WO2008060449A2 - Détecteur microfluidique - Google Patents

Détecteur microfluidique Download PDF

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Publication number
WO2008060449A2
WO2008060449A2 PCT/US2007/023595 US2007023595W WO2008060449A2 WO 2008060449 A2 WO2008060449 A2 WO 2008060449A2 US 2007023595 W US2007023595 W US 2007023595W WO 2008060449 A2 WO2008060449 A2 WO 2008060449A2
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Prior art keywords
cells
cell
immunoassay
microfluidic
channel
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PCT/US2007/023595
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English (en)
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WO2008060449A3 (fr
Inventor
Manish J. Butte
Amy P. Wong
Arlene H. Sharpe
George M. Whitesides
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President And Fellows Of Harvard College
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Publication of WO2008060449A2 publication Critical patent/WO2008060449A2/fr
Publication of WO2008060449A3 publication Critical patent/WO2008060449A3/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56966—Animal cells
    • G01N33/56972—White blood cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54366—Apparatus specially adapted for solid-phase testing
    • G01N33/54373—Apparatus specially adapted for solid-phase testing involving physiochemical end-point determination, e.g. wave-guides, FETS, gratings
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
    • G01N2333/08—RNA viruses
    • G01N2333/15—Retroviridae, e.g. bovine leukaemia virus, feline leukaemia virus, feline leukaemia virus, human T-cell leukaemia-lymphoma virus
    • G01N2333/155—Lentiviridae, e.g. visna-maedi virus, equine infectious virus, FIV, SIV
    • G01N2333/16—HIV-1, HIV-2
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/70503—Immunoglobulin superfamily, e.g. VCAMs, PECAM, LFA-3
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/24—Immunology or allergic disorders

Definitions

  • the present invention relates generally to articles and methods for determining an analyte and, in particular, to the determination of an analyte indicating a disease condition.
  • An accurate early and ongoing determination of a disease condition is important for the prevention and treatment of human and animal diseases.
  • One class of diagnostic techniques uses reactions to detect the number of a particular type of cells in a sample taken from a subject.
  • the number of cells (e.g., T cells) in the sample may be indicative of a disease condition such as severe combined immune deficiency (SCID) or human immunodeficiency virus (HIV).
  • SCID severe combined immune deficiency
  • HCV human immunodeficiency virus
  • the CD4 T cell count in a patient is a critical tool in the diagnosis of HIV/ AIDS, in its initial management with antiretro viral therapy, in monitoring the efficacy of and viral resistance to therapy, and in determining the need for prophylactic medications to prevent opportunistic infections.
  • Articles and methods for determining an indicating a disease condition are provided.
  • a series of methods are provided.
  • a method comprises determining a disease condition.
  • the method comprises passing a fluid sample comprising cells over a surface, allowing a plurality of cells to bind to antibodies and adhesion molecules disposed on the surface, and determining the number of cells bound to the surface, wherein the number of cells bound is indicative of a disease condition.
  • a method comprises determining immobilization of a cell at a surface. The comprises allowing a cell to bind to both at least one antibody and at least one adhesion molecule disposed on the surface, and determining immobilization of the cell at the surface.
  • a method comprises flowing a fluid sample over a surface of a microfluidic channel, allowing a cell to bind with an adhesion protein and an antibody disposed on the surface of the microfluidic channel, and accumulating an opaque material on a portion of the surface of the microfluidic channel.
  • an immunoassay comprises a microfluidic chamber having a surface, at least one cell disposed on a portion of the chamber surface, and an opaque layer associated with the portion of the chamber.
  • an immunoassay comprises a microfluidic chamber having a surface, at least one adhesion protein and at least one antibody disposed on a portion of the chamber surface, and an opaque layer associated with the portion of the chamber.
  • FIG. 1 shows a schematic diagram of a microfluidic system for determining a disease condition according to one embodiment of the invention
  • FIG. 2A is a micrograph showing fluorescent detection of T cells according to one embodiment of the invention
  • FIG. 2B is a micrograph showing the absence of binding of immune cells other than T cells according to one embodiment of the invention.
  • FIGs. 3A-3E are micrographs showing detection of T-cells using silver staining according to one embodiment of the invention.
  • FIGs. 3F-3J are micrographs showing channels incubated with other cell types (non T cells) that did not stain with silver according to one embodiment of the invention.
  • FIG. 4 is a schematic diagram of an optical detection system according to one embodiment of the invention.
  • FIG. 5 is a schematic diagram illustrating serial dilution according to one embodiment of the invention
  • FIGs. 6 A and 6B are diagrams illustrating the result of serially diluting a sample of B cells using a microfluidic dilutor network according to one embodiment of the invention.
  • the present invention relates generally to articles and methods for determining an analyte and, in particular, for determining an analyte indicative of a disease condition.
  • articles and methods described herein can be used for determining a presence, qualitatively or quantitatively, of a component, such as a particular type of cell, in a fluid sample.
  • a low-cost microfluidic system for rapid detection of T cells is provided.
  • the microfluidic system may use immobilized antibodies and adhesion molecules in a channel to capture T cells from a fluid sample such as a small volume of blood.
  • the captured T cells may be labeled with a metal colloid (e.g., gold nanoparticles) using an antibody specific for the T Cell Receptor (TCR), and metallic silver can be catalytically precipitated onto the cells.
  • TCR T Cell Receptor
  • the number of T cells captured can be counted and may indicate a disease condition of a patient such as severe combined immune deficiency or human immunodeficiency virus.
  • the microfluidic systems described herein may be portable, easy-to-use, battery-powered, disposable, inexpensive, and/or can allow high throughput handling of samples. Moreover, only a few drops of fluid sample may be required for determination of a diseased condition, thereby eliminating the need for trained phlebotomists or technicians. The sample may also be obtained non-invasively, thus providing for a safer and more patient-friendly analytical procedure. Finally, the sample may be analyzed with little or no sample preparation.
  • a sample is flowed over a surface associated with a prospective binding partner of a sample component.
  • the assay can be performed in a channel of a microfluidic device allowing the sample to be flowed over one or more binding partners, for example, an antibody and/or an adhesion protein.
  • binding partners for example, an antibody and/or an adhesion protein.
  • all, or portions of, surface 20 of microfluidic channel 12 may be coated with antibodies 26 and adhesion proteins 28 to form one or more reactive portions 14.
  • antibodies 26 may include T cell-surface proteins (e.g., anti-CD3, anti-CD4, anti-CD8, and anti-CD5) and adhesion proteins 28 may include ICAM-I and/or LFA-3 adhesion molecules, which can interact with cell-surface integrins on T cells.
  • adhesion proteins 28 may include ICAM-I and/or LFA-3 adhesion molecules, which can interact with cell-surface integrins on T cells.
  • the channel may be optionally treated with a substance (e.g., a silane such as 3- (cyanopropyl)dimethylchlorosilane) prior to coating of the prospective binding partner.
  • a sample such as a biological sample taken from a subject, is flowed through microchannels 12.
  • the sample may be a liquid sample, but in some embodiments need not be diluted, purified or treated prior to analysis.
  • the sample may be flowed through the microchannel at a rate sufficient to allow component 30 of the sample (e.g., a T cell) to bind with one or more binding partners (e.g., antibodies 26 and adhesion proteins 28) immobilized on the surface of the channel.
  • component 30 of the sample e.g., a T cell
  • binding partners e.g., antibodies 26 and adhesion proteins 28
  • a sampled is flowed through the channel to introduce the sample into the channel, and the flow is decreased or stopped to allow binding of the sample component to the binding partners.
  • the binding partners immobilized on the channel are specific for T cells, only T cells may be captured from the biological sample.
  • Cells may optionally be exposed to a fixing agent to cause stronger attachment of the cell to the surface. "Fixing" the cell may cause covalent binding or formation of intermolecular bridges on the cell surface.
