is removed from the reader assembly.
  • a method of manufacturing a fluidic device for detecting an analyte in a biological fluid of a subject comprises providing a plurality of layers of a material, wherein at least one of said layers comprises a sample collection unit, wherein at least one of said layers comprises a filtration site, wherein at least one of said layers comprises a reactant chamber, wherein at least one of said layers comprises a fluidic channel, wherein at least one of said layers comprises a reaction site, wherein at least one of said layers comprises a waste chamber; and ultrasonically welding said layers together such that a fluidic network of channels exists between said sample collection unit, said reactant chambers, said filtration site, said reaction sites, said fluidic channel, and said waste chamber.
  • the different layers of the fluidic device are ultrasonically welded together according to methods known in the art.
  • the layers may also be coupled together using other methods, including without limitation stamping, thermal bonding, adhesives or, in the case of certain substrates, for example, glass, or semi-rigid and non-rigid polymeric substrates, a natural adhesion between the two components
  • the subject system provides an effective means for high throughput and real-time detection of analytes present in a bodily fluid from a subject.
  • the detection methods may be used in a wide variety of circumstances including identification and quantification of analytes that are associated with specific biological processes, physiological conditions, disorders or stages of disorders.
  • the subject apparatus and systems have a broad spectrum of utility in, e.g. drug screening, disease diagnosis, phylogenetic classification, parental and forensic identification.
  • the subject apparatus and systems are also particularly useful for advancing preclinical and clinical stage of development of therapeutics, improving patient compliance, monitoring ADRs associated with a prescribed drug, and developing individualized medicine.
  • the term “subject” or “patient” is used interchangeably herein, which refers to a vertebrate, preferably a mammal, more preferably a human. Mammals include, but are not limited to, murines, simians, humans, farm animals, sport animals, and pets.
  • a sample of bodily fluid can first be provided to the fluidic device by any of the methods described herein.
  • the fluidic device can then be inserted into the reader assembly.
  • An identification detector housed within the reader assembly can detect an identifier of the fludic device and communicate the identifier to a communication assembly, which is preferably housed within the reader assembly.
  • the communication assembly then transmits the identifier to an external device which transmits a protocol to run on the fluidic device based on the identifier to the communication assembly.
  • a controller preferably housed within the reader assembly controls actuating elements including at least one pump and one valve which interact with the fluidic device to control and direct fluid movement within the device.
  • the fist step of the assay is a wash cycle where all the surfaces within the fluidic device are wetted using a wash buffer.
  • the fluidic device is then calibrated using a calibration assembly by running the same reagents as will be used in the assay through the calibration reaction sites, and then a luminescence signal from the reactions sites is detected by the detection means, and the signal is used in calibrating the fluidic device.
  • the sample containing the analyte is introduced into the fluidic channel.
  • the sample may be diluted and further separated into plasma or other desired component at a filter.
  • the separated sample now flows through the reaction sites and analytes present therein will bind to reactants bound thereon.
  • the plasma of sample fluid is then flushed out of the reaction wells into a waste chamber.
  • analytes includes without limitation drugs, prodrugs, pharmaceutical agents, drug metabolites, biomarkers such as expressed proteins and cell markers, antibodies, serum proteins, cholesterol, polysaccharides, nulceic acids, biological analytes, biomarker, gene, protein, or hormone, or any combination thereof.
  • biomarkers such as expressed proteins and cell markers, antibodies, serum proteins, cholesterol, polysaccharides, nulceic acids, biological analytes, biomarker, gene, protein, or hormone, or any combination thereof.
  • the analytes can be polypeptide glycoprotein, polysaccharide, lipid, nucleic acid, and a combination thereof.
  • biomarkers are associated with a particular disease or with a specific disease stage.
  • Such analytes include but are not limited to those associated with autoimmune diseases, obesity, hypertension, diabetes, neuronal and/or muscular degenerative diseases, cardiac diseases, endocrine disorders, any combinations thereof.
  • biomarkers that are present in varying abundance in one or more of the body tissues (i.e., tissue-specific) including heart, liver, prostate, lung, kidney, bone marrow, blood, skin, bladder, brain, muscles, nerves, and selected tissues that are affected by various disease, such as different types of cancer (malignant or non-metastatic), autoimmune diseases, inflammatory or degenerative diseases.
  • body tissues i.e., tissue-specific
  • diseases such as different types of cancer (malignant or non-metastatic), autoimmune diseases, inflammatory or degenerative diseases.
