Electrochemical aptamer sensor for detection of phenol-soluble modulins

Abstract

The present disclosure provides, in part, electrochemical sensors and aptamers for detecting phenol-soluble modulins (PSMs) and methods of preparing and using the same, e.g., for detecting Staphylococcus spp.

Description

Attorney Docket No.: QSM-004PC / 126665-5004ELECTROCHEMICAL APTAMER SENSOR FOR DETECTION OF PHENOL-SOLUBLE MODULINSFIELD

[0001] The present disclosure relates to, in part, electrochemical sensors and aptamers for detecting phenol-soluble modulins (PSMs) and methods of preparing and using the same, e.g., for detecting Staphylococcus spp.CROSS REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of and priority to U.S. Provisional Application No. 63 / 706,097, filed October 11 , 2024, the entire contents of which are incorporated by reference.SEQUENCE LISTING

[0003] The instant application contains a sequence listing, which has been submitted in XML format via Patentcenter. The contents of the XML file named “126665-5004_Sequence_Listing,” which was created on October 9, 2025, and is approximately 5,776 bytes in size, are incorporated herein by reference in their entirety.BACKGROUND

[0004] Quorum sensing molecules (QSMs) are small signaling molecules that bacteria use to communicate and coordinate their behavior within a population. Detecting bacteria through QSMs generally involves using molecules which bind and / or detect the QSMs to identify and monitor bacterial presence. Phenol-soluble modulins (PSMs) are a category of bacterial-synthesized QSM molecules. PSMs are amphipathic peptides produced by almost all pathogenic strains of the bacterial genus Staphylococcus and play multiple roles in pathogenesis, such as functioning as cytotoxins and pro-inflammatory agents. Staphylococcus are common pathogens in humans, companion pets, livestock, etc. There remains a need for accurate and sensitive PSM sensors for detecting the presence of Staphylococcus for clinical, diagnostic, and commercial purposes.SUMMARY

[0005] Accordingly, the present disclosure provides, in part, aptamer sensors for binding phenol-soluble modulins (PSMs). In embodiments, the aptamer comprises a DNA sequence having about or at least about 80% sequence identity to a DNA sequence selected from one or more of SEQ ID NOs: 1-5. In embodiments, the aptamer comprises a DNA sequence having about or at least about 85%, about or at least about 90%,DBl / 163111002.1 1Attorney Docket No.: QSM-004PC / 126665-5004 about or at least about 95%, about or at least about 96%, about or at least about 97%, about or at least about 98%, or about or at least about 99% sequence identity to a DNA sequence selected from one or more of SEQ ID NOs: 1-5. In embodiments, the aptamer comprises a DNA sequence selected from one or more of SEQ ID NOs: 1-5. In embodiments, the aptamer has a DNA sequence of SEQ ID NO: 3.

[0006] In embodiments, the aptamer binds one or more phenol-soluble modulins (PSMs). In embodiments, the one or more PSMs are from one or more Staphylococcus bacteria.

[0007] In embodiments, the aptamer is functionalized with a carbon-based linker molecule, polyethylene glycol (PEG) linker molecule, polyethylene oxide (PEG) linker molecule, and / or polyoxyethylene (POE) linker molecule. In embodiments, the linker molecule is functionalized with one or more thiol group (sulfhydrylreactive PEGylated crosslinkers), amine group (amine-reactive PEGylated crosslinkers), biotin group (biotinylated PEGylated crosslinkers), optionally at an end of the linker. In embodiments, the PEG linker is or comprises one or more of PEG 200, PEG 300, PEG 500, PEG 600, PEG 1000, PEG 2000, PEG 3350, PEG 6000, PEG 8000, PEG 10000, PEG 15000, and PEG 20000.

[0008] In embodiments, the aptamer is suitable for attachment to a surface, optionally a metal surface. In embodiments, the surface is or comprise a gold surface, and the aptamer is optionally attached using goldthiol chemistry.

[0009] In embodiments, the aptamer is labeled with a redox molecule. In embodiments, the redox molecule is or comprises methylene blue (MB), azure B, and / or azurin, optionally the redox molecule is fluorescent or fluorescently labeled.

[0010] In aspects, described herein is an electrochemical sensor. In embodiments, the electrochemical sensor comprises a surface having disposed thereon one or more aptamers, as described herein, where the one or more aptamers binds one or more phenol-soluble modulins (PSMs).

[0011] In embodiments, the one or more aptamers comprise a DNA sequence having about or at least about 80% sequence identity to a DNA sequence selected from one or more of SEQ ID NOs: 1-5. In embodiments, the one or more aptamers comprises a DNA sequence having about or at least about 85%, about or at least about 90%, about or at least about 95%, about or at least about 96%, about or at least about 97%, about or at least about 98%, or about or at least about 99% sequence identity to a DNA sequence selected from one or more of SEQ ID NOs: 1-5. In embodiments, the one or more aptamers comprises aDBl / 163111002.1 2Attorney Docket No.: QSM-004PC / 126665-5004DNA sequence selected from one or more of SEQ ID NOs: 1-5. In embodiments, the electrochemical sensor comprises an aptamer having a DNA sequence of SEQ ID NO: 3.

[0012] In embodiments, the PSMs are synthesized by one or more Staphylococcus bacteria. In embodiments, the surface having disposed thereon the one or more aptamers comprises one or more metals. In embodiments, the metal comprises gold. In embodiments, the one or more aptamers are attached to the surface via gold-thiol chemistry.

[0013] In embodiments, the electrochemical sensor is configured to provide and / or measure one or more signals as a function of binding the one or more PSMs. In embodiments, the one or more signals comprise a solution resistance, impedance (impedimetric detection), capacitance, and / or porosity. In embodiments, the one or more signals comprise a solution resistance, impedance (impedimetric detection), capacitance, and / or porosity.

[0014] In embodiments, the electrochemical sensor detects the presence of bacteria as a function of binding one or more PSMs.

[0015] In aspects, described herein are methods of identifying and / or generating one or more aptamers that target one or more phenol-soluble modulins (PSMs), the method comprising (i) tagging one or more PSMs with histidine, (ii) immobilizing the one or more PSMs onto nickel-coated magnetic beads to generate one or more PSM-labeled magnetic beads, (iii) contacting the one or more PSM-labeled magnetic beads with a first library of aptamers, (iv) separating, using a magnetic field, one or more aptamers from the first library of aptamers as a function of binding to the PSM-labeled magnetic beads, and (v) sequencing the one or more bound aptamers to determine the nucleic acid sequence that bound the one or more PSMs.

[0016] In embodiments, the method comprises eluting the one or more bound aptamers from the PSM- labeled magnetic beads, optionally by changing the pH (decrease or increase), changing the temperature (increase or decrease), or removing from the magnetic beads (e.g., via imidazole).

[0017] In embodiments, the method comprises repeating one or more of steps (iii), (iv), and (v) using a second library of aptamers.

[0018] In embodiments, the method further comprises synthesizing the one or more bound aptamers from the sequencing. In embodiments, the method further comprises attaching the synthesized aptamers to a surface of an electrochemical sensor, optionally using gold-thiol chemistry.DBl / 163111002.1 3Attorney Docket No.: QSM-004PC / 126665-5004

[0019] In aspects, described herein are methods of detecting the presence of bacteria in a biological sample comprising providing the biological sample, contacting the biological sample with a surface of an electrochemical sensor as described herein, where the electrode surface comprises one or more aptamers disposed thereon (as described herein), and wherein the one or more aptamers binds one or more phenol- soluble modulins (PSMs), and detecting the presence of bacteria in the biological sample as a function of measuring aptamer binding of one or more phenol-soluble modulins (PSMs) on the surface of the electrochemical sensor.

[0020] In embodiments, the bacteria is one or more Staphylococcus bacteria. In embodiments, the biological sample is or comprises urine.

[0021] In embodiments, the aptamer is labeled with a redox molecule, optionally methylene blue (MB), azure B, and / or azurin, optionally the redox molecule is fluorescent or fluorescently labeled.

[0022] In embodiments, the aptamer is not labeled with a redox molecule. In embodiments, the method further comprises adding a redox molecule to the electrochemical sensor during the contacting step. In embodiments, the redox molecule is methylene blue (MB) azure B, and / or azurin, optionally fluorescently labeled, and wherein the redox molecule is optionally added at a concentration of about or at least about 1 pM, about or at least about 5 pM, about or at least about 10 pM, about or at least about 20 pM, about or at least about 50 pM, or about or at least about 100 pM.

[0023] In embodiments, measuring aptamer binding comprises measuring one or more electrochemical impedance spectroscopy (EIS) parameters at the surface of the electrochemical surface at one or more frequencies. In embodiments, the one or more frequencies comprises about 3714 Hz, about 1834 Hz, and about 68 Hz. In embodiments, the detecting further comprises utilizing one or more machine-learning processes with the one or more EIS parameters. In embodiments, the one or more machine-learning processes comprises a support vector machine (SVM) and / or ensemble model. In embodiments, the one or more machine-learning processes classifies the biological sample as either Staphy / ococcus-positive or Stap / ry / ococcus-negative.

[0024] In aspects, described herein are kits comprising one or more aptamers, as described herein, and / or one or more electrochemical sensors, as described herein, and optionally one or more redox molecules (e.g., methylene blue (MB), azure B, and / or azurin). In embodiments, the kits are useful for performing one or more methods, as described herein.DBl / 163111002.1 4Attorney Docket No.: QSM-004PC / 126665-5004DESCRIPTION OF THE DRAWINGS

[0025] Figs. 1A-1 B depict non-limiting, illustrative diagrammatic representations of two different sensing platforms for impedimetric detection of QSMs in bodily fluids. Fig. 1A illustrates a methylene blue (MB)-labeled aptamer and Fig. 1B illustrates a redox label-free aptamer.

[0026] Fig. 2 depicts a graphical representation of the results of phenol-soluble modulin (PSM) detection using an electrochemical sensor using a MB-labeled aptamer.

[0027] Fig. 3 depicts a graphical representation of the results of PSM detection in diluted urine (with a 1 :9 urine to 1x phosphate buffered saline (PBS) ratio) using the first platform with MB-labeled aptamer (e.g., as shown in Fig. 1A).

[0028] Fig. 4 depicts a graphical representation of the results of PSM detection in urine using the second platform with label-free aptamer (e.g., as shown in Fig. 1 B) with 19.2 pM MB in 1x PBS.

[0029] Fig. 5 depicts a graphical representation of a comparison of electrochemical impedance spectroscopy (EIS) parameters between Staphylococcus-pos\ \\ie and Staphy / ococcus-negative samples at a phase angle at 3714 Hz (top left), a phase angle at 1834 Hz (top right), and a Z real at 68 Hz (bottom).

[0030] Fig. 6 depicts a graphical representation of a comparison of EIS parameters between Sfaphy / ococcus-positive (left) and Sfaphy / ococcus-negative (right) samples at a phase angle at 3714 Hz.

[0031] Fig. 7 depicts a graphical representation of scatterplots showing the correlation between key variables that distinguish Sfaphy / ococcus-positive and Sfaphy / ococcus-negative samples using machinelearning processes.

[0032] Fig. 8 depicts a graphical representation of results for a support vector machine (SVM) model with the top showing confusion matrix results (e.g., as percentages), the bottom left showing confusion matrix results (e.g., as number of observations), and bottom right showing an receiver operating characteristic curve (ROC curve).

[0033] Fig. 9 depicts a graphical representation of the average accuracy, specificity, and sensitivity of the SVM and ensemble models. In each parameter, the left bar represents SVM and the right bar represents Ensemble.DBl / 163111002.1 5Attorney Docket No.: QSM-004PC / 126665-5004DETAILED DESCRIPTION

[0034] In embodiments, QSMs, such as PSMs, are small signaling molecules that are synthesized and / or secreted by bacteria to communicate and coordinate their behavior within a population. In embodiments, detecting bacteria through QSMs involves using these molecules to identify and monitor bacterial presence. In embodiments, aptamer sensors offer several advantages over traditional immunosensors and are suitable to be utilized for detecting bacteria. In embodiments, the advantages include high affinity, specificity, ease of synthesis and modification, high stability, and low cost. In embodiments, aptamer sensors are an effective tool for selective detection of a wide range of proteins, nucleic acids, and small molecule QSMs.

[0035] In embodiments, aptamers undergo conformational changes when they bind to target molecules. These conformational changes, in embodiments, are induced by the target QSM (e.g., PSMs), and are detectable and measurable using various electrochemical techniques, such as electrochemical impedance spectroscopy (EIS). In embodiments, to obtain a potentiostatic EIS measurement, an alternating current (AC) potential is applied, and the resulting current is measured. In embodiments, by analyzing EIS data at different frequencies, subtle changes in the electrochemical properties of the surface, including the conformational changes of aptamers induced by QSM target binding, are capturable and are used as the signal for the aptamer sensor.Phenol-Soluble Modulin (PSM)-Specific Aptamers and Electrochemical Sensors

[0036] Described herein are aptamers and electrochemical sensors for detecting phenol-soluble modulins (PSMs). In embodiments, PSMs are amphipathic peptides produced by almost all pathogenic strains of staphylococcus and play multiple roles in pathogenesis, such as acting as cytotoxins and pro- inflammatory agents. In embodiments, aptamers and / or electrochemical sensors herein are designed for identifying Staphylococcus. In embodiments, Staphylococcus is a bacterial genus which includes one or more bacterial species (or subspecies, spp.), bacterial isolates, or bacterial strains present in urine samples (e.g, of humans, companion pets, livestock, laboratory animals, etc.).Table 1 : Illustrative DNA aptamer sequences for PSM detection.DBl / 163111002.1 6Attorney Docket No.: QSM-004PC / 126665-5004

[0037] In embodiments, the aptamer comprises a DNA sequence having about or at least about 80% sequence identity to a DNA sequence selected from one or more of SEQ ID NOs: 1-5 (e.g., having 1, 2, 3, 4, 5, 6, 7, 8, 9, about 10, or about 15 DNA base differences relative to any one of SEQ ID NOs: 1-5). In embodiments, the aptamer comprises a DNA sequence having about or at least about 85%, about or at least about 90%, about or at least about 95%, about or at least about 96%, about or at least about 97%, about or at least about 98%, or about or at least about 99% sequence identity to a DNA sequence selected from one or more of SEQ ID NOs: 1-5. In embodiments, the aptamer comprises a DNA sequence selected from one or more of SEQ ID NOs: 1-5. In embodiments, the aptamer has a DNA sequence of SEQ ID NO: 3. Persons skilled in the art, with the benefit of this disclosure in its entirety, will understand how to measure which aptamers bind PSMs and have a DNA sequence within about 80% sequence identity to the sequences provided herein.

[0038] In embodiments, the aptamer binds one or more phenol-soluble modulins (PSMs). In embodiments, the one or more PSMs are from one or more Staphylococcus bacteria.

[0039] In embodiments, the aptamer is functionalized with a carbon-based linker molecule (e.g., a hydrophilic or amphipathic linker), polyethylene glycol (PEG) linker molecule, polyethylene oxide (PEO) linker molecule, and / or polyoxyethylene (POE) linker molecule. In embodiments, the linker molecule is functionalized with one or more thiol group (sulfhydryl-reactive PEGylated crosslinkers), amine groups (amine-reactive PEGylated crosslinkers), biotin groups (biotinylated PEGylated crosslinkers). In embodiments, the functionalization is applied at an end of the linker (e.g., at a terminal end of the linker molecule for binding to a surface or another molecule). In embodiments, aptamers are functionalized with one or more PEGs using maleimide chemistry, streptavidin-biotin linkage, etc.

[0040] In embodiments, the PEG linker is or comprises one or more of PEG 200, PEG 300, PEG 500, PEG 600, PEG 1000, PEG 2000, PEG 3350, PEG 6000, PEG 8000, PEG 10000, PEG 15000, and PEG 20000.DBl / 163111002.1 7Attorney Docket No.: QSM-004PC / 126665-5004

[0041] In embodiments, the aptamer is suitable for attachment to a surface, optionally a metal surface (e.g., gold, silver, copper, iron, nickel, lead, tin, platinum, palladium, rhodium, and alloys and oxides thereof). In embodiments, the surface is a gold surface. In embodiments, the aptamer is attached using gold-thiol chemistry.

[0042] In embodiments, the aptamer is labeled with a redox molecule (e.g., as shown in Fig. 1A). In embodiments, the redox molecule is or comprises methylene blue (MB), azure B, and / or azurin (e.g., the redox molecule being fluorescent or fluorescently labeled). In embodiments, the aptamer is not conjugated to a redox molecule (e.g., as shown in Fig. 1B).

[0043] In aspects, described herein are electrochemical sensors which have a surface with a plurality of aptamers (of one or more DNA sequences) conjugated to or otherwise coated onto a surface of the electrochemical sensor. In embodiments, the electrochemical sensor comprises a surface having disposed thereon one or more aptamers (as described herein), where the one or more aptamers binds one or more phenol-soluble modulins (PSMs). In embodiments, the surface is comprised of a material that is compatible with exposure to urine samples.

[0044] In embodiments, the electrochemical sensor comprises one or more aptamers having a DNA sequence of about or at least about 80% sequence identity to a DNA sequence selected from one or more of SEQ ID NOs: 1-5 (e.g., having about 1 , 2, 3, 4, 5, about 10, or about 15 DNA base differences relative to any one of SEQ ID NOs: 1-5). In embodiments, the electrochemical sensor comprises one or more aptamer having a DNA sequence having about or at least about 85%, about or at least about 90%, about or at least about 95%, about or at least about 96%, about or at least about 97%, about or at least about 98%, or about or at least about 99% sequence identity to a DNA sequence selected from one or more of SEQ ID NOs: 1-5. In embodiments, the electrochemical sensor comprises one or more aptamer having a DNA sequence selected from one or more of SEQ ID NOs: 1-5. In embodiments, the electrochemical sensor comprises one or more aptamer having a DNA sequence of SEQ ID NO: 3.

[0045] In embodiments, the electrochemical sensor has a surface comprised of a metal material (e.g., gold, silver, copper, iron, nickel, lead, tin, platinum, palladium, rhodium, and alloys and oxides thereof). In embodiments, the electrochemical sensor has a surface with a surface area of about or at least about 1 pm2to about or at least about 1 cm2. In embodiments, the electrochemical sensor surface is or comprises gold. In embodiments, the one or more aptamers are attached to the electrochemical sensor surface via gold-thiol chemistry. In embodiments, the electrochemical sensor is configured to provide and / or measure one or moreDBl / 163111002.1 8Attorney Docket No.: QSM-004PC / 126665-5004 signals as a function of binding the one or more PSMs. In embodiments, the one or more signal comprises, or is related to, a solution resistance, impedance (impedimetric detection), capacitance, and / or porosity (e.g., as described in Example 1). In embodiments, the signal is related to a measured solution resistance, impedance (impedimetric detection), capacitance, and / or porosity.

[0046] In embodiments, the electrochemical sensor is suitable to detect the one or more phenol-soluble modulins (PSMs) by electrochemical impedance spectroscopy (EIS). In embodiments, the electrochemical sensor detects the presence of bacteria as a function of binding one or more PSMs.Methods of Identifying and / or Generating PSM-Specific Aptamers

[0047] The present disclosure provides methods of identifying and / or generating one or more aptamers that bind one or more phenol-soluble modulins (PSMs) and / or that detect the presence of bacteria (e.g., Staphylococcus). In embodiments, the method comprises tagging one or more PSMs with histidine, immobilizing the one or more PSMs onto nickel-coated magnetic beads to generate one or more PSM-labeled magnetic beads, contacting the one or more PSM-labeled magnetic beads with a first library of aptamers, separating, using a magnetic field, one or more aptamers from the first library of aptamers as a function of binding to the PSM-labeled magnetic beads, and sequencing the one or more bound aptamers to determine the nucleic acid sequence that bound the one or more PSMs.

[0048] In embodiments, the method comprises (i) identification of aptamers against the target QSM (e.g., PSM) from a pool of DNA molecules, (ii) attachment of the aptamers to an electrode surface, (iii) testing the target QSM (e.g., PSMs) in, e.g., about a 1x phosphate buffered saline (PBS) and / or about a 10% urine solution, and (iv) differentiating between Staphylococcus-positive and Staphylococcus-negative urine samples from various patients (e.g., humans and / or non-human animals) using the PSM aptamer to detect PSMs.

[0049] In embodiments, aptamer identification is performed through a bead-mediated systematic evolution of ligands by exponential enrichment (SELEX) platform. In embodiments, this process includes functionalization and immobilization of the target QSMs (e.g., PSMs), which includes tagging the PSM peptides with histidine and immobilizing the histidine-tagged PSMs onto nickel-coated magnetic beads. In embodiments, the magnetic beads, now functionalized with the target QSMs (PSM), are incubated with a first library of aptamers. In embodiments, one or more unbound aptamers are removed through a series of rinse / wash steps. In embodiments, one or more bound aptamers are eluted by changing the pH (e.g., decreasing or increasing), changing the temperature (e.g., decreasing or increasing the temperature to forDBl / 163111002.1 9Attorney Docket No.: QSM-004PC / 126665-5004 denaturation of the aptamers), or removing from the magnetic beads (e.g., via imidazole competition to elute the PSM and / or aptamer). In embodiments, the aptamers that bound PSMs are then DNA sequenced for identification. In embodiments, the process (including any one or more of the steps) is repeated for at least a second library of aptamer sequences (e.g., designed from the initially captured aptamers, to further refine and enhance for high-affinity aptamers). In embodiments, a library of aptamers is a population of a single aptamer (distinct aptamers separated into individual reactions), or two or more distinct aptamers in a single reaction (e.g., about 2, 3, 4, 5, 10, 15, 20, 30, 40, 50, 100, or 1000 or more aptamers in a heterogenous sample). For example, in embodiments, a first library is a first reaction with a single aptamer (or more than a single aptamer) from a collection of aptamers to be characterized, and a second library is a second reaction with a second single aptamer (or more than a single aptamer) from the collection of aptamers to be characterized.

[0050] In embodiments, the aptamer is functionalized with a linker molecule (e.g., carbon-based or PEG-based linker molecule). In embodiments, the linker molecule comprises a thiol group (e.g., ending in a terminal thiol group), which enables its attachment to a metal surface (e.g., to a gold sensor surface). Consequently, in embodiments, the aptamer sensor is suitable for modifying a gold surface (e.g., of an electrochemical sensor surface) with one or more aptamers using gold-thiol chemistry.

[0051] For example, in embodiments, the process of attaching the aptamer to a surface includes preparing / cleaning the gold surface (e.g., by using a cotton-tipped swab with ethanol) and then attaching the aptamer to a surface by first disposing, for example about 50 mM H2SO4, onto the metal electrode, incubating for about 5 minutes, and then subsequently rinsing and drying the metal electrode. For example, in embodiments, attaching the aptamer to the electrochemical sensor surface includes depositing it at the desired concentration onto the surface, incubating for about 1 hour in a humidity chamber, protected from light, and then rising and drying the sensor.

[0052] In embodiments, the aptamer and electrochemical surface is functionalized by attaching mercaptohexanol spacer molecules by applying mercaptohexanol to the surface (e.g., about 3 mM solution). In embodiments, the sensor is incubated for about 1 hour in a humidity chamber, protected from light, followed by rinsing and drying. In embodiments, aptamers herein are attached to surfaces (e.g., to a surface of an electrochemical sensor) using gold-thiol chemistry.

[0053] In embodiments, the aptamer and / or electrochemical sensor are labeled with a redox molecule, e.g., like methylene blue (MB), azure B, and / or azurin, optionally fluorescent or fluorescently labeled (e.g.,DBl / 163111002.1 10Attorney Docket No.: QSM-004PC / 126665-5004 as shown in Fig. 1A). In embodiments, the aptamer is label-free, with a redox molecule (e.g., MB, azure B, and / or azurin) present in the electrolyte solution (e.g., as shown in Fig. 1B) when using the sensor. In embodiments, the redox molecule includes any colorimetric, fluorescent, or phosphorescent redox molecule used as a benchtop or laboratory reagent and / or for clinical assays.Methods of Using Aptamers and / or Electrochemical Sensors

[0054] The present disclosure provides methods of using aptamers and / or electrochemical sensors herein to detect PSM and / or the presence of bacteria. In embodiments, the method comprises providing the biological sample, contacting the biological sample with a surface of an electrochemical sensor (as described herein), where the electrode surface comprises one or more aptamers disposed thereon (as described herein) and where the one or more aptamers binds one or more phenol-soluble modulins (PSMs), and detecting the presence of bacteria in the biological sample as a function of measuring aptamer binding of one or more phenol-soluble modulins (PSMs) on the surface of the electrochemical sensor.

[0055] In embodiments, the bacteria is one or more Staphylococcus bacteria. In embodiments, the biological sample is or comprises urine. In embodiments, the urine is from a human or non-human animal. In embodiments, the urine is canine orfeline. In embodiments, Staphylococcus infections are common to a wide range of animals, including livestock (e.g., cattle, sheep, goats, pigs, horses, etc.), pets and companion animals (e.g., dogs, cats, rabbits, racoons, squirrels, etc.), to various wildlife (e.g., bears, zebras, dolphins, seals, boars, deer, monkeys and apes, etc.), and the aptamers, electrochemical sensors, kits, and / or methods herein are suitable for use with a wide range of urine samples from a variety of animals. In embodiments, the aptamers, electrochemical sensors, kits, and / or methods herein are useful for veterinary purposes. In embodiments, the aptamers, electrochemical sensors, kits, and / or methods herein are useful for canine and / or feline use.

[0056] In embodiments, the aptamer is labeled with a redox molecule, optionally methylene blue (MB), azure B, and / or azurin (e.g., fluorescently labeled). In embodiments, the aptamer is not labeled with a redox molecule, and thus the method comprises adding a redox molecule to the electrochemical sensor during the contacting step. In such embodiments, the redox molecule is methylene blue (MB), azure B, and / or azurin, optionally fluorescent or fluorescently labeled, and the redox molecule is added at a concentration of about or at least about 1 pM, about or at least about 5 pM, about or at least about 10 pM, about or at least about 20 pM, about or at least about 50 pM, or about or at least about 100 pM.DBl / 163111002.1 11Attorney Docket No.: QSM-004PC / 126665-5004

[0057] In embodiments, upon exposure to a sample containing the PSM, the PSMs bind specifically to the binding sites of aptamers, altering their surface structures. In embodiments, upon the binding results in restructuring which induces changes in the electrochemical processes on the surface, which are quantifiable by fitting electrochemical impedance spectroscopy (EIS) data with an equivalent circuit model (e.g., as described in Figs. 1A-1 B). In embodiments, the circuit model for the aptamer surface includes the use of three resistors and two capacitors, where Rsrepresents the solution resistance, RAptamer accounts for the resistance caused by the aptamer layer on the surface, and CPEAptamer represents the capacitance of this resistor (e.g., as shown in Figs. 1A-1 B and described in Eq. 1). Additionally, in embodiments, R Charge Transfer signifies the resistance of the faradaic current resulting from the redox reactions of MB on the electrodeelectrolyte interface.Normalized change i Eq. 1

[0058] In embodiments, described herein are two different aptamer sensors and / or methods, where one uses a redox label (e.g., MB-labeled aptamer) and the other uses a label-free aptamer, where each is suitable for PSM detection. In embodiments, detection is suitable from a biological sample. In embodiments, the biological sample is or comprises urine, phosphate-buffered saline (PBS) buffer, or diluted urine.

[0059] In embodiments, the impedance of the initial sensor surfaces (e.g., baselines) is measured when no PSM is present (e.g., by using blank solutions at various time intervals such as 10 minutes and 50 minutes). In embodiments, this duration allows sufficient time for the folding of aptamers to reach an equilibrium state. Subsequently, in embodiments, the impedance is measured after exposing the surfaces to various concentrations of the PSM after 10 min and 50 min time intervals. In embodiments, the EIS data is fitted with a circuit model (e.g., as shown in Figs. 1A-1B) and the normalized changes in different circuit elements are calculated and plotted against the PSM concentration. In embodiments, normalized change in RAptamer (e.g., Eq. 1) is selected as the sensor signal as it shows a linear relationship with the PSM concentration. In embodiments, the aptamer sensor is exposed to a solution containing PSM (the target QSM molecule), where PSM molecules bind with the binding sites of aptamers and the aptamers undergo structural changes (e.g., conformational changes). In embodiments, the rearrangement of the aptamers from the structural changes on the surface causes some changes in the porosity of the layer and affects the resistance to the non-faradaic current (RAptamer). As a result, in embodiments, by increasing the concentration of PSM (e.g., in PBS as shown in Fig. 2, and in diluted urine as shown in Fig. 3) the normalized change in RAptamerDBl / 163111002.1 12Attorney Docket No.: QSM-004PC / 126665-5004 increases indicating that upon the binding of PSM molecules with the aptamers, the surface becomes less porous.

[0060] In embodiments, and in reference to Figs. 1 A, 2, and 3, the redox (e.g., MB) molecules are attached to the aptamers and the faradaic current in this system is directly influenced by the proximity of redox (e.g., MB) molecules to the surface. In embodiments, as the redox (e.g., MB) labels approach the surface, the distance between them and the surface decreases, resulting in an increase in the faradaic current (e.g., a decrease in Rcharge Transfer). Conversely, in embodiments, when the redox (e.g., MB) labels move away from the surface, the distance increases, leading to a decrease in the faradaic current (e.g., an increase in Rcharge Transfer). In embodiments, and in reference to Figs. 1 A, 2, and 3, because no significant correlation between R charge Transfer and PSM concentration was observed, the rearrangement of aptamers notably impacted the layer’s porosity and the flow of non-faradaic current, but it did not significantly alter the proximity of the redox (e.g., MB) molecules to the surface. Therefore, in embodiments, there was not a significant change in the amount of faradaic current. In embodiments, by altering both the length / distance of the aptamer relative to the electrochemical sensor surface, and the positioning of redox molecule (e.g., MB), it is possible to observe a detectable alteration in the faradaic current. Thus, in embodiments, a subsequent change in Rcharge Transfer upon the binding of QSM molecules with the aptamer surface is detectable.

[0061] In embodiments, PSM detection is performed using non-redox-labeled aptamers (e.g., “label- free aptamer). In embodiments, to confirm that the observed RAptamer in the first platform (e.g., depicted in Figs. 1 A, 2, and 3), with MB-labeled aptamer is due to the changes in surface porosity, a second platform using MB-free aptamer (e.g., as illustrated in Fig. 1B) was used to measure PSM and compare (e.g., as shown in Fig. 4). In embodiments, this sensor configuration utilizes aptamers that are label-free. In embodiments, both the blank and sample solutions contain a redox molecule (e.g., MB).

[0062] In embodiments, this mechanism uses the faradaic current increases that occur when MB ions in the solution diffuse through the aptamer surface, where changes in Rcharge transfer indicate alterations in the porosity of the aptamer layer. In embodiments, methods using the MB-labeled aptamer show an increase in RAptamer and a decrease in aptamer layer porosity upon PSM binding. In embodiments, methods using label- free aptamer demonstrate an increase in Rcharge transfer. In embodiments, and in reference to Fig. 4, increasing concentration of PSM in PBS demonstrates a directly proportional increase in Rcharge transfer.

[0063] In embodiments, the redox molecule-labeled aptamer, and the methods herein, are suitable to detect Staphylococcus in urine samples. In embodiments, the Staphylococcus infections are tested in urineDBl / 163111002.1 13Attorney Docket No.: QSM-004PC / 126665-5004 samples from a wide range of animals, including livestock (e.g., cattle, sheep, goats, pigs horses, etc.), pets and companion animals (e.g., dogs, cats, racoons, squirrels, etc.), to various wildlife (e.g., bears, zebras, dolphins, seals, boars, deer, monkeys and apes, etc.). In embodiments, the urine belongs to a companion animal, such as a cat or dog. In embodiments, methods herein allow evaluation of the performance of an aptamer sensor and distinguish between Staphylococcus-positive and Staphylococcus-negative samples. In embodiments, the methods herein are veterinary methods.

[0064] In embodiments, measuring aptamer binding comprises measuring one or more electrochemical impedance spectroscopy (EIS) parameters at the surface of the electrochemical surface at one or more frequencies (e.g., as shown in Figs. 5-6). In embodiments, the EIS frequencies range from about 100 kHz to about 0.1 Hz, or about 10 Hz to about 5000 Hz, or about 50 Hz to about 4000 Hz. In embodiments, one or more frequencies comprises about 3714 Hz, about 1834 Hz, and about 68 Hz. In embodiments, detecting further comprises utilizing one or more machine-learning processes with the one or more EIS parameters. In embodiments, the one or more machine-learning processes comprises a support vector machine (SVM) and / or ensemble model (e.g., illustrative outputs as shown in Figs. 7-9). In embodiments, the one or more machine-learning processes classifies the biological sample as either Staphylococcus-positive or Staphylococcus-negative. In embodiments, methods herein utilize machine-learning (e.g., SVM and / or Ensemble models) which exhibit an accuracy, specificity, and / or sensitivity of about or at least about 70%, about or at least about 75%, about or at least about 80%, about or at least about 85%, about or at least about 90%, or about or at least about 95%.Kits

[0065] In embodiments, compositions of the present disclosure are assembled into a kit. In embodiments, the kit comprises one or more aptamers (e.g., in one or more vials, cryovials, tubes, etc.) for application in the field, laboratory, and / or for use in one or more methods as described herein. In embodiments, the kit comprises one or more electrochemical sensors, as described herein, for application in the field, laboratory, and / or for use in one or more methods as described herein. In embodiments, the kit is designed for off-the-shelf bacterial detection and / or urinalysis.

[0066] The kit described herein may include one or more containers housing components for performing the methods described herein and optionally instructions for use. Any of the kits described herein may further comprise components needed for performing the manufacturing methods described herein. Each component of the kits, where applicable, may be provided in liquid form (e.g., aptamers in aqueous solution,DBl / 163111002.1 14Attorney Docket No.: QSM-004PC / 126665-5004 electrochemical sensors in a preservative or buffer). In embodiments, some of the components are reconstitutable or otherwise processible (e.g., compositions of lyophilized aptamers and are reconstitutable), for example, by the addition of a suitable solvent, media, or other species, which may or may not be provided with the kit.

[0067] The kits may have a variety of forms, such as a blister pouch, a shrinkwrapped pouch, a vacuum sealable pouch, a sealable thermoformed tray, or a similar pouch or tray form, with the accessories loosely packed within the pouch, one or more tubes, containers, a box, or a bag.

[0068] In embodiments, the kits may optionally include instructions and / or promotion for use of the components provided. As used herein, "instructions" can define a component of instruction and / or promotion, and typically involve written instructions on or associated with packaging of the disclosure. Instructions also can include any oral or electronic instructions provided in any manner such that a user will clearly recognize that the instructions are to be associated with the kit, for example, audiovisual (e.g., videotape, DVD, etc.), Internet, and / or web-based communications, etc. As used herein, "promoted" includes all methods of doing business including methods of education, engineering instruction, scientific inquiry, discovery or development, academic research, manufacturing, chemical, cosmetic, and pharmaceutical industry activity including sales, and any advertising or other promotional activity including written, oral, and electronic communication of any form, associated with the disclosure. Additionally, the kits may include other components depending on the specific application, as described herein.

[0069] Without further elaboration, it is believed that one skilled in the art can, based on the above description, utilize the present disclosure to its fullest extent. The following specific embodiments are, therefore, to be construed as merely illustrative, and not limiting of the remainder of the disclosure in anyway whatsoever.DEFINITIONS

[0070] The following definitions are used in connection with the disclosure disclosed herein. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of skill in the art to which this disclosure belongs.

[0071] As used herein, “a,” “an,” or “the” can mean one or more than one.

[0072] In embodiments, the term “about” in reference to numbers or numerical ranges relates to + / -10%, e.g., where “about 50,” relates to 45 to 55. In embodiments, the term “about,” in reference to wholeDBl / 163111002.1 15Attorney Docket No.: QSM-004PC / 126665-5004 numbers where + / - 10% of the numerical value results in non-whole numbers, relates to the next consecutive rounded whole numbers, e.g., where “about 25” refers to 23, 24, 25, 26, 27, or 28, and the like. In embodiments, where the term “about,” in reference to whole numbers where + / - 10% of the numerical value results in non-whole numbers and where rounding does not result in separate whole numbers, the term relates to the next consecutive whole numbers, e.g., where “about 1” refers to 0, 1 , or 2, or “about 5” refers to 4, 5, or 6, and the like.

[0073] As referred to herein, all compositional percentages are by weight or amount of the total composition, unless otherwise specified. As used herein, the word “include,” and its variants, is intended to be non-limiting, such that recitation of items in a list is not to the exclusion of other like items that may also be useful in the compositions and methods of this technology. Similarly, the terms “can” and “may” and their variants are intended to be non-limiting, such that recitation that an embodiment can or may comprise certain elements or features does not exclude other embodiments of the present technology that do not contain those elements or features.

[0074] Although the open-ended term “comprising,” as a synonym of terms such as including, containing, or having, is used herein to describe and claim the disclosure, the present disclosure, or embodiments thereof, may alternatively be described using alternative terms such as “consisting of” or “consisting essentially of.”

[0075] In embodiments, as used herein, the words “preferred” and “preferably” refer to embodiments of the technology that afford certain benefits, under certain circumstances. However, other embodiments may also be preferred, under the same or other circumstances. Furthermore, the recitation of one or more preferred embodiments does not imply that other embodiments are not useful, and is not intended to exclude other embodiments from the scope of the technology.EXAMPLESExample 1: Detection of Bacterial Phenol-Soluble Modulins (PSMs) using Aptamer Electrochemical Sensors

[0076] Aptamer identification was performed through a bead-mediated systematic evolution of ligands by exponential enrichment (SELEX) platform through the following steps. Functionalization and Immobilization: the target QSMs PSMs were tagged with histidine and immobilized onto nickel-coated magnetic beads. Incubation with Aptamer Library: the magnetic beads, now functionalized with the target QSMs (PSM), were incubated with a library of aptamers. Rinsing: unbound aptamers were removed throughDBl / 163111002.1 16Attorney Docket No.: QSM-004PC / 126665-5004 a series of rinse steps. Elution and Sequencing: bound aptamers are eluted by changing the pH, where the aptamers that were eluted from PSM-coated magnetic beads were sequenced for identification. Iterative Screening: the process was repeated for 1-3 additional cycles, using a secondary library derived from the initially captured aptamers, to further refine and enhance for high-affinity aptamers. The results of the highest- affinity aptamers are summarized in Table 1 (SEQ ID NOs: 1-5).

[0077] The aptamers were then functionalized with a carbon-based or PEG-based linker molecule ending in a thiol group, enabling its attachment to the gold sensor surface. Consequently, the aptamer sensor was developed by modifying a gold surface with the aptamer using gold-thiol chemistry through the following steps: Cleaning the Gold Surface: the surface was first cleaned by rubbing with ethanol using a cotton-tipped swab. Then, 50 mM H2SO4 was deposited on the electrode, incubated for 5 minutes, and subsequently rinsed and dried. Aptamer Modification: the aptamer was attached by depositing it onto the gold surface. The sensor was then incubated for 1 hour in a humidity chamber, protected from light. After incubation, the sensor was rinsed and dried. Attachment of Mercaptohexanol Spacer Molecules: A 3 mM solution of mercaptohexanol was prepared and deposited on the gold surface. The sensor was incubated for 1 hour in a humidity chamber, protected from light, followed by rinsing and drying.

[0078] This example tested two different aptamer sensors - one using a methylene blue (MB)-labeled aptamer and the other a label-free aptamer for PSM detection in 1x phosphate buffered saline (PBS) buffer and diluted urine.

[0079] PSM Detection Using MB-labeled Aptamer: first the impedance of initial sensor surfaces (baselines) was measured when no PSM was present by using blank solutions at various time intervals (10 minutes and 50 minutes). This duration allowed sufficient time for the folding of aptamers to reach an equilibrium state. Subsequently, the impedance after exposing the surfaces to various concentrations of the PSM after 10 min and 50 min time intervals was measured. Then all the electrochemical impedance spectroscopy (EIS) data was fitted with the circuit model shown in Fig. 1 A or 1 B and the normalized changes in different circuit elements were calculated and plotted against the PSM concentration. Normalized change in RAptamer (e.g., as in Eq. 1) was selected as the sensor signal as it showed a linear relationship with the PSM concentration.

[0080] When the aptamer sensor was exposed to a solution containing PSM (the target QSM molecule), PSM molecules bound within the aptamer binding sites, triggering structural changes (e.g., conformational changes). The rearrangement of the aptamers on the electrochemical sensor surface caused changes in theDBl / 163111002.1 I7Attorney Docket No.: QSM-004PC / 126665-5004 porosity of the layer and affected the resistance to the non-faradaic current (RAptamer). As a result, by increasing the concentration of PSM in 1x PBS (e.g., as shown in Fig. 2) and diluted urine (e.g., as shown in Fig. 3), the normalized change in RAptamer increases indicated that upon the binding of PSM molecules within the aptamers, that the surface became less porous.

[0081] As shown in Fig. 1A, MB molecules were attached to the aptamers and the faradaic current in this system was directly influenced by the proximity of the MB molecules to the surface. As the MB labels approach the surface, the distance between them and the surface decreased, resulting in an increase in the faradaic current (decrease in R charge Transfer). Accordingly, when the MB labels moved away from the surface, the distance increased, leading to a decrease in the faradaic current (increase in R charge Transfer). Because no significant correlation between Rc arge Transfer and PSM concentration was observed, it could be inferred that while the rearrangement of aptamers notably impacted the layer’s porosity and the flow of non-faradaic current, it did not significantly alter the proximity of MB molecules to the surface. Therefore, there was not a significant change in the amount of faradaic current. However, altering both the length of the aptamer and the positioning of MB, we result in a detectable alteration in the faradaic current, and thus, a subsequent change in Rcharge Transfer upon the binding of QSM molecules with the aptamer surface.

[0082] PSM Detection Using Label-Free Aptamer: to confirm that the observed RAptamer in the first platform (e.g., as depicted in Figs. 1A, 2, and 3), with MB-labeled aptamer was due to the changes in surface porosity, a second platform using MB-free aptamer was utilized (e.g., as illustrated in Fig. 1B) to measure PSM in 1x PBS (e.g., as shown in Fig. 4). In this sensor, the aptamers were label-free, and both blank and sample solutions contained MB. The concentration of MB was consistent in both blank and sample solutions. MB concentrations at 4.8 pM, 9.6 pM, and 19.2 pM were tested. 19.2 M MB provided the highest signal to noise ratio.

[0083] In this mechanism, the faradaic current increased when MB ions in the solution diffused through the aptamer surface, where changes in Rcharge transfer indicated alterations in the porosity of the aptamer layer. The data with MB-labeled aptamers showed an increase in RAptamer and a decrease in aptamer layer porosity upon PSM binding, with an increased Rcharge transfer using label-free aptamer. The data with the label-free aptamer confirmed the previous results with the MB-labeled aptamer. As shown in Fig. 4, increasing the concentration of PSM in the 1x PBS solution leads to an increase in Rcharge transfer.

[0084] Urine samples from different animals were tested for the presence of Staphylococcus by detecting phenol-soluble modulins (PSMs) using the aptamer-coated electrochemical sensor. Each urineDBl / 163111002.1 18Attorney Docket No.: QSM-004PC / 126665-5004 sample was tested with different pH levels, blood content, crystals, and other characteristics, which were collected from companion pets (e.g., as described in Table 2). Cytology and aerobic bacterial tests were performed to determine the presence of Staphylococcus or other bacteria in the urine samples. Table 2 lists all 14 Sfaphy / ococcus-positive urine samples received, along with 13 Sfaphy / ococcus-negative samples randomly selected from the 52 Sfaphy / ococcus-negative samples received.Table 2: Illustrative characteristics of urine specimens tested with PSM aptamer sensor.DBl / 163111002.1 19Attorney Docket No.: QSM-004PC / 126665-5004

[0085] The urine samples were tested using the MB-labeled PSM aptamer (e.g., as illustrated in Fig. 1A) with an n = 4 after a 12-hour incubation at 37°C according to the following sample preparation steps:

[0086] Step 1 : Sample Preparation: added 100 pL of urine sample to 900 pL of Luria broth (LB) in artificial urine (1 .25 g LB / 50 mL artificial urine). Incubated the sample in an incubator at 37°C for 12-14 hours. Then a 10% test sample was made by adding 100 pL of overnight cultured urine sample to 900 pL of 1X PBS. Step 2: Testing the 10% Urine Sample Using the PSM Aptamer: the PSM aptamer was inserted, 100 pL of blank 1X PBS was added, and EIS was run to measure the initial surface impedance. Then, 100 pL of the 10% test sample was added to the surface and EIS was run to measure the final surface impedance.

[0087] Equivalent circuit analysis was highly accurate for analyzing EIS data of simple buffer solutions. Interpreting impedance data in complex biological solutions using equivalent circuit models was challenging. The varying characteristics of different biological components complicated the circuit fit, which made it difficult for a single circuit to accurately represent the surface interactions in all types of biological solutions (16).DB1 / 163111002.1 20Attorney Docket No.: QSM-004PC / 126665-5004Therefore, to analyze the EIS data of urine samples, a percentage change in different EIS parameters at different frequencies resulting from the addition of the urine sample and the structural changes on the surface was calculated. The two groups of Staphy / ococcus-positive and Staphy / ococcus-negative samples were compared, revealing significant differences (p-value < 0.005) in percentage changes in phase angle at 3714 Hz, phase angle at 1834 Hz, and Z real at 68 Hz (e.g., as shown in Fig. 5). This indicated that the observed differences were due to the presence of PSM in the positive samples and their binding with the PSM aptamer, indicating that the PSM aptamer successfully differentiated between positive and negative samples.

[0088] To further explore sample-to-sample variations and the effect of different urine characteristics, changes in phase angle at 3714 Hz for all positive and negative urine samples were individually analyzed (e.g., as percentage change in phase angle as shown in Fig. 6). As shown in Fig. 6, the data indicated that the Sfapfiy / ococcus-negative samples with too many bacterial cells in cytology (TNTC) are more likely to cause false positives. Additionally, Staphylococcus-negative samples with high or medium amounts of blood may also generate false positive results.

[0089] The scatter plots in Fig. 7 illustrate the correlation between the variables that were significantly different between Staphylococcus-positive and Staphylococcus-negative samples. Each scatter plot demonstrates the relationship between pairs of these features, providing a visual representation of how they distinguish between positive and negative samples. Leveraging these three features, two machine learning algorithms for analysis of urine samples data were utilized. The first model that utilized a simple support vector machine (SVM) and the second model utilized a more complex ensemble model. Each of the models utilized the distinct patterns revealed in the scatter plots in Fig. 7 to analyze the samples and predict their classification as either positive or negative.

[0090] Fig. 8 shows the 5-fold cross validation confusion matrix of the SVM model. In 5-fold cross- validation, the dataset was randomly divided into five parts. Each part took a turn as the test set while the others were used fortraining. The model's accuracy, specificity, and sensitivity were scored for each iteration, and the overall performance was averaged from these scores.

[0091] The 5-fold cross-validation for the SVM and the ensemble model was repeated five times, and as shown in Fig. 9 demonstrated the average accuracy, specificity, and sensitivity of these models. The error bars represent the standard deviation across these repetitions. As shown in Fig. 9, the SVM model demonstrated slightly better performance, with an accuracy of 79.48%, specificity of 76%, and sensitivity of 82.68%.DBl / 163111002.1 21Attorney Docket No.: QSM-004PC / 126665-5004

[0092] The data demonstrated, inter alia, that the aptamers were capable of binding PSMs and that the aptamer-coated electrochemical sensors were able to detect the presence of bacteria with high accuracy, specificity, and sensitivity.

[0093] While the disclosure has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any variations, uses, or adaptations of the disclosure following, in general, the principles of the disclosure and including such departures from the present disclosure as come within known or customary practice within the art to which the disclosure pertains and as may be applied to the essential features hereinbefore set forth and as follows in the scope of the appended claims.REFERENCES

[0094] Naga NG, El-Badan DE, Ghanem KM, Shaaban Ml. It is the time for quorum sensing inhibition as alternative strategy of antimicrobial therapy. Cell Communication and Signaling. 2023;21 (1):133.

[0095] Sismaet HJ, Pinto AJ, Goluch ED. Electrochemical sensors for identifying pyocyanin production in clinical Pseudomonas aeruginosa isolates. Biosens Bioelectron. 2017;97:65-9.

[0096] Arishi WA, Eissa S, Al-Kattan K, Zourob M. Aptamer-based label-free electrochemical biosensors for the diagnosis of sickle cell anemia. Biosens Bioelectron X. 2023; 14: 100389.

[0097] Long LL, Hu WX, Wang X, Yuan R, Chai YQ. Antibody-Protein-Aptamer Electrochemical Biosensor based on Highly Efficient Proximity-Induced DNA Hybridization on Tetrahedral DNA Nanostructure for Sensitive Detection of Insulin-like Growth Factor-1 . Anal Chem. 2024 Mar 5;96(9):3837- 43.

[0098] Li L, Chen Z. Electrochemical aptamer biosensor for DNA detection based on label-free aptamers. Bioelectrochemistry. 2023; 153: 108494.

[0099] Fan J, Yang W. Electrochemical DNA / aptamer biosensors based on SPAAC for detection of DNA and protein. Sens Actuators B Chem. 2022;353: 131100.

[0100] Capatina D, Lupoi T, Feier B, Blidar A, Hosu O, Tertis M, et al. Label-Free Electrochemical Aptasensor for the Detection of the 3-O-C12-HSL Quorum-Sensing Molecule in Pseudomonas aeruginosa. Biosensors (Basel). 2022; 12(7).DBl / 163111002.1 22Attorney Docket No.: QSM-004PC / 126665-5004

[0101] Duan N, Ye M, Lu M, Chen X, Wu S. DNA aptamers selection and characterization for development of impedimetric aptasensor for Bacillus cereus at different growing stages. Advanced Agrochem. 2023;2(3):284-90.

[0102] Panahi Z, Ren T, Halpern JM. Nanostructured Cyclodextrin-Mediated Surface for Capacitive Determination of Cortisol in Multiple Biofluids. ACS Appl Mater Interfaces. 2022 Sep 21 ;14(37):42374— 87.

[0103] Otto M. Phenol-soluble modulins. International Journal of Medical Microbiology. 2014; 304(2): 164-9.

[0104] Peschel A, Otto M. Phenol-soluble modulins and staphylococcal infection. Nat Rev Microbiol. 2013;11 (10):667— 73.

[0105] Kim DM, Go MJ, Lee J, Na D, Yoo SM. Recent Advances in Micro / Nanomaterial-Based Aptamer Selection Strategies. Molecules. 2021 ;26(17).

[0106] Tomita Y, Morita Y, Suga H, Fujiwara D. DNA Module Platform for Developing Colorimetric Aptamer Sensors. Biotechniques. 2016 Jun 1 ;60(6):285-92.

[0107] Cecchetto J, Fernandes FCB, Lopes R, Bueno PR. The capacitive sensing of NS1 Flavivirus biomarker. Biosens Bioelectron. 2017;87:949-56.

[0108] Fernandes FCB, Santos A, Martins DC, Goes MS, Bueno PR. Comparing label free electrochemical impedimetric and capacitive biosensing architectures. Biosens Bioelectron. 2014;57:96-102.

[0109] Rong Y, Padron A V, Hagerty KJ, Nelson N, Chi S, Keyhani NO, et al. Post hoc support vector machine learning for impedimetric biosensors based on weak protein-ligand interactions. Analyst. 2018; 143(9):2066— 75.EQUIVALENTS

[0110] While the disclosure has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any variations, uses, or adaptations of the disclosure following, in general, the principles of the disclosure and including such departures from the present disclosure as come within known or customary practice within the art to which the disclosure pertains and as may be applied to the essential features hereinbefore set forth and as follows in the scope of the appended claims.DBl / 163111002.1 23Attorney Docket No.: QSM-004PC / 126665-5004

[0111] Those skilled in the art will recognize, or be able to ascertain, using no more than routine experimentation, numerous equivalents to the specific embodiments described specifically herein. Such equivalents are intended to be encompassed in the scope of the following claims.INCORPORATION BY REFERENCE

[0112] All patents and publications referenced herein are hereby incorporated by reference in their entireties.

[0113] The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present disclosure is not entitled to antedate such publication by virtue of prior disclosure.

[0114] As used herein, all headings are simply for organization and are not intended to limit the disclosure in any manner. The content of any individual section may be equally applicable to all sections.DBl / 163111002.1 24

Claims

Attorney Docket No.: QSM-004PC / 126665-5004CLAIMSWhat is claimed is:1 . An aptamer comprising a DNA sequence having about or at least about 80% sequence identity to a DNA sequence selected from one or more of SEQ ID NOs: 3, 1-2, and 4-5.

2. The aptamer of claim 1 , comprising a DNA sequence having about or at least about 85%, about or at least about 90%, about or at least about 95%, about or at least about 96%, about or at least about 97%, about or at least about 98%, or about or at least about 99% sequence identity to a DNA sequence selected from one or more of SEQ ID NOs: 3, 1-2, and 4-5.

3. The aptamer of claim 1 or 2, comprising a DNA sequence selected from one or more of SEQ I D NOs: 3, 1-2, and 4-5.

4. The aptamer of claim 3, wherein the aptamer has a DNA sequence of SEQ ID NO: 3.

5. The aptamer of any one of claims 1-3, wherein the aptamer binds one or more phenol-soluble modulins (PSMs).

6. The aptamer of claim 5, wherein the one or more PSMs are from one or more Staphylococcus bacteria.

7. The aptamer of any one of claims 1-6, wherein the aptamer is functionalized with a carbon-based linker molecule, polyethylene glycol (PEG) linker molecule, polyethylene oxide (PEO) linker molecule, and / or polyoxyethylene (POE) linker molecule.

8. The aptamer of claim 7, wherein the linker molecule is functionalized with one or more thiol group, sulfhydryl-reactive PEGylated crosslinker, amine group, amine-reactive PEGylated crosslinker, biotin group, biotinylated PEGylated crosslinker, optionally at an end of the linker molecule.

9. The aptamer of claim 7 or 8, wherein the linker molecule comprises a PEG linker that is or comprises one or more of PEG 200, PEG 300, PEG 500, PEG 600, PEG 1000, PEG 2000, PEG 3350, PEG 6000, PEG 8000, PEG 10000, PEG 15000, and PEG 20000.

10. The aptamer of any one of claims 1 -9, wherein the aptamer is suitable for attachment to a surface, optionally a metal surface.

11. The aptamer of claim 10, wherein the surface is or comprises a gold surface, and the aptamer is optionally attached using gold-thiol chemistry.DBl / 163111002.1 25Attorney Docket No.: QSM-004PC / 126665-500412. The aptamer of any one of claims 1-11 , wherein the aptamer is labeled with a redox molecule.

13. The aptamer of claim 12, wherein the redox molecule is or comprises methylene blue (MB), azure B, and / or azurin, optionally wherein the redox molecule is fluorescent or fluorescently labeled.

14. An electrochemical sensor comprising a surface having disposed thereon one or more aptamers of any one of claims 1-13, wherein the one or more aptamers binds one or more phenol-soluble modulins (PSMs).

15. The electrochemical sensor of claim 14, wherein the one or more aptamers comprise a DNA sequence having about or at least about 80% sequence identity to a DNA sequence selected from one or more of SEQ ID NOs: 1-5.

16. The electrochemical sensor of claim 14 or 15, wherein the one or more aptamers comprises a DNA sequence having about or at least about 85%, about or at least about 90%, about or at least about 95%, about or at least about 96%, about or at least about 97%, about or at least about 98%, or about or at least about 99% sequence identity to a DNA sequence selected from one or more of SEQ ID NOs: 1-5.

17. The electrochemical sensor of any one of claims 14-16, wherein the one or more aptamers comprises a DNA sequence selected from one or more of SEQ ID NOs: 3, 1-2, and 4-5.

18. The electrochemical sensor of claim 17, wherein the electrochemical sensor comprises an aptamer having a DNA sequence of SEQ ID NO: 3.

19. The electrochemical sensor of any one of claims 14-18, wherein the PSMs are synthesized by one or more Staphylococcus bacteria.

20. The electrochemical sensor of any one of claims 14-19, wherein the surface having disposed thereon the one or more aptamers comprises one or more metals.21 . The electrochemical sensor of claim 20, wherein the metal is or comprises gold.

22. The electrochemical sensor of any one of claims 14-21 , wherein the one or more aptamers are attached to the surface via gold-thiol chemistry.

23. The electrochemical sensor of any one of claims 14-22, wherein the electrochemical sensor is configured to provide and / or measure one or more signals as a function of binding the one or more PSMs.DBl / 163111002.1 26Attorney Docket No.: QSM-004PC / 126665-500424. The electrochemical sensor of claim 23, wherein one or more signal comprises, or is related to, a solution resistance, impedance, impedimetric detection, capacitance, and / or porosity.

25. The electrochemical sensor of any one of claims 14-24, wherein the electrochemical sensor is suitable to detect the one or more phenol-soluble modulins (PSMs) by electrochemical impedance spectroscopy (EIS).

26. The electrochemical sensor of any one of claims 14-25, wherein the electrochemical sensor detects the presence of bacteria as a function of binding one or more PSMs.

27. A method of identifying and / or generating one or more aptamers that bind one or more phenol- soluble modulins (PSMs), the method comprising: i. tagging one or more PSMs with histidine; ii. immobilizing the one or more PSMs onto nickel-coated magnetic beads to generate one or more PSM-labeled magnetic beads; ill. contacting the one or more PSM-labeled magnetic beads with a first library of aptamers; iv. separating, using a magnetic field, one or more aptamers from the first library of aptamers as a function of binding to the PSM-labeled magnetic beads; and v. sequencing the one or more bound aptamers to determine the nucleic acid sequence that bound the one or more PSMs.

28. The method of claim 27, further comprising eluting the one or more bound aptamers from the PSM- labeled magnetic beads, optionally by changing the pH (decrease or increase), changing the temperature (increase or decrease), or removing from the magnetic beads, optionally using imidazole.

29. The method of claim 27 or 28, further comprising repeating one or more of steps (iii), (iv), and (v) using a second library of aptamers.

30. The method of any one of claims 27-29, further comprising synthesizing the one or more bound aptamers from the sequencing.

31. The method of claim 30, further comprising attaching the synthesized aptamers to a surface of an electrochemical sensor, optionally using gold-thiol chemistry.

32. A method of detecting the presence of bacteria in a biological sample comprising: i. providing the biological sample;DBl / 163111002.1 27Attorney Docket No.: QSM-004PC / 126665-5004 ii. contacting the biological sample with a surface of an electrochemical sensor of any one of claims 14-26, wherein the electrode surface comprises one or more aptamers disposed thereon, and wherein the one or more aptamers binds one or more phenol-soluble modulins (PSMs); and iii. detecting the presence of bacteria in the biological sample as a function of measuring aptamer binding of one or more phenol-soluble modulins (PSMs) on the surface of the electrochemical sensor.

33. The method of claim 32, wherein the bacteria is one or more Staphylococcus bacteria.

34. The method of claim 32 or 33, wherein the biological sample is or comprises urine.

35. The method of claim 34, wherein the urine is non-human, optionally canine or feline.

36. The method of any one of claims 32-35, wherein the aptamer is labeled with a redox molecule, optionally methylene blue (MB), azure B, and / or azurin, and optionally wherein the redox molecule is fluorescent or fluorescently labeled.

37. The method of any one of claims 32-35, wherein the aptamer is not labeled with a redox molecule.

38. The method of claim 37, further comprising adding a redox molecule to the electrochemical sensor during the contacting step.

39. The method of claim 38, wherein the redox molecule is methylene blue (MB), azure B, and / or azurin, optionally wherein the redox molecule is fluorescent or fluorescently labeled, and wherein the redox molecule is optionally added at a concentration of about or at least about 1 pM, about or at least about 5 pM, about or at least about 10 pM, about or at least about 20 pM, about or at least about 50 pM, or about or at least about 100 pM.

40. The method of any one of claims 32-39, wherein measuring aptamer binding comprises measuring one or more electrochemical impedance spectroscopy (EIS) parameters at the surface of the electrochemical surface at one or more frequencies.41 . The method of claim 40, wherein the one or more frequencies comprises about 3714 Hz, about 1834 Hz, and about 68 Hz.

42. The method of claim 40 or 41 , wherein detecting further comprising utilizing one or more machinelearning processes with the one or more EIS parameters.DBl / 163111002.1 28Attorney Docket No.: QSM-004PC / 126665-500443. The method of claim 42, wherein the one or more machine-learning processes comprises a support vector machine (SVM) and / or ensemble model.

44. The method of claim 42 or 43, wherein the one or more machine-learning processes classifies the biological sample as either Staphytococcus-positive or Staphy / ococcus-negative.

45. A kit comprising:(i) one or more aptamers from any one of claims 1 -13; and / or(ii) one or more electrochemical sensors of any one of claims 14-26, and optionally one or more redox molecules, optionally methylene blue (MB), azure B, and / or azurin.DBl / 163111002.1 29

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