Patent Application
20220317082
The present application relates to biosensors, and in particular to biosensors and methods for detecting analytes.
This application contains a Sequence Listing in computer readable form. The computer readable form is incorporated herein by reference.
Given the rapid emergence of various infectious disease pandemics, point of care (POC) devices have gained significant interest due to their applicability in clinical decision making for rapid, simple, and early screening, diagnosis, and treatment monitoring, for example, for early diagnosis and monitoring in resource limiting settings.
In addition, antibiotic consumption rates are at a record high, contributing significantly to the increased emergence of antibiotic resistance. Advanced diagnostic technologies for identifying infection-causing pathogens enable antibiotics to be prescribed more effectively. Such systems can decrease the sample-to-result time of infectious disease testing, allowing clinicians to intervene and practice more precision medicine. Although molecular diagnostic tests are improving in certain areas, such diagnostic technologies may be costly and more operationally complex to implement at primary health care facilities, particularly in the context of replacing the large volume of cultures performed in the day-to-day diagnosis of common infections such as urinary tract infections (UTIs).
The background herein is included solely to explain the context of the application. This is not to be taken as an admission that any of the material referred to was published, known, or part of the common general knowledge as of the priority date.
In accordance with an aspect, there is provided a biosensor for detecting an analyte comprising: a) a first and second working electrode; b) a detection probe functionalized on the first working electrode, the detection probe comprising a reporter moiety and a recognition moiety for an analyte; c) a capture probe functionalized on the second working electrode; and d) a counter electrode; wherein each working electrode is configured to provide a change in signal if the analyte is present.
With respect to the biosensor, there is provided: in another aspect, wherein the reporter moiety is coupled to the recognition moiety. In another aspect, wherein the capture probe is for recognizing and coupling to the reporter moiety. In another aspect, whereby coupling of the analyte to the detection probe results in delocalization of the reporter moiety from the first working electrode to the second working electrode. In another aspect, whereby the reporter moiety is for delocalizing from the first working electrode to the second working electrode in the presence of the analyte. In another aspect, wherein the first and second working electrodes each, independently, comprise conductive materials, semi-conductive materials, or combinations thereof. In another aspect, wherein the first and second working electrodes each, independently, comprise metals, metal alloys, metal oxides, superconductors, semi-conductors, carbon-based materials, conductive polymers, or combinations thereof. In another aspect, wherein the first and second working electrodes each, independently, comprise metals, metal alloys, carbon-based materials, or combinations thereof. In another aspect, wherein the first and second working electrodes each, independently, comprise metals. In another aspect, the first and second working electrodes are each, independently, any shape, geometry and/or pattern. In another aspect, wherein the first and second working electrodes are each, independently, squares, rectangles, parallelograms, rhombuses, stars, spiral, serpentine, circles, triangles, and/or jigsaw-shaped. In another aspect, wherein each of the working electrodes have the same shape, geometry, and/or pattern. In another aspect, wherein the working electrodes are selected from interdigitated, intermeshed, interleaved and/or intertwined. In another aspect, wherein the working electrodes are intertwined. In another aspect, wherein the first and second working electrodes are selected from concentric spiral electrodes, serpentine line electrodes, brush electrodes, intertwined star electrodes, and/or multiple intertwined electrodes. In another aspect, wherein the first working electrode and the second working electrode are in proximity to each other such that the reporter moiety is capable of transporting from the detection probe to the capture probe. In another aspect, wherein the working electrodes are adjacent to one another. In another aspect, wherein there is a gap between at least a portion of the first working electrode and at least a portion of the second working electrode, wherein the reporter moiety is capable of transporting from the detection probe to the capture probe. In another aspect, wherein the gap is from about 10 μm to about 2 mm, about 10 μm to about 1 mm, about 10 μm to about 500 μm, about 10 μm to about 400 μm, about 10 μm to about 300 μm, about 10 μm to about 200 μm, or about 10 μm to about 100 μm. In another aspect, wherein at least one of the first and second working electrodes is a nanostructured electrode. In another aspect, wherein at least one of the first and second working electrodes has edges. In another aspect, wherein at least one of the first and second working electrodes has sharp edges and/or points. In another aspect, wherein at least one of the first and second working electrodes has a high-aspect ratio. In another aspect, wherein the high-aspect ratio is from about 2 to about 10, about 3 to about 10, about 4 to about 10, about 5 to about 10, about 6 to about 10, about 7 to about 10, about 8 to about 10, about 2 to about 9, about 3 to about 9, about 4 to about 9, about 5 to about 9, about 6 to about 9, about 7 to about 9, about 8 to about 9, about 2 to about 8, about 3 to about 8, about 4 to about 8, about 5 to about 8, about 6 to about 8, about 7 to about 8, about 2 to about 7, about 3 to about 7, about 4 to about 7, about 5 to about 7, or about 6 to about 7. In another aspect, wherein surface areas of at least one of the first and second working electrodes is from about 1.0 cm2 to 5.0 cm2, about 1.5 cm2 to 5.0 cm2, about 1.5 cm2 to 4.5 cm2, about 1.5 cm2 to 4.0 cm2, about 1.5 cm2 to 3.9 cm2, about 1.7 cm2 to 3.8 cm2, or about 1.7 cm2 to 3.7 cm2, about 1.7 cm2 to 3.6 cm2, about 1.7 cm2 to 3.5 cm2, about 1.7 cm2 to 3.4 cm2, about 1.7 cm2 to 3.3 cm2, or about 1.7 cm2 to 3.2 cm2. In another aspect, wherein a limit-of-detection of the biosensor is about 1 to about 3 orders of magnitude higher compared to planar counterparts In another aspect, wherein the change in signal for the first working electrode comprises a change in current, potential or impedance. In another aspect, wherein the change in signal for the second working electrode comprises a change in current, potential or impedance. In another aspect, wherein the change in signal is a change in current. In another aspect, wherein the first working electrode is configured to provide a decrease in the signal and the second working electrode is configured to provide an increase in the signal in the presence of the analyte. In another aspect, wherein the decrease in the signal is about 0.1 to about 0.6 fold decrease and the increase in the signal is about 50 to about 200 fold increase. In another aspect, wherein the first working electrode is configured to provide an increase in the signal and the second working electrode is configured to provide a decrease in the signal in the presence of the analyte. In another aspect, wherein the increase in the signal is about 50 to about 200 fold increase and the decrease in the signal is about 0.1 to about 0.6 fold decrease. In another aspect, wherein the reporter moiety comprises a moiety with a redox, photoelectrochemical, semi-conductive and/or conductive species. In another aspect, wherein the reporter moiety comprises a redox species. In another aspect, wherein the redox species is selected from methylene blue, methylene blue succinymide, methylene blue maleimide, Atto MB2 maleimide (Sigma Aldrich) and other methylene blue derivatives; 3,7-Bis-[(2-Ammoniumethyl) (methyl)amino]phenothiazin-5-ium trifluoroacetate; 3,7-Bis-(piperazin-4-ium-1-yl)phenothiazin-5-ium trifluoroacetate; 3,7-Bis-[(2-ammoniumethyl)(methyl)amino]phenothiazin-5-ium chloride; and 3,7-Bis-(piperazin-4-ium-1-yl)phenothiazin-5-ium chloride, and/or ferrocene. In another aspect, wherein the redox species is methylene blue and/or ferrocene. In another aspect, wherein the reporter moiety comprises a biopolymer modified with the redox species. In another aspect, wherein the reporter moiety comprises a moiety with a passivating species. In another aspect, wherein the reporter moiety is for transducing the presence of an analyte recognized by the recognition moiety to a detectable signal. In another aspect, wherein the reporter moiety comprises an antibody, an antigen, oligonucleotide, oligopeptide, and/or oligosaccharide. In another aspect, wherein the recognition moiety comprises a DNAzyme, aptamer, antibody, and/or nucleic acid that is able to recognize the presence of the analyte. In another aspect, wherein the recognition moiety is a DNAzyme that cleaves RNA in the presence of the analyte, an aptamer that changes conformation in presence of the analyte, or an antibody. In another aspect, wherein the recognition moiety is a DNAzyme that cleaves RNA in the presence of the analyte to release the reporter moiety from the first working electrode. In another aspect, wherein the capture probe comprises ssDNA, ssPNA, dsDNA, ssRNA, and/or dsRNA. In another aspect, wherein the capture probe comprises ssDNA. In another aspect, wherein the analyte is a toxin, biopolymer, cell, tissue, and/or pathogen. In another aspect, wherein the analyte is a microorganism and/or virus. In another aspect, wherein the microorganism is a gram-negative bacterium. In another aspect, wherein the microorganism is Escherichia coli, Salmonella typhimurium, Pseudomonas peli, Brevundimonas diminuta, Hafnia alvei, Yersinia ruckeri, Ochrobactrum grignonese, Achromobacter xylosoxidans, Moraxella osloensis, Acinetobacter lwoffi, and Serratia fonticola. In an embodiment, the microorganism is a gram-positive bacterium, for example Listeria monocytogenes, Bacillus subtilis, Clostridium difficile, Actinomyces orientalis, Pediococcus acidilactici, Leuconostoc mesenteroides, Lactobacillus planturum, or a combination thereof. In another aspect, wherein the microorganism is a pathogenic bacterium. In another aspect, the biosensor further comprising a reference electrode. In another aspect, wherein the counter electrode is both a counter electrode and a reference electrode.
In accordance with another aspect, there is provided a device comprising the biosensor described herein above.
With respect to the device, there is provided: in another aspect, wherein the device is used for clinical and agricultural diagnostics, agri-food quality control, environmental monitoring, health monitoring, and/or pharmaceutical development. In another aspect, wherein the device is used for screening. In another aspect, wherein the device is a hand-held device.
In accordance with another aspect, there is provided a kit for detection of an analyte comprising a biosensor as described herein above or device as described herein above and instructions for use.
In accordance with another aspect, there is provided a method of determining the presence of an analyte in a sample comprising: exposing the biosensor described herein above to the sample to delocalize the reporter moiety from the first working electrode to the second working electrode in the presence of the analyte, producing a change in signal at each of the first working electrode and the second working electrode.
With respect to the method, there is provided: in another aspect, the method further comprises measuring the signal at the first and second working electrodes. In another aspect, wherein the biosensor is exposed to the sample under conditions to delocalize the reporter moiety from the first working electrode to the second working electrode. In another aspect, wherein the change in signal from the first working electrode is a decrease in signal and the change in signal from the second working electrode is an increase in signal. In another aspect, wherein the change in signal from the first working electrode is an increase in signal and the change in signal from the second working electrode is a decrease in signal. In another aspect, wherein prior to exposing the biosensor to the sample, an initial signal is measured at each of the first and second working electrodes and after exposing the biosensor to the analyte, a final signal is measured at each of the first and second working electrodes, whereby differential change in signal between the initial signal and the final signal of the first working electrode and a change in signal between the initial signal and the final signal of the second working electrode, indicates the presence of the analyte in the sample. In another aspect, wherein the change in signal for the first working electrode and/or the second working electrode comprises a change in current, potential or impedance. In another aspect, wherein the change in signal for the first working electrode and/or the second working electrode is a change in current. In another aspect, wherein the analyte is a toxin, biopolymer, cell, tissue, and/or pathogen. In another aspect, wherein the analyte is a microorganism and/or virus. In another aspect, wherein the microorganism is a gram-negative bacterium. In another aspect, wherein the microorganism is Escherichia coli, Salmonella typhimurium, Pseudomonas peli, Brevundimonas diminuta, Hafnia alvei, Yersinia ruckeri, Ochrobactrum grignonese, Achromobacter xylosoxidans, Moraxella osloensis, Acinetobacter lwoffi, and Serratia fonticola. In an embodiment, the microorganism is a gram-positive bacterium, for example Listeria monocytogenes, Bacillus subtilis, Clostridium difficile, Actinomyces orientalis, Pediococcus acidilactici, Leuconostoc mesenteroides, Lactobacillus planturum, or a combination thereof. In another aspect, wherein the microorganism is a pathogenic bacterium. In another aspect, wherein the pathogen is E. coli. In another aspect, wherein exposing the biosensor to the sample comprises contacting the biosensor with the sample. In another aspect, wherein the sample is a urine or blood sample.
In accordance with another aspect, there is provided nanostructured working electrode for functionalizing with a detection probe comprising a reporter moiety and a recognition moiety for an analyte.
With respect to the electrode, there is provided: in another aspect, wherein the reporter moiety is coupled to the recognition moiety. In another aspect, whereby coupling of the analyte to the detection probe results in delocalization of the reporter moiety from the electrode. In another aspect, wherein the reporter moiety comprises a moiety with a redox, a passivating species, photoelectrochemical, semi-conductive and/or conductive species. In another aspect, wherein the moiety comprises an antibody, an antigen, oligonucleotide, oligopeptide, and/or oligosaccharide. In another aspect, wherein the recognition moiety comprises a DNAzyme, aptamer, antibody, and/or nucleic acid that is able to recognize the presence of the analyte. In another aspect, wherein the recognition moiety is a DNAzyme that cleaves RNA in the presence of the analyte, an aptamer that changes conformation in presence of the analyte, or an antibody. In another aspect, wherein the recognition moiety is a DNAzyme that cleaves RNA in the presence of the analyte to release the reporter moiety.
In accordance with another aspect, there is provided nanostructured working electrode for functionalizing with a capture probe.
With respect to the electrode, there is provided: in another aspect, wherein the capture probe is for recognizing and coupling to the reporter moiety. In another aspect, wherein the capture probe comprises ssDNA, ssPNA, dsDNA, ssRNA, and/or dsRNA. In another aspect, wherein the electrode comprises conductive materials, semi-conductive materials, or combinations thereof. In another aspect, wherein the electrode comprises metals, metal alloys, metal oxides, superconductors, semi-conductors, carbon-based materials, conductive polymers, or combinations thereof. In another aspect, wherein the electrode comprises metals, metal alloys, carbon-based materials, or combinations thereof. In another aspect, wherein the electrode comprise metals. In another aspect, wherein the electrode is any shape, geometry and/or pattern. In another aspect, wherein the electrode is a square, rectangle, parallelogram, rhombus, star, spiral, serpentine, circle, triangle, and/or jigsaw-shaped. In another aspect, wherein the electrode has edges. In another aspect, wherein the electrode has sharp edges and/or points. In another aspect, wherein the electrode has a high-aspect ratio. In another aspect, wherein the high-aspect ratio is from about 2 to about 10, about 3 to about 10, about 4 to about 10, about 5 to about 10, about 6 to about 10, about 7 to about 10, about 8 to about 10, about 2 to about 9, about 3 to about 9, about 4 to about 9, about 5 to about 9, about 6 to about 9, about 7 to about 9, about 8 to about 9, about 2 to about 8, about 3 to about 8, about 4 to about 8, about 5 to about 8, about 6 to about 8, about 7 to about 8, about 2 to about 7, about 3 to about 7, about 4 to about 7, about 5 to about 7, or about 6 to about 7. In another aspect, wherein surface area of the electrode is from about 1.0 cm2 to 5.0 cm2, about 1.5 cm2 to 5.0 cm2, about 1.5 cm2 to 4.5 cm2, about 1.5 cm2 to 4.0 cm2, about 1.5 cm2 to 3.9 cm2, about 1.7 cm2 to 3.8 cm2, or about 1.7 cm2 to 3.7 cm2, about 1.7 cm2 to 3.6 cm2, about 1.7 cm2 to 3.5 cm2, about 1.7 cm2 to 3.4 cm2, about 1.7 cm2 to 3.3 cm2, or about 1.7 cm2 to 3.2 cm2. In another aspect, wherein a limit-of-detection of the electrode is about 1 to about 3 orders of magnitude higher compared to a planar counterpart electrode.
In accordance with another aspect, there is provided a biosensor comprising the electrode as described herein above.
In accordance with another aspect, there is provided a device comprising the biosensor as described herein above.