  • analytes that are indicative of a microorganism include but are not limited to bacterium, virus, fungus and protozoa.
  • Analytes that can be detected by the subject method also include blood-born pathogens selected from a non-limiting group that consists of Staphylococcus epidermidis, Escherichia coli, methicillin-resistant Staphylococcus aureus (MSRA), Staphylococcus aureus, Staphylococcus hominis, Enterococcus faecalis, Pseudomonas aeruginosa, Staphylococcus capitis, Staphylococcus warneri, Klebsiella pneumoniae, Haemophilus influnzae, Staphylococcus simulans, Streptococcus pneumoniae and Candida albicans.
  • blood-born pathogens selected from a non-limiting group that consists of Staphylococcus epidermidis, Escherichia coli
  • Analytes that can be detected by the subject method also encompass a variety of sexually transmitted diseases selected from the following: gonorrhea ( Neisseria gorrhoeae ), syphilis ( Treponena pallidum ), clamydia ( Clamyda tracomitis ), nongonococcal urethritis ( Ureaplasm urealyticum ), yeast infection ( Candida albicans ), chancroid ( Haemophilus ducreyi ), trichomoniasis ( Trichomonas vaginalis ), genital herpes (HSV type I & II), HIV I, HIV II and hepatitis A, B, C, G, as well as hepatitis caused by TTV.
  • gonorrhea Neisseria gorrhoeae
  • syphilis Treponena pallidum
  • clamydia Clamyda tracomitis
  • Additional analytes that can be detected by the subject methods encompass a diversity of respiratory pathogens including but not limited to Pseudomonas aeruginosa, methicillin-resistant Staphlococccus aureus (MSRA), Klebsiella pneumoniae, Haemophilis influenzae, Staphlococcus aureus, Stenotrophomonas maltophilia, Haemophilis parainfluenzae, Escherichia coli, Enterococcus faecalis, Serratia marcescens, Haemophilis parahaemolyticus, Enterococcus cloacae, Candida albicans, Moraxiella catarrhalis, Streptococcus pneumoniae, Citrobacter freundii, Enterococcus faecium, Klebsella oxytoca, Pseudomonas fluorscens, Neiseria meningitidis, Streptococcus py
  • a variety of assays may be performed on a fluidic device according to the present invention to detect an analyte of interest in a sample.
  • labels are available in the art that can be employed for conducting the subject assays.
  • labels are detectable by spectroscopic, photochemical, biochemical, immunochemical, or chemical means.
  • useful nucleic acid labels include 32P, 35S, fluorescent dyes, electron-dense reagents, enzymes, biotin, dioxigenin, or haptens and proteins for which antisera or monoclonal antibodies are available.
  • a wide variety of labels suitable for labeling biological components are known and are reported extensively in both the scientific and patent literature, and are generally applicable to the present invention for the labeling of biological components.
  • Suitable labels include radionucleotides, enzymes, substrates, cofactors, inhibitors, fluorescent moieties, chemiluminescent moieties, bioluminescent labels, calorimetric labels, or magnetic particles.
  • Labeling agents optionally include, for example, monoclonal antibodies, polyclonal antibodies, proteins, or other polymers such as affinity matrices, carbohydrates or lipids. Detection proceeds by any of a variety of known methods, including spectrophotometric or optical tracking of radioactive or fluorescent markers, or other methods which track a molecule based upon size, charge or affinity.
  • a detectable moiety can be of any material having a detectable physical or chemical property.
  • a label includes without limitation any composition detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical thermal, or chemical means.
  • the label is coupled directly or indirectly to a molecule to be detected such as a product, substrate, or enzyme, according to methods well known in the art.
  • a molecule to be detected such as a product, substrate, or enzyme
  • Non radioactive labels are often attached by indirect means.
  • a ligand molecule is covalently bound to a polymer.
  • the ligand then binds to an anti-ligand molecule which is either inherently detectable or covalently bound to a signal system, such as a detectable enzyme, a fluorescent compound, or a chemiluminescent compound.
  • a signal system such as a detectable enzyme, a fluorescent compound, or a chemiluminescent compound.
  • a number of ligands and anti-ligands can be used.
  • a ligand has a natural anti-ligand, for example, biotin, thyroxine, and cortisol, it can be used in conjunction with labeled, anti-ligands.
  • a haptenic or antigenic compound can be used in combination with an antibody.
  • the label can also be conjugated directly to signal generating compounds, for example, by conjugation with an enzyme or fluorophore.