  • Methods of fixing cells to surfaces including the use of paraformaldehyde solutions or organic solvents, are known in the art and may be used in accordance with the present invention. Many species are known which can bind a cell and immobilize a cell at a surface. The prior art describes many such species and cell immobilization techniques.
  • one aspect of the present invention involves the recognition that a particular combination of species for cell adhesion and immobilization, namely, a combination of at least one adhesion molecule and at lease one antibody, can immobilize specific cells at a surface in a manner that is particularly robust and/or particularly resistive to subsequent flow of fluid against the cells immobilized at the surface which may be necessary for certain analytical techniques.
  • the combination of the adhesion molecule and antibody also can be useful in immobilizing a cell, at a surface, from a fluid that is flowing relative to the surface.
  • the inventors believe that the combination of the adhesion molecule and antibody provides a uniquely favorable combination of both recognition specificity and binding strength, and/or causes changes of the cell itself which allow the cell to better adhere to the surface.
  • the cell can be effectively captured from a flowing stream and/or can be immobilized at a surface from a flowing or stagnant stream and subjected to further flow of fluid without detachment.
  • Further flow of fluid can involve, for example, a flow of a fixing solution, flow with buffer or cleaning solutions to remove non-immobilized cells, flow of fluorescent-labeled antibodies, metallic staining solutions or the like for cell identification, or other detection techniques described further herein.
  • the unique adhesive properties of the adhesion molecule/antibody combination described herein find particular use in microfluidic environments in which fluid flow can cause particularly strong shear forces at surfaces which otherwise can readily detach weakly-immobilized cells.
  • the immobilization technique of the invention can be particularly useful in microfluidic environments where the technique provides good adhesion of desired cells, while allowing the strong shear forces in a microfluidic environment to remove from the surface cells that are not adhered in the manner of the invention, for better specificity of identification of adhered cells versus cells that are not adhered.
  • the captured sample component is stained with a fluorescent antibody.
  • T cells captured in a channel can be stained with anti-CD3, anti-CD4, and/or anti-CD8 fluorescent antibodies, and detected with fluorescence microscopy.
  • FIG. 2A is a micrograph showing fluorescent detection of T-cells. Cell nuclei 50 of T cells were stained with DAPI and the T cells 52 were stained with phycoerythrin-anti-CD3. As illustrated in the embodiment of FIG. 2B, only T cells 52 were captured in the channel. Other immune cells did not significantly bind to the channels, as shown by flow cytometric analysis of the unspecific cells that were washed out the channel.
  • the captured sample components can be labeled with a metal colloid 34 (e.g., gold nanoparticles) as shown in the embodiment illustrated in FIG. 1.
  • Metal colloid 34 may specifically label sample component 30 using, for example, binding partner 36 specific to sample component 30.
  • Metallic silver 38 can be catalytically precipitated onto the sample component and measured by various methods, as described in more detail below.
  • T cells are labeled with gold nanoparticles using an antibody specific for the T Cell Receptor (TCR), and metallic silver is catalytically precipitated onto the cells.
  • TCR T Cell Receptor
  • biotin-labeled anti-TCR- ⁇ antibody may be flowed in the channel and can bind to a surface of the T cells.
  • a fluid containing a metal colloid (e.g., streptavidin that had been conjugated with 1 nm gold nanoparticles) associated with a second binding partner (e.g., biotin-labeled anti-TCR- ⁇ antibody) of the sample component can then be flowed through the microchannel, allowing the metal colloid to bind with any sample component that has been associated with a reactive portion of the microchannel.
  • a metal colloid e.g., streptavidin that had been conjugated with 1 nm gold nanoparticles
  • a second binding partner e.g., biotin-labeled anti-TCR- ⁇ antibody
  • a metal precursor can be flowed through channel 12 in a similar manner as was the metal colloid.
  • the metal precursor can be flowed through the microchannel at a concentration and a rate that allows an opaque layer to be formed wherever a threshold number of metal colloids have been associated with the surface.
  • a gold-conjugated binding partner is used as a metal colloid
  • a silver nitrate solution may be used to electrolessly deposit a silver layer on the portion of the channel associated with the gold-conjugated antibody.
  • all, or portion of, surface 20 of the microfluidic network may include, in successive layers, antibodies and adhesion molecules such as those specific for T-cells, a sample component (e.g., T cells) obtained from a subject, a binding partner of the sample component such as biotin-labeled anti-TCR- ⁇ antibody, a metal colloid such as gold- labeled streptavidin, and an opaque layer of metal, such as silver, that has been electrolessly deposited on the metal colloid.
  • Rinsing solutions may be flowed through the channel before or after each of the steps.
  • FIGs. 3A-3J are micrographs showing detection of T-cells using silver staining.
  • FIGs. 3A-3E are DIC images taken at two minute intervals of the T cells as silver 56 precipitates upon the gold colloid conjugated anti-CD3 antibody.
  • FIGs. 3F-3 J show channels incubated with other cell types (e.g., not T cells) that were stained with the same gold-conjugated anti-CD3 antibody; these cells showed no silver precipitation.
  • the metal precursor may also be deposited on metal that has previously been electrolessly deposited on the gold-conjugated binding partner.
  • an opaque material may be formed over some or all of portion 14 allowing for detection by, for example, the unaided eye or an optical detection device.
  • the opaque material may be a continuous material rather than, for example, independent particles, and may include a horizontal dimension that, in a dimension measured in substantially the same plane as surface 20, measures greater than 1 micron, greater than 10 microns, or greater than 100 microns.
  • an opaque layer may form a web or honeycomb of material that includes passages allowing light to be transmitted therethrough. As additional material is deposited, these passages may become smaller, allowing less and less light to be transmitted through the material. As the passages disappear, the amount of light transmittance may be reduced to zero, providing for a completely opaque material. Any cell/binding partner complex that forms may be associated with a metal colloid that provides a catalytic surface for the deposition of an opaque material, such as a layer of metal.
  • any opaque layer that is formed in the microfluidic channel can be detected optically, for example, by measuring a reduction in light transmittance through a portion of the microfluidic channel compared to a portion of the channel that does not include the cell.
  • the opaque layer may provide an increase in assay sensitivity when compared to techniques that do not form an opaque layer.
  • FIG. 4 illustrates microfluidic device 10, as shown in FIG. 1.
  • light source 60 e.g., an oscillator-modulated laser diode emitting 432 nm light
  • detector 62 such as an optical integrated circuit (IC).
  • detector 62 is a potentiometer.
  • the detector signal may be amplified and passed through a bandpass filter centered at the same frequency as the oscillator controlling the light source.
  • the output may then be passed to an A/D converter which can then provide an output on a readout, such as an LCD display.
  • Both the light source and the detector may be powered by a 9 volt battery, such as the type typically used in portable hand-held radios. Using such a system, absorption of light by silver can signify the presence of T cells, whereas the unimpeded transmission of light signified the absence of T cells.
  • detector 62 of the circuit may allow selection of a clinically appropriate clinical cut-off value of a particular number of cells per unit volume of sample. A range of cut-off values may be chosen depending on the particular disease condition.
  • the cut-off value can be set at about 1000 cells/ ⁇ L, the 1 st percentile of the normal range of T cell counts for infants, for the detection severe combined immune deficiency as described in more detail below. Above this density of cells captured in a channel may indicate normal T cell counts in an infant; below this density of captured cells may indicate a diseased condition. In another embodiment, the cut-off value may be set to 200 cells/ ⁇ L; HIV-positive patients with a CD4 count less than 200 cells/ ⁇ L, for example, indicates that the patient should undergo antiretroviral therapy.
  • a cut-off value of cells captured in a channel may be chosen to be, for example, 100 cells/ ⁇ L, 200 cells/ ⁇ L, 300 cells/ ⁇ L, 400 cells/ ⁇ L, 500 cells/ ⁇ L, 600 cells/ ⁇ L, 700 cells/ ⁇ L, 800 cells/ ⁇ L, 900 cells/ ⁇ L, 1,000 cells/ ⁇ L, 1,100 cells/ ⁇ L, 1,200 cells/ ⁇ L, 1,300 cells/ ⁇ L, 1,400 cells/ ⁇ L, or 1,500 cells/ ⁇ L.
  • other cut-off values may also be chosen, e.g., depending on the particular disease condition.
  • fluid samples can be serially diluted in a microfluidic dilutor network, generating decreasing numbers of cells with each dilution step.
  • the cell count in at least one of the detection areas will be in the linear range of the detection system.
  • cell dilution can result in decreasing silver precipitation 82 in reaction sites 84 in the direction of arrow 80 across the dilutor. As depicted in FIG.
  • FIG. 5A too much dilution can result in too few cells for accurate detection of a disease condition. Typical cell counts are depicted in FIGs. 5B- 5C.
  • FIG. 5D depicts too many cells for accurate detection of a disease condition. Additional examples of dilutors that can be used in accordance with the present invention include those described in U.S. Patent Publication No. 2004/0258571 , which is incorporated herein by reference. Serial dilution can allow quantitative tests to be carried out, in addition to presence/absence type tests. Such a quantitative test may be of interest, for example, to those monitoring levels of a component (e.g., a T cell) in a patient during treatment.
  • FIG. 6A shows the result of a microfluidic dilutor network delivering serially diluted B cells to six channels 90, 92, 94, 96, 98, and 100.
  • Microfluidic channels were coated with antibodies targeting mouse B cells.
  • the A20 cell line was expanded in tissue culture and loaded into a microfluidic dilutor network that delivered diluted cells into the six channels.
  • Channels 92 and 94 show saturated capture of cells; subsequent channels 96, 98, and 100 show stepwise dilution, which may be suitable for determination of a disease condition.
  • Counts of cells in each lane by microscopy are shown in FIG. 6B.
  • a dilutor network with three output lanes, initially using a 10-fold serial dilution ratio, is used for detection of a disease condition.
  • mouse T cells can be used for prototype counting and calibration of the dilutor.
  • Captured cells can be counted by light microscopy or by other suitable methods. Cell counts in samples can be estimated by comparison to a calibration curve. Subsequently, stages of HIV progression and CD4 cell loss can be modeled using whole blood from severely immunodeficient (SCID) mice, which lack T and B cells, mixed in varying proportions with whole blood from wild-type mice. Finally, adult health human blood can be tested in the dilutor network.
  • SCID severely immunodeficient mice
  • a disease condition may include any condition that is abnormal in the body or mind of a subject that causes discomfort, dysfunction, or distress to the subject afflicted or those in contact with the subject.
  • Disease conditions can include, for example, infections (e.g., viral infections), immune deficiencies, injuries, disabilities, disorders, syndromes, symptoms, deviant behaviors, and atypical variations of structure and/or function.
  • SCID severe combined immune deficiency
  • HIV Complete DiGeorge Syndrome
  • PCP Pneumocystis jiroveci pneumonia
  • MAC Mycoplasma avium complex
  • CMV Cytomegaloviral disease
  • Toxoplasmosis and allergic rhinitis. It should be understood that methods and apparatuses described herein can be used to detect other disease conditions and that the invention is not limited in this respect.
  • methods and apparatuses described herein are used for the detection of severe combined immune deficiency, a collection of primary immunodeficiency disorders that results in extreme susceptibility to infection because of profound T cell failure.
  • the estimated incidence of SCID of 1/50,000 live births probably underestimates the true incidence since some infants die prior to being diagnosed.
  • SCID hematopoietic stem cell transplantation
  • patients with SCID succumb to infection and death in the first two years of life. Because patients are often asymptomatic from the time of birth due to protection from maternal antibodies, diagnosis typically occurs after severe or opportunistic infections at a mean age of six to seven months.
  • Diagnosis of SCID may rely on expensive flow cytometric testing, which at $650 per test, for example, is too expensive for broad screening.
  • flow cytometry may be limited by the requirements of a large amount of blood (2-5 mL), trained technicians, expensive equipment, and fluorescent antibodies that have a limited shelf-life. Since lymphocyte precursors fail to develop, SCID patients demonstrate the principal feature of low or absent T cells in the peripheral blood; other laboratory features can include low or absent B cells and low immunoglobulin levels. Early detection of SCID would facilitate early access to antibiotic prophylaxis, intravenous immunoglobulin, improved nutrition, and curative HSCT before the onset of life-threatening or chronic infections.
  • helper T cell attrition directly and indirectly results in the general state of immunodeficiency conferred by HIV. Consequently, HIV infected patients suffer from increased susceptibility to infection proportional to the fall in CD4 cell count.
  • the CD4 cell count rises in HIV patients treated with antiretroviral drugs and falls when viral resistance to therapy develops. Thus, the CD4 count is a critical tool for monitoring patients with HIV/ AIDS.
  • the CD4 T cell count has been extensively studied in its predictive role in HIV/AIDS diagnosis.
  • the CD4 T cell count is part of the recommended initial assessment of all patients with HIV/ AIDS to help define the need for antiretroviral treatment as it helps detect patients with asymptomatic or sub-clinical disease (WHO stage II disease).
  • Antiretroviral therapy is currently recommended for all patients with a CD4 count of less than 200 cells/ ⁇ L, because this level of CD4 T cells indicates advanced viral infection, or for those with AIDS-defining illnesses regardless of CD4 count.
  • An asymptomatic, newly diagnosed, HIV-positive patient with a CD4 count greater than 400 cells/ ⁇ L should defer treatment with antiretroviral medications, but is recommended to have CD4 counts performed every 3-6 months to assess the need to start treatment.
  • CD4 count Treatment of HIV infection with antiretroviral medications often results in an increase in the CD4 count of approximately 100-150 cells/ ⁇ L per year, though this increase can vary greatly. Access to antiretroviral medications has been dramatically improving in the developing world thanks to increased funding via the World Health Organization (WHO) "3 by 5" program (3 million patients on therapy by 2005). Tracking the efficacy of treatment requires following the CD4 count. Immunological treatment failure is defined as having either a CD4 T cell count that falls below the patient's own baseline count, or one that falls by more than 50% after an initial increase. Because the costs for CD4 T cell counts in resource-poor settings can be higher than the cost of therapy, many clinics simply do not obtain CD4 counts following initiation of treatment.
  • WHO World Health Organization
  • HIV develops resistance to antiretroviral infection because its defective reverse transcriptase allows a high rate of mutations. Many recent studies demonstrate that resistance during therapy can develop in less than six months. Immunological failure with falling CD4 T cell count occurs after HIV develops resistance, so the US standard of care is to obtain viral genotyping studies to assess resistance patterns. These genotyping studies are far too expensive (e.g., over $300 per test) for screening use in the developing world, thus falling CD4 counts present the most useful sign of viral resistance to therapy.
  • CD4 T cell count may be the best predictor of risk of opportunistic infections such as Pneumocystis jiroveci pneumonia, Mycoplasma avium complex, Cytomegaloviral disease, and Toxoplasmosis.
  • Current US and recommendations suggest that CD4 counts be obtained every 3-6 months to monitor need for prophylaxis against opportunistic infections and define the trigger points of CD4 counts for initiating prophylactic medications against these opportunistic infections.
  • the CD4 T cell count is a critical tool in the diagnosis of HIV/ AIDS, in its initial management with antiretroviral therapy, in monitoring the efficacy of, and viral resistance to, therapy, and in determining the need for prophylactic medications to prevent opportunistic infections. Accordingly, methods and apparatuses described herein may be used to count T cells, or other cells indicative of a disease condition, in the settings described above. Some conventional methods of counting T cells in the United States, such as flow cytometry, may be far too expensive to be used in resource-poor settings, such as the developing countries where, ironically, HIV infection is the worst. For over a decade, researchers have sought ways of providing lower-cost flow cytometry or other counting strategies to CD4 counting.
