Apparatus and methods for rapid detection of bacteria

Abstract

Various embodiments are described herein for a device and method for detecting bacteria. Included is a needle apparatus for detecting a bacteria comprising a plurality of working electrodes, each working electrode of the plurality having a detecting face and extending from the detecting end to the connecting end and comprising: an electrically conductive surface the detecting face of each working electrode, a peptidic probe immobilized on at least a portion of the electrically conductive surface, the peptidic probe comprising an electroactive moiety and a cleavable peptide, the cleavable peptide optionally comprising at least one linker; a reference electrode; an electrode spacer configured to support and align the plurality of working electrodes and the reference electrode each inserted or insertable therein; a syringe hub counter-electrode, optionally configured to mate with and receive the electrode spacer.

Description

Apparatus and methods for rapid detection of bacteriaCROSS-REFERENCE TO RELATED APPLICATION

[0001] This application claims the benefit of priority of United States Provisional Application No. 63 / 742,266, filed January 6, 2025, the entire contents of which is incorporated by reference in its entirety.SEQUENCE LISTING

[0002] A computer readable form of the Sequence Listing “P93249722WO01 Sequence Listing” (62416 bytes) created on January 6, 2026, is herein incorporated by reference.FIELD

[0003] The various embodiments described herein generally relate to peptidic probes, and various apparatuses for detecting bacteria, particularly for rapid detection of bacteria and methods of use thereof.BACKGROUND

[0004] Infections such as bacterial infections can be a serious issue which can require immediate medical attention. Bacterial diseases such as necrotizing fasciitis or wound infections can result in significant morbidity and even death.

[0005] Rapid or real time identification of bacterial species would allow for immediate intervention if necessary. Devices and methods that permit rapid or real time identification would be useful in a variety of settings including a health clinic, hospital, or emergency room, for example.

[0006] Rapidly identifying the causative bacteria in infectious diseases, such as necrotizing fasciitis (NF), can be crucial for limiting morbidity and determining optimal surgical and antibiotic strategies. The current standard for diagnosis is bacterial culture analysis - a process which can take days or weeks. Other methods include histological or molecular analyses of aspirate and / or tissue biopsy, such as polymerase chain reaction (PCR) analysis. However, PCR analysis is limited by inhibitors, contamination, and high cost. Enzyme-linked immunosorbent assay (ELISA) offers relatively quick results but is also time-consuming and costly. Chromatography combined with mass spectrometry is another option but requires specialized facilities and expertise. In many cases, these results are inconclusive, leaving patients in critical need of care.

[0007] Electrochemical systems when utilized for analysis can enable techniques which provide rapid measurements (seconds to minutes), high sensitivity (<pM), and low costs. They also have great adaptability to small size, making them suitable for integration into diagnostic devices. Operating via electron transfer between molecules of interest and theelectrode surface at a specific voltage, these systems require no labels, tags, or extensive sample preparation. Selectivity can be adjusted to target specific molecules, and the equipment is compact and portable, requiring no expensive infrastructure. Electrochemical cells typically consist of three types of electrodes: the working electrode (WE), reference electrode (RE), and counter electrode (CE). Arrays of WE can be combined with the same RE and CE for simultaneous measurements, allowing for multianalyte detection.

[0008] Furthermore, electrode surfaces can be functionalized to include nanostructures, such as nanospikes, and even further functionalized to immobilize peptidic probes on the surface of said nanospikes. Said peptidic probes could include, for example, cleavable peptide sequences that are selectively cleaved by enzymes which are known to be secreted by specific bacteria so as to increase selectivity of the electrochemical system. Without wishing to be bound to theory, the nanospikes on the surface of the electrode can increase the effective e.g., electrochemical active surface area, allowing for example for improved peptidic probe immobilization, by offering more binding sites for peptidic probes, and / or improving their exposure to the surrounding medium. This increased surface area also allows for enhanced mass transport, thus enabling peptidic probes and enzymes to interact more effectively, which may improve enzyme cleavage efficiency. Moreover, the sharp features of the nanospikes can influence peptidic probe conformation such that it may adopt a more favorable orientation, increasing enzyme accessibility. Nanospikes may also enhance electron transfer rates, which enable more sensitive electrochemical monitoring of enzymatic reactions.

[0009] Despite the existence of bacterial biosensors, they often lack specificity, especially in the presence of biofilms, which are prevalent in over 80% of human bacterial infections.

[0010] There is an urgent need for faster and / or more specific diagnostic approaches for bacterial infections.SUMMARY OF VARIOUS EMBODIMENTS

[0011] An aspect of the disclosure provides a working electrode for detecting a bacteria comprisinga body having a detecting face and an electrically conductive surface on at least the detecting face, anda peptidic probe immobilized on at least a portion of the electrically conductive surface, the peptidic probe comprising an electroactive moiety and a cleavable peptide, the cleavable peptide optionally comprising at least one linker.

[0012] Another aspect of the disclosure provides a needle apparatus for detecting a bacteria comprisinga detecting end and a connecting end,a plurality of working electrodes, each working electrode of the plurality having a detecting face and extending from the detecting end to the connecting end and comprising:an electrically conductive surface on at least the detecting face of each working electrode,a peptidic probe immobilized on at least a portion of the electrically conductive surface, the peptidic probe comprising an electroactive moiety and a cleavable peptide, the cleavable peptide optionally comprising at least one linker;a reference electrode;an electrode spacer configured to support and align the plurality of working electrodes and the reference electrode each inserted or insertable therein;a syringe hub counter-electrode, optionally configured to mate with and receive the electrode spacer;wherein each working electrode of the plurality having a detecting face and extending from the detecting end to the connecting end; and wherein the plurality of working electrodes is substantially parallel at least at and approaching the detecting end.

[0013] Also provided is a microelectrode apparatus, methods of making and using, as well as isolated peptides, peptidic probes, compositions, kits and packages.

[0014] Other features and advantages of the present application will become apparent from the following detailed description. It should be understood, however, that detailed description and 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 with the description as a whole.BRIEF DESCRIPTION OF THE DRAWINGS