  • Enzymes of interest as labels will primarily be hydrolases, particularly phosphatases, esterases and glycosidases, or oxidoreductases, particularly peroxidases.
  • Fluorescent compounds include fluorescein and its derivatives, rhodamine and its derivatives, dansyl, and umbelliferone.
  • Chemiluminescent compounds include luciferin, and 2,3-dihydrophthalazinediones, such as luminol.
  • means for detection include a scintillation counter or photographic film as in autoradiography.
  • the label is a fluorescent label, it may be detected by exciting the fluorochrome with the appropriate wavelength of light and detecting the resulting fluorescence by, for example, microscopy, visual inspection, via photographic film, by the use of electronic detectors such as digital cameras, charge coupled devices (CCDs) or photomultipliers and phototubes, or other detection device.
  • enzymatic labels are detected by providing appropriate substrates for the enzyme and detecting the resulting reaction product.
  • simple colorimetric labels are often detected simply by observing the color associated with the label. For example, conjugated gold often appears pink, while various conjugated beads appear the color of the bead.
  • the detectable signal may be provided by luminescence sources.
  • Luminescence is the term commonly used to refer to the emission of light from a substance for any reason other than a rise in its temperature.
  • atoms or molecules emit photons of electromagnetic energy (e.g., light) when then move from an “excited state” to a lower energy state (usually the ground state); this process is often referred to as “radioactive decay”.
  • electromagnetic energy e.g., light
  • photoluminescence the luminescence process
  • the exciting cause is an electron
  • the luminescence process is referred to as “electroluminescence”.
  • electroluminescence results from the direct injection and removal of electrons to form an electron-hole pair, and subsequent recombination of the electron-hole pair to emit a photon.
  • Luminescence which results from a chemical reaction is usually referred to as “chemiluminescence”.
  • Luminescence produced by a living organism is usually referred to as “bioluminescence”.
  • photoluminescence is the result of a spin-allowed transition (e.g., a single-singlet transition, triplet-triplet transition)
  • the photoluminescence process is usually referred to as “fluorescence”.
  • fluorescence emissions do not persist after the exciting cause is removed as a result of short-lived excited states which may rapidly relax through such spin-allowed transitions.
  • photoluminescence is the result of a spin-forbidden transition (e.g., a triplet-singlet transition)
  • the photoluminescence process is usually referred to as “phosphorescence”.
  • phosphorescence emissions persist long after the exciting cause is removed as a result of long-lived excited states which may relax only through such spin-forbidden transitions.
  • a “luminescent label” may have any one of the above-described properties.
  • Suitable chemiluminescent sources include a compound which becomes electronically excited by a chemical reaction and may then emit light which serves as the detectible signal or donates energy to a fluorescent acceptor.
  • a diverse number of families of compounds have been found to provide chemiluminescence under a variety or conditions.
  • One family of compounds is 2,3-dihydro-1,4-phthalazinedione.
  • a frequently used compound is luminol, which is a 5-amino compound.
  • Other members of the family include the 5-amino-6,7,8-trimethoxy- and the dimethylamino[ca]benz analog. These compounds can be made to luminesce with alkaline hydrogen peroxide or calcium hypochlorite and base.
  • Chemiluminescent analogs include para-dimethylamino and -methoxy substituents. Chemiluminescence may also be obtained with oxalates, usually oxalyl active esters, for example, p-nitrophenyl and a peroxide such as hydrogen peroxide, under basic conditions. Other useful chemiluminescent compounds that are also known include -N-alkyl acridinum esters and dioxetanes. Alternatively, luciferins may be used in conjunction with luciferase or lucigenins to provide bioluminescence.
  • immunoassays are run on the fluidic device. While competitive binding assays, which are well known in the art, may be run in some embodiments, in preferred embodiments a two-step method is used which eliminates the need to mix a conjugate and a sample before exposing the mixture to an antibody, which may be desirable when very small volumes of sample and conjugate are used, as in the fluidic device of the present invention.
  • a two-step assay has additional advantages over the competitive binding assays when use with a fluidic device as described herein. It combines the ease of use and high sensitivity of a sandwich (competitive binding) immunoassay with the ability to assay small molecules.
  • the sample containing analyte (“Ag”) first flows over a reaction site containing antibodies (“Ab”).
  • the antibodies bind the analyte present in the sample.
  • a solution with analyte conjugated to a marker (“labeled Ag”) at a high concentration is passed over the surface.