  • Methods and apparatuses described herein may be low-cost (e.g., about $2 per device), disposable, and/or compact (palm-sized).
  • methods of detection do not require fluorescence microscopy or highly trained personnel to operate.
  • the presence, absence, or amount of an analyte (e.g., a cell) in a sample may be indicated by the formation of an opaque material.
  • the opaque material may be used to refract light or may be excited to emit light at a similar or different wavelength than the light to which the layer is exposed, the measurement of light transmission may be preferred due to, for example, lower equipment and operating costs, and ease of use.
  • an opaque layer may be visible to the naked eye and, in particular if reflective, may be detected without the use of instrumentation.
  • Any opaque material that forms can be a series of discontinuous independent particles, but in one embodiment is a continuous material that takes on a generally planar shape.
  • the opaque material may have a dimension greater than, for example, 1 micron or greater than 10 microns.
  • the opaque layer may have a thickness of, for example, less than 1 micron, of less than 100 nanometers, or less than 10 nanometers. Even at these small thicknesses, a detectable change in transmittance can be obtained.
  • the opaque material may be a metal and is preferably a metal that can be electrolessly deposited. These metals include, for example, copper, nickel, cobalt, palladium, and platinum.
  • a metal precursor is a material that can provide the source of the elemental metal for depositing on, for example, a metal colloid.
  • a metal precursor may be a metallic salt solution such as silver nitrate.
  • a metal precursor may include 0.1% silver nitrate, 1.7% hydroquinone and 0.1 M citrate buffer at a pH of 3.5.
  • Some other examples of electrolessly deposited materials can be found in Modern Electroplating, 4 th Edition, Schlesinger and Paunovic, Wiley, 2000. Metal precursors can be stored for long periods of time and may be stable for several years whereas optically-active compounds may have much shorter shelf lives.
  • any metal colloid associated with a surface may be widely scattered over a portion of the surface.
  • gold-conjugated antibodies may be bound to sample components that are associated with the portion of the surface but spaces may exist between the gold-conjugated antibodies, making them discontinuous.
  • the precursor When a metal precursor is first exposed to these gold-conjugated antibodies, the precursor may form particulates centered around individual metal colloids. As metal, e.g., silver, is deposited on these metal colloids, the particles become larger and soon the metal precursor may deposit metal not only on gold colloids but on metal that has been previously electrolessly deposited.
  • a silver nitrate solution may deposit silver metal onto silver metal particles that have previously been deposited on gold-conjugated antibodies.
  • the opaque material may have a thickness greater than 1, 10, 100 or 1000 nanometers. For some opaque materials, the material may become completely opaque at thicknesses greater than about 100 nm. However, in some embodiments, such as when a honeycomb or similar structure is formed, thicknesses in some portions may be much greater while still allowing some light to be transmitted.
  • Determination techniques may include optically-based techniques such as light transmission, light absorbance, light scattering, light reflection and visual techniques. Determination techniques may also measure conductivity. For example, microelectrodes placed at opposite ends of a portion of a microfluidic channel may be used to measure the deposition of a conductive material, for example an electrolessly deposited metal. As a greater number of individual particles of metal grow and contact each other, conductivity may increase and provide an indication of the amount of conductor material, e.g., metal, that has been deposited on the portion. Therefore, conductivity or resistance may be used as a quantitative measure of analyte concentration.
  • optically-based techniques such as light transmission, light absorbance, light scattering, light reflection and visual techniques. Determination techniques may also measure conductivity. For example, microelectrodes placed at opposite ends of a portion of a microfluidic channel may be used to measure the deposition of a conductive material, for example an electrolessly deposited metal. As a greater number of individual particles of
  • Another analytical technique may include measuring a changing concentration of a precursor from the time the precursor enters the microfluidic channel until the time the precursor exits the channel.
  • a silver sensitive electrode may be capable of measuring a loss in silver concentration due to the deposition of silver in a channel as the precursor passes through the channel.
  • the measurement of transmission or absorbance means that light can be detected at the same wavelength at which it is emitted from a light source.
  • the light source can be a narrow band source emitting at a single wavelength, it may also may be a broad spectrum source, emitting over a range of wavelengths, as many opaque materials can effectively block a wide range of wavelengths.
  • the system may be operated with a minimum of optical components.
  • the determining device may be free of a photo multiplier, may be free of a wavelength selector such as a grating, prism or filter, or may be free of a device to direct or columnate light such as a columnator.
  • optical component can include passive elements or devices that do not produce electromagnetic radiation but rather diffract or refract or otherwise change a property of electromagnetic radiation or light.
  • an optical component can, for example, be a prism, a mirror, a diffractive lens, a refractive lens, reflective lens, a spherically-shaped lens, an aspherically-shaped lens, a non-spherically- shaped lens, a plano-convex-shaped lens, a polygonal convex-shaped lens or a graded- index optical fiber or fiber optical component.
  • An “optical component” also includes active elements or devices that produce electromagnetic radiation including, for example, lasers or light-emitting diodes.
  • a “light-focusing element” is an optical element that is capable of refracting, bending or changing the direction of the propagating of waves of electromagnetic radiation so that the waves can converge, or diverge, on or near a preferred plane, location or region.
  • a light source can be pulse modulated, for example, at a frequency of 1 ,000 Hz.
  • a detector may include a filter operating at the same frequency.
  • the light source may be a laser diode.
  • the photodetector may be any device capable of detecting the transmission of light that is emitted by the light source.
  • One type of photodetector is an optical integrated circuit including a photodiode having a peak sensitivity at 700 nm, an amplifier and a voltage regulator. If the light source is pulse modulated, the photodetector may include a filter to remove the effect of light that is not at the selected frequency.
  • a waveguide can be used for detection. Systems and methods of detection are further described in
  • a suitable analyte includes one that can preferentially bind to both at least one antibody (or antigen) and at least one adhesion molecule.
  • An example of such an analyte is a cell.
  • analytes include specific cell types, a characteristic of which can indicate a disease condition. For example, the absence, abundance, or change in physical property of a particular cell type may indicate the diagnosis or progression of a disease condition, and/or the efficacy of a therapeutic agent in treating the disease condition.
  • an analyte is an eosinophil (e.g., from nasal aspirates), the amount of which may indicate diagnosis of allergic rhinitis in a patient.
  • an analyte is a CD4 T cell, the amount of which may be an indicator of risk of opportunistic infections such as Pneumocystis jiroveci pneumonia, mycoplasma avium complex, cytomegaloviral disease, and toxoplasmosis, or an indicator for HIV.
  • the specific cell types may have corresponding specific binding partners (e.g., antibodies and adhesion molecules) that can be disposed on a surface, for example, to allow specific binding between the respective cell and binding partner.
  • suitable analytes can preferentially bind to two different types of binding partners as described below.
  • Typical sample fluids that may contain an analyte include physiological fluids such as human or animal whole blood, blood serum, blood plasma, semen, tears, urine, sweat, saliva, mucus, cerebro-spinal fluid, and vaginal secretions, in-vitro fluids used in research, or environmental fluids such as aqueous liquids suspected of being contaminated by the analyte.
  • physiological fluids such as human or animal whole blood, blood serum, blood plasma, semen, tears, urine, sweat, saliva, mucus, cerebro-spinal fluid, and vaginal secretions
  • in-vitro fluids used in research or environmental fluids such as aqueous liquids suspected of being contaminated by the analyte.
  • the low level of detection capable with the invention allows for the use of samples that typically contain low concentrations analyte, such as certain cells in blood from diseased patients having a lower number of cells than that of healthy patients.
  • a biological sample may be obtained noninvasively. By allowing samples to be obtained
  • the methods and apparatuses of the present invention may be capable of obtaining limits of detection (LOD) comparable to those achievable by, for example, immunochromatographic assays, ELISA, as well as flow cytometry.
  • LOD limits of detection
  • concentrations below 1 nM and even in the 100 pM range can be detected.
  • the assay can be qualitative, quantitative, or both.
  • concentration of analyte increases, the apparent absorbance of the opaque material increases accordingly.
  • binding refers to the interaction between a corresponding pair of components that exhibit mutual affinity or binding capacity, typically specific or non-specific binding or interaction, including biochemical, physiological, and/or pharmaceutical interactions.
  • Biological binding defines a type of interaction that occurs between pairs of components including proteins, (e.g., adhesion proteins), nucleic acids, glycoproteins, carbohydrates, hormones and the like.
  • cell e.g., cell surface receptor
  • adhesion protein/integrin e.g., cell surface receptor
  • antibody/antigen e.g., antibody/hapten
  • enzyme/substrate enzyme/inhibitor
  • enzyme/cofactor binding protein/substrate
  • carrier protein/substrate e.g., lectin/carbohydrate
  • receptor/hormone e.g., receptor/effector
  • complementary strands of nucleic acid e.g., cell surface receptor
  • protein/nucleic acid repressor/inducer e.g., ligand/cell surface receptor
  • virus/ligand e.g., virus/ligand, etc.
  • binding of a sample component includes interaction between two or more different corresponding pairs of components that exhibit mutual affinity or binding capacity, such as two or more of the corresponding pairs listed above.
  • the interaction of at least two corresponding pairs of binding partners e.g., binding of both an adhesion molecule and an antibody with the sample component, can cause effective recognition and capture of the sample component from a solution, resulting in immobilization of the component at a surface.
  • binding of a sample component may include interaction between an integrin (a receptor in the plasma membrane of the cell) and an adhesion molecule (e.g., ICAM-I and/or LFA-3), as well as interaction between an antibody of the cell (e.g., anti-CD3, anti-CD4, anti-CD8, and/or anti-CD5) and its corresponding antigenic binding partner (e.g., CD3, CD4, CD8, and/or CD5, respectively).
  • an integrin a receptor in the plasma membrane of the cell
  • an adhesion molecule e.g., ICAM-I and/or LFA-3
  • an antibody of the cell e.g., anti-CD3, anti-CD4, anti-CD8, and/or anti-CD5
  • its corresponding antigenic binding partner e.g., CD3, CD4, CD8, and/or CD5, respectively.
  • An "opaque material” is a substance that interferes with the transmittance of light at one or more wavelengths.
  • An opaque material does not merely refract light, but reduces the amount of transmission through the material by, for example, absorbing or reflecting light. Different opaque materials or different amounts of an opaque material may allow transmittance of less than 90, 80, 70, 60, 50, 40, 30, 20, 10 or 1 percent of the light illuminating the opaque material. Examples of opaque materials include molecular layers of elemental metal and polymeric layers.
  • Colloids as used herein, means nanoparticles, i.e., very small, self-suspendable or fluid-suspendable particles including those made of material that is, e.g., inorganic or organic, polymeric, ceramic, semiconductor, metallic (e.g., gold), non-metallic, crystalline, amorphous, or a combination.
  • colloid particles used in accordance with the invention are of less than 250 nm cross section in any dimension, more typically less than 100 nm cross section in any dimension, and in most cases are of about 1-30 nm cross section.
  • One class of colloids suitable for use in the invention is 10-30 nm in cross section, and another about 1-10 nm in cross section.
  • Colloids may be associated with a binding partner, for example, an antibody.
  • a component that is "immobilized relative to" another component either is fastened to the other component or is indirectly fastened to the other component, e.g., by being fastened to a third component to which the other component also is fastened, or otherwise is transitionally associated with the other component.
  • a signaling entity is immobilized with respect to a binding species if the signaling entity is fastened to the binding species, is fastened to a colloid particle to which the binding species is fastened, is fastened to a dendrimer or polymer to which the binding species is fastened, etc.
  • Signaling entity means an entity that is capable of indicating its existence in a particular sample or at a particular location.
  • Signaling entities of the invention can be those that are identifiable by the unaided human eye, those that may be invisible in isolation but may be detectable by the unaided human eye if in sufficient quantity (e.g., colloid particles), entities that absorb or emit electromagnetic radiation at a level or within a wavelength range such that they can be readily detected visibly (unaided or with a microscope including an electron microscope or the like), optically, or spectroscopically, entities that can be detected electronically or electrochemically, such as redox-active molecules exhibiting a characteristic oxidation/reduction pattern upon exposure to appropriate activation energy (“electronic signaling entities”), or the like.
  • Examples include dyes, pigments, electroactive molecules such as redox-active molecules, fluorescent moieties (including, by definition, phosphorescent moieties), up- regulating phosphors, chemiluminescent entities, electrochemiluminescent entities, or enzyme-linked signaling moieties including horseradish peroxidase and alkaline phosphatase.
  • "Precursors of signaling entities” are entities that, by themselves, may not have signaling capability but, upon chemical, electrochemical, electrical, magnetic, or physical interaction with another species, become signaling entities.
  • An example includes a chromophore having the ability to emit radiation within a particular, detectable wavelength only upon chemical interaction with another molecule.
  • Precursors of signaling entities are distinguishable from, but are included within the definition of, "signaling entities” as used herein.
  • Binding partners immobilized at a region or portion of a region can be immobilized in essentially any manner, and many immobilization techniques suitable for use with the invention are known in the art. See U.S. Patent Application No. 10/654,587 and U.S. Patent No. 6,686,184, which are incorporated by reference in their entirety herein. Immobilization can be done in a way such that the species are randomly oriented relative to the surface (i.e., each immobilized species can be oriented, relative to the surface, randomly), or greater control of the orientation of species relative to the surface can be provided. For example, where proteins are immobilized at the surface, they can be oriented such that their binding sites for the assay are oriented generally away from the surface, maximizing their binding capacity or availability.
  • One technique for doing so involves synthesizing the protein with a polyamino acid tag such as, for example, a sequence of 6 histidines, at a location generally opposite the protein's relevant binding site, providing a metal chelate, such as nitrilotriacetic acid, chelating a metal ion such as nickel in such a way that at least two coordination sites on nickel are free for binding to the polyamino acid tag, and allowing the tag to coordinate to the metal ions, thus immobilizing the protein at the region or portion of a region in an advantageous orientation.
  • a metal chelate such as this can be immobilized at the region in any of a number of ways.
  • One way involves forming a self-assembled monolayer (SAM) at the region, terminating in the metal chelate, as described in the above-referenced U.S. Pat. No. 5,620,850.
  • SAM self-assembled monolayer
  • a thin, essentially transparent thin gold layer can be deposited at the region, and S AM-forming alkyl thiols, terminating in a metal chelate, can be deposited on the gold layer as a SAM.
  • Other chemistry described in U.S. Pat. No. 5,620,850 and other references, and known to those of ordinary skill in the art, can be used to form such a SAM on a region defined by a variety of base materials.
  • two or more parallel assays may be run.
  • a single sample may be physically split into two or more samples using a microfluidic device.
  • a microfluidic device may have a single input channel that branches into two, three, or more parallel channels.
  • Parallel analysis may be performed at different threshold levels of a similar or identical analyte, or for different analytes at the same or different thresholds.
  • a control may also be performed in parallel.
  • a sample can be analyzed for two or more analytes at any number of threshold concentrations.
  • a control may also be run concurrently and may be useful in calibrating and/or verifying the detection method that is used.
  • the assay may be stable for an extended period of time, for example, greater than one month or one year, so that assays may be collected and analyzed or re-analyzed at a later date.
  • Reagents and samples may be supplied to the assay using methods known to those skilled in the art or by using delivery methods described herein.
  • a microfluidic device can be used in conjunction with a vessel designed to contain, store, protect and/or transport two or more fluids.
  • vessels include cartridges and tubes.
  • a vessel may contain two or more distinct fluids separated by a third fluid that can be immiscible with both. Any number of distinct fluids may be contained in a vessel.
  • the vessel may be a tube that includes a series of fluid plugs such as a reagent solution plug followed by an air plug, followed by a rinse solution plug. An additional air plug may separate the first rinse solution plug from a second rinse solution plug.
  • the ends of the tube may be sealed, for example, to retain the fluid plugs and to prevent contamination from external sources.
  • the liquid plugs may retain their relative positions in the tube and may be prevented from contacting each other by the interspaced air plugs.
  • the tube dimensions and materials of construction may be chosen to help fluid plugs retain their position and remain unmixed. For example, see the cartridge systems described in Vincent Linder, Samuel K. Sia, and George M. Whitesides "Reagent-Loaded Cartridges for Valveless and Automated Fluid Delivery in Microfluidic Devices," Anal. Chem.; 2005; 77(1) pp 64-71.
  • Reagents and other fluids may be stored for extended lengths of time in the vessel. For example, reagents may be stored for greater than 1 day, 1 week, 1 month or 1 year. By preventing contact between fluids, fluids containing components that would typically react or bind with each other are prevented from doing so, while being maintained in a continuous chamber.
  • Fluids may be transferred from the vessel to the assay by applying pressure or vacuum after removing or piercing a seal at an end of the tube.
  • the vessel need not be sealed and fluid flow can be started by applying an external force, such as a pressure differential.
  • One end of the vessel for example, can be in, or can be placed in, fluid communication with an assay or another device that will receive the fluids from the vessel.
  • Fluid may be flowed to the reaction site by, for example, pushing or pulling the fluid through the vessel. Fluids can be pushed to the reaction site using, for example, a pump, syringe, pressurized vessel, or any other source of pressure.
  • fluids can be pulled to the reaction site by application of vacuum or reduced pressure on a downstream side of the reaction site.
  • Vacuum may be provided by any source capable of providing a lower pressure condition than exists upstream of the reaction site.
  • sources may include vacuum pumps, Venturis, syringes and evacuated containers.
  • a vessel may contain fluid plugs in linear order so that as fluids flow from the vessel to a reaction site they are delivered in a predetermined sequence.
  • an assay may receive, in series, an antibody fluid, a rinse fluid, a labeled-antibody fluid and a rinse fluid.
  • an immiscible fluid a separation fluid
  • the assay fluids can be delivered in sequence from a single vessel while avoiding contact between any of the assay fluids.
  • Any immiscible fluid that can separate assay fluids may be applied to the reaction site without altering the conditions of the reaction site. For instance, if antibody-antigen binding has occurred at a reaction site, air can be applied to the site with minimal or no effect on any binding that has occurred.
  • Pre-filling of the vessel with reagents may allow the reagents to be dispensed in a predetermined order for a downstream process.
  • the amount of each fluid in the vessel may be proportional to the amount of time the reagent is exposed to a downstream reaction site. For example, if the desired exposure time for a first reagent is twice the desired exposure time for a second reagent, the volume of the first reagent in the vessel may be twice the volume of the second reagent in the vessel.
  • the exposure time of each fluid at a specific point, such as a reaction site may be proportional to the relative volume of the fluid.
  • Factors such as vessel geometry, pressure or viscosity can also be altered to change flow rates of specific fluids from the vessel.
  • microfluidic refers to a device, apparatus or system including at least one fluid channel having a cross-sectional dimension of less than 1 mm, and a ratio of length to largest cross-sectional dimension of at least 3:1.
  • a “microfluidic channel,” as used herein, is a channel meeting these criteria. The "cross-sectional dimension" of the channel is measured perpendicular to the direction of fluid flow. Most fluid channels in components of the invention have maximum cross-sectional dimensions less than 2 mm, and in some cases, less than 1 mm.
  • all fluid channels containing embodiments of the invention are microfluidic or have a largest cross sectional dimension of no more than 2 mm or 1 mm.
  • the fluid channels may be formed in part by a single component (e.g., an etched substrate or molded unit).
  • larger channels, tubes, chambers, reservoirs, etc. can be used to store fluids in bulk and to deliver fluids to components of the invention.
  • the maximum cross-sectional dimension of the channel(s) containing embodiments of the invention are less than 500 microns, less than 200 microns, less than 100 microns, less than 50 microns, or less than 25 microns.
  • a “channel,” as used herein, means a feature on or in an article (substrate) that at least partially directs the flow of a fluid.
  • the channel can have any cross-sectional shape (circular, oval, triangular, irregular, square or rectangular, or the like) and can be covered or uncovered. In embodiments where it is completely covered, at least one portion of the channel can have a cross-section that is completely enclosed, or the entire channel may be completely enclosed along its entire length with the exception of its inlet(s) and outlet(s).
  • a channel may also have an aspect ratio (length to average cross sectional dimension) of at least 2:1, more typically at least 3:1, 5:1, or 10:1 or more.
  • An open channel generally will include characteristics that facilitate control over fluid transport, e.g., structural characteristics (an elongated indentation) and/or physical or chemical characteristics (hydrophobicity vs. hydrophilicity) or other characteristics that can exert a force (e.g., a containing force) on a fluid.
  • the fluid within the channel may partially or completely fill the channel.
  • the fluid may be held within the channel, for example, using surface tension (i.e., a concave or convex meniscus).
  • the channel may be of any size, for example, having a largest dimension perpendicular to fluid flow of less than about 5 mm or 2 mm, or less than about 1 mm, or less than about 500 microns, less than about 200 microns, less than about 100 microns, less than about 60 microns, less than about 50 microns, less than about 40 microns, less than about 30 microns, less than about 25 microns, less than about 10 microns, less than about 3 microns, less than about 1 micron, less than about 300 nm, less than about 100 nm, less than about 30 nm, or less than about 10 nm.
  • the dimensions of the channel may be chosen such that fluid is able to freely flow through the article or substrate.
  • the dimensions of the channel may also be chosen, for example, to allow a certain volumetric or linear flowrate of fluid in the channel.
  • the number of channels and the shape of the channels can be varied by any method known to those of ordinary skill in the art. In some cases, more than one channel or capillary may be used. For example, two or more channels may be used, where they are positioned inside each other, positioned adjacent to each other, positioned to intersect with each other, etc.
  • the geometry of the microfiuidic channel may provide for the laminar flow of fluids through the channel, even at relatively high flow rates.
  • turbulent flow may be employed for example, by using even faster flow rates, wider channels, or devices such as microfiuidic mixers. Such mixing may provide for a greater amount of contact between potential binding partners.
  • Microfiuidic systems described herein may optionally include one or more platforms for performing chemical reactions, combining and separating fluids, diluting samples, and generating gradients, such as those described in US Patent No. 6,645,432, hereby incorporated by reference herein.
  • a microfluidic device described herein can be fabricated of a polymer, for example an elastomeric material such as poly(dimethylsiloxane) (PDMS) using rapid prototyping and soft lithography.
  • PDMS poly(dimethylsiloxane)
  • a high resolution laser printer may be used to generate a mask from a CAD file that represents the channels that make up the fluidic network.
  • the mask may be a transparency that may be contacted with a photoresist, for example, SU-8 photoresist (MicroChem), to produce a negative master of the photoresist on a silicon wafer.
  • a photoresist for example, SU-8 photoresist (MicroChem)
  • a positive replica of PDMS may be made by molding the PDMS against the master, a technique known to those skilled in the art.
  • a flat substrate for example, a glass slide, silicon wafer, or polystyrene surface may be placed against the PDMS surface and may be held in place by van der Waals forces, or may be fixed to the PDMS using an adhesive.
  • holes e.g., 1 millimeter in diameter
  • tubing for example of polyethylene
  • the connection may be sealed with a sealant or adhesive such as epoxy glue.
  • Microfluidic channels This example shows a method for preparing microfluidic channels according to one embodiment of the invention.
  • Microfluidic channels (e.g., with dimensions 50 ⁇ m wide x 100 ⁇ m tall x 1 cm long) were fabricated by molding uncured poly-dimethylsiloxane (PDMS) polymer from a silicon-photoresist master, following standard photolithographic methods.
  • PDMS poly-dimethylsiloxane
  • SU-8 negative photoresist (MicroChem) was layered onto silicon masters and exposed to UV light through an aligned transparency mask. Unpolymerized photoresist was removed by agitating for 20 min in a dish containing the solvent l-methoxy-2-propanol acetate (PGMEA, Sigma).
  • the silicon wafer and photoresist features were silanized to facilitate subsequent molding steps with tridecafluoro 1,1,2,2 tetrahydroctyl tricholorsilane.
  • PDMS was prepared in a 1 : 10 ratio of crosslinker to pre-polymer, degassed for 30 min in a vacuum chamber, poured onto the silicon master, and cured in a 70 °C oven for at least 4 hours.
  • the glass slide No. 1 borosilicate glass slides, Fisher
  • the glass slides were then baked for 5 h at 200 0 C to remove any residual water. After curing, the PDMS mold was peeled from the master and plasma sealed to the glass slides.
  • the microfluidic channels were prepared as follows. Rat anti-mouse CD3 antibody (clone 17A2), rat anti-mouse CD4 antibody (clone L3T4), rat anti-mouse CD8 ⁇ antibody (clone 53-6.7), and rat anti-mouse CD5 antibody (clone 53-7.3), all from BD Biosciences, were added to the channel at 30 ⁇ g/mL, 30 ⁇ g/mL, 30 ⁇ g/mL, and 30 ⁇ g/mL respectively, diluted in PBS from their stock solutions.
  • the adhesion molecule ICAM-I CD54
  • R&D Systems recombinant Fc chimera
  • mice anti-human CD3 (clone HIT3a), mouse anti-human CD4 (clone RPA-T4), mouse anti-human CD8 (clone RPA-78) mouse anti-human CD5 (clone UCHT2) were added at 30 ⁇ g/mL, 50 ⁇ g/mL, 50 ⁇ g/mL, and 10 ⁇ g/mL respectively, diluted in PBS from their stock solutions.
  • adhesion molecules ICAM-I CD54
  • 10 ⁇ g/mL 10 ⁇ g/mL
  • LFA-3 CD58
  • 30 ⁇ g/mL in the form of recombinant Fc chimeras
  • This example shows a method for performing an assay involving detection of T cells according to one embodiment of the invention.
  • the resulting cells were resuspended in 50 mL Dulbecco's phosphate buffered saline (PBS, Gibco), counted using light microscopy (Kova Glasstic Slide, Hycor), and centrifuged at 40Og (Beckman GS-6KR centrifuge) to a pellet. T cells were then purified using anti-CD4 and anti-CD8 MACS beads following the manufacturer's protocol (Miltenyi Biotec).
  • MACS buffer PBS with 0.5% (7.5 mM) BSA (Sigma Aldrich) and 2 mM EDTA (Fluka), pH 7.2 was added at 90 ⁇ L per 10 7 cells to the pellet, along with MACS anti-CD4 and anti-CD8 antibody superparamagnetic beads (Miltenyi Biotec) at a ratio of 10 ⁇ L per 10 7 cells.
  • the cells and beads were refrigerated (8 0 C) for 15 min, washed with 20 mL MACS buffer, centrifuged at 40Og to a pellet, and resuspended in 1 mL of MACS buffer. The cells were loaded onto a MACS LS column suspended in its magnetic holder.
  • the column was washed with 3 mL MACS buffer three times, then the column was removed from its magnetic holder. To elute the T cells, 5 mL of MACS buffer was forced through the column. The cells resulting from this purification process were counted.
  • Human whole blood Use of blood from healthy adult human volunteers was approved by the Harvard Medical School Institutional Review Board and the Harvard Office for Research Subject Protection. Whole blood was drawn using sterile venipuncture technique into sodium heparin coated vacuum tubes (Vacutainer) and used directly without further manipulation.
  • Channels were prepared by flowing a solution of 1% BSA in PBS buffer through the channels for 1 h at room temperature.
  • Experiments involved introducing samples such as Human or mouse blood or blood cells purged of T cells (above) (10-20 ⁇ L) into the prepared channel (e.g., a serial dilution system) by hand vacuum and incubating them in the channels for 1-2 h at room temperature. This time allowed cells from the blood to settle and adhere to the antibodies and adhesion molecules.
  • Cells were then fixed by washing the channel with 10-20 ⁇ L of 4% vol/vol paraformaldehyde(Polysciences) with 0.1% Triton X, then incubated at 37 °C for 30 min. Washing the channels with phosphate buffered saline removed unadherent cells and left behind mostly T cells in the channel.
  • rat anti-mouse CD3 (clone 17A2) was used at 30 ⁇ g/mL, diluted in PBS from the stock solution, to coat surfaces of the microfluidic channel, followed by goat anti-rat IgG gold conjugated antibody (Sigma). Channels were rinsed with copious amounts of PBS (over 100 ⁇ L), then 10-20 ⁇ L of secondary anti- CD3 antibodies (specifics above in the antibody section) were added at 10 ⁇ g/mL and allowed to incubate at room temperature for one hour.
  • the channel was rinsed with 10- 20 ⁇ L of PBS, then 10-20 ⁇ L of tertiary gold-conjugated antibody (Sigma) diluted 1 :300 from stock in PBS was introduced and allowed to incubate at room temperature for one hour.
  • the channels were rinsed with distilled water to prevent AgCl precipitation.
  • Silver nitrate and hydroquinone (the reducing agent; Silver Enhancer Kit, Sigma) were introduced into the channel.
  • the gold nanoparticles catalyzed an amplified reduction of silver nitrate to metallic silver, and generated an opaque precipitate of silver in the region of the T cells. In pilot experiments utilizing this protocol, silver precipitation on T cells was observed in roughly 10 minutes. The reaction was halted after 20 minutes by washing the channel with water.
  • Human T cells were detected using the following procedure. 20 ⁇ L of biotin- conjugated anti-human TCR- ⁇ (Ebioscience, clone IP26) at 10 ⁇ g/mL was added, diluted in PBS from the stock solution, followed by Streptavidin that had been conjugated with 1 nm gold nanoparticles diluted 1 :100 from stock in 1% BSA. The detection process was performed hours or days later and gave similar results.
  • Human T cells were counted by using flow cytometry, which was performed on blood from healthy human volunteers by following the same protocol above with the exception that 50 ⁇ L CountBright beads (Invitrogen) were added to the final mixture as per the manufacturer's directions. T cells were labeled with FITC anti-CD3 and at least 10000 events were counted for both the T cells and the counting beads. Ratios and cell counts were calculated as per the manufacturer's directions.
  • pre-channel blood samples
  • post-channel blood at the outlet port
  • Pre-channel blood samples and post-channel blood samples were mixed with fluorescein isothiocyanate labeled anti-CD3 antibody (BD Biosciences), phycoerythrin labeled anti-CD 19 (BD Biosciences).
  • fluorescein isothiocyanate labeled anti-CD3 antibody BD Biosciences
  • phycoerythrin labeled anti-CD 19 BD Biosciences
  • the labeled species were allowed to incubate with the cells in the dark at 8 0 C for 20 minutes.
  • the cells were washed by pelleting at 15,000 rpm in a Microfuge, discarding the supernatant, then adding 200 ⁇ L of PBS and repeating twice.
  • the cells were then fixed by adding 4% paraformaldehyde (Polysciences, Inc.). Red blood cells were lysed. Flow cytometry was performed on a BD Facscalibur device with CellQuest software (BD Biosciences), and analyzed in FlowJo (TreeStar Software).
  • EXAMPLE 3 The following is a prophetic example of counting T cells using silver staining and transmission measurements.
  • Example 2 the same procedures as in Example 2 will be performed except the cells will be detected using a detector system that measures light transmission (or absorbance).
  • a detector system that measures light transmission (or absorbance).
  • the detector system will allow quantitative counting of cells within a certain range. (E.g., it was previously observed by microscopy that the number of cells captured was monotonic with the number of cells inputted).
  • a specimen of blood will be serially diluted into a series of microfluidic channels, and the cells will be captured and stained in the channels using the procedures described in Example 2.
  • Each of the channels will have an optical fiber embedded therein and red light from a LED or laser can shine through the optical fiber, through the channels that contain the cells, and out to a conventional photoresistor that is driven by a simple circuit.
  • the absorption of light by silver will signify the presence of T cells, whereas the unimpeded transmission of light will signify the absence of T cells.
  • Absorbance/transmission measurements of each of the channels will be obtained, plotted, and calibrated with a known sample to obtain the number of cells in the sample.
  • potentiometers in the circuit design can allow selection of a clinically appropriate cut-off value, which can be set at, for example, 1000 cells/ ⁇ L, the 1st percentile of the normal range of T cell counts for infants. This can allow determination of whether a sample of blood from an infant contains a normal T cell count.
  • Such a system can also be used for counting CD4 T cells for HI V/ AID S patients.
  • a clinically appropriate cut-off value can be set at, for example, 350 CD4 T cells/ ⁇ L, below which the CDC guidelines suggest offering anti-retroviral treatment.
  • a reference to "A and/or B", when used in conjunction with open-ended language such as “comprising” can refer, in one embodiment, to A only (optionally including elements other than B); in another embodiment, to B only (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); etc.
  • the phrase "at least one,” in reference to a list of one or more elements, should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed within the list of elements and not excluding any combinations of elements in the list of elements.
  • This definition also allows that elements may optionally be present other than the elements specifically identified within the list of elements to which the phrase "at least one" refers, whether related or unrelated to those elements specifically identified.
  • “at least one of A and B" can refer, in one embodiment, to at least one, optionally including more than one, A, with no B present (and optionally including elements other than B); in another embodiment, to at least one, optionally including more than one, B, with no A present (and optionally including elements other than A); in yet another embodiment, to at least one, optionally including more than one, A, and at least one, optionally including more than one, B (and optionally including other elements); etc.
  • the order of the steps or acts of the method is not necessarily limited to the order in which the steps or acts of the method are recited.

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Abstract

L'invention concerne des articles et des procédés permettant de déterminer un analyte indiquant une condition de maladie. Dans certains modes de réalisation, les articles et procédés décrits ici peuvent être utilisés pour déterminer, qualitativement ou quantitativement, une présence d'un composant, tel qu'un type de cellule particulier, dans un échantillon de fluide. Dans un mode de réalisation particulier, on propose un système microfluidique à faible coût permettant une détection rapide de cellules T. Le système microfluidique peut utiliser des anticorps immobilisés et des molécules d'adhérence dans un canal pour capturer les cellules T à partir d'un échantillon de fluide tel qu'un petit volume de sang. Les cellules T capturées peuvent être étiquetées avec un colloïde métallique (par exemple des nanoparticules d'or) en utilisant un anticorps spécifique au récepteur de cellules T (TCR), et de l'argent métallique peut être précipité de manière catalytique sur les cellules. Le nombre de cellules T capturées peut être compté et peut indiquer une condition de maladie d'un patient telle qu'une déficience immune combinée sévère ou un virus d'immunodéficience humaine.
PCT/US2007/023595 2006-11-09 2007-11-09 Détecteur microfluidique WO2008060449A2 (fr)

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010003188A1 (fr) 2008-07-11 2010-01-14 Monash University Procédé de fabrication de systèmes microfluidiques
US9011798B2 (en) 2008-08-14 2015-04-21 Monash University Switches for microfluidic systems
WO2016112245A1 (fr) * 2015-01-08 2016-07-14 Hackensack University Medical Center Procédés ex vivo pour minimiser les risques et maximiser les effets bénéfiques d'une transfusion de sang allogénique et greffe de moelle
WO2016209775A1 (fr) * 2015-06-20 2016-12-29 The Regents Of The University Of California Dosage microfluidique basé sur des intégrines de monocytes destiné à l'évaluation de maladies coronaires

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20040248205A1 (en) * 2003-04-16 2004-12-09 Stern Lawrence J. Major histocompatibility complex (MHC)-peptide arrays
US20050042623A1 (en) * 2002-10-30 2005-02-24 Dana Ault-Riche Systems for capture and analysis of biological particles and methods using the systems
WO2005066613A1 (fr) * 2003-12-31 2005-07-21 President And Fellows Of Harvard College Dispositif et procede de dosage

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20050042623A1 (en) * 2002-10-30 2005-02-24 Dana Ault-Riche Systems for capture and analysis of biological particles and methods using the systems
US20040248205A1 (en) * 2003-04-16 2004-12-09 Stern Lawrence J. Major histocompatibility complex (MHC)-peptide arrays
WO2005066613A1 (fr) * 2003-12-31 2005-07-21 President And Fellows Of Harvard College Dispositif et procede de dosage

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
BERG N N ET AL: "Characterization of intercellular adhesion molecule-1 (ICAM-1)-augmented degranulation by cytotoxic T cells. ICAM-1 and anti-CD3 must be co-localized for optimal adhesion and stimulation." JOURNAL OF IMMUNOLOGY (BALTIMORE, MD. : 1950) 15 AUG 1995, vol. 155, no. 4, 15 August 1995 (1995-08-15), pages 1694-1702, XP002478490 ISSN: 0022-1767 *
BUTTE ET AL: "Microfluidic Device Allows Low-Cost Newborn Screening for Severe Combined Immune Deficiency (SCID)" JOURNAL OF ALLERGY AND CLINICAL IMMUNOLOGY, MOSBY - YEARLY BOOK, INC, US, vol. 117, no. 2, February 2006 (2006-02), page S325, XP005360657 ISSN: 0091-6749 *
MISHRA ET AL: "On-chip micro-biosensor for the detection of human CD4<+> cells based on AC impedance and optical analysis" BIOSENSORS & BIOELECTRONICS, ELSEVIER SCIENCE PUBLISHERS, BARKING, GB, vol. 21, no. 5, 15 November 2005 (2005-11-15), pages 696-704, XP005122213 ISSN: 0956-5663 *
STONE D J ET AL: "HLA-restricted epitope identification and detection of functional Tcell responses by using MHC-peptide and costimulatory microarrays" PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF USA, NATIONAL ACADEMY OF SCIENCE, WASHINGTON, DC, US, vol. 102, no. 10, 8 March 2005 (2005-03-08), pages 3744-3749, XP002383140 ISSN: 0027-8424 *

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010003188A1 (fr) 2008-07-11 2010-01-14 Monash University Procédé de fabrication de systèmes microfluidiques
US8852526B2 (en) 2008-07-11 2014-10-07 Monash University Method of fabricating microfluidic systems
US9011798B2 (en) 2008-08-14 2015-04-21 Monash University Switches for microfluidic systems
WO2016112245A1 (fr) * 2015-01-08 2016-07-14 Hackensack University Medical Center Procédés ex vivo pour minimiser les risques et maximiser les effets bénéfiques d'une transfusion de sang allogénique et greffe de moelle
EP3242671A4 (fr) * 2015-01-08 2018-08-08 Hackensack University Medical Center Procédés ex vivo pour minimiser les risques et maximiser les effets bénéfiques d'une transfusion de sang allogénique et greffe de moelle
WO2016209775A1 (fr) * 2015-06-20 2016-12-29 The Regents Of The University Of California Dosage microfluidique basé sur des intégrines de monocytes destiné à l'évaluation de maladies coronaires

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