Patent Application
20160327510
There is a significant interest in the development of paper point-of-care (POC) devices that are cheap, user friendly, robust, sensitive, and portable. Such devices pose an effective solution to the existing economic and healthcare accessibility problems in underdeveloped countries, as well as the growing trend in more affluent societies to become better informed in terms of its health. Although commercial paper-based sensors have been around for about 25 years (e.g., pregnancy test and glucose test strips), few paper POC devices have been successfully commercialized. Such failure to produce trustworthy paper POC devices is a combination of many factors, including poor limits of detection (LOD), high non-specific adsorption (NSA), unstable reagents, long analysis time, complex user-technology interface, detection method, and poor sensitivity.
Provided herein are methods for the electrochemical detection of analytes. Methods for detecting an analyte can comprise flowing fluid along a channel to accumulate the analyte conjugated to a metal particle (i.e., an analyte conjugate) in a region of the channel in electrochemical contact with a working electrode. The channel can be, for example, a microfluidic channel. The analyte conjugate can be accumulated in the region of the channel in electrochemical contact with a working electrode by a localization element. The localization element can be any feature that is configured to increase the concentration of the analyte conjugate in the region of the channel in electrochemical contact with the working electrode in the presence of fluid flow through the channel. For example, the localization element can be a physical barrier disposed in the region of the channel in electrochemical contact with the working electrode (e.g., a material configured to physically entrap the analyte conjugate), one or more localization electrodes configured to apply an electric field to the region of the channel in electrochemical contact with the working electrode (e.g., configured to electrophoretically localize the analyte conjugate), a magnet configured to apply a magnetic field in the region of the channel in electrochemical contact with the working electrode, or a combination thereof.
Once the analyte conjugate is accumulated in the region of the channel in electrochemical contact with a working electrode, fluid flow along the channel can be interrupted. The metal particle can then be oxidized, forming a population of metal ions in the region of the channel in electrochemical contact with the working electrode. The metal particle can be oxidized by any suitable method, such as by contacting the metal particle with a suitable oxidant (e.g., permanganate or hypochlorite) or by direct electrochemical oxidation of the metal particle by a potential applied at the working electrode. In some embodiments, the localization element (e.g., the magnet) can be translocated from an incubation region, where the conjugate analyte is accumulated, to an oxidation region comprising an oxidant, thereby bringing the metal particle into contact with the oxidant. Once formed, the metal ions (and by extension the analyte) can then be electrochemically detected and/or quantified, for example, using the working electrode (e.g., by anodic stripping voltammetry).
Also provided are devices for the electrochemical detection of analytes. The devices can comprise a channel defining a path for fluid flow from a fluid inlet to a fluid outlet, a working electrode positioned in electrochemical contact with a region of the channel, and a localization element configured to accumulate the analyte conjugated to the metal particle (i.e., the analyte conjugate) in the region of the channel in electrochemical contact with the working electrode.
As described above, the localization element can be any feature that is configured to increase the concentration of the analyte conjugate in the region of the channel in electrochemical contact with the working electrode in the presence of fluid flow through the channel. For example, the localization element can be a physical barrier disposed in the region of the channel in electrochemical contact with the working electrode, one or more localization electrodes configured to apply an electric field to the region of the channel in electrochemical contact with the working electrode, a magnet configured to apply a magnetic field in the region of the channel in electrochemical contact with the working electrode, or a combination thereof.
In certain embodiments, the localization element can comprise a magnet configured to apply a magnetic field in the region of the channel in electrochemical contact with the working electrode. In these embodiments, the devices can comprise a channel defining a path for fluid flow from a fluid inlet to a fluid outlet, an electrode positioned in electrochemical contact with a region of the channel, and a magnet configured to apply a magnetic field to the region of the channel positioned in electrochemical contact with the electrode.
Devices can further include a counter electrode, a reference electrode, or combinations thereof in electrochemical contact with the channel. Optionally, if desired for a particular detection method, the devices can further include a second working electrode positioned in electrochemical contact with a second region of the channel, and a second localization element configured to accumulate an analyte conjugated to a metal particle (i.e., an analyte conjugate) in the second region of the channel in electrochemical contact with the second working electrode.
Devices can further comprise an engageable platform that can be translocated from a retracted position to a deployed position. When the engageable platform is in the retracted position, the engageable platform is fluidly independent from the channel. When the engageable platform is in the deployed position, the engageable platform is in fluid contact with the region of the channel in electrochemical contact with the working electrode. An oxidant, such as potassium permanganate or hypochlorite, can be disposed on the engageable platform. In these embodiments, the oxidant can be introduced to the region of the channel in electrochemical contact with the working electrode by translocation of the engageable platform (e.g., to oxidize the metal particle).
The devices and methods described herein are inexpensive, user friendly (they employ electrochemical detection without any washing steps or electrode modification), sensitive, portable, robust (they employ metal particles for signal amplification as opposed to enzymes), efficient, rapid (completion of analysis in 4.6 min), and can detect low concentrations (767 fM). As such, the device and methods are well suited for use in numerous applications including point-of-care (POC) diagnostics.
The methods and devices described herein may be understood more readily by reference to the following detailed description of specific aspects of the disclosed subject matter, figures and the examples included therein.
Before the present devices and methods are disclosed and described, it is to be understood that the aspects described below are not intended to be scope by the specific devices and methods described herein, which are intended as illustrations. Various modifications of the devices and methods in addition to those shown and described herein are intended to fall within the scope of that described herein. Further, while only certain representative devices and method steps disclosed herein are specifically described, other combinations of the devices and method steps also are intended to fall within the scope of that described herein, even if not specifically recited. Thus, a combination of steps, elements, components, or constituents may be explicitly mentioned herein or less, however, other combinations of steps, elements, components, and constituents are included, even though not explicitly stated.
The term “comprising” and variations thereof as used herein is used synonymously with the term “including” and variations thereof and are open, non-limiting terms. Although the terms “comprising” and “including” have been used herein to describe various examples, the terms “consisting essentially of” and “consisting of” can be used in place of “comprising” and “including” to provide for more specific examples of the invention and are also disclosed. Other than in the examples, or where otherwise noted, all numbers expressing quantities of ingredients, reaction conditions, and so forth used in the specification and claims are to be understood at the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, to be construed in light of the number of significant digits and ordinary rounding approaches.
As used in the description and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a composition” includes mixtures of two or more such compositions, reference to “an agent” includes mixtures of two or more such agents, reference to “the component” includes mixtures of two or more such components, and the like.
“Optional” or “optionally” means that the subsequently described event or circumstance can or cannot occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.
It is understood that throughout this specification the identifiers “first” and “second” are used solely to aid in distinguishing the various components and steps of the disclosed subject matter. The identifiers “first” and “second” are not intended to imply any particular order, amount, preference, or importance to the components or steps modified by these terms.
Also, throughout this specification, various publications are referenced. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art to which the disclosed matter pertains. The references disclosed are also individually and specifically incorporated by reference herein for the material contained in them that is discussed in the sentence in which the reference is relied upon.
Methods
Provided herein are methods for the electrochemical detection of analytes. The methods employ metal particles (e.g., metal nanoparticles) conjugated to analytes. The metal particles can serve as an electrochemical label for the analyte to which they are conjugated. Specifically, the metal particles can be oxidized to form metal ions that can subsequently be electrochemically detected and/or quantified. For example, the metal ions can be electrodeposited as metal on a working electrode. The potential applied at the working electrode can then be varied to reoxidize the deposited metal to metal ions. The intensity of the resulting voltammetric peak reflects the amount of metal deposited on the working electrode, and therefore the amount of metal nanoparticle label (and by extension analyte) present in a sample. Sensitivity can be improved by selectively localizing the analyte-metal particle conjugate in the vicinity of the working electrode. Using this method, analytes can be detected at concentrations as low as 767 fM via anodic stripping voltammetry, with no washing steps or electrode modifications.
Methods for detecting an analyte can comprise flowing fluid along a channel to accumulate the analyte conjugated to a metal particle (i.e., an analyte conjugate) in a region of the channel in electrochemical contact with a working electrode. The channel can be, for example, a microfluidic channel. The analyte conjugate can be accumulated in the region of the channel in electrochemical contact with a working electrode by a localization element. The localization element can be any feature that is configured to increase the concentration of the analyte conjugate in the region of the channel in electrochemical contact with the working electrode in the presence of fluid flow through the channel. For example, the localization element can be a physical barrier disposed in the region of the channel in electrochemical contact with the working electrode (e.g., a material configured to physically entrap the analyte conjugate), one or more localization electrodes configured to apply an electric field to the region of the channel in electrochemical contact with the working electrode (e.g., configured to electrophoretically localize the analyte conjugate), a magnet configured to apply a magnetic field in the region of the channel in electrochemical contact with the working electrode, or a combination thereof.
Once the analyte conjugate is accumulated in the region of the channel in electrochemical contact with a working electrode, fluid flow along the channel can be interrupted. The metal particle can then be oxidized, forming a population of metal ions in the region of the channel in electrochemical contact with the working electrode. The metal particle can be oxidized by any suitable method, such as by contacting the metal particle with a suitable oxidant or by direct electrochemical oxidation of the metal particle by a potential applied at the working electrode. Once formed, the metal ions (and by extension the analyte) can then be electrochemically detected and/or quantified, for example, using the working electrode.
The analyte can be, for example, an antibody, peptide (natural, modified, or chemically synthesized), protein (e.g., a glycoprotein, a lipoprotein, or a recombinant protein), polynucleotide (e.g, DNA or RNA, an oligonucleotide, an aptamer, or a DNAzyme), lipid, polysaccharide, small molecule organic compound (e.g., a hormone, a prohormone, a narcotic, or a small molecule pharmaceutical), pathogen (e.g., bacteria, virus, or fungi, or protozoa), or combination thereof.
In some embodiments, the analyte can be a molecule of interest present in a fluid sample that is introduced into the channel. By way of example, the fluid sample can be a bodily fluid. “Bodily fluid”, as used herein, refers to a fluid composition obtained from or located within a human or animal subject. Bodily fluids include, but are not limited to, urine, whole blood, blood plasma, serum, tears, semen, saliva, sputum, exhaled breath, nasal secretions, pharyngeal exudates, bronchoalveolar lavage, tracheal aspirations, interstitial fluid, lymph fluid, meningal fluid, amniotic fluid, glandular fluid, feces, perspiration, mucous, vaginal or urethral secretion, cerebrospinal fluid, and transdermal exudate. Bodily fluid also includes experimentally separated fractions of all of the preceding solutions, as well as mixtures containing homogenized solid material, such as feces, tissues, and biopsy samples. The molecule of interest can be, for example, a biomarker (i.e., a molecular indicator associated with a particular pathological or physiological state) present in the bodily fluid that can be assayed to identify risk for, diagnosis of, or progression of a pathological or physiological process in a subject. Examples of biomarkers include proteins, hormones, prohormones, lipids, carbohydrates, DNA, RNA, and combinations thereof.
When the analyte is a molecule of interest present in the fluid sample that is introduced into the channel, methods can further involve conjugating the molecule of interest to a metal particle to form an analyte complex (e.g., for example by contacting the molecule of interest with a metal nanoparticle bound to a recognition element for the molecule of interest, as described in more detail below). Conjugation can occur in the fluid sample prior to introduction into the channel, such that the resulting analyte complex is introduced into the channel. Alternatively, conjugation can occur in situ within the device (e.g., by contacting with by contacting the molecule of interest with a metal nanoparticle bound to a recognition element that is deposited on or within the channel or a fluid inlet fluidly connected thereto).
In other embodiments, the analyte can be a surrogate for the molecule of interest. The surrogate can be an analyte whose concentration in the fluid flowing through the channel is proportional to the concentration of the molecule of interest in the fluid sample, such that by detecting and/or quantifying the surrogate using the electrochemical methods described herein, the molecule of interest can be detected and/or quantified. By way of example, a fixed analyte support (e.g., an aptamer that specifically binds a molecule of interest) can immobilized on or within the channel or a fluid inlet fluidly connected thereto. A surrogate (e.g., a recognition element for the aptamer such as a polynucleotide probe having a complementary sequence to a portion of the aptamer) can be bound to the fixed analyte support. When the surrogate-fixed analyte support conjugate is contacted with the molecule of interest, the molecule of interest binds to the fixed analyte support, displacing the surrogate. The surrogate then functions as the analyte in the detection methods described above.
The metal particle can be, for example, a metal nanoparticle. The metal particle comprise any suitable metal, such as gold, silver, copper, platinum, rhodium, palladium, iridium, nickel, iron, bismuth, cadmium, cobalt, or combinations thereof. The metal particle can also comprise a suitable metal compound, such as, for example, a metal oxide, halide, and/or chalcogenide, such as Ag2O, AgI, Bi2O5, CuO, Cd3P2, CdS, CdSe, CdTe, Co2O3, CrO3, Cu2S, HgI2, MnO2, PbS, PbO2, SnO2, TiO2, RuO2, ZnO, ZnS or ZnO2. Suitable metal particles can be selected in view of a number of factors, including the nature of the oxidation process employed, the presence or absence of other species present in the fluid sample flowing through the channel, the nature of the electrochemical techniques employed, the desired stability of the metal particle towards environmental conditions (e.g., stability in air), compatibility with a desired means of conjugation to the analyte, and combinations thereof. For example, in some embodiments, the metal particle can be formed from a metal or metal compound that is not present (or is only present at low levels) in the fluid sample flowing through the channel. In some cases, the metal particle can be selected such that it can be reduced by an oxidant (e.g., the metal particle can be selected such that it has a reduction potential that is more negative than the oxidant).
The analyte can be conjugated to the metal particle by any suitable covalent or non-covalent means. In some embodiments, the analyte can be bound to the metal particle by a recognition element. For example, the metal particle can be bound (via any non-covalent or covalent means) to a recognition element for the analyte, which can be bound to the analyte.
Recognition elements for particular analytes are known in the art. An appropriate recognition element for the formation of an analyte conjugate can be selected in view of a number of considerations including analyte identity, analyte concentration, and the nature of the sample in which the analyte is to be bound. Suitable recognition elements include antibodies, antibody fragments, antibody mimetics (e.g., engineered affinity ligands such as AFFIBODY® affinity ligands), peptides (natural or modified peptides), proteins (e.g., recombinant proteins, host proteins), polynucleotides (e.g, DNA or RNA, oligonucleotides, aptamers, or DNAzymes), receptors, ligands, antigens, organic small molecules (e.g., antigen or enzymatic co-factors), and combinations thereof.
In some embodiments, the recognition element selectively associates with the analyte. The term “selectively associates”, as used herein when referring to a recognition element, refers to a binding reaction which is determinative for the analyte in a heterogeneous population of other similar compounds. Generally, the interaction is dependent upon the presence of a particular structure (e.g., an antigenic determinant or epitope) on the binding partner. By way of example, an antibody or antibody fragment selectively associates to its particular target (e.g., an antibody specifically binds to an antigen) but it does not bind in a significant amount to other proteins present in the sample or to other proteins to which the antibody may come in contact in an organism.
In some embodiments, a recognition element can be a molecule that has an affinity constant (Ka) greater than about 105 M−1 (e.g., greater than about 106 M−1, greater than about 107 M−1, greater than about 108 M−1, greater than about 109 M−1, greater than about 1010 M−1, greater than about 1011 M−1, greater than about 1012 M−1, or more) with that analyte.
In certain embodiments, the recognition element comprises an antibody. The term “antibody” refers to natural or synthetic antibodies that selectively bind a target antigen. The term includes polyclonal and monoclonal antibodies. In addition to intact immunoglobulin molecules, also included in the term “antibodies” are fragments or polymers of those immunoglobulin molecules, and human or humanized versions of immunoglobulin molecules that selectively bind the target antigen. The term encompasses intact and/or full length immunoglobulins of types IgA, IgG (e.g., IgG1, IgG2, IgG3, IgG4), IgE, IgD, IgM, IgY, antigen-binding fragments and/or single chains of complete immunoglobulins (e.g., single chain antibodies, Fab fragments, F(ab′)2 fragments, Fd fragments, scFv (single-chain variable), and single-domain antibody (sdAb) fragments), and other proteins that include at least one antigen-binding immunoglobulin variable region, e.g., a protein that comprises an immunoglobulin variable region, e.g., a heavy (H) chain variable region (VH) and optionally a light (L) chain variable region (VL). The light chains of an antibody may be of type kappa or lambda.
An antibody may be polyclonal or monoclonal. A polyclonal antibody contains immunoglobulin molecules that differ in sequence of their complementarity determining regions (CDRs) and, therefore, typically recognize different epitopes of an antigen. Often a polyclonal antibody is derived from multiple different B cell lines each producing an antibody with a different specificity. A polyclonal antibody may be composed largely of several subpopulations of antibodies, each of which is derived from an individual B cell line. A monoclonal antibody is composed of individual immunoglobulin molecules that comprise CDRs with the same sequence, and, therefore, recognize the same epitope (i.e., the antibody is monospecific). Often a monoclonal antibody is derived from a single B cell line or hybridoma. An antibody may be a “humanized” antibody in which for example, a variable domain of rodent origin is fused to a constant domain of human origin or in which some or all of the complementarity-determining region amino acids often along with one or more framework amino acids are “grafted” from a rodent, e.g., murine, antibody to a human antibody, thus retaining the specificity of the rodent antibody.
An appropriate analyte conjugate and localization element can be selected in combination so as to facilitate accumulation of the analyte conjugate in the region of the channel in electrochemical contact with a working electrode. For example, in some embodiments, the analyte conjugate is charged (e.g., the analyte itself is charged, the metal particle is charged, or the analyte and/or the metal particle is conjugated to a charged moiety such as a charged molecule or charged particle), and the localization element comprises a localization electrode configured to apply an electric field to the region of the channel, so as to increase the concentration of the charged analyte conjugate in the region of the channel in electrochemical contact with the working electrode. In these embodiments, methods of detecting the analyte can comprise flowing fluid comprising the charged analyte conjugated to the metal particle along the channel, and applying electric field via one or more localization electrodes to accumulate the charged analyte conjugated to the metal particle in the region of the channel in electrochemical contact with a working electrode.
In some embodiments, the localization element can comprise a physical barrier disposed in the region of the channel in electrochemical contact with the working electrode. The physical barrier can be any suitable material configured to physically entrap the analyte conjugate. For example, the physical barrier can be a porous hydrophilic material (e.g., paper) or a matrix of polymer beads disposed within the fluid flow path formed by the channel that can physically entrap the analyte conjugate. In some embodiments, the analyte conjugate can further include a steric particle (e.g., a microbead) conjugated to the analyte and/or the metal particle to increase the hydrodynamic volume of the analyte, thereby facilitating entrapment of the analyte conjugate in the physical barrier. In these embodiments, methods of detecting the analyte can comprise flowing fluid comprising the analyte conjugated to the metal particle along the channel to contact the physical barrier such that the analyte accumulates in the region of the channel in electrochemical contact with a working electrode.
In certain embodiments, the localization element can comprise a magnet configured to apply a magnetic field in the region of the channel in electrochemical contact with the working electrode, and the analyte conjugate can comprise a magnetic moiety. For example, the analyte conjugate can comprise an analyte conjugated to a metal particle and a magnetic particle.
The magnetic particle can be any magnetic particle that can be conjugated to the analyte and which can provide for localization of the bound analyte under an applied magnetic field. For example, the magnetic particle can be a magnetic microbead. Magnetic microbeads are superparamagnetic, monodisperse, polymer beads that comprise a dispersion of a magnetic material (e.g., gamma Fe2O3 and Fe3O4) throughout the polymer bead. The microbeads are coated with a thin polymer shell which encases the magnetic material and provides a defined surface area for the adsorption or coupling of various molecules. Suitable magnetic microbeads are known in the art, and are commercially available from Life Technologies under the tradename DYNABEADS®.
The analyte can be conjugated to the magnetic particle by any suitable covalent or non-covalent means. In some embodiments, the analyte can be bound to the magnetic particle by a recognition element, as described above. For example, the magnetic particle can be bound (via any non-covalent or covalent means) to a recognition element for the analyte that can be bound to the analyte.
In certain embodiments, the localization element can comprise a magnet configured to apply a magnetic field in the region of the channel in electrochemical contact with the working electrode, and the analyte conjugate can comprise an analyte bound to a first antibody and a second antibody, wherein a metal particle is bound to the first antibody and a magnetic particle is bound to the second antibody.
In certain embodiments, the localization element can comprise a magnet configured to apply a magnetic field in the region of the channel in electrochemical contact with the working electrode, and the analyte conjugate can comprise an analyte bound to a first polynucleotide and a second polynucleotide, wherein a metal particle is bound to the first polynucleotide and a magnetic particle is bound to the second polynucleotide. In some of these embodiments, the analyte comprises a polynucleotide.
In these embodiments, methods of detecting the analyte can comprise flowing fluid comprising the analyte conjugated to the metal particle and the magnetic particle along the channel, and applying the magnetic field to accumulate the analyte conjugated to the metal particle and the magnetic particle in the region of the channel.
As described above, once the analyte conjugate is accumulated in the region of the channel in electrochemical contact with a working electrode, the metal particle can then be oxidized to form a population of metal ions in the region of the channel in electrochemical contact with the working electrode. The metal particle can be oxidized by any suitable oxidation method, such as by contacting the metal particle with a suitable oxidant or by direct electrochemical oxidation of the metal particle by a potential applied at the working electrode. In certain embodiments, oxidizing the metal particle comprises contacting the metal particle with a suitable oxidant. In certain embodiments, the region of the channel in electrochemical contact with the working electrode can comprise an incubation region and an oxidation region, wherein the oxidation region can comprise an oxidant. In these embodiments, the localization element (e.g., a magnet) can be used to accumulate the analyte conjugate in the incubation region, then the localization element can be translocated from the incubation region to the oxidation region thereby bringing the metal particle into contact with the oxidant. The oxidant can be any suitable oxidant known in the art. Examples of oxidants include, but are not limited to, nitric acid, sulfuric acid, peroxides (e.g., hydrogen peroxide), peroxydisulfuric acid, peroxymonosulfuric acid, halogen compounds (e.g., chlorite, chlorate, perchlorate), hypohalite compounds (e.g., hypochlorites such as sodium hypochlorite), hexavalent chromium compounds (e.g., chromate and dichromate salts), permanganate compounds (e.g. potassium permanganate), sodium perborate, nitrous oxide, silver oxide, osmium tetroxide, cerium(IV) oxide, potassium nitrate, and combinations thereof. In some embodiments, the oxidant comprises potassium permanganate. In other embodiments, the oxidant comprises an oxidant that is electrogenerated in situ in the channel. A suitable oxidant can be selected in view of a number of factors, including the desired stability of the oxidant towards environmental conditions (e.g., stability in air) and the composition of the metal particle to be oxidized. For example, the oxidant can be selected such that it can effectively oxidize the metal particle to produce a population of metal ions (e.g., the oxidant can be selected such that it has a reduction potential that is more positive than the metal particle).
Once formed, the metal ions (and by extension the analyte) can then be electrochemically detected and/or quantified, for example, using the working electrode. Various techniques of electrochemical analysis may be used to assay the dissolved metal ions. They are preferentially anodic stripping voltammetry with a potential scan which may be linear, cyclic, square-wave, normal pulse or differential pulse, or with a superimposed sinusoidal voltage, or else anodic stripping chronopotentiometry. However, other techniques may be used, such as ion exchange voltammetry, adsorptive cathodic stripping voltammetry (or polarography) with a scan which may be linear, cyclic, square-ware, normal pulse or differential pulse, or with a superimposed sinusoidal voltage, or else chronoamperometry, chronocoulometry or linear, cyclic, square-wave, normal pulse or differential pulse voltammetry (or polarography) or voltammetry (or polarography) with a superimposed sinusoidal voltage. These techniques require a possibly two-electrode or even three-electrode assembly, e.g., an assembly comprising the working electrode, a reference electrode, and a counter electrode.
A variety of potential assays (e.g., sandwich-type assays, competitive binding assays, etc.) can be envisioned that employ the electrochemical detection methods described above for analyte detection and/or quantification. The precise design of such assays will vary based on, for example, the nature of the analyte and the localization element used. By way of example, several methods of detection that employ magnetic localization are described below. While these methods include particular method steps, components, and analyte conjugates based on the use of a magnetic localization (e.g., an analyte conjugate comprising an analyte conjugated to a metal particle and a magnetic particle), it will be understood that these methods can be adapted to employ method steps, components, and analyte conjugates based on the use of an alternative localization element.
An example method for the electrochemical detection of an analyte is schematically illustrated in
An example of another method for the electrochemical detection of an analyte is schematically illustrated in
An example of a method for the electrochemical detection of a polynucleotide (e.g., DNA) is schematically illustrated in
An example of a method for the electrochemical detection of a molecule of interest via a surrogate is schematically illustrated in
Also provided are methods for the simultaneous electrochemical detection of multiple analytes.
Devices
Also provided are devices for the electrochemical detection of analytes. The devices can be used to practice the electrochemical detection methods described above. The devices can comprise a channel defining a path for fluid flow from a fluid inlet to a fluid outlet, a working electrode positioned in electrochemical contact with a region of the channel, and a localization element configured to accumulate the analyte conjugated to the metal particle (i.e., the analyte conjugate) in the region of the channel in electrochemical contact with the working electrode. As described above, the localization element can be any feature that is configured to increase the concentration of the analyte conjugate in the region of the channel in electrochemical contact with the working electrode in the presence of fluid flow through the channel. For example, the localization element can be a physical barrier disposed in the region of the channel in electrochemical contact with the working electrode (e.g., a material configured to physically entrap the analyte conjugate), one or more localization electrodes configured to apply an electric field to the region of the channel in electrochemical contact with the working electrode (e.g., configured to electrophoretically localize the analyte conjugate), a magnet configured to apply a magnetic field in the region of the channel in electrochemical contact with the working electrode, or a combination thereof. Devices can further include a counter electrode, a reference electrode, or combinations thereof in electrochemical contact with the channel. Optionally, if desired for a particular detection method, the devices can further include a second working electrode positioned in electrochemical contact with a second region of the channel, and a second localization element configured to accumulate an analyte conjugated to a metal particle (i.e., an analyte conjugate) in the second region of the channel in electrochemical contact with the second working electrode.