In accordance with another aspect, there is provided a kit for detection of an analyte comprising a biosensor as described herein above or a device as described herein above and instructions for use.
In accordance with another aspect, there is provided the biosensor as described herein above, wherein the biosensor is an electrochemical chip.
In accordance with another aspect, there is provided the device as described herein above, wherein the biosensor is an electrochemical chip.
In accordance with another aspect, there is provided the kit as described herein above, wherein the biosensor is an electrochemical chip.
In accordance with another aspect, there is provided the method as described herein above, wherein the biosensor is an electrochemical chip.
In accordance with another aspect, there is provided use of the biosensor as described herein above to determine the presence of an analyte in a sample.
In accordance with another aspect, there is provided use of the device as described herein above to determine the presence of an analyte in a sample.
In accordance with another aspect, there is provided use of the kit as described herein above to determine the presence of an analyte in a sample.
In accordance with another aspect, there is provided a biosensor for detecting an analyte comprising: a) at least a first and second working electrode; b) a detection probe functionalized on the first working electrode that comprises a reporter moiety linked to a recognition moiety for an analyte; c) a capture probe functionalized on the second working electrode; d) a counter electrode; and e) a reference electrode, wherein current flows between each of the working electrodes and the counter electrode.
With respect to the biosensor, there is provided: in another aspect, wherein binding of the analyte to the detection probe results in delocalization of the reporter moiety from the first to second working electrode. In another aspect, wherein change in current, potential or impedance is measured from both the first and second working electrodes. In another aspect, wherein change in current is measured from both the first and second working electrodes. In another aspect, wherein the first and second working electrodes are in close proximity to one another. In another aspect, wherein the electrodes comprise nanostructures. In another aspect, wherein the nanostructures comprise high aspect ratio geometries. In another aspect, wherein the nanostructures comprise conductive or semiconductive materials selected from the group consisting of noble metals, alloys, carbon-based materials, metal oxides, or semiconductors. In another aspect, wherein the nanostructures comprise gold. In another aspect, wherein the nanostructures are deposited on the electrodes using electrodeposition, electroless deposition or printing. In another aspect, wherein the recognition moiety is a DNAzyme that cleaves RNA in presence of the analyte, an aptamer that changes conformation in presence of the analyte, or an antibody. In another aspect, wherein the recognition moiety is a DNAzyme that cleaves RNA in presence of the analyte to release the linked reporter moiety from the first working electrode. In another aspect, wherein the reporter moiety comprises a biopolymer modified with a redox, photoelectrochemical, semiconductive or conductive species. In another aspect, wherein the reporter moiety comprises a biopolymer modified with a redox species. In another aspect, wherein the biopolymer comprises an oligonucleotide, oligopeptide or oligosaccharide. In another aspect, wherein the redox species is methylene blue or ferrocene. In another aspect, wherein the redox species is methylene blue. In another aspect, wherein the analyte is a pathogen. In another aspect, wherein the pathogen is E. coli.
In accordance with another aspect, there is provided a kit for detection of an analyte comprising a biosensor described herein above.
In accordance with another aspect, there is provided a method of determining the presence of an analyte in a test sample comprising: a) exposing the biosensor of any one of claims 1 to 20 to the sample under conditions to delocalize the reporter moiety from the first to second working electrode in presence of the analyte; b) measuring current from both the first and second working electrode, wherein a reduction in current from the first electrode and an increase in current from the second electrode indicates the presence of the analyte in the sample.
With respect to the method, there is provided: in another aspect, wherein the analyte is a pathogen. In another aspect, wherein the pathogen is E. coli.
Other features and advantages of the present application will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating embodiments of the application, are given by way of illustration only and the scope of the claims should not be limited by these embodiments, but should be given the broadest interpretation consistent with the description as a whole.
Certain embodiments of the application will now be described in greater detail with reference to the drawings in which:
Unless otherwise indicated, the definitions and embodiments described in this and other sections are intended to be applicable to all embodiments and aspects of the present application herein described for which they are suitable as would be understood by a person skilled in the art. It is also to be understood that the terminology used herein is for the purpose of describing particular aspects only and is not intended to be limiting.
The term “sample” or “test sample” as used herein may refer to any material in which the presence or amount of a target analyte is unknown and can be determined in an assay. The sample may be from any source, for example, any biological (e.g. human or animal samples, including clinical samples), environmental (e.g. water, soil or air) or natural (e.g. plants) source, or from any manufactured or synthetic source (e.g. food or drinks). The sample may be comprised or is suspected of comprising one or more analytes. The sample may be a “biological sample” comprising cellular and non-cellular material, including, but not limited to, tissue samples, urine, blood, serum, other bodily fluids. In an embodiment, the sample comprises urine.
The term “target”, “analyte” or “target analyte” as used herein may refer to any agent, including, but not limited to, a small inorganic molecule, small organic molecule, metal ion, biomolecule, toxin, biopolymer (such as a nucleic acid, carbohydrate, lipid, peptide, protein), cell, tissue, microorganism and virus, for which one would like to sense or detect. In an embodiment, the analyte is either isolated from a natural source or is synthetic. The analyte may be a single compound or a class of compounds, such as a class of compounds that share structural or functional features. The term analyte also includes combinations (e.g. mixtures) of compounds or agents such as, but not limited, to combinatorial libraries and samples from an organism or a natural environment. In an embodiment, the analyte comprises a microorganism target.
The term a “microorganism target” as used herein may be a molecule, compound or substance that is present in or on a microorganism or is generated, excreted, secreted or metabolized by a microorganism. In an embodiment, a microorganism target is present in the extracellular matrix of a microorganism. In another embodiment, a microorganism target is present in the intracellular matrix of a microorganism. In another embodiment, a microorganism target comprises a protein, a nucleic acid, a small molecule, extracellular matrix, intracellular matrix, a cell of the microorganism, or any combination thereof. In an embodiment, a microorganism target is a crude or purified extracellular matrix or a crude or purified intracellular matrix. In another embodiment, a microorganism target is specific to a particular species or strain of microorganism.
The term “microorganism” as used herein may refer to a microscopic organism that comprises either a single cell or a cluster of single cells including, but not limited to, bacteria, fungi, archaea, protists, algae, plankton and planarian. In an embodiment, the microorganism is a gram-negative bacterium, for example Escherichia coli, Salmonella typhimurium, Pseudomonas peli, Brevundimonas diminuta, Hafnia alvei, Yersinia ruckeri, Ochrobactrum grignonese, Achromobacter xylosoxidans, Moraxella osloensis, Acinetobacter lwoffi, and Serratia fonticola. In an embodiment, the microorganism is a gram-positive bacterium, for example Listeria monocytogenes, Bacillus subtilis, Clostridium difficile, Actinomyces orientalis, Pediococcus acidilactici, Leuconostoc mesenteroides, and Lactobacillus planturum. In another embodiment, the microorganism is a pathogenic bacterium (for example, a bacterium that causes bacterial infection), such as Escherichia coli O157:H7, Listeria monocytogenes, Salmonella typhimurium or Clostridium difficile.
The term “detection probe” as used herein may refer to a probe that comprises a recognition moiety and a reporter moiety. In an embodiment, the reporter moiety is coupled to a recognition moiety. “Coupled” is used herein to include, for example, covalent binding interactions and/or noncovalent binding interactions (e.g. ionic bonds, Van Der Waals forces, hydrogen bonds, etc.). In an embodiment, the reporter moiety hybridizes to the recognition moiety. In another embodiment, the recognition moiety and the reporter moiety comprise a single molecule.
The term “recognition moiety” as used herein may refer to a moiety comprising a molecule (e.g. compound) such as, but not limited to, a DNAzyme, aptamer, antibody, and/or nucleic acid that is able to recognize the presence of an analyte (e.g. bind to the analyte). In an embodiment, the recognition moiety comprises a DNAzyme.
The term “reporter moiety” as used herein may refer to a moiety comprising a molecule (e.g. compound) for reporting the presence of an analyte. For example, the moiety is used for transducing the presence of an analyte recognized by the recognition moiety to a detectable signal. In an embodiment, the reporter moiety comprises a molecule modified with a redox, photoelectrochemical, passivating, semi-conductive and/or conductive species. In embodiments, the redox, photoelectrochemical, semi-conductive and/or conductive species decreases a signal on a first working electrode and increases a signal on the second working electrode in the presence of an analyte. In other embodiments, the passivating species increases a signal on a first working electrode and decreases a signal on the second working electrode in the presence of an analyte. In a specific embodiment, the reporter moiety comprises a biopolymer modified with a redox, photoelectrochemical, passivating, semi-conductive, and/or conductive species. In an embodiment, the reporter moiety comprises a nucleic acid sequence modified with a redox, photoelectrochemical, passivating, semi-conductive, and/or conductive species. In an embodiment, the reporter moiety comprises a specific DNA barcode modified with a redox, photoelectrochemical, passivating, semi-conductive, and/or conductive species. Examples of redox species include, for example, methylene blue, methylene blue succinymide, methylene blue maleimide, Atto MB2 maleimide (Sigma Aldrich) and other methylene blue derivatives; 3,7-Bis-[(2-Ammoniumethyl) (methyl)amino]phenothiazin-5-ium trifluoroacetate; 3,7-Bis-(piperazin-4-ium-1-yl)phenothiazin-5-ium trifluoroacetate; 3,7-Bis-[(2-ammoniumethyl)(methyl)amino]phenothiazin-5-ium chloride; and 3,7-Bis-(piperazin-4-ium-1-yl)phenothiazin-5-ium chloride, and/or ferrocene, etc. Examples of photoelectrochemical, semi-conductive and/or conductive species include, for example, photo-active dyes, quantum dots, surface plasmon resonance (SPR) nanoparticles, semi-quantum dots, metal oxides, polymer nanoparticles, and/or metal nanoparticles.
Examples of passivating species include insulating compounds/molecules such as insulating polymers and/or insulating metal oxides. These may include particles and beads such as silica beads and/or polystyrene beads.
The term “nucleic acid” as used herein may refer to a polynucleotide, such as deoxyribonucleic acid (DNA), ribonucleic acid (RNA), modified nucleotides and/or nucleotide derivatives, and may be either double stranded (ds) or single stranded (ss). In some embodiments, modified nucleotides may contain one or more modified bases (e.g. tritiated bases and unusual bases such as inosine), modified backbones (e.g. peptide nucleic acid, PNA) and/or other chemically, enzymatically, or metabolically modified forms.
The term “antibody” as used herein may refer to a glycoprotein, or antigen-binding fragments thereof, that has specific binding affinity for an antigen as the target analyte. Antibodies may be monoclonal and/or polyclonal antibodies.
The term “aptamer” as used herein may refer to a short, chemically synthesized nucleic acid molecule or oligonucleotide sequence which can be generated by in vitro selection to fold into specific three-dimensional (3D) structures that bind to a specific analyte with dissociation constants, for example, in the pico- to nano-molar range. Aptamers may be single-stranded DNA, and may include RNA, modified nucleotides and/or nucleotide derivatives. Aptamers may also be naturally occurring RNA aptamers termed “riboswitches”.
The term “DNAzyme” as used herein may refer to a nucleic acid molecule or oligonucleotide sequence for specifically recognizing and binding to an analyte. For example, it can catalyze or initiate a reaction in response to specifically recognizing to a target analyte. The DNAzyme may recognize a target analyte and release a reporter moiety upon interaction of the DNAzyme with the target. DNAzymes may be single-stranded DNA, and may include RNA, modified nucleotides and/or nucleotide derivatives. In an embodiment, the DNAzyme catalyzes the cleavage of a particular substrate, for example a nucleic acid sequence comprising one or more ribonucleotides. In an embodiment, the DNAzyme cleaves a single ribonucleotide (RNA) linkage in a nucleic acid sequence wherein the remaining nucleotides are deoxyribonucleotides (DNA). In an embodiment, the DNAzyme cleaves a nucleic acid sequence at a single ribonucleotide linkage thereby producing a nucleic acid cleavage fragment. In an embodiment, the DNAzyme recognizes a microorganism target and cleaves a nucleic acid sequence at a single ribonucleotide linkage upon interaction of the DNAzyme with the target thereby releasing the reporter moiety as the cleavage fragment.
The term “capture probe” as used herein may refer to a probe that recognizes and binds to a reporter moiety. In an embodiment, the capture probe comprises a molecule that recognizes and binds (e.g. hybridizes) to a reporter moiety comprising a molecule modified with a redox, photoelectrochemical, passivating, semi-conductive and/or conductive species. In an embodiment, the capture probe comprises a biopolymer. In an embodiment, the capture probe comprises a nucleic acid sequence that hybridizes to the reporter moiety. Examples include ssDNA, ssPNA, dsDNA, ssRNA, and/or dsRNA.
The term “hybridizes” or “hybridization” as used herein refers to the sequence specific non-covalent binding interaction with a complementary nucleic acid sequence.
The term “functionalizing” or “functionalizing on” as used herein may refer to various common approaches for functionalizing a material, which can be classified as mechanical, physical, chemical and biological. Any suitable form of coupling may be utilized (e.g. coating, binding, etc.).
The term “edge” as used herein may be understood to be an area where two plane surfaces meet. The angle between the two plane surfaces may be any suitable range. In particular, in an embodiment, the range is between about 70° and 120°. The area where the two plane surfaces meet may be blunt, in that the intersection between the two planes is not precise or well defined and the transition from one plane to the other may be gradual. A “sharp edge” used herein may be understood to be an edge that has not been rounded. For example, the area where the two plane surfaces meet, in the intersection between the two planes is precise or well defined.
In understanding the scope of the present application, the term “comprising” and its derivatives, as used herein, are intended to be open ended terms that specify the presence of the stated features, elements, components, groups, integers, and/or steps, but do not exclude the presence of other unstated features, elements, components, groups, integers and/or steps. The foregoing also applies to words having similar meanings such as the terms, “including”, “having” and their derivatives. The term “consisting” and its derivatives, as used herein, are intended to be closed terms that specify the presence of the stated features, elements, components, groups, integers, and/or steps, but exclude the presence of other unstated features, elements, components, groups, integers and/or steps. The term “consisting essentially of”, as used herein, is intended to specify the presence of the stated features, elements, components, groups, integers, and/or steps as well as those that do not materially affect the basic and novel characteristic(s) of features, elements, components, groups, integers, and/or steps.
Terms of degree such as “substantially”, “about” and “approximately” as used herein mean a reasonable amount of deviation of the modified term such that the end result is not significantly changed. These terms of degree should be construed as including a deviation of at least 5% of the modified term if this deviation would not negate the meaning of the word it modifies.
As used in this application, the singular forms “a”, “an” and “the” include plural references unless the content clearly dictates otherwise.
In embodiments comprising an “additional” or “second” component, the second component as used herein is chemically different from the other components or first component. A “third” component is different from the other, first, and second components, and further enumerated or “additional” components are similarly different.
Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of this disclosure, suitable methods and materials are described below.
The abbreviation, “e.g.” is derived from the Latin exempli gratia and is used herein to indicate a non-limiting example. Thus, the abbreviation “e.g.” is synonymous with the term “for example.” The word “or” is intended to include “and” unless the context clearly indicates otherwise.
The term “and/or” as used herein means that the listed items are present, or used, individually or in combination. In effect, this term means that “at least one of” or “one or more” of the listed items is used or present.
It will be understood that any component defined herein as being included may be explicitly excluded from the claimed invention by way of proviso or negative limitation.
In addition, all ranges given herein include the end of the ranges and also any intermediate range points, whether explicitly stated or not.
Biosensors can bring together signal transduction and biorecognition elements to analyze biologically-relevant targets. These devices can have a wide range of applications in clinical and agricultural diagnostics, agri-food quality control, environmental monitoring, health monitoring, and pharmaceutical development. Furthermore, these devices can be used in screening. There are several transduction mechanisms that can be used for biosensing (e.g. generating a single signal for each target capture). Using a single signal response in a complex analyte sample may be inefficient due to potential degradation of the biorecognition elements, as well as the interference of background biological materials with the biorecognition and transducer elements, leading to false positive and/or negative results.
In an embodiment, a biosensor is provided for detecting an analyte. The biosensor comprises a first and second working electrode, a detection probe functionalized on the first working electrode, a capture probe functionalized on the second working electrode, and a counter electrode. The detection probe functionalized on the first working electrode comprises a reporter moiety linked to a recognition moiety for an analyte. Each working electrode is configured to provide a change in signal if the analyte is present. In a specific embodiment, each of the working electrodes and the counter electrode are configured for current to flow therebetween. In embodiments, the biosensors described herein can allow bacterial analysis to be performed with minimal user intervention in a single incubation step and without, for example, the use of external reagents or labels. In embodiments, the biosensor further comprises a reference electrode. In certain embodiments, the counter electrode acts as both the counter and reference electrode.
In embodiments, the biosensors described herein can be used for label-free, and reagent-free detection of analytes. For example, additional labels and/or reagents do not need to be added after the sample is exposed (e.g. introduced, contacted, etc.) to the biosensor. In other embodiments, the biosensors can be used for rapid detection of analytes, including rapid, label-free, and reagent-free detection of analytes.
In embodiments of the biosensor, the biosensor has a first working electrode functionalized with biorecognition elements, such as DNAzymes, for the detection of target analytes by generating dual signal responses from a single redox species and another working electrode functionalized with a capture probe for receiving the reporter moiety with the single redox species. The electrodes can allow signals to be gathered from two channels (e.g. signal-on and signal-off channels) to increase the sensitivity, specificity and reliability of the biosensor. The nature of the electrodes can also facilitate the use of a single redox species as part of the reporter moiety to generate a dual response by its release from one electrode and spatial movement to another electrode, where it can be captured. The signal changes of the two electrodes are correlated: analyte capture causes a signal decrease in the first electrode and a signal increase in the second electrode. In embodiments, the geometries of the working electrodes are such that the diffusion length of the reporter moiety comprising the redox species between electrodes can be minimized, and the response time shortened. The multi-electrode biosensor may reduce the signal variation between the electrodes using common on-chip reference and counter electrodes.
In an embodiment, binding of the analyte to the detection probe results in delocalization of the reporter moiety from the first to second working electrode and the signal (e.g. current, potential, impedance, etc.) is measured from both the first and second working electrodes. In a further embodiment, the reporter moiety comprises a biopolymer modified with a redox, photoelectrochemical, passivating, semi-conductive and/or conductive species. In an embodiment, the recognition moiety comprises a DNAzyme that cleaves RNA in the presence of the analyte, an aptamer that changes conformation in the presence of the analyte, or an antibody. In a further embodiment, the first working electrode is functionalized with DNAzymes ligated to redox-tagged DNA oligomers and the second working electrode is functionalized with single-stranded DNA (ssDNA) capture probes. When the chip is exposed to the target analyte, the DNAzyme on the first working electrode cleaves at a ribonucleotide site in its sequence to release its redox-DNA reporter segment. This reporter then diffuses towards the second working electrode and hybridizes with the capture probe immobilized on this electrode.
The dual signal approaches can reduce the possibility of false results by providing a secondary signal to validate a primary signal. The dual signal approach is used in fluorescence (Wernette D P, Mead C, Bohn P W, Lu Y. Surface immobilization of catalytic beacons based on ratiometric fluorescent DNAzyme sensors: A systematic study. Langmuir. 2007; 23(18): 9513-9521; Wang J, Wang X, Tang H, et al. A ratiometric magnesium sensor using DNAzyme-templated CdTe quantum dots and Cy5. Sensors Actuators, B Chem. 2018; 272 (October 2017): 146-150; Ling Y, Zhou J, Zhang X F, Wang X H, Li N B, Luo H Q. A ratiometric fluorescent sensor for sensitive detection of UDG using poly(thymine)-templated copper nanoclusters and DAPI with exonuclease III assisted amplification. Sensors Actuators, B Chem. 2019; 286 (January): 46-51; Huang P J J, Vazin M, Liu J. In vitro selection of a new lanthanide-dependent DNAzyme for ratiometric sensing lanthanides. Anal Chem. 2014; 86(19):9993-9999) based sensing, electrochemiluminescence (Wang Y, Shan D, Wu G, et al. A novel “dual-potential” ratiometric electrochemiluminescence DNA sensor based on enhancing and quenching effect by G-quadruplex/hemin and Au-Luminol bifunctional nanoparticles. Biosens Bioelectron. 2018; 106 (January): 64-70; Cheng Y, Huang Y, Lei J, Zhang L, Ju H. Design and biosensing of Mg2+-dependent DNAzyme-triggered ratiometric electrochemiluminescence. Anal Chem. 2014; 86(10):5158-5163) based sensing, and electrochemical (Li X, Dou B, Yuan R, Xiang Y. Mismatched catalytic hairpin assembly and ratiometric strategy for highly sensitive electrochemical detection of microRNA from tumor cells. Sensors Actuators, B Chem. 2019; 286 (January): 191-197; Gao F, Du L, Zhang Y, Tang D, Du Y. Molecular beacon mediated circular strand displacement strategy for constructing a ratiometric electrochemical deoxyribonucleic acid sensor. Anal Chim Acta. 2015; 883:67-73) sensing. The dual-signaling approach can be applied in electrochemical biosensing using two different redox moieties (Gao F, Du L, Zhang Y, Tang D, Du Y. Molecular beacon mediated circular strand displacement strategy for constructing a ratiometric electrochemical deoxyribonucleic acid sensor. Anal Chim Acta. 2015; 883:67-73) in two modes i.e. “signal on-off” where the two electrochemical processes respond in an opposite way in the presence of the target (Shakir I, Sarfraz M. Evaluation of Electrochemical Charge Storage Mechanism and Structural Changes in Intertwined MoO3-MWCNTs Composites for Supercapacitor Applications. Electrochim Acta. 2014; 147:380-384), and “signal on/off” where the one signal reports on the presence of the target and the other signal reports on the interference present in the sample (Cheng Y, Huang Y, Lei J, Zhang L, Ju H. Design and biosensing of Mg2+-dependent DNAzyme-triggered ratiometric electrochemiluminescence. Anal Chem. 2014; 86(10):5158-5163). However, as it may be difficult to find multiple redox species that are stable under aqueous conditions, a single stable redox species can be used in the embodiments described herein, signal-off and signal-on information can be transmitted as one differential signal by adding or subtracting the two complementary signals to reduce the background and enhance the signal-to-noise ratio.
Any suitable electrodes and multiple electrode designs may be used in the biosensors described herein, in particular, in the differential signaling approach. Two or more working electrodes may be used. The electrodes may be any suitable shape, geometry or pattern such as, and without being limited thereto, squares, rectangles, parallelograms, rhombuses, stars, spiral, serpentine, circles, triangles, jigsaw-shaped, etc. In addition, the electrodes may be interdigitated, intermeshed, interleaved or intertwined geometries or patterns such as fingers, dendritic or sawtooth electrode patterns, or indeed any of a number of possibilities that will now become apparent to those skilled in the art, including irregular patterns. Further examples include concentric spiral and serpentine line electrodes, brush electrodes, and intertwined star electrodes and multiple intertwined electrodes such as, and without being limited thereto, multi-star electrodes (
In embodiments, the electrodes are nanostructures. In other embodiments, the electrodes have high-aspect ratios. The high-aspect ratio may be any suitable ratio. Examples of the high-aspect ratio include a vertical-to-lateral ratio ranging from about 2 to about 10, about 3 to about 10, about 4 to about 10, about 5 to about 10, about 6 to about 10, about 7 to about 10, about 8 to about 10, about 2 to about 9, about 3 to about 9, about 4 to about 9, about 5 to about 9, about 6 to about 9, about 7 to about 9, about 8 to about 9, about 2 to about 8, about 3 to about 8, about 4 to about 8, about 5 to about 8, about 6 to about 8, about 7 to about 8, about 2 to about 7, about 3 to about 7, about 4 to about 7, about 5 to about 7, or about 6 to about 7. In embodiments, the electrodes have multiple edges and/or points (see e.g.
In embodiments, the nanostructuring of the electrodes enhanced their limit-of-detection in detecting electroactive oligonucleotides by about 1 to about 3 orders of magnitude compared to their planar counterparts.
Specifically, with respect to intertwined electrodes, intertwining may be used to enhance the proximity of the two or more electrodes and maximize interaction over a smaller area. (Shakir I, Sarfraz M. Evaluation of Electrochemical Charge Storage Mechanism and Structural Changes in Intertwined MoO3-MWCNTs Composites for Supercapacitor Applications. Electrochim. Acta. 2014; 147:380-384; Hou Z Q, Wang Z Y, Yang L X, Yang Z G. Nitrogen-doped reduced graphene oxide intertwined with V2O3 nanoflakes as self-supported electrodes for flexible all-solid-state supercapacitors. RSC Adv. 2017; 7(41): 25732-25739; Lee D U, Park W, Park M G, Ismayilov V, Chen Z. Synergistic Bifunctional Catalyst Design based on Perovskite Oxide Nanoparticles and Intertwined Carbon Nanotubes for Rechargeable Zinc-Air Battery Applications. ACS Appl Mater Interfaces. 2015; 7:58.) The electrodes can be intertwined and define a gap therebetween. In an embodiment, the distance between the working electrodes (e.g. E1 and E2) is such that the reporter moiety is capable of transporting (e.g. diffusing and/or active transport) from the detection probe to the capture probe. In embodiments, the electrodes are adjacent to one another and do not contact each other. The gap may be generally constant along the lengths of the electrodes depending on the geometry and shape, for example, such as in spiral and serpentine line electrodes. In embodiments, the gap between the first working electrode and the second working electrode is generally constant along its' length (e.g. serpentine example in
The electrodes may be constructed of any suitable conductive and/or semi-conductive material(s) comprising a metal, metal alloy, metal oxides, superconductor, semi-conductor, carbon-based materials, conductive polymer, or combinations thereof. The metal may be selected from aluminum (Al), antimony (Sb), bismuth (Bi), boron (B), cadmium (Cd), carbon (C), cerium (Ce), chromium (Cr), cobalt (Co), copper (Cu), dysprosium (Dy), erbium (Er), europium (Eu), gadolinium (Gd), germanium (Ge), gold (Au), graphite (C), hafnium (Hf), holmium (Ho), indium (In), iridium (Ir), iron (Fe), lanthanum (La), lutetium (Lu), magnesium (Mg), manganese (Mn), molybdenum (Mo), neodymium (Nd), nickel (Ni), niobium (Nb), palladium (Pd), platinum (Pt), praseodymium (Pr), rhenium (Re), ruthenium (Ru), samarium (Sm), selenium (Se), scandium (Sc), silver (Ag), silicon (Si), tantalum (Ta), terbium (Tb), thulium (Tm), tin (Sn), titanium (Ti), tungsten (W), vanadium (V), ytterbium (Yb), yttrium (Y), zirconium (Zr) and/or zinc (Zn). Typically, the metals are selected from gold, other noble metals, or combinations thereof. The metal alloy may be selected from the group consisting of aluminum copper (AlCu), aluminum chromium (AlCr), aluminum magnesium (AlMg), aluminum silicon (AlSi), aluminum silver (AlAg), cerium gadolinium (CeGd), cerium samarium (CeSm), chromium silicon (CrSi), cobalt chromium (CoCr), cobalt iron (CoFe), cobalt iron boron (CoFeB), copper cobalt (CuCo), copper gallium (CuGa), copper indium (CuIn), copper nickel (CuNi), copper zirconium (CuZr), hafnium iron (HfFe), iron boron (FeB), iron carbon (FeC), iron manganese (FeMn), iridium manganese (IrMn), iridium rhenium (IrRe), indium tin (InSn), molybdenum silicon (MoSi), nickel aluminum (NiAl), nickel chromium (NiCr), nickel chromium silicon (NiCrSi), nickel iron (NiFe), nickel niobium titanium (NiNbTi), nickel titanium (NiTi), nickel vanadium (NiV), samarium cobalt (SmCo), silver copper (AgCu), silver tin (AgSn), tantalum aluminum (TaAl), terbium dysprosium iron (TbDyFe), terbium iron alloy (TbFe), titanium aluminum (TiAl), titanium nickel (TiNi), titanium chromium (TiCr), tungsten rhenium (WRe), tungsten titanium (WTi), zirconium aluminum (ZrAl), zirconium iron (ZrFe), zirconium nickel (ZrNi), zirconium niobium (ZrNb), zirconium titanium (ZrTi), zirconium yttrium (ZrY), zinc aluminum (ZnAl) and/or zinc magnesium (ZnMg).
The electrodes, and more specifically, the nanostructured electrodes may be made from any suitable method, for example, a seed layer for the nanostructured electrodes may be made by sputter-coating, evaporation, chemical vapor deposition, or a pulsed laser method, ink jet printing. The nanostructures may be made using direct solution based deposition using covalent bonding, electroless or electroplating, electrospinning, template based synthesis, electrophoretic deposition, etching, printing, self-assembly of nanoparticles, etc. In an example, the method comprises applying a patterned mask to a substrate, depositing a conductive material on the mask forming a conductive layer, exposing the conductive layer to pre-conductive material, and using a potentiostatic technique to convert the pre-conductive material to the conductive material, forming the nanostructured electrodes.
Functionalizing of the electrode with the detection probe or capture probe may be done by any suitable techniques, for example, as described in Putzbach, W., & Ronkainen, N. J. (2013). Immobilization techniques in the fabrication of nanomaterial-based electrochemical biosensors: A review. In Sensors (Switzerland) (Vol. 13, Issue 4, pp. 4811-4840). MDPI AG. The method may include binding a detection probe molecule to the surface of the electrode.
It is understood that in the biosensor described herein, the detection probe, the reporter moiety, the recognition moiety, and/or the capture probe may be any suitable species as described above under the definitions section.
In another embodiment, a biosensor comprises a multi-electrode electrochemical chip. The chip has nanostructured intertwined electrodes E1 and E2. A detection probe (e.g. biorecognition elements and redox moieties) is functionalized on E1 and a capture probe is functionalized on E2 (e.g. redox moieties capture probes) for the detection of target analytes. In an embodiment, this biosensor is an integrated four electrode chip including reference and counter electrodes, as well as two working electrodes (E1=release channel and E2=capture channel). In a further embodiment, current flows between each of the working electrodes and the counter electrode. In another embodiment, a detection probe comprises a reporter moiety (e.g. redox moiety), which is located on E1 before target introduction and generates a measurable electrochemical current in response to a potential applied to both E1 and E2 electrodes with respect to the reference electrode. Current is still measured as a signal from E1 to provide a quality control checkpoint for the functionality of the assay. Once the target analyte is introduced, the redox moiety moves from E1 to E2, the signal at E1 decreases (signal off), whereas the signal at E2 increases (signal on).
In embodiments, the biosensors, kits and methods of use described herein can be used for detecting any suitable analyte, such as, and without being limited thereto, a wide range of small molecule, protein and nucleic acid analytes, including infection-causing pathogens in point-of-care diagnostics and health monitoring. In a further embodiment, the biosensor and/or method are used to detect E. coli in buffer and urine test samples. In embodiments, the biosensors enable an ultra-sensitivity (10 CFU) and high specificity against a panel of bacteria.
In an embodiment of the kit, the kit comprises a biosensor described herein and instructions for use.
The biosensors described herein can be used in any suitable device, including a hand-held device and/or diagnostic device. For example, the device may resemble the glucose monitor in terms of ease-of-use, portability, cost, and/or amplification-free bacterial detection based, for example, on an electrochemical readout. In an embodiment, the device may be a handheld electrical reader comprises the biosensor (e.g. self-contained e-differential chip).
In further embodiments, the biosensors described herein may be used in a handheld screening and/or diagnostic device that performs benchtop testing and generates test results in less than about 30 minutes, less than about 20 minutes, or less than about 15 minutes, for example, while matching the clinical sensitivity and specificity offered by comparative tests (e.g. urine cultures that require days to complete). In embodiments, the biosensor may be used without the need for sample pre-treatment, target labeling, and/or amplification.
Urinary tract infections, despite being the second most common bacterial infectious disease, lacks the developments towards affirmative early diagnosis albeit the challenges of antibiotic resistance associated with it. The existing in-patient or outpatient approaches to urine analysis suffer from false positive results and sample-to-answer time of 1-2 days causing delays in the disease treatment, and an increased cost of care. The devices described herein may increase the accuracy and decrease the timeline for diagnosis.
In an embodiment of a method for determining the presence of an analyte in a sample. The biosensor described herein is exposed to the sample. The reporter moiety delocalizes from the first working electrode to the second working electrode in the presence of the analyte, providing a change in signal (e.g. current, potential, impedance, etc.) at each of the first working electrode and the second working electrode. The change in signal indicates the presence of the analyte. In various embodiments, the change in signal for the first working electrode is from about 0.1 to about 0.6 fold decrease and the change in signal for the second working electrode is from about 50 to about 200 fold increase or the change in signal for the first working electrode is from about 50 to about 200 fold increase and the change in signal for the second working electrode is from about 0.1 to about 0.6 fold decrease. In embodiments, the signal is current. With respect to the fold decrease, the ranges may be from about 0.1 to about 0.6 fold decrease, about 0.1 to about 0.5 fold decrease, about 0.1 to about 0.4 fold decrease, about 0.1 to about 0.3 fold decrease, about 0.1 to about 0.2 fold decrease, about 0.2 to about 0.6 fold decrease, about 0.3 to about 0.6 fold decrease, about 0.4 to about 0.6 fold decrease, or about 0.5 to about 0.6 fold decrease. With respect to the fold increase, the ranges may be from about 50 to about 200 fold increase, from about 50 to about 150 fold increase, from about 50 to about 100 fold increase, from about 50 to about 90 fold increase, from about 50 to about 80 fold increase, from about 50 to about 70 fold increase, from about 50 to about 60 fold increase, from about 60 to about 200 fold increase, from about 70 to about 200 fold increase, from about 80 to about 200 fold increase, from about 90 to about 200 fold increase, from about 100 to about 200 fold increase, from about 125 to about 200 fold increase, from about 150 to about 200 fold increase, or from about 175 to about 200 fold increase.
In embodiments, if there is no measurable signal change, the analyte is not present in the sample. In another embodiment, if the change in signal from the first working electrode is a decrease in signal and the change in signal from the second working electrode is an increase in signal, the analyte is present. In another embodiment, if the change in signal from the first working electrode is an increase in signal and the change in signal from the second working electrode is a decrease in signal, the analyte is present.
In embodiments, if no measurable current change, the analyte is not present in the sample. In another embodiment, the change in current from the first working electrode is a decrease in current and the change in current from the second working electrode is an increase in current, the analyte is present. In another embodiment, if the change in current from the first working electrode is an increase in current and the change in current from the second working electrode is a decrease in current, the analyte is present.
In embodiments, the biosensor described herein is exposed to the sample under conditions to delocalize the reporter moiety from the first working electrode to the second working electrode in the presence of the analyte, providing the change in signal.
In other embodiments described herein, the signal is measured from each of the working electrodes. For example, the current is measured from each of the working electrodes.
In other specific embodiments of the method described herein, prior to exposing the biosensor to the sample, an initial signal is measured at each of the first and second working electrodes. After exposing the biosensor to the analyte, a final signal is measured at each of the first and second working electrodes. A change in signal between the initial signal and the final signal of the first working electrode and a change in signal between the initial signal and the final signal of the second working electrode, indicates the presence of the analyte in the sample.
In other specific embodiments of the method described herein, prior to exposing the biosensor to the sample, an initial current is measured at each of the first and second working electrodes. After exposing the biosensor to the analyte, a final current is measured at each of the first and second working electrodes. A change in current between the initial current and the final current of the first working electrode and a change in current between the initial current and the final current of the second working electrode, indicates the presence of the analyte in the sample.
The above disclosure generally describes the present invention. A more complete understanding can be obtained by reference to the following specific Examples. These Examples are described solely for purposes of illustration and are not intended to limit the scope of the invention. Changes in form and substitution of equivalents are contemplated as circumstances may suggest or render expedient. Although specific terms have been employed herein, such terms are intended in a descriptive sense and not for purposes of limitation.
A four-electrode electrochemical chip was fabricated on pre-stressed polystyrene (PS) sheets (Graphix Shrink Film, Graphix). The PS was cleaned with ethanol and DI water, after which a vinyl mask (FDC 4304, FDC graphic films) was applied onto the sheet. The vinyl mask was then cut into the star-shaped working electrode pattern (as defined by Adobe Illustrator) using a Robo Pro CE5000-40-CRP cutter (Graphtec America Inc.). The vinyl was then peeled off, and a 100 nm gold (Au) layer was sputtered on top using DC sputtering (MagSput™, Torr International). The chip comprises two star-shaped working electrodes (WEs; release channel, E1 and capture channel, E2) and both counter (CE) and reference (RE) electrodes. This chip with the conductive layer served as a seed layer for the nucleation and growth of high-aspect ratio nanostructures using an electrodeposition technique. A 10 mM gold chloride (HAuCl4) in 5 mM HCl solution was bubbled with N2, before use, for about 10 minutes. The conductive layer containing the electrodes was dipped in this solution and gold nanostructures were electrodeposited on the two working electrodes by applying a static potential of about −0.6V (anodic negative) for about 600 s using a potentiostatic technique to reduce the gold ions. Both E1 and E2 were deposited with gold (Au) nanostructures. The resulting chip comprised two nanostructured star-shaped working electrodes (WE; release channel, E1 and capture channel, E2) and both counter (CE) and reference (RE) electrodes. The nanostructuring of the electrodes enhanced their limit-of-detection in detecting electroactive oligonucleotides by three orders of magnitudes compared to their planar counterparts (
A truncated version of the RNA-cleaving fluorescent DNAzyme for E. coli detection (RFD-EC1) as described in Ali et al. (Ali M M, Aguirre S D, Lazim H, Li Y. Fluorogenic DNAzyme Probes as Bacterial Indicators. Angew. Chem. Int. Ed. 2011; 50:3751-3754) was used. The truncated DNAzyme has a secondary structure for better release of the cleaved redox-tagged DNA segment. The shorter sequence increased the loading capacity on the electrode and reduced cost. The truncated DNAzyme had similar activity and specificity to the RFD-EC1 (
The e-DNAzymes were prepared by T4 DNA ligase-mediated DNA ligation of the biotinylated DNAzyme sequence (BD) and labeled DNA reporter (DR) in the presence of a ligation template (LT) as the template to complete the biotinylated DNAzyme reporter (BDR). The methylene blue labeled DNA was purchased from Biosearch Inc, and all other DNA oligonucleotides (including the thiolated probe, TP) were purchased from Integrated DNA Technologies. All DNA oligonucleotides were purified via standard 10% denaturing (7 M urea) polyacrylamide gel electrophoresis (dPAGE), with their concentrations determined spectroscopically. The sequences and functions of the synthetic oligonucleotides used herein are provided in Table 1. ATP, T4 DNA ligase, T4 polynucleotide kinase (PNK), and T4 DNA ligase, along with their respective buffers, were purchased from Thermo Scientific. Water used in the experiments (ddH2O) was purified with a Milli-Q Synthesis A10 water-purification system. All other chemicals were purchased from either Sigma-Aldrich or Bioshop Canada and were used without further purification.
E. coli
1 nmol of BD was phosphorylated in 100 μL of 1×PNK buffer A containing 2 mM ATP (final concentration) with 20 U (units) of PNK enzyme at 37° C. for 40 min. The reaction was quenched by heating the mixture at 90° C. for 5 min. Then, an equal number (1 nmol) of MS and LT were added to the reaction mixture. The mixture was then heated at 90° C. for 1 min before being cooled over 15 min until it reached room temperature. Once the mixture had cooled, 20 μL of 10×DNA ligase buffer and 20 U of T4 DNA Ligase was added, and the final volume was adjusted to 200 μL by adding ddH2O. After incubation at room temperature for 2 h, the DNA molecules were isolated by ethanol precipitation and the ligated DNA molecules were purified via 10% dPAGE. After being dissolved in ddH2O, the DNAzyme concentration was measured using a Nanovue and the DNAzyme solutions were stored at −20° C. until use. Fluorophore-labeled (using FAM in place of methylene blue) DNAzyme was also prepared by T4 DNA ligase-mediated DNA ligation of BD and DR in the presence of LT as the template using the same ligation protocol.
The e-differential chips were rinsed in isopropanol and ddH2O. This was followed by electrochemical cleaning using cyclic voltammetry (CV) in 0.1 M H2SO4 (0 V-1.5 V, 100 mV/s, 40 cycles). After cleaning of the e-differential chips, the release and capture channels were functionalized with their respective biorecognition elements. To functionalize the release channel (E1), 50 mM cysteamine hydrochloride was drop-deposited for 20 hours followed by 10% glutaraldehyde (pH=7) for 12 hours in the dark at room temperature. 1 μM streptavidin was then deposited on the cysteamine-glutaraldehyde functionalized release channel for 4 hours at 4° C., followed by washing in buffer and the deposition of 2 μM of biotinylated DNAzyme reporter (BDR; sequence in
The electrochemical surface area was calculated by performing a cyclic voltammetry scan from 0 to 1.5V (cathodic positive) in 0.1 M H2SO4 using a scan rate of 50 mV/s. The area under the reduction peak was integrated to calculate the electrochemical charge involved in the redox process, which was subsequently divided by the surface charge density involved in forming a monolayer of AuOx (386 μC/cm2).
The immobilization of BDR on release channel E1 was characterized by square wave voltammetry (SWV) over a voltage range of 0 V to −0.6 V (anodic negative) before and after the DNAzyme deposition steps in 25 mM PBS and 25 mM NaCl buffer (25:25 buffer). As the DNAzyme has the redox species attached (before target addition), before the incubation there was no peak whereas after the incubation the peak emerges. Thus, the methylene blue moieties on the chemically immobilized e-DNAzymes participated in charge transfer with the release channel and resulted in a current peak in SWVs, validating DNAzyme immobilization on the electrode surface (
To assess the ability of the e-DNAzyme test to detect clinically relevant amounts of bacteria, various known concentrations of E. coli were tested in buffer and spiked in urine. E. coli was chosen as the target due to its predominance in urinary tract infections (UTIs), which is one of the most prevalent bacterial infections in clinical practice; however, the e-DNAzyme strategy could in principle be applied to any relevant bacteria.
For testing the assay in buffer or urine samples, pathogenic samples comprised a crude intracellular mixture (CIM) of E. coli (test sample containing target analyte) or B. subtilis, Y. ruckeri, C. difficile, H. alvei or L. monocytogenes bacteria (control samples). Escherichia coli K12 (E. coli K12; MG1655) was used herein. To measure the colony-forming units (CFU/mL) of E. coli cells, a single colony freshly grown on Luria Broth (LB) agar plate inoculated into 2 mL of LB and grown for 14 h at 37° C. with continuous shaking at 250 rpm. Following incubation, a 10-fold serial dilution of bacterial culture was conducted, and 100 μL of the diluted solutions were then spread onto LB agar plates (in triplicate) and incubated at 37° C. for 16 h. Finally, the colonies were counted and averaged to obtain the number of CFU/mL. For the preparation of bacterial CIM samples for testing, 1 mL of each dilution was centrifuged at 11,000 g for 5 min at 4° C. The clear supernatant was then discarded, and the cell pellet was resuspended in 100 μL of ddH2O and heated at 65° C. for 10 min to release the DNAzyme-activating target. The heat-treated cell suspension was then vortexed to dissolve the cell pellet completely and stored at −20° C. Different concentrations of bacterial CIM were spiked into both the 25:25 buffer and healthy human urine samples. After chip fabrication and functionalization, 10 μL of bacteria-doped samples were incubated on top of the chip for 30 minutes at 37° C. The signal generated with E. coli was compared to a panel of the five control samples containing CIM from the other bacteria (B. subtilis, Y. ruckeri, C. difficile, H. alvei and L. monocytogenes) to assess the specificity toward E. coli. A small volume (10 μL) of these samples were dropped on the two working electrodes (E1 and E2) for 30 min at 37° C. The signal quantification was performed using SWV over a voltage range of 0 V to −0.6 V (anodic negative) in 25:25 buffer and urine.
E. coli samples having a bacterial load of 0-105 CFU were introduced on the biosensor and the current generated on the release and capture channels were measured using SWV (
The e-DNAzyme test could sensitively detect E. coli in both buffer and urine, the specificity of this assay was assessed against a panel of gram-negative and gram-positive bacterial targets: B. subtilis, Y. ruckeri, C. difficile, H. alvei and L. monocytogenes (
To demonstrate the utility of the e-DNAzyme test in a practical clinical context, the RCDs specific to Escherichia coli were used to assess the operation of the e-DNAzyme test towards the identification of this pathogen in UTIs using clinical human urine samples that were bacteria negative, bacteria positive but E. coli negative, and E. coli positive.
A set of 41 urine samples including 20 clinically-significant E. coli-only, 10 clinically-insignificant E. coli-only, 2 K. pneumonie-only, 2 E. faecalis-only, 1 S. aureus-only, and 1 P. mirabilis-only urine samples, and 5 culture-negative (i.e. no bacterial growth) urine samples were obtained from Hamilton General Hospital. The urine samples were heated at 55° C. for 15 minutes followed by centrifugation at 11,000×g for 5 minutes. A 10 μL of the suspension of the urine was then incubated on the chip for 30 minutes at 37° C., after which SWV over a voltage range of 0 V to −0.6 V (anodic negative) was conducted in 25:25 buffer. Clinical sensitivity was calculated by dividing “True Positive” results from a sum of “True Positive” and “False Negative” results, while clinical specificity was calculated by dividing “True Negative” results from a sum of “True Negative” and “False Positive” results.
The disclosed biosensor was used to analyze the 41 urine samples collected from patients who were suspected of having UTIs due to the presence of clinical symptoms. A handheld electrical reader comprises the self-contained e-differential chip to demonstrate the potential of the e-DNAzyme test for point-of-care analysis (
The biosensor allowed the analysis of clinical urine samples using a handheld reader without the need for sample pre-treatment, target labeling, enrichment or amplification and readout reagents within 30 minutes while matching the clinical sensitivity and specificity offered by urine cultures that require days to complete.
The design and fabrication of a highly specific and sensitive electrochemical assay based on the integration of RNA-cleaving DNAzyme technology, differential electrochemical signal readout, and nanostructured electrodes for rapidly identifying bacteria without time-consuming growth cultures is disclosed herein. Differential electrochemical signaling is made possible due to the programmable nature of electroactive RNA-cleaving DNAzymes combined with a two-channel electrochemical chip. Each of the building blocks of the e-DNAzyme assay contributes to enhanced sensitivity, specificity, or speed of operation. This system can be engineered to target and identify multiple bacteria in parallel for performing rapid, multiplexed, and reagent-free bacterial analysis.
While the present application has been described with reference to examples, it is to be understood that the scope of the claims should not be limited by the embodiments set forth in the examples, but should be given the broadest interpretation consistent with the description as a whole.
All publications, patents and patent applications are herein incorporated by reference in their entirety to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated by reference in its entirety. Where a term in the present application is found to be defined differently in a document incorporated herein by reference, the definition provided herein is to serve as the definition for the term.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/CA2020/050375 | 3/20/2020 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 62821219 | Mar 2019 | US |