  • the conjugate saturates any of the antibodies that have not yet bound the analyte.
  • the high-concentration conjugate solution is washed off.
  • the amount of conjugate bound to the surface is then measured by the appropriate technique, and the detected conjugate is inversely proportional to the amount of analyte present in the sample.
  • the marker can be a commercially available marker such as dioxitane-phosphate, which is not luminescent but becomes luminescent after hydrolysis by, for example, alkaline phosphatase.
  • An enzyme such as alkaline phosphatase is also passed over the substrate to cause the marker to luminesce.
  • the substrate solution is supplemented with enhancing agents such as, without limitation, fluorescein in mixed micelles, soluble polymers, or PVC which create a much brighter signal than the luminophore alone.
  • FIG. 12 shows that for analytes in the picomolar concentration, TOSCA is able to provide a more robust signal (higher sensitivity) than a competitive binding assay. Use of a two-step binding assay thus contributes to higher sensitivity capabilities of the present invention.
  • unbound conjugates may need to be washed from a reaction site to prevent unbound conjugates from activating the substrate and producing and inaccurate signal. It may be difficult to remove conjugates sticking to the edges of the reaction sites in such a fluidic device if, for example, there is not an excess of a wash solution. To decrease the signal contributed from unbound conjugates stuck to the edge of a reaction site, it may be advantageous to expand the reaction site edge or wall radius in order to distance stuck conjugate from the desired actual detection area, represented by bound probes.
  • FIGS. 14A and 14B illustrates this concept.
  • Reaction site 6 contains reaction surface 24 and edge or wall surface 26 . An edge surface 26 is shown at a greater distance from the center of the reaction site 6 than is the edge surface of the prior art design. This allows unbound conjugates to adhere to the edge surfaces and be distanced from bound conjugates, which are concentrated closer to the center of the reaction site 6 .
  • the fluidic device includes at least one waste chamber to trap or capture all liquids after they have been used in the assay.
  • there is more than one waste chamber at least one of which is to be used with a calibration assembly described herein below.
  • On-board waste chambers also allow the device to be easily disposable.
  • the waste chamber is preferably in fluidic communication with at least one reaction site.
  • the subject system is capable of detecting a plurality of analytes.
  • the system can be used to identify and quantify analytes present varying concentrations that differ by more than 3 orders of magnitude.

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US11/388,723 US20060264779A1 (en) 2005-05-09 2006-03-24 Fluidic medical devices and uses thereof
US11/554,509 US8741230B2 (en) 2006-03-24 2006-10-30 Systems and methods of sample processing and fluid control in a fluidic system
PCT/US2006/042563 WO2007111651A2 (en) 2006-03-24 2006-10-31 Systems and methods of sample processing and fluid control in a fluidic system
US12/625,430 US9075046B2 (en) 2005-05-09 2009-11-24 Fluidic medical devices and uses thereof
US14/270,618 US9176126B2 (en) 2006-03-24 2014-05-06 Systems and methods of sample processing and fluid control in a fluidic system
US14/727,547 US9772291B2 (en) 2005-05-09 2015-06-01 Fluidic medical devices and uses thereof
US14/868,208 US10533994B2 (en) 2006-03-24 2015-09-28 Systems and methods of sample processing and fluid control in a fluidic system
US15/689,905 US11630069B2 (en) 2005-05-09 2017-08-29 Fluidic medical devices and uses thereof
US15/951,641 US11287421B2 (en) 2006-03-24 2018-04-12 Systems and methods of sample processing and fluid control in a fluidic system
US17/706,102 US20230035908A1 (en) 2006-03-24 2022-03-28 Systems and Methods of Sample Processing and Fluid Control in a Fluidic System
US18/301,835 US20240085338A1 (en) 2005-05-09 2023-04-17 Fluidic medical devices and uses thereof

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US11/388,823 Continuation-In-Part US8841076B2 (en) 2005-05-09 2006-03-24 Systems and methods for conducting animal studies
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US11/389,410 Abandoned US20060264783A1 (en) 2005-05-09 2006-03-24 Systems and methods for monitoring pharmacological parameters
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US12/986,954 Active US9182388B2 (en) 2005-05-09 2011-01-07 Calibration of fluidic devices
US13/286,168 Abandoned US20120052561A1 (en) 2005-05-09 2011-10-31 Systems and methods for analyzing bodily fluids
US13/647,325 Pending US20130115685A1 (en) 2005-05-09 2012-10-08 Point-of-care fluidic systems and uses thereof
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