Devices can further comprise an engageable platform that can be translocated from a retracted position to a deployed position. When the engageable platform is in the retracted position, the engageable platform is fluidly independent from the channel (e.g., engageable platform is positioned in a region of the device such that the engageable platform is not in fluid contact with the channel). When the engageable platform is in the deployed position, the engageable platform is in fluid contact with the region of the channel in electrochemical contact with the electrode (e.g., engageable platform is positioned in fluid contact with the region of the channel in electrochemical contact with the electrode). In some embodiments, when the engageable platform is in retracted position, the path for fluid flow from the fluid inlet to the fluid outlet is continuous, such that fluid can flow from the fluid inlet to the fluid outlet, and when the engageable platform is in the deployed position, the path for fluid flow from the fluid inlet to the fluid outlet is interrupted, such that fluid cannot flow from the fluid inlet to the fluid outlet.
The engageable platform can be provided, for example, as a portion of a translocatable layer of a multilayer microfluidic device, as described in more detail below. Alternatively, the engageable platform can be provided independently from one or more layers that combine to form a microfluidic device (e.g., as part of a translocatable region within a stationary layer of a multilayer microfluidic device). The engageable platform can be formed from a porous, hydrophilic material, such as paper. An oxidant can be disposed on the engageable platform (e.g., adsorbed or absorbed so the engageable platform). The oxidant can be any suitable oxidant as described above (e.g., potassium permanganate).
The devices described herein can be fabricated from any suitable material or combination of materials. In some embodiments, the devices are paper-based microfluidic devices. Paper-based microfluidic devices include a channel (i.e., a path such as a conduit, through which one or more fluids can flow) formed within a layer of a porous, cellulosic substrate. The channel can be a void space through which a fluid can flow (i.e., a hollow channel), a porous hydrophilic substrate such as paper through which fluid flows by wicking (i.e., a filled channel), or a combination thereof. The dimensions of the channel within the layer of porous, cellulosic substrate are defined by a hydrophobic boundary that substantially permeates the thickness of the porous, cellulosic substrate, so as to form a boundary for fluid flow from the channel to a region on the porous, cellulosic substrate outside of the channel, thereby directing fluid flow along the channel.
The channel can be patterned within a layer of a porous, cellulosic substrate using any suitable method known in the art. For example, the channel can be patterned by wax printing. In these methods, an inkjet printer is used to pattern a wax material on the porous, cellulosic substrate. Many types of wax-based solid ink are commercially available and are useful in such methods as the ink provides a visual indication of the location of the channels. However, it should be understood, that the wax material used to form the channels does not require an ink to be functional. Examples of wax materials that maybe used include polyethylene waxes, hydrocarbon amide waxes or ester waxes. Once the wax is patterned, the porous, cellulosic substrate is heated (e.g., by placing the substrate on a hot plate with the wax side up at a temperature of 120° C.) and cooled to room temperature. This allows the wax material to substantially permeate the thickness of the porous, cellulosic substrate, so as to form a hydrophobic boundary that defines the dimensions of the channel. At this point, the resulting channel is a filled channel, as the channel defined by the hydrophobic boundary includes a porous hydrophilic substrate (the porous, cellulosic substrate) through which fluid can flow by wicking. If desired for device design, a hollow channel can be formed by removing the porous, cellulosic substrate within the hydrophobic boundary, thereby forming a void space through which a fluid can flow.
In some embodiments, the porous, cellulosic substrate used to form the paper-based microfluidic device is flexible. For certain applications, it is preferable that the cellulosic substrate can be folded, creased, or otherwise mechanically shaped to impart structure and function to the paper-based device formed from the cellulosic substrate. Examples of suitable porous, cellulosic substrates for the fabrication of paper-based microfluidic devices include cellulose; derivatives of cellulose such as nitrocellulose or cellulose acetate; paper (e.g., filter paper, chromatography paper); woven cellulosic materials; and non-woven cellulosic materials.
In some embodiment, the porous, cellulosic substrate is paper. Paper is inexpensive, widely available, readily patterned, thin, lightweight, and can be disposed of with minimal environmental impact. Furthermore, a variety of grades of paper are available, permitting the selection of a paper substrate with the weight (i.e., grammage), thickness and/or rigidity and surface characteristics (i.e., porosity, hydrophobicity, and/or roughness), desired for the fabrication of a particular paper-based device. Suitable papers include, but are not limited to, chromatography paper, card stock, filter paper, vellum paper, printing paper, wrapping paper, ledger paper, bank paper, bond paper, blotting paper, drawing paper, fish paper, tissue paper, paper towel, wax paper, and photography paper.
In certain embodiments, the localization element can comprise a magnet configured to apply a magnetic field in the region of the channel in electrochemical contact with the working electrode. In these embodiments, the devices can comprise a channel defining a path for fluid flow from a fluid inlet to a fluid outlet, an electrode positioned in electrochemical contact with a region of the channel, and a magnet configured to apply a magnetic field to the region of the channel positioned in electrochemical contact with the electrode.
An example device employing a magnetic localization element is illustrated in
Referring now to
The device is assembled by aligning the four layers as shown in
Referring now to
If desired, a reagent for the detection of a molecule of interest can be deposited at the fluid inlet. Optionally, an indicator can be disposed on the sink, the port in the third layer, the port in the second layer, or combinations thereof. The indicator can be a dye that is transported to the fluid outlet by the fluid flowing through the device, thereby indicating completion of an assay. In certain embodiments, the first layer, the second layer, and the fourth layer are fabricated from a single (integral) piece of paper that is folded to form the device.
Referring now to
The fluid inlet comprises a porous hydrophilic substrate, such as paper, onto which reagents for the detection of the molecule of interest (e.g., an analyte bound to a metal particle and an antibody for the analyte bound to a magnetic particle in the case of the method schematically illustrated in
Referring now to
If desired, the devices described herein can be affixed to or secured within a polymer, metal, glass, wood, or paper support structure to facilitate handling and use of the device. In some embodiments, the devices described herein are affixed to or secured within an inert, non-absorbent polymer such as a polyether block amide (e.g., PEBAX®, commercially available from Arkema, Colombes, France), a polyacrylate, a polymethacrylate (e.g., poly(methyl methacrylate)), a polyimide, polyurethane, polyamide (e.g., Nylon 6,6), polyvinylchloride, polyester, (HYTREL®, commercially available from DuPont, Wilmington, Del.), polyethylene (PE), polyether ether ketone (PEEK), fluoropolymers such as polytetrafluoroethylene (PTFE), perfluoroalkoxy, fluorinated ethylene propylene, or a blend or copolymer thereof. Silastic materials and siliconbased polymers can also be used.
The devices described herein can be coupled to a power supply and optionally to one or more additional suitable features including, but not limited to, a voltmeter, an ammeter, a multimeter, an ohmmeter, a signal generator, a pulse generator, an oscilloscope, a frequency counter, a potentiostat, or a capacitance meter. The devices described herein can also be coupled to a computing device that performs arithmetic and logic operations necessary to process the electrochemical signals produced by the device (e.g., to determine analyte concentration, etc.).
The devices and methods described herein are inexpensive, user friendly (they employ electrochemical detection without any washing steps), sensitive, portable, robust (they employ metal particles for signal amplification as opposed to enzymes), efficient, rapid (completion of analysis in 4.6 min), and can detect low concentrations (767 fM). As such, the device and methods are well suited for use in numerous sensing applications.
For example, the devices and methods described herein can be used in clinical and healthcare settings to detect and/or quantify biomarkers to identify risk for, diagnosis of, or progression of a pathological or physiological process in a subject. Examples of biomarkers include proteins, hormones, prohormones, lipids, carbohydrates, DNA, RNA, and combinations thereof.
The devices and methods described herein can be used in POC applications to diagnose infections in a patient (e.g., by measuring serum antibody concentrations or detect antigens). For example, the devices and methods described herein can be used to diagnose viral infections (e.g., HIV, hepatitis B, hepatitis C, rotavirus, influenza, polio, measles, yellow fever, rabies, dengue, or West Nile Virus), bacterial infections (e.g., E. coli, C. tetani, cholera, typhoid, diphtheria, tuberculosis, plague, Lyme disease, or H. pylori), and parasitic infections (e.g., toxoplasmosis, Chagas disease, or malaria). The devices and methods described herein can be used to rapidly assesses the immune status of people or animals against selected vaccine-preventable diseases (e.g. anthrax, human papillomavirus (HPV), diphtheria, hepatitis A, hepatitis B, haemophilus influenzae type b (Hib), influenza (flu), Japanese encephalitis (JE), measles, meningococcal, mumps, pertussis, pneumococcal, polio, rabies, rotavirus, rubella, shingles (herpes zoster), smallpox, tetanus, typhoid, tuberculosis (TB), varicella (chickenpox), yellow fever). The devices and methods described herein can be used to rapidly screen donated blood for evidence of viral contamination by HIV, hepatitis C, hepatitis B, and HTLV-1 and -2. The devices and methods described herein can also be used to measure hormone levels. For example, the devices and methods described herein can be used to measure levels of human chorionic gonadotropin (hCG) (as a test for pregnancy), Luteinizing Hormone (LH) (to determine the time of ovulation), or Thyroid Stimulating Hormone (TSH) (to assess thyroid function). The devices and methods described herein can be used to diagnose or monitor diabetes in a patient, for example, by measuring levels of glycosylated hemoglobin, insulin, or combinations thereof. The devices and methods described herein can be used to detect protein modifications (e.g., based on a differential charge between the native and modified protein and/or by utilizing recognition elements specific for either the native or modified protein). The devices and methods described herein can be used to administer personalized medical therapies to a subject (e.g., in a pharmacogenomic assay performed to select a therapy to be administered to a subject).
The devices and methods described herein can also be used in other commercial applications. For example, the devices and methods described herein can be used in the food and beverage industry, for example, in quality control applications or to detect potential food allergens, such as milk, peanuts, walnuts, almonds, and eggs. The devices and methods described herein can be used to detect and/or measure the levels of proteins of interest in foods, cosmetics, nutraceuticals, pharmaceuticals, and other consumer products. The devices and methods described herein can also be used to rapidly and accurately detect narcotics and biothreat agents (e.g., ricin).
The examples below are intended to further illustrate certain aspects of the systems and methods described herein, and are not intended to limit the scope of the claims.
The following examples are set forth below to illustrate the methods and results according to the disclosed subject matter. These examples are not intended to be inclusive of all aspects of the subject matter disclosed herein, but rather to illustrate representative methods and results. These examples are not intended to exclude equivalents and variations of the present invention which are apparent to one skilled in the art.
Efforts have been made to ensure accuracy with respect to numbers (e.g., amounts, temperature, etc.) but some errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, temperature is in ° C. or is at ambient temperature, and pressure is at or near atmospheric. There are numerous variations and combinations of reaction conditions, e.g., component concentrations, temperatures, pressures and other reaction ranges and conditions that can be used to optimize the product purity and yield obtained from the described process.
Overview
Provided herein is a ready-to-use paper point-of-care platform that exploits a versatile electrochemical method for the detection and/or quantification of analytes. Specifically, it is shown that an oxidizing agent (potassium permanganate) loaded into a device can serve to spontaneously oxidize a metal nanoparticle label bound to an analyte (e.g., a silver nanoparticle (AgNP) bound to an analyte) to form metal ions in the vicinity of a working electrode. The metal ions (Ag+) can subsequently be electrodeposited as metal (Ag) on the working electrode. The potential can then be varied to reoxidize the deposited metal (Ag) to metal ions (AO. The intensity of the resulting voltammetric peak reflects the amount of metal deposited on the working electrode, and therefore the amount of metal nanoparticle label (and by extension analyte) present in the solution. Using this method, POC devices are provided that can detect molecules of interest at concentrations as low as 767 fM via anodic stripping voltammetry, with no washing steps or electrode modifications.
Background
There is an interest in the development of paper POC devices that are cheap, user friendly, robust, sensitive, and portable. Such devices pose an effective solution to the existing economic and healthcare accessibility problems in underdeveloped countries, as well as the growing trend in more affluent societies to become better informed in terms of its health. Although commercial paper-like sensors have been previously described (e.g., pregnancy test and glucose test strips), few paper POC devices have been successfully commercialized. Such failure to produce trustworthy paper POC devices is a combination of many factors, including poor limits of detection (LOD), high non-specific adsorption (NSA), unstable reagents, long analysis time, complex user-technology interface, detection method, and poor sensitivity. Herein, a robust and easily fabricated (no electrode modifications) paper-based platform is presented that offers a simple user-device interface (ready-to-use type of device).
Experimental
Chemicals and Materials
Sodium phosphate monobasic, sodium phosphate dibasic, biotin (5-fluorescein) conjugate, microtater plates (Corning 3650), and potassium permanganate (KMnO4) were purchased from Sigma-Aldrich (St. Louis, Mo.). Sodium chloride (NaCl), sodium hydroxide (NaOH), Whatman grade 1 chromatography paper (180 μm thick, 20 cm×20 cm, linear flow rate (water) of 13 cm/30 min), microcentrifuge tubes, razor blade, two part 5-min epoxy, polytetrafluoroethylene (PTFE) tape, Parafilm paper, and Kimwipes were purchased from Fisher Scientific (Pittsburg, Pa.). AlexaFluor-647/streptavidin conjugate was purchased from Life Technologies (Grand Island, N.Y.). Streptavidin-coated magnetic microbeads (2.8 μm in diameter) were obtained from Bangs Laboratories (Fishers, Ind.). Citrate-capped silver nanoparticles (AgNP, 20.0 nm in diameter) and conductive copper tape (6.3 mm thick) were bought from Ted Pella (Redding, Calif.). Erioglaucine disodium salt (blue dye) was obtained from Acros Organics (Pittsburgh, Pa.). Conductive carbon paste (Cl-2042) was purchased from Engineered Conductive Materials (Delaware, Ohio). Neodymium cylindrical magnets ( 1/16″×½″, N48) were purchased from Apex Magnets (Petersburg, W.V.). Acrylic plates (0.6 cm thick) were obtained from Evonik Industries (Acrylite®FF). Clear nail polish was purchased from Electron Microscopy Sciences (Hatfield, Pa.). Copper epoxy (EPO-TEK 430) was acquired from Epoxy Technology (Billerica, Mass.). A4 transparency films were purchased from Office Depot. A PTFE electrochemical cell was used for all bulk-solution experiments (see
Instrumentation
All electrochemical measurements were performed with a bipotentiostat (700E, CH Instruments, Austin, Tex.). The citrate-capped AgNP diameter reduction in the presence of KMnO4 was characterized via Nanoparticle Tracking Analysis (NS500, NanoSight, Malvern Instruments). The microtiter plates were analyzed for fluorescence using an EnVision Multilabel reader (Perkin Elmer). UV-Vis measurements were made with a Hewlett-Packard HP8453 spectrometer. A Sorvall Legend Micro 21R Centrifuge (Thermo Scientific) was used in the synthesis of biotinylated AgNPs during the washing procedure. Mixing of all solutions was performed with a Mini Vortexer 945300 (VWR Scientific Products). Carving of the stencil was performed with an Epilog laser engraving system (Zing 16). Vacuum centrifugation was achieved with a Thermo Savant DNA120 SpeedVac Concentrator. Wax printing was obtained using a Xerox ColorQube 8570DN inkjet printer. The capture efficiency of fluorescein magnetic microbeads on the WE described below and in
oSlip Fabrication
The oSlip patterns (
Before depositing the carbon electrodes on the device, the carbon paste was thickened by adding a 1.0 cm thick layer of carbon paste on a glass vial and placing it in an oven at 65° C. for 30 min. Next, the paste was removed from the oven, mixed with a glass rod, and re-heated in the oven at 65° C. for 5 min. The re-heating step was performed a total of two times. The thickened paste was left to cool at 25° C. until used. In addition, the stencil was designed (
Modification of Citrate-Capped AgNPs with Biotin/DNA.
The citrate-capped AgNPs were biotinylated following a protocol by Alivisatos and co-workers (Sonnichsen et al. Nat. Biotech. 2005, 23, 741-745). Briefly, 10.0 μL of 100.0 μM biotin/DNA and 600.0 μL of 0.75 nM citrate-capped AgNPs were incubated at 25° C. while vortexing (level 3) for 24 h. Then, the solution's salt concentration was slowly increased to 70.0 mM NaCl and 7.0 mM phosphate buffer by adding one aliquot of a solution containing 2.5 μL of 5.0 M NaCl and 25.0 μL of 50.0 mM phosphate buffer (pH 7.0) every day for 4 days. Next, the solution's volume was slowly reduced to 250.0 μL at 40° C. using vacuum centrifugation for 3 hours. The resulting solution was centrifuged at 16,000 g for 20 min and the supernatant was removed. The silver nanoparticles were washed by re-suspending them in 600.0 μL of a solution containing 100.0 mM NaCl and 10.0 mM phosphate buffer (pH 7.0), centrifuging at 16,000 g for 20 min, and discarding the supernatant. This last washing procedure was repeated a total of three times. In order to confirm the biotinylation of the AgNPs, an aqueous solution of AlexaFluor-647/streptavidin conjugate (50.0 μg/mL final concentration) was incubated with the resulting AgNP solution at 25° C. for 30 minutes and then washed three times by re-suspending them in 600.0 μL of a solution containing 100.0 mM NaCl and 10.0 mM phosphate buffer (pH 7.0), centrifuging at 16,000 g for 20 min, and discarding the supernatant. At the same time, a control experiment was performed where 600.0 μL of 0.75 nM citrate-capped AgNPs were subjected to the same protocol described above but in the absence of biotin/DNA and finally incubated with AlexaFluor-647/streptavidin conjugate. Then, aliquots of each experiment (test and control) were placed in a microtiter plate and their fluorescence was read using a plate reader (λex=652 nm, λem=688 nm). The fluorescence recorded for the test experiment was 87% higher than that of the control experiment, confirming the biotinylation of the AgNPs.
Preparation of AgNP/Biotin/Streptavidin/Magnetic Microbeads Composite
100.0 μL of stock streptavidin-coated magnetic microbeads (1.11 pM, 2.8 μm in diameter) were placed in a microcentrifuge tube and a magnet was held close to the tube for 30 s, followed by the removal of the supernatant. The microbeads were washed three times with 50.0 μL of 10.0 mM phosphate buffer (pH 7.4, PB) by placing the magnet close to the tube for 30 s and removing the supernatant between washes. After the third wash the magnetic beads were re-suspended in 200.0 μL of the previously synthesized biotinylated AgNPs solution. The AgNPs and magnetic microbeads were incubated for 30 min at 25° C. while vortexing (level 3) and then washed three times with 100.0 μL of 10.0 mM PB containing 100.0 mM NaCl by placing the magnet close to the tube for 30 s and removing the supernatant between washes. The composite formation (AgNP/biotin/streptavidin/magnetic microbead) was confirmed using UV-Vis spectroscopy (
Modification of Streptavidin-Coated Magnetic Microbeads with Biotin/Fluorescein Conjugate
18.0 μL of stock streptavidin-coated magnetic microbeads were placed in a microcentrifuge tube and washed three times with 100.0 μL of 10.0 mM PB by placing a magnet close to the tube for 30 s. After the third wash, the microbeads were re-suspended in an aqueous solution containing 100.0 μL of 62.0 μM biotin/fluorescein conjugate and incubated at 25° C. under vortexing (level 3) for 30 min. Next, the resulting fluorescein/magnetic microbeads were washed three times with 100.0 μL of 10.0 mM PB by placing a magnet close to the tube for 30 s. After the last wash, the beads were re-suspended in 100.0 μL of 10.0 mM PB and stored at 4° C. in the dark until used.
Optimization of the Electrochemical Signal
The maximum charge obtained in both the oSlip and bulk solution-based experiments was optimized by changing the number of moles of KMnO4 added (while keeping the same number of moles of Ag) and then measuring the resulting charge. For the oSlip experiment, different amounts of KMnO4 moles were dried by nitrogen flow on the square reservoir of Layer 3 (
MnO4−+2H2O+3eMnO2+4OH−
Therefore, the higher the KMnO4 concentration used, the more MnO2 is produced and the more the electrode is electronically isolated.
Calibration Curve in Bulk Solution and Characterization of the Composite's AgNP Content
Before implementing the proposed electrochemical method (using KMnO4) on paper, we first studied it in bulk solution. In this case, a glassy carbon working electrode (GCE, 1.0 mm in diameter), Ag/AgCl reference electrode, and platinum wire counter electrode were used in the Facing Up setup (
The concentration of AgNPs present in the composite was calculated by adding 125.0 μL of 100.0 mM PB containing 100.0 mM NaCl, 50.0 μL of 187.0 μM KMnO4, 3.0 μL of composite, and 47.0 μL of deionized water to the PTFE cell while in the Facing Up electrochemical setup (
Spontaneous Oxidation of AgNPs by KMnO4
Two experiments were performed in order to confirm the spontaneous oxidation of AgNPs in the presence of KMnO4. In the first one (the test), 625.0 μL of 100.0 mM PB containing 100.0 mM NaCl and 250.0 μL of 75.0 pM citrate-capped AgNPs were placed in a microcentrifuge tube. Next, 250.0 μL of 187.0 μM KMnO4 was added and, after 30 s, the tube was placed in the nanoparticle tracking instrument (NS500, Nanosight) for analysis. In the second experiment (the control), 625.0 μL of 100.0 mM PB containing 100.0 mM NaCl, 250.0 μL of 75.0 pM citrate-capped AgNPs, and 250.0 μL of deionized water were placed in a microcentrifuge tube and analyzed. For each experiment, the solution was evaluated a total of three times. Citrate-capped AgNP diameters of 13±3 and 21±1 nm were obtained for the experiments with and without KMnO4, respectively (one example of each experiment is provided in
Optimization of KMnO4 Resolvation Time in the oSlip
The waiting time between slipping Layer 3 into position 2 and the initiation of the electrodeposition step was optimized in order to maximize the amount of KMnO4 that reaches the electrode surface for the oxidation of the AgNPs. This was done by drying 4.0 μL of 934.0 μM KMnO4 on the engageable platform of Layer 3 (see
Effect of NaCl in the Electrochemical Signal
The effect of NaCl on the electrochemical signal was demonstrated by performing two bulk solution-based experiments. In the first experiment, 50.0 μL of 75.0 pM AgNP, 125.0 μL of 100.0 mM PB containing 100.0 mM NaCl, and 50.0 μL of 187.0 μM KMnO4 were added to the PTFE cell in the facing up setup (Ag E°=0.7991 V vs NHE; AgCl+e
Ag++Cl−E°=0.2223 V vs NHE), the charges measured are not statistically different. This result can be attributed to the presence of KMnO4 keeping Ag+ in solution even in the presence of high Cl− concentrations that should precipitate Ag+ as AgCl. The experimental setups (facing up vs. facing down,
Results and Discussion
Herein, a robust and easily fabricated (no electrode modifications) paper-based platform is presented that offers a simple user-device interface (ready-to-use type of device). The detection method consists of the signal amplification of AgNP labels via their spontaneous oxidation by KMnO4 and the subsequent electrodeposition of Ag+ onto the device's working electrode (WE). This deposited Ag can be later stripped off to obtain an anodic current transient that is directly proportional to the concentration of AgNP labels present. KMnO4 is a well-known oxidizing agent (E°=1.70 V vs. NHE at neutral pH) used in many important organic and inorganic redox reactions and water disinfection. KMnO4 was selected, among other strong oxidizing agents, due to its stability under the conditions needed for this invention.
The proposed paper platform, called oSlip, is illustrated in
Prior to the initiation of the experiment, biotin-modified citrate-capped AgNP (20.0 nm in diameter) and commercially available streptavidin-coated magnetic microbeads (2.8 μm in diameter) are incubated to obtain a composite of the form AgNP/biotin/streptavidin/magnetic microbead. The AgNPs concentration present in the stock composite solution was calculated to be 533.4 pM from a bulk solution-based calibration curve of charge as a function of citrate-capped AgNP concentration (
Once the reagents are loaded to their respective reservoirs and the device is assembled and placed in the holder, 50.0 μL of 100.0 mM phosphate buffer (PB) at pH 7.4 containing 100.0 mM NaCl and different concentrations of composite were introduced to the fluid inlet (Inlet) while Layer 3 is in position 1 (
Two experiments were performed to calculate the capture efficiency of the composite at the WE. In the first experiment (Control), 2.0 μL of 10.0 mM PB containing 0.2 pM fluorescein-labeled magnetic microbeads (2.8 μm in diameter) were placed on the WE of an unassembled device and left to dry at room temperature. Once the solution was dry, its fluorescence was measured. In the second experiment (Test), 50.0 μL of 100.0 mM PB containing 100.0 mM NaCl and the same amount of fluorescein-magnetic microbead moles as in the Control experiment were added to the Inlet of an assembled device. After flow stopped (indicated by the fluid outlet turning blue), the device was left at 25° C. for 2 h, until the sample was dry. At this point, the device was opened and the fluorescence at the WE was measured. The fluorescence intensity observed at the Control WE (
Once flow through the device is halted, the blue color at the fluid inlet indicates that Layer 3 needs to be pulled into position 2 (Ag,
The conditions under which the proof-of-concept experiment was carried out (neutral pH and salt concentration of 100.0 mM) mirror those present in human urine, which is the potential sample matrix for a wide variety of bioassays. The Ksp of Ag+ in the presence of 100.0 mM NaCl is 1.8×10−9 M; however, no experimental evidence was found showing the precipitation of Ag+ as AgCl (see
In conclusion, a paper platform was developed that is ideal for POC applications because it is cheap (˜$1.22/device), user friendly (electrochemical detection and no washing steps), sensitive, portable, robust (AgNPs instead of enzymes), efficient (composite capture efficiency of 36±10% and charge collection efficiency of 13±2%), fast (completion of analysis in 4.6 min), and can detect low concentrations (767 fM). In addition, the proposed platform facilitates the timed introduction of reagents and it permits the integration of all the steps necessary for the automatic production of the signal, with the only requirements from the user being the injection of the sample and the slipping of a paper layer to activate the sensor. The proposed platform can be used to detect a myriad of analytes without having to change the source of the signal or the signal amplification method because the AgNPs and magnetic microbeads can be modified with various binding agents.
Other advantages which are obvious and which are inherent to the invention will be evident to one skilled in the art. It will be understood that certain features and sub-combinations are of utility and may be employed without reference to other features and sub-combinations. This is contemplated by and is within the scope of the claims. Since many possible embodiments may be made of the invention without departing from the scope thereof, it is to be understood that all matter herein set forth or shown in the accompanying drawings is to be interpreted as illustrative and not in a limiting sense.
This invention was made with government support under Contracts No. HDTRA-1-10-1-0031 and HDTRA-1-11-1-0005 awarded by the Department of Defense/Defense Threat Reduction Agency (DTRA). The government has certain rights in this invention.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US14/71389 | 12/19/2014 | WO | 00 |
| Number | Date | Country | |
|---|---|---|---|
| 61923713 | Jan 2014 | US |