[0015] For a better understanding of the various embodiments described herein, and to show more clearly how these various embodiments may be carried into effect, reference will be made, by way of example, to the accompanying drawings which show at least one example embodiment, and in which:FIG. 1 A shows a diagram of exemplary peptidic probes with different PEG spacer lengths, where Fc refers to a ferrocenyl electroactive moiety and PEP refers to peptide.FIG. 1B shows exemplary electroactive moieties, including Formulas l-VI.FIG. 2A shows an electron microscopy image of the gold working electrode after to gold nanospike surface modification prepared according to the method described in Example 2 at low magnification of 6500X.FIG. 2B shows an electron microscopy image of the gold working electrode with gold nanospike-modified surface prepared according to the method described in Example 2 at a high magnification of 50000X.FIG. 3 shows a cyclic voltammetry graph comparing peptidic probe immobilization incubation times as described further in Example 3.FIG. 4A shows a cyclic voltammetry graph comparing different scan rates (0.05-1.0 V / s) after peptidic probe immobilization as described further in Example 3.FIG. 4B shows the relationship between anodic peak current and the square root of the scan rate, as described further in Example 3.FIG. 5A shows a cyclic voltammetry graph comparing gold working electrodes with gold nanospike-functionalized modified surface with peptidic probe immobilized on the surface of the gold nanospikes before and after the addition of metalloprotease SepA with a scan rate of 0.5 V / s as described further in Example 4.FIG. 5B shows a square wave voltammetry plot comparing gold working electrodes with gold nanospike-functionalized modified surface with peptidic probe immobilized on the surface of the gold nanospikes both before and after the addition of metalloprotease SepA with a pulse height of 25 mV, step height of 10 mV, and frequency of 100 Hz as described further in Example 4.FIG. 6 shows the relationship between the logarithm of SepA concentration (log[SepA (ng / mL)]) and the percentage in peak current (lo-l / lo) at 37°C, with a linear range between 0.01 and 1 ng / mL, as described further in Example 4.FIG. 7A shows a cyclic voltammetry graph comparing different scan rates ranging from 0.1 V / s to 2 V / s in 10mM PBS applied to the gold working electrode with Ferrocenylcarbonyl-Phe-Gly-p-(2-furyl)-Ala-PEG2-Cys immobilized on the surface as described further in Example 5.FIG. 7B shows a linear calibration curve of the redox peaks against the scan rates as seen in FIG. 7A, with an adjusted R2of 0.9915, as described further in Example 5.FIG. 7C shows a cyclic voltammetry graph comparing the gold working electrode with Ferrocenylcarbonyl-Phe-Gly-p-(2-furyl)-Ala-PEG2- Cys immobilized on the surface (dashes), the gold nanostructure-modified gold working electrode with Ferrocenylcarbonyl-Phe-Gly-p-(2-furyl)-Ala-PEG2-Cys immobilized on the surface (black line), as described further in Example 5.FIG. 7D shows cyclic voltammetry graph of different peptide incubation duration, showing the relationship between immobilization and current gain as described further in Example 5.FIG. 8A shows a square wave voltammetry graph of the gold nanostructure-modified gold working electrode with Ferrocenylcarbonyl-Phe-Gly-p-(2-furyl)-Ala-PEG2-Cys immobilized on the surface in 10 mM PBS at pH 6.8 over a potential range of 0.15 to 0.75 V that was completed in triplicate, as described further in Example 5.FIG. 8B shows the average decrease percentage of the current of the gold nanostructure-modified gold working electrode with Ferrocenylcarbonyl-Phe-Gly-p-(2-furyl)-Ala-PEG2-Cys immobilized on the surface when in contact with 10 mM PBS buffer after 15, 30, 40, and 60 minutes as measured with square wave voltammetry in 10 mM PBS at pH 6.8, as described further in Example 5.FIG. 9A shows a cyclic voltammetry graph of the Ferrocenylcarbonyl-Phe-Gly-P-(2-furyl)-Ala-PEG2-Cys modified electrode in contact with 0.0003 mg / mL of aureolysin after 0, 15, 30, and 45 minutes over a potential range of 0.12 to 0.8 V at 1 V / s in 10 mM PBS at pH 6.8, as described further in Example 5.FIG. 9B shows a square wave voltammetry graph showing the average current decrease versus the logarithm of different concentrations of aureolysin for the gold nanostructure-modified gold working electrode with Ferrocenylcarbonyl-Phe-Gly-p-(2-furyl)-Ala-PEG2-Cys immobilized on the surface in 10 mM PBS at pH 6.8, with an R2of 0.9941, as described further in Example 5.FIG. 10A shows a schematic of a needle apparatus as described further in Example 7.FIG. 10B shows a schematic of a top perspective view of the detecting end of a needle apparatus as described further in Example 7.FIG. 10C shows a schematic of the 3D-printed electrode spacer as described further in Example 7.FIG. 10D shows a schematic of the 3D-printed support assembly as described further in Example 7.FIG. 10E show cyclic voltammetry graphs comparing individual (dotted lines) and simultaneous (full lines) electrochemical responses of four electrodes in 5 mM ferrocyanide solution as described further in Example 7.FIG. 11 A shows images of the method of using the needle apparatus to detection pyocyanin in a cadaver pig leg as described further in Example 8.FIG. 11 B shows cyclic voltammetry graphs resulting from applying range of 0.8 to -0.8V at 0.05V / S in cadaver pig leg without and with a solution of NaCI, or pyocyanin at 1mM as described further in Example 8.FIG. 12A shows cyclic voltammetry graphs resulting from applying a voltage range of 0.0 to 0.6 V at 0.05 V / s in 1 mM ferrocyanide solution, as described further in Example 11.FIG. 12B shows a cyclic voltammetry graph for device electrodes recorded in 10mM Fe(C5H5)2and 10mM KOI, at 0.5V / s from 1 to -1V for electrode set up as shown in FIG.10A with 4 working electrodes as described further in Example 7 and Example 11.FIG. 13A is a side cross section view of an embodiment of the needle apparatus, prior to embedding wires and polishing the detecting end.FIG. 13B is a side cross section view of an embodiment of the embedded and polished needle apparatus.FIG. 13C is a front view of the needle apparatus in an embodiment, showing 8 working electrodes.FIG. 13D is a front view of the needle apparatus in an embodiment, showing 16 working electrodes.FIG. 14A is a side cross section view of an embodiment of the needle apparatus, wherein one of the working electrodes is larger than other working electrodes.FIG. 14B is a front view of FIG. 14A.FIG. 15A is a side cross section view of an embodiment of the needle apparatus wherein the detecting end is polished at an angle.FIG. 15B is a front view of FIG. 15A.FIG. 16A is a side cross section view of an embodiment of the microelectrode apparatus showing a plurality of connectors connected to the connecting end of the plurality of working electrodes.FIG. 16B is a front view of FIG. 16A.FIG. 17A shows four plots of peak current density versus the scan rate of nanospiked sensors (e.g. working electrodes) functionalized with peptidic probes comprising various spacer lengths (PEG2, PEG4, PEG6, and PEG12) in Ferrocenylcarbonyl-Phe-Gly-P-(2-furyl)-Ala-PEGx-Cys as described further in Example 12.FIG. 17B shows the plot of current change over time using square wave voltammetry recorded after 15, 30, 45, and 60 minutes of incubation at room temperature in PBS 1X pH 7 as described further in Example 12.FIG. 17C shows the square wave voltammetry graphs and corresponding calibration curves of nanospiked sensors functionalized with PEG-peptides (e.g. peptidic probes) in 1X PBS as described further in Example 12.FIG. 18A shows a plot of the current decrease from square wave voltammetry responses of the nanospiked sensor functionalized with PEG2 in different bacterial culture supernatants as described further in Example 13.FIG. 18B shows a calibration curve obtained by square wave voltammetry in PBS 1X pH 7 in staphylococcus aureus supernatant as described further in Example 13.FIG. 19A shows a square wave voltammetry graph with varying pulse heights as described further in Example 14.FIG. 19B shows a square wave voltammetry graph with varying frequencies as described further in Example 14.FIG. 20A shows a cyclic voltammetry graph comparing Cys-PEG2-Phe-Met-Ser-Leu-Leu-Ser-Met-Lys-(Ferrocenylcarbonyl) (SEQ ID NO: 68) modified gold nanospiked working electrodes before and after exposure to metalloprotease SepA as described further in Example 15.FIG. 20B shows a square wave voltammetry plot comparing Cys-PEG2-Phe-Met-Ser-Leu-Leu-Ser-Met-Lys-(Ferrocenylcarbonyl) (SEQ ID NO: 68) modified gold nanospiked working electrodes before and after exposure to metalloprotease SepA as described further in Example 15.FIG. 200 shows a cyclic voltammetry graph comparing different scan rates (0.2-1.0 V / s) of Cys-PEG2-Phe-Met-Ser-Leu-Leu-Ser-Met-Lys-(Ferrocenylcarbonyl) (SEQ ID NO: 68) modified gold nanospiked working electrodes, as described further in Example 15.FIG. 20D shows the relationship between peak current density and the scan rate as described further in Example 15.FIG. 20E shows a calibration curve based on the square wave voltammetry responses of Cys-PEG2-Phe-Met-Ser-Leu-Leu-Ser-Met-Lys-(Ferrocenylcarbonyl) (SEQ ID NO: 68) modified gold nanospiked working electrodes in PBS with commercial enzyme as described further in Example 15.FIG. 20F shows a linear calibration model created from square wave voltammetry responses of Cys-PEG2-Phe-Met-Ser-Leu-Leu-Ser-Met-Lys-(Ferrocenylcarbonyl) (SEQ ID NO: 68) modified gold nanospiked working electrodes, where the percentage signal change (Al / lo) was plotted against the bacterial concentration as described further in Example 15.FIG. 20G shows a polynomial calibration model created from square wave voltammetry responses of Cys-PEG2-Phe-Met-Ser-Leu-Leu-Ser-Met-Lys-(Ferrocenylcarbonyl) (SEQ ID NO: 68) modified gold nanospiked working electrodes, where the percentage signal change (Al / lo) was plotted against the bacterial concentration as described further in Example 15.FIG. 20H shows the square wave voltammetry responses of Cys-PEG2-Phe-Met-Ser-Leu-Leu-Ser-Met-Lys-(Ferrocenylcarbonyl) (SEQ ID NO: 68) modified gold nanospiked working electrodes in supernatants obtained from the target bacterium, Staphylococcus epidermidis, and three non-target species, including Staphylococcus aureus, Pseudomonas aeruginosa, and Escherichia coli as described further in Example 15.FIG. 21 A shows a cyclic voltammetry graph comparing Cys-PEG2-Gly-Ala-Thr-Leu-Thr-Tyr-Thr-Lys-(Ferrocenylcarbonyl) (SEQ ID NO: 69) modified gold nanospiked working electrodes before and after exposure to metalloprotease SepA as described further in Example 16.FIG. 21 B shows a square wave voltammetry plot comparing Cys- PEG2-Gly-Ala-Thr-Leu-Thr-Tyr-Thr-Lys-(Ferrocenylcarbonyl) (SEQ ID NO: 69) modified gold nanospiked working electrodes before and after exposure to metalloprotease SepA as described further in Example 16.FIG. 210 shows a cyclic voltammetry graph comparing different scan rates (0.2-1.0 V / s) of Cys-PEG2-Gly-Ala-Thr-Leu-Thr-Tyr-Thr-Lys-(Ferrocenylcarbonyl) (SEQ ID NO: 69) modified gold nanospiked electrodes, as described further in Example 16.FIG. 21 D shows the relationship between peak current density and the scan rate as described further in Example 16.FIG. 21 E shows a calibration curve based on the square wave voltammetry responses of Cys-PEG2-Gly-Ala-Thr-Leu-Thr-Tyr-Thr-Lys-(Ferrocenylcarbonyl) (SEQ ID NO: 69) modified gold nanospiked working electrodes in standard SepA solutions as described further in Example 16.FIG. 21 F shows a linear calibration model created from square wave voltammetry responses of Cys-PEG2-Gly-Ala-Thr-Leu-Thr-Tyr-Thr-Lys-(Ferrocenylcarbonyl) (SEQ ID NO: 69) modified gold nanospiked working electrodes, where the percentage signal change (Al / lo) was plotted against the bacterial concentration as described further in Example 16.FIG. 21 G shows the square wave voltammetry responses of Cys- PEG2-Gly-Ala-Thr-Leu-Thr-Tyr-Thr-Lys-(Ferrocenylcarbonyl) (SEQ ID NO: 69) modified gold nanospiked working electrodes in supernatants obtained from the target bacterium, Staphylococcusepidermidis, and three non-target species, including Staphylococcus aureus, Pseudomonas aeruginosa, and Escherichia coli as described further in Example 16.FIG. 22A shows the changes in the electrochemical signal before and after immersion in different media (phosphate-buffered saline, staphylococcus epidermidis 24 h culture supernatant, and tryptic soy broth) for the Cys-PEG2-Phe-Met-Ser-Leu-Leu-Ser-Met-Lys-(Ferrocenylcarbonyl) (SEQ ID NO: 68) modified gold nanospiked working electrode as described further in Example 17.FIG. 22B shows the changes in the electrochemical signal before and after immersion in different media (phosphate-buffered saline, staphylococcus epidermidis 24 h culture supernatant, and tryptic soy broth) for the Cys-PEG2-Gly-Ala-Thr-Leu-Thr-Tyr-Thr-Lys-(Ferrocenylcarbonyl) (SEQ ID NO: 69) modified gold nanospiked working electrode as described further in Example 17.FIG. 23A shows cyclic voltammetry graphs resulting from bare gold electrodes recorded in 0.5 M H2SO4from 0.1 to 1.4 V at 0.5 V s'1as described further in Example 18.FIG. 23B shows cyclic voltammetry graphs of the electrodes after modifications of one electrode with gold (Au) nanostructures as described further in Example 18.FIG. 24A shows raw square wave voltammetry graphs obtained from three independent devices measures in PBS with pyocyanin concentrations ranging from 10 M to 600 M in PBS 1X as described further in Example 19.FIG. 24B show a calibration curve showing the average response ± standard deviation of the three independent devices as described further in Example 19.FIG. 24C shows an image of using the needle apparatus to detect pyocyanin in chicken samples as described further in Example 19.FIG. 24D shows a square wave voltammetry graph for the detection of pyocyanin in chicken samples within a potential range of -0.6 V to -0.2 V as described further in Example 19.FIG. 25A shows a cyclic voltammogram recorded in 0.5 M H2SO4from 0.1 to 1.4 V at a scan rate of 0.5 V s'1, comparing the nanospiked gold working electrode and the bare gold electrode as described further in Example 20.FIG. 25B shows a cyclic voltammetry graph recorded from 0.1 to 0.8 V at 0.5 Vs-1, comparing the bare gold electrode (dotted and dashed line), the nanospiked gold electrode with no peptide immobilized (dashed line), and the gold electrode with Ferrocenylcarbonyl-Phe-Gly-p-(2-furyl)-Ala-PEG2-Cys functionalized in 1X PBS either on the bare gold electrode (dotted line) or on the nanospiked gold working electrode (black line) as described further in Example 20.FIG. 26A shows square wave voltammograms of Cys-PEG2-Phe-Met-Ser-Leu-Leu-Ser-Met-Lys-(Ferrocenylcarbonyl) (SEQ ID NO: 68) modified gold nanospiked electrodes recorded at concentrations ranging from 31.25 to 1000 g / mL as described further in Example 21.FIG. 26B shows square wave voltammograms of Cys-PEG2-Gly-Ala-Thr-Leu-Thr-Tyr-Thr-Lys-(Ferrocenylcarbonyl) (SEQ ID NO: 69) modified AuNP electrodes recorded at concentrations ranging from 20 to 1000 g / ml_ as described further in Example 21.FIG. 27 shows square wave voltammetry characterization of peptide-modified bare working electrodes (e.g. lacking nanostructures) before (top line) and after 15 and 30 mins of exposure to enzyme SpyCEP as described further in Example 22.FIG. 28 shows the structures of exemplary modified amino acid residues that can be used in the peptidic probes described herein.DETAILED DESCRIPTION OF THE EMBODIMENTS

[0016] Various apparatuses or processes will be described below to provide an example of an embodiment of each claimed invention. No embodiment described below limits any claimed invention and any claimed invention may cover processes or apparatuses that differ from those described below. The claimed inventions are not limited to apparatuses or processes having all of the features of any one apparatus or process described below or to features common to multiple or all of the apparatuses or processes described below. It is possible that an apparatus or process described below is not an embodiment of any claimed invention. Any invention disclosed in an apparatus or process described below that is not claimed in this document may be the subject matter of another protective instrument, for example, a continuing patent application, and the applicants, inventors or owners do not intend to abandon, disclaim or dedicate to the public any such invention by its disclosure in this document.

[0017] Furthermore, it will be appreciated that for simplicity and clarity of illustration, where considered appropriate, reference numerals may be repeated among the figures to indicate corresponding or analogous elements. In addition, numerous specific details are set forth in order to provide a thorough understanding of the embodiments described herein. However, it will be understood by those of ordinary skill in the art that the embodiments described herein may be practiced without these specific details. In other instances, well-known methods, procedures and components have not been described in detail so as not to obscure the embodiments described herein. Also, the description is not to be considered as limiting the scope of the embodiments described herein.

[0018] 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 presentapplication herein described for which they are suitable as would be understood by a person skilled in the art.

[0019] As used in this application and claim(s), the words “comprising” (and any form of comprising, such as “comprise” and “comprises”), “having” (and any form of having, such as “have” and “has”), “including” (and any form of including, such as “include” and “includes”) or “containing” (and any form of containing, such as “contain” and “contains”), are inclusive or open-ended and do not exclude additional, unrecited elements or process steps.

[0020] As used in this application and claim(s), the word “consisting” and its derivatives, are intended to be close ended terms that specify the presence of stated features, elements, components, groups, integers, and / or steps, and also exclude the presence of other unstated features, elements, components, groups, integers and / or steps.

[0021] 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 these features, elements, components, groups, integers, and / or steps.

[0022] The terms “about”, and “substantially” as used herein means 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 or about ±5% of the modified term if this deviation would not negate the meaning of the word it modifies.

[0023] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range is encompassed within the description. Ranges from any lower limit to any upper limit are contemplated. The upper and lower limits of these smaller ranges which may independently be included in the smaller ranges is also encompassed within the description, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the description.

[0024] As used in this application, the singular forms “a”, “an” and “the” include plural references unless the content clearly dictates otherwise. For example, an embodiment including “a bacteria” should be understood to include two or more different bacteria.

[0025] In embodiments comprising an “additional” or “second” component, such as an additional or second polymer, 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.

[0026] 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.

[0027] The present description refers to a number of terms and abbreviations used by those skilled in the art. Nevertheless, definitions of selected terms are provided for clarity and consistency.

[0028] Embodiments of the present disclosure will employ, unless otherwise indicated, techniques of biochemistry, electrochemistry, materials chemistry, and the like, which are within the skill of the art. Such techniques are explained fully in the literature.

[0029] The term “electrode” as used herein refers to a conductor through which electricity enters or leaves an object, substance, or region. For example, it may be composed of a metal such as a noble metal, for example gold, platinum, or silver, or a non-metal such as an allotrope of carbon.

[0030] The term “electrically conductive” as used herein means any material which conducts electricity. This may include, for example, a material which has an electrical conductivity of or about at least 3.3x102S / m, for example at least or about 1 x103S / m or at least or about 1 x104S / m or at least or about 1 x105S / m or at least or about 1 x106S / m.

[0031] The term “cleavable peptide” as used herein means a compound consisting of at least two amino acids, each amino acid being linked together via a peptide bond, wherein at least one of the peptide bonds is cleavable by an enzyme. The cleavable peptide can include additional residues necessary for enzyme recognition. Further, the cleavable peptide is a component of the peptidic probe and can comprise a linker. The non-linker portion of the cleavable peptide (e.g. when a linker is present) is the portion comprising the cleavage site and is cleavable by the enzyme.

[0032] The term “nanostructure” as used herein means a structure with at least one dimension between 1-100 nm, which includes for example nanospikes and / or nanorods.

[0033] The term “linker” as used herein means a chemical compound attached via one or more chemical bonds to for example extend the length of a peptidic probe or provide attachment chemistry. This includes, for example, a linker capable of chemically bonding (directly or indirectly) to both an amino acid and an electroactive moiety (e.g. an electroactive moiety linker) or to an amino acid and electrically conductive surface (e.g. an electrode linker). A linker can include for example polymer units such as polyethylene glycol (PEG) units or amino acids. The linker can be or have a N and / or C terminable attachment residue such as a cysteine or a lysine residue capable reacting to attach for example the electroactive moiety or to the electrically conductive surface.

[0034] A "conservative amino acid substitution" as used herein, is one in which one amino acid residue is replaced with another amino acid residue without abolishing the protein's desired properties. Suitable conservative amino acid substitutions can be made by substituting amino acids with similar hydrophobicity, polarity, and R-chain length for one another. Examples of conservative substitutions include the substitution of one non-polar (hydrophobic) residue such as alanine, isoleucine, valine, leucine or methionine for another, the substitution of one polar (hydrophilic) residue for another such as between arginine and lysine, between glutamine and asparagine, between glycine and serine, the substitution of one basic residue such as lysine, arginine or histidine for another, or the substitution of one acidic residue, such as aspartic acid or glutamic acid for another. The phrase “conservative substitution” also includes the use of a chemically derivatized residue or non-natural amino acid in place of a non-derivatized residue provided that such polypeptide displays the requisite activity.

[0035] As demonstrated herein, the inventors have prepared a sensor apparatus that can be used for real time detection and identification of bacterial species. The apparatuses, systems, kits and methods described herein allow for rapid and cost-effective detection of bacteria and point of care detection.

[0036] The apparatuses, systems, kits and methods described herein can also be useful in the presence biofilms. For example, the cleavable peptides are designed to be cleaved by secreted bacterial enzymes or virulent factors. The virulent factors diffuse through biofilms and are able to be detected. Other biosensors which for example use antibodies to detect a bacterial surface protein, get trapped in biofilms and their ability to properly detect or quantify the target bacteria is therefore compromised.

[0037] Accordingly, an aspect provided herein relates to a working electrode comprising:a body having a detecting face and an electrically conductive surface on at least the detecting face, anda peptidic probe immobilized on at least a portion of the electrically conductive surface, the peptidic probe comprising an electroactive moiety and a cleavable peptide, the cleavable peptide optionally comprising at least one linker.

[0038] The working electrode can be a plurality of working electrodes.

[0039] The plurality can be comprised in an apparatus or kit described herein. For example, the plurality can be comprised in a needle apparatus or microelectrode apparatus described herein.

[0040] Another aspect provided herein relates to a needle apparatus 100 for detecting a bacteria comprising:i) a detecting end 102 and a connecting end,ii) a plurality of working electrodes, each working electrode 108 of the plurality having a detecting face and extending from the detecting end 102 to the connecting end and comprising:an electrically conductive surface on at least the detecting face of each working electrode 108,a peptidic probe immobilized on at least a portion of the electrically conductive surface, the peptidic probe comprising an electroactive moiety and a cleavable peptide, the cleavable peptide optionally comprising at least one linker,iii) a reference electrode 106,iv) an electrode spacer 110 configured to support and align the plurality of working electrodes and the reference electrode 106 each inserted or insertable therein, and v) a syringe hub counter-electrode 104, optionally configured to mate with and receive the electrode spacer 110.

[0041] As shown in for example FIGs. 10C, 13B, 14A, 15A and 16A, the electrode spacer 110 as well as a binder, for example epoxy, keep each of the working electrodes as well as the reference electrode 106 substantially parallel to each other at least at and approaching the detecting end 102. For example, the working electrodes as well as the reference electrode 106 are substantially parallel within the electrode spacer 110 and at the detecting end 102. Working electrodes comprising peptidic probes can be referred to as sensors or functionalized working electrodes interchangeably herein.

[0042] In some embodiments, the detecting end 102 is configured to detect one or more bacteria. For example, the working electrodes can each be functionalized to detect different bacteria or two or more of the working electrodes can be functionalized to detect the same bacteria.

[0043] In some embodiments, the reference electrode 106 is centrally located and the working electrodes are regularly spaced around the reference electrode 106. In further embodiments, each of the working electrodes are approximately equidistant from the reference electrode 106. Examples are shown in for example FIGs. 13C, 13D, 14B and 15B.

[0044] In some embodiments, the detecting face is disc shaped or elliptically shaped. In one embodiment, the detecting face is disc shaped. In one embodiment, the detecting face is elliptically shaped. As shown in FIGs. 15A and B, the detecting end 102 can be polished on an angle such as 45°, providing an elliptically shaped detecting face.

[0045] In some embodiments, each working electrode 108 is flush, recessed, or protruding from the detecting end 102.

[0046] The number of working electrodes can for example be 2-16, or more for example 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16 working electrodes. They are regularly spaced. FIG. 13C shows an embodiment of an apparatus comprising 8 working electrodes and FIG. 13D shows an embodiment of an apparatus comprising 16 working electrodes.

[0047] Another aspect of the disclosure relates to a microelectrode apparatus 114 comprisingi) a needle apparatus 100 described herein andii) a plurality of connectors configured to connect to the connecting end of the plurality of working electrodes and the reference electrode 106, wherein detection of the presence of bacteria is measured by electrochemical signal, for example, the voltage or current, at one or more of each working electrode 108 of the plurality of working electrodes following cleavage of the cleavable peptide of the peptidic probe by a bacterial enzyme which releases the electroactive moiety.

[0048] The apparatuses can detect more than one bacterial simultaneously, should the sample or tissue being analysed comprise multiple bacterial populations.

[0049] The electrochemical signal can for example be voltage, current charge, impedance or capacitance or a combination thereof.

[0050] Voltage or current can be measured. For example, a potential can be applied to the working electrodes and a current measured.

[0051] In some embodiments, one or more of each of the plurality of working electrodes is or comprises gold, carbon, platinum, silver or a combination thereof. In one embodiment, one or more of each of the plurality of working electrodes is or comprises gold.

[0052] In some embodiments, the electrically conductive surface comprises a carbon material, gold, platinum, silver or a combination thereof.

[0053] In some embodiments, the electrically conductive surface comprises nanostructures. In one embodiment, the nanostructures are nanospikes. The electrically conductive surface may be a coating, or it may be the entire body of the working electrode 108. For example, each working electrode has an electrically conductive surface and a body. For example, the body of the working electrode and the electrically conductive surface may be a unit composed of the same material. For example, the working electrode body and conductive surface may be entirely gold or composed of another conductive material. In other embodiments, the conductive surface may be a distinct layer or may comprise a coating that is gold or other conductive material. Hence the conductive layer may be the same material as the electrode body, it may be a different material and / or it may be distinct or manufactured as a single unit.

[0054] In some embodiments, the electrically conductive surface has a peptidic probe density of about or at least 1013molecules / cm2, of about or at least 1014molecules / cm2, or of about or at least 1015molecules / cm2.

[0055] In some embodiments, the electroactive moiety comprises ferrocene, such as captured in ferrocenylcarbonyl, methylene blue, anthraquinone, tetrathiafulvalene, Ruthenium complexes, Osmium complexes, quinones such as hydroquinone or benzoquinone, phenothiazines such as toluidine blue. In one embodiment, the electroactive moiety comprises ferrocene. Any compound that includes a moiety or functional group that can be undergo reduction and oxidation at an electrode surface thereby producing electrical signal, can be used as the electroactive moiety.

[0056] In some embodiments, the peptidic probe (e.g., the cleavable peptide) comprises a linker. The linker in some embodiments, is an electroactive moiety linker, optionally comprising Lys (e.g. C-terminal ultimate residue or N-terminal ultimate residue). The Lys residue can be used for conjugating the electroactive moiety. The linker in some embodiments, is an attachment residue such as a Phe at the N terminus of the peptidic probe. The electroactive moiety can be attached to a NH2 group as shown for Phe. Accordingly, in some embodiments, the peptidic probe comprises an electroactive moiety (e.g. Ferrocenylcarbonyl).

[0057] In some embodiments, the apparatus, further comprises one or more of i) a support assembly 112 configured to mate with the electrode spacer 110, wherein the support assembly 112 comprises support and alignment of the plurality of connectors and ii) a binder configured to immobilize the plurality of working electrodes and the reference electrode 106 enclosed in the syringe hub counter-electrode 104.

[0058] The apparatus can also include a positive control electrode.

[0059] For example, the positive control can be 1 electrode used with 3 peptidic probes. The peptide sequences of the peptidic probes can be from 1 or more, for example 3 bacteria modified with one of each electroactive moiety, for example, anthraquinone (potential at -0.54 V vs Ag / AgCI), methylene blue (-0.27 V) and Ferrocene (+0.18 V). An electrochemical scan can be performed in PBS to provide a baseline. A further electrochemical scan can be performed in PBS with the 3 markers secreted by bacteria in PBS. This can provide confirmation that the apparatus is working.

[0060] The peptidic probe can for example be or comprise 3 amino acid residues or 4 amino acid residues (e.g. 3 or 4 contiguous amino acids in a cleavable peptide), optionally with a linker (e.g. a spacer) such as a PEG linker or additional amino acids. The peptidic probe may include one or more attachment linkers (e.g. residues such as Cys, Phe or Lys). The peptidic probe may be up to about 16 amino acids, for example, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16 amino acids or any range therein. This can include one or more linkers, optionally a PEGspacer linker and / or one or more attachment residues. The attachment residues in some embodiments are included in the up to 16 amino acids.

[0061] The length of the peptidic probe can be for example 16 residues, or any range therein for example, 2, 4, 6, or 12 PEG units and upto 14 amino acid residues (e.g. for example 2 PEG units and 14 amino acid residues, 12 PEG units and 3 or 4 amino acid residues). Example schematic structures are shown in Fig. 1 A and can comprise for example, any of the peptides in Tables 3, 4, 5, 6, 7 or 8, or described herein.

[0062] In some embodiments, the peptidic probe comprises an electrode linker (LE), optionally comprising an attachment residue such as cysteine (Cys) (e.g., N-terminal ultimate residue or C-terminal ultimate residue) and / or PEGn with n = 2, 4, 6, or 12, and / or 2-14 amino acid residues such as but not limited to glycine, alanine, p-alanine, isoleucine, leucine, methionine, serine or valine. The Cys residue or other C terminal amino acids may be amidated, for example to cap and stabilize the carboxy terminus. The Cys can be at the C-terminal end for example as shown herein in peptidic probes for Staphylococcus Aureus.

[0063] 2-(2-Aminoethoxy) ethoxyacetic acid (AEEAc) has the same structure as two PEG units. Accordingly, the terms AEEAc or PEG2 (i.e. 2 PEG units) are used interchangeably herein.

[0064] Linkers with Cys attachment residues can for example be used in peptidic probes for attaching them to the working electrode 108, e.g., the gold working electrode 108. For example, Cys can be included at the N terminus and used to attach to the electrode, for example through its sulfhydryl moiety. The electroactive moiety in such cases, would be incorporated onto or attached to the C terminus of the peptidic probe or cleavable peptide, for example via a Lys residue. As shown herein the alternate configuration is also possible with for example Cys included at the C terminus and the electroactive moiety incorporated or attached to the N-terminus, optionally via a N- terminal Lys residue.

[0065] The linker can comprise additional amino acids in addition to the attachment residues. Any amino acid residues can be used including for example glycine, alanine, isoleucine, leucine, methionine and / or valine. The linker can comprise an amino acid spacer, for example 2 amino acids, 3 amino acids, 4 amino acids, 5 amino acids, 6 amino acids, 7 amino acids, 8 amino acids, 9 amino acids, 10 amino acids, 11 amino acids or 12 amino acids.

[0066] The linker can comprise a PEG spacer. The PEG spacer can be for example 2 PEG units, 3 PEG units, 4, PEG units, 5 PEG units, 6 PEG units, 7 PEG units, 8 PEG units, 9 PEG units, 10 PEG units, 11 PEG units, or 12 PEG units.

[0067] Other compounds that can be used as or in a linker, include for example other polymers like PVA or PEI, alkyl chains like C6 or C10 or C12, carbohydrate chains like dextran, alkanethiols like mercaptohexanol, and aminoalkyl silanes like APTES.

[0068] In some embodiments, the bacteria is selected from Staphylococcus Aureus, Staphylococcus epidermidis, Escherichia Coli, Streptococcus pyogenes or Pseudomonas Aeruginosa or combinations thereof.

[0069] In some embodiments, the bacteria is or comprises Escherichia Coli.

[0070] In some embodiments, the peptidic probe comprises Phe-Phe-Arg-Arg (SEQ ID NO:1), Asp-Phe-Phe-Arg-Arg (SEQ ID NO:2), Gly-Asp-Phe-Phe-Arg-Arg (SEQ ID NO:3), Gly-Leu-Leu-Gly-Asp-Phe-Phe-Arg-Arg (SEQ ID NO:4), Arg-Trp-Ala-Arg (SEQ ID NO:5), Gly-Arg-Trp-Ala-Arg (SEQ ID NO:6), Gly-Gly-Arg-Trp-Ala-Arg (SEQ ID NO:7), or Ala-Arg-Arg-Leu (SEQ ID NO:8) or any of the above comprising at least one linker, optionally an electrode linker comprising Cys and / or two or more PEG units, for example, 2, 4, 6, or 12 and / or a electroactive moiety linker comprising lysine for conjugating the electroactive moiety.

[0071] In some embodiments, the peptidic probe is or comprises Cys-Gly-Leu-Leu- Gly-Asp-Phe-Phe-Arg-Arg-Lys-(Ferrocenylcarbonyl) (SEQ ID NO: 72), Cys-Gly-Gly-Arg-Trp-Ala-Arg-Lys-(Ferrocenylcarbonyl) (SEQ ID NO: 73); or Cys-Ala-Arg-Arg-Leu-Lys-(Ferrocenylcarbonyl) (SEQ ID NO: 74).

[0072] The peptidic probe can comprise an amino acid sequence having at least 90% sequence identity to any one of SEQ ID NO: 1-8 or 72-74 where the Arg-Arg, Ala-Arg or Arg-Leu cut site residues (e.g. bolded residues in sequences) are maintained.

[0073] The peptidic probe can comprise an amino acid sequence having at least 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence identity to any one of SEQ ID NO: 1-8 or 72-74 where the Arg-Arg, Ala-Arg or Arg-Leu cut site residues (e.g. bolded residues in sequences) are maintained.

[0074] Sequence identity can for example be determined by BLAST, for example the XBLAST program or Gapped BLAST, optionally using parameters for short sequences.

[0075] In some embodiments, the bacteria is or comprises Staphylococcus epidermidis.

[0076] In some embodiments, the peptidic probe comprises X1-X2-X3-Leu-Thr-X4- X5, where when X2 is present, X1 is either not present or is selected from Gly, Ser, or Ala, X2 is either not present or is selected from Ala, Vai, Ser, or Gly, X3 is selected from Thr, Ser, or Vai, X4 is either not present or is selected from Tyr, Trp, or Phe, and when X4 is present, X5 is either not present or selected from Thr, Ser, or Vai. For example, the peptidic probe can comprise of Gly-Ala-Thr-Leu-Thr-Tyr-Thr (SEQ ID NO: 15).

[0077] In further embodiments, the peptidic probe is or comprises Cys-AEEAc-Gly- Ala-Thr-Leu-Thr-Tyr-Thr-Lys-(Ferrocenylcarbonyl) (SEQ ID NO: 15).

[0078] In some embodiments, the peptidic probe comprises X1-X2-X3-Leu-Leu-X4- X5, where when X2 is present, X1 is either not present or is selected from Phe, Trp, or Tyr, X2 is either not present or is selected from Met, Leu, or lie, X3 is selected from Ser, Thr, or Gly, or Ala, X4 is either not present or is selected from Ser, Thr, or Gly, or Ala, and when X4 is present, X5 is either not present or selected from Met, Leu, or lie. For example, the peptidic probe can comprise of Phe-Met-Ser-Leu-Leu-Ser-Met (SEQ ID NO: 22).

[0079] In further embodiments, the peptidic probe is or comprises Cys-AEEAc-Phe- Met-Ser-Leu-Leu-Ser-Met-Lys-(Ferrocenylcarbonyl) (SEQ ID NO: 22).

[0080] In some embodiments, the peptidic probe comprises Ala-Thr-Leu-Thr (SEQ ID NO:9), Thr-Leu-Thr-Tyr (SEQ ID NO: 10), Ala-Thr-Leu-Thr-Tyr (SEQ ID NO: 11), Gly-Ala-Thr-Leu-Thr (SEQ ID NO:12), Gly-Ala-Thr-Leu-Thr-Tyr (SEQ ID NO:13), Ala-Thr-Leu-Thr-Tyr-Thr (SEQ ID NO: 14), Gly-Ala-Thr-Leu-Thr-Tyr-Thr (SEQ ID NO: 15), Met-Ser-Leu-Leu (SEQ ID NO:16), Ser-Leu-Leu-Ser (SEQ ID NO:17), Phe-Met-Ser-Leu-Leu (SEQ ID NO:18), Met-Ser-Leu-Leu-Ser (SEQ ID NO:19), Phe-Met-Ser-Leu-Leu-Ser (SEQ ID NO:20), Met-Ser-Leu-Leu-Ser-Met (SEQ ID NO:21), Phe-Met-Ser-Leu-Leu-Ser-Met (SEQ ID NO:22), Phe-Met-Thr-Leu-Leu-Ser-Met (SEQ ID NO:23), Met-Thr-Leu-Leu (SEQ ID NO:24), Trp-Met-Thr-Leu-Leu (SEQ ID NO:25), Trp-Met-Thr-Leu-Leu-Cys (SEQ ID NO:26), Phe-Met-Gly-Leu-Leu-Ser-Met (SEQ ID NO:27), Met- Gly -Leu-Leu-Ser-Met (SEQ ID NO:28), Trp-Met-Gly-Leu-Leu-Ser-Met (SEQ ID NO:29), Met-Gly-Leu-Leu-Cys-Met (SEQ ID NO:30), Phe-Met-Gly-Leu-Leu-Cys-Met (SEQ ID NO:31), Phe-Met-Gly-Leu-Leu-Thr-Met (SEQ ID NO:32), Met-Gly-Leu-Leu-Thr-Met (SEQ ID NO:33), Met-Ala-Leu-Leu-Thr-Met (SEQ ID NO:34), Tyr-Met-Ser-Leu-Leu-Ser-Met (SEQ ID NO:35), Phe-Leu-Ser-Leu-Leu-Ser-Met (SEQ ID NO: 56), Phe-lle-Ser-Leu-Leu-Ser-Met (SEQ ID NO: 57), Phe-Met-Ser-Leu-Leu-Ala-Met (SEQ ID NO: 58), Phe-Met-Ser-Leu-Leu-Ser-Leu (SEQ ID NO: 59), or Phe-Met-Ser-Leu-Leu-Ser-lle (SEQ ID NO: 60) or any of the above comprising at least one linker, optionally an electrode linker comprising Cys and / or two or more PEG units, for example, 2, 4, 6, 8, 10 or 12 and / or a electroactive moiety linker comprising or consisting of lysine for conjugating the electroactive moiety.

[0081] For example, the peptidic probe can comprise Phe-Met-Ser-Leu-Leu-Ser- Met-Lys (SEQ ID NO: 68) or Gly-Ala-Thr-Leu-Thr-Tyr-Thr-Lys (SEQ ID NO: 69).

[0082] In further embodiments, the peptidic probe is or comprises Cys-PEG2-Phe- Met-Ser-Leu-Leu-Ser-Met-Lys-(Ferrocenylcarbonyl) (SEQ ID NO: 68) or Cys-PEG2-Gly-Ala-Thr-Leu-Thr-Tyr-Thr-Lys-(Ferrocenylcarbonyl) (SEQ ID NO: 69).

[0083] The electroactive moiety can be as described herein, attached at the N terminus end or the Cterminus end. For example, the ferrocenyl electroactive moiety can beattached to the amino end of a peptidic probe or the carboxy end of a peptidic probe as described for example in the Examples, such as Example 1 and 27.

[0084] The linker can comprise amino acids that are non-proteinogenic, which can be used for attaching the electroactive moiety.

[0085] For example, ferrocene NHS esters (e.g CAS RN: 115223-09-1 | Product Number: S0820 / V-Succinimidyl Ferrocenecarboxylate) can react with NH2, for example an a-amino group at the N-terminus and s-amino groups of lysine, other amino acids or other compounds comprising primary NH2 (e.g., ornithine, 2,4-diaminobutyric acid, ,3-diaminopropionic acid). Ferrocene electroactive moiety can be attached to the C-terminus of a peptidic probe, for example by activating a COOH group and coupling with an amine-functional ferrocene (any ferrocene comprising compound also comprising a primary amine group.

[0086] The electroactive moiety can be incorporated, as a carboxyl-functional electroactive compound, such as ferrocenylcarboxylic acid or an activated derivative (e.g., NHS ester). It is coupled in the same manner either as a terminal capping group or at a selected position within the sequence following Fmoc deprotection of the corresponding amine (Phe or Lys for example).

[0087] The peptidic probe can comprise an amino acid sequence having at least 90% sequence identity to any one of SEQ ID NO: 9-60 or 68-69, or X1-X2-X3-Leu-Thr-X4-X5 as defined herein orX1-X2-X3-Leu-Leu-X4-X5 as defined herein, where the Leu-Leu or Leu-Thr cut site residues (e.g. bolded residues in sequences) are maintained.

[0088] The peptidic probe can comprise an amino acid sequence having at least 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence identity to any one of SEQ ID NO: 9-60 or 68-69 or X1-X2-X3-Leu-Thr-X4-X5 as defined herein or X1-X2-X3-Leu-Leu-X4-X5 as defined herein, where the Leu-Leu or Leu-Thr cut site residues (e.g. bolded residues in sequences) are maintained.

[0089] In some embodiments, the peptidic probe comprises 1, 2 or 3 alternate amino acids outside the cut site residues. The alternate amino acid may be a conservative substitution.

[0090] In some embodiments, the bacteria is or comprises Streptococcus pyogenes.

[0091] In some embodiments, the peptidic probe comprises X1-X2-X3-Gln-Arg-X4- X5, where X1 is either not present or is selected from Asn, Gin, Ser, or Asp, X2 is selected from Trp, Tyr, or Phe, X3 is selected from Vai, lie, or Leu, X4 is either not present or is selected from Vai, lie, or Leu, and when X4 is present, X5 is either not present or is selected from Vai, lie, or Leu. For example, the peptidic probe can comprise of Asn-Trp-Val-GIn-Arg-Val-Val (SEQ ID NO: 39).

[0092] In further embodiments, the peptidic probe is or comprises Cys-AEEAc-Asn- Trp-Val-Gln-Arg-Val-Val-Lys-(Ferrocenylcarbonyl) (SEQ ID NO: 39).

[0093] In some embodiments, the peptidic probe comprises P-X2-X3-Lys-Lys-X4-X5, where X2 and X3 are independently selected from Vai, lie, or Leu and X4 and X5 are independently either not present or selected from Vai, lie, or Leu. For example, the peptidic probe can comprise of Pro-lle-Val-Lys-Lys-lle-lle (SEQ ID NO: 52).

[0094] In further embodiments, the peptidic probe is or comprises Cys-AEEAc-Pro-lle-Val-Lys-Lys-lle-lle-Lys-(Ferrocenylcarbonyl) (SEQ ID NO: 53).

[0095] In some embodiments, the peptidic probe comprises Asn-Trp-Val-GIn-Arg (SEQ ID NO:36), Asn-Trp-Val-GIn-Arg-Val (SEQ ID NO:37), Trp-Val-GIn-Arg-Val (SEQ ID NO:38), Asn-Trp-Val-GIn-Arg-Val-Val (SEQ ID NO:39), Gln-Trp-Val-Gln-Arg-Val-Val (SEQ ID NO:40), Ser-Trp-Val-Gln-Arg-Val-Val(SEQ ID NO:41), Asp-Trp-Val-GIn-Arg-Val-Val (SEQ ID NO:42), Asn-Tyr-Val-GIn-Arg-Val-Val (SEQ ID NO:43), Asn-Phe-Val-GIn-Arg-Val-Val (SEQ ID NO:44), Asn-Trp-lle-GIn-Arg-Val-Val (SEQ ID NO:45), Asn-Trp-Leu-Gln-Arg-Val-Val (SEQ ID NO:46), Asn-Trp-Val-GIn-Arg-lle-Leu (SEQ ID NO:47), Pro-lle-Val-Lys-Lys (SEQ ID NO:48), Pro-lle-Val-Lys-Lys-Leu (SEQ ID NO:49), Pro-lle-Val-Lys-Lys-Val (SEQ ID NO:50), Pro-Val-Leu-Lys-Lys-lle-lle (SEQ ID NO:51), Pro-lle-Val-Lys-Lys-lle-lle (SEQ ID NO:52), Pro-lle-Val-Lys-Lys-lle-lle-Lys (SEQ ID NO:53), Pro-lle-Leu-Lys-Lys-Leu-Val (SEQ ID NO:54), Pro-Val-lle-Lys-Lys-Val-Val (SEQ ID NO:55), Asn-Trp- Asn -Gln-Arg-Val-Val (SEQ ID NO: 61), Asn-Trp-Val-GIn-Arg-Val-lle (SEQ ID NO: 62), or Pro-Leu-lle-Lys-Lys-Val-Leu (SEQ ID NO: 63) or any of the above comprising at least one linker, optionally an electrode linker comprising Cys and / or two or more PEG units, for example, 2, 4, 6, or 12 and / or a electroactive moiety linker comprising lysine for conjugating the electroactive moiety.

[0096] For example, the peptidic probe can comprise Cys-PEG2-Asn-Trp-Val-Gln- Arg-Val-Val-Lys (SEQ ID NO: 70) or Cys-PEG2-Pro-lle-Val-Lys-Lys-lle-lle-Lys (SEQ ID NO: 53).

[0097] In further embodiments, the peptidic probe is or comprises Cys-PEG2-Asn- Trp-Val-Gln-Arg-Val-Val-Lys-(Ferrocenylcarbonyl) (SEQ ID: 70) or Cys-PEG2-Pro-lle-Val-Lys-Lys-lle-lle-Lys-(Ferrocenylcarbonyl) (SEQ ID NO: 53).

[0098] The peptidic probe can comprise an amino acid sequence having at least 90% sequence identity to any one of SEQ ID NO: 36-55, 61-63, or 70 or X1-X2-X3-Gln-Arg-X4-X5 as defined herein, or P-X2-X3-Lys-Lys-X4-X5 as defined herein, where the Gin-Arg or Lys-Lys cut site residues (e.g. bolded residues in sequences) are maintained.

[0099] The peptidic probe can comprise an amino acid sequence having at least 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence identity to any one of SEQ ID NO: 36-55, 61-63, or 70 or X1-X2-X3-Gln-Arg-X4-X5 as defined herein, or P-X2-X3-Lys-Lys-X4-X5 as defined herein, where the Gin-Arg or Lys-Lys cut site residues (e.g. bolded residues in sequences) are maintained.

[0100] In some embodiments, the bacteria is or comprises Staphylococcus Aureus.

[0101] In some embodiments, the peptidic probe sequence can comprise a furyl modified amino acid residue, for example 2-amino-3-(furan-2-yl)propanoic acid (p-(2-furyl)-Ala), 3-amino-3-(furan-2-yl)propanoic acid (3-(furan-2-yl)-p-Ala)), 2-amino-2-(furan-2-yl)acetic acid (2-(furan-2-yl)-Gly) [2-amino-3-(furan-2-yl)butanoic acid (p-(furan-2-yl)-Val), or2-amino-4-(furan-2-yl)pentanoic acid (y-(furan-2-yl)-Leu). Representative structures are shown for example in FIG. 28.

[0102] The furyl amino acid residue can be a (2-furyl) or a (3-furyl) modification. In further embodiments, the furyl modified residue is p-(2-furyl)-Ala.

[0103] In some embodiments, the peptidic probe comprises X-Gly-R-Ls, where X is selected from Phe, Leu, lie, Vai, Met, Trp or Tyr, R is a furyl modified amino acid, for example p-(2-furyl)-Ala, and LE is an electrode linker, for example PEGn, where n = 2, 4, 6, 12, or small amino acids like Gly, Ala, p-Ala, Ser. For example, the peptidic probe can comprise Ferrocenylcarbonyl-Phe-Gly-p-(2-furyl)-Ala-PEG2 or Ferrocenylcarbonyl-Phe-Gly-p-(2-furyl)-Ala-PEG2-Cys. The Fc can be attached to the free amino group at a N terminus of the peptidic probe.

[0104] In some embodiments, the peptidic probe comprises Phe-Gly-P-(2-furyl)-Ala-Cys (SEQ ID NO: 71), Phe-Gly-P-(2-furyl)-Ala, Tyr-Gly-P-(2-furyl)-Ala, Trp-Gly-P-(2-furyl)-Ala, Val-Gly-P-(2-furyl)-Ala, Met-Gly-P-(2-furyl)-Ala, lle-Gly-P-(2-furyl)-Ala, Leu- Gly-p-(2-furyl)-Ala, Phe-Gly-3-(furan-2-yl)-P-Ala, Ala-Gly-3-(furan-2-yl)-P-Ala, Gly-Gly-3-(furan-2-yl)-P-Ala, Val-Gly-3-(furan-2-yl)-P-Ala Met- Gly-3-(furan-2-yl)-P-Ala, lie- Gly-3-(furan-2-yl)-P-Ala, Leu-Gly-3-(furan-2-yl)-P-Ala, Phe-Gly-P-(furan-2-yl)-Val, Ala-Gly-P-(furan-2-yl)-Val, Gly-Gly-P-(furan-2-yl)-Val, Val-Gly-P-(furan-2-yl)-Val, Met-Gly-P-(furan-2-yl)-Val, lle-Gly-P-(furan-2-yl)-Val, Leu-Gly-P-(furan-2-yl)-Val, Phe-Gly-y-(furan-2-yl)-Leu, Ala-Gly-y-(furan-2-yl)-Leu, Gly-Gly-y-(furan-2-yl)-Leu, Val-Gly-y-(furan-2-yl)-Leu, Met-y-(furan-2-yl)-Leu, lle-Gly-y-(furan-2-yl)-Leu, or Leu-Gly-y-(furan-2-yl)-Leu or any of the above comprising at least one linker, optionally an electrode linker comprising Cys and / or two or more PEG units, for example, 2, 4, 6, or 12 and / or a electroactive moiety linker comprising lysine or phenylalanine for conjugating the electroactive moiety.

[0105] In some embodiments, the peptidic probe comprises Phe-Gly-p-(2-furyl)-Ala-PEG2, Phe-Gly-P-(2-furyl)-Ala-PEG4, Phe-Gly-P-(2-furyl)-Ala-PEG6, or Phe-Gly-P-(2-furyl)-Ala-PEG12.

[0106] In further embodiments, the peptidic probe is or comprises Ferrocenylcarbonyl-Phe-Gly-p-(2-furyl)-Ala-PEG2-Cys, Ferrocenylcarbonyl-Phe-Gly-p-(2-furyl)-Ala-PEG4-Cys, Ferrocenylcarbonyl-Phe-Gly-p-(2-furyl)-Ala-PEG6-Cys, or Ferrocenylcarbonyl-Phe-Gly-p-(2-furyl)-Ala-PEG12-Cys.

[0107] The peptidic probe can comprise an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 71 where the Gly-P-(2-furyl)-Ala cut site residues (e.g. bolded residues in sequences) are maintained.

[0108] The peptidic probe can comprise an amino acid sequence having at least 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence identity to SEQ ID NO: 71 where the Gly- P-(2-furyl)-Ala cut site residues (e.g. bolded residues in sequences) are maintained.

[0109] In some embodiments the peptidic probes comprise a C terminal or N terminal lysine. When on the C terminus, lysine can be used to incorporate the ferrocene based electroactive moieties.

[0110] In some embodiments, the peptidic probe comprises a C terminal or N terminal cysteine. The SH group in cysteine or other SH containing molecules can be used to attach to a working electrode 108, e.g., a gold working electrode 108. For example, hexanethiol, mercaptoethanol, mercaptopropionic acid or dithiols ( 1 ,2-Ethanedithiol (EDT) or 1,3-Propanedithiol) or PEG-thiol, can also be used.

[0111] In some embodiments, the reference electrode 106 comprises an Ag / AgCI electrode.

[0112] The electrochemical potential window for biological systems is generally -0.6 to +0.8 V vs. Ag / AgCI, due to the limits imposed by water electrolysis reactions. Working within this window avoids hydrogen and oxygen evolution, preserving biomolecule stability.

[0113] In some embodiments, the electrode spacer 110 comprises between 2 to 16 holes, each of the holes for housing one of the plurality of working electrodes. The electrode spacer 110 can also include one or more additional holes, for example for housing a reference electrode 106.

[0114] In some embodiments, the syringe hub counter-electrode 104 is a stainless-steel syringe hub.

[0115] In some embodiments, the binder comprises medical grade epoxy.

[0116] In another aspect of the disclosure, is provided a package comprising the apparatus described herein and inert gas, wherein the package is hermetically sealed.

[0117] In another aspect of the disclosure, is provided a detection system comprising the apparatus or package described herein; and a potentiostat configured to connect to theplurality of connectors, wherein the potentiostat is configured to measure a difference of potential between the reference electrode 106 and the syringe hub counter-electrode 104.

[0118] In another embodiment, the system further comprises a data processor and / or a display. In another embodiment, the system is a unit.

[0119] In some embodiments, the needle apparatus 100 or microelectrode apparatus 114 are a unit and the potentiostat is a separate unit.

[0120] The needle apparatus 100 or the microelectrode apparatus 114 can for example be provided for easy connection to a potentiostat. For instance, circular, pinned, keyed, insulation-displacement, ribbon connectors, etc, can be provided, that are male or female which are optionally crimped, soldered, bound, screwed or glued to the individual electrodes, possibly through wires or bespoke circuits and connection strategies. In yet another aspect, is provided a method for detecting bacteria, the method comprising i) inserting the detecting end 102 of the needle or microelectrode apparatus 114, package or the system described herein in a test sample or tissue suspected of bacterial infection, optionally in a wound and ii) measuring electrochemical signal, wherein the electrochemical signal comprises one or more of current, voltage, charge, impedance, capacitance, or a change therein produced upon cleavage of a peptidic probe.

[0121] The test sample can for example be a patient sample such as an aspirate, a food or an environmental sample such as wastewater.

[0122] The tissue can be a wound or the apparatus’ described can be used to pierce a surface such as skin or a mucosal surface.

[0123] In some embodiments, the apparatus is an in situ use device or for use in situ.

[0124] In some embodiments, the needle is contacted with the sample or tissue, optionally wound, for at least 5 minutes or at least 10 minutes. In some embodiments, the needle is contacted with the test sample or tissue, optionally wound for between about 10 minutes to about 20 minutes. In another embodiment, the needle is contacted with the test sample or tissue, optionally wound for about 15 minutes.

[0125] In some embodiments, measuring the electrochemical signal comprises calibrating the microelectrode apparatus 114. In further embodiments, measuring the voltage comprises using square wave voltammetry (SWV).

[0126] In some embodiments, the apparatus is functionalized for detecting the bacteria that causes necrotizing fasciitis (NF).

[0127] Another aspect of the invention is a method of making a working electrode 108, comprising i) obtaining an electrode comprising having a detecting face, the electrode being a gold, platinum, silver or carbon or combination thereof electrode, ii) mechanical polishing thedetecting face of the electrode; the mechanical polishing comprising polishing with an alumina slurry; ultrasonic cleaning; rinsing and drying, iii) chemical polishing the detecting face in an oxidative solution, optionally a Piranha solution, rinsing, drying and immersion in ethanol, rinsing and drying, iv) electropolishing the detecting face via cyclic voltammetry, v) optionally electrodepositing a nanostructure layer, optionally a gold nanostructure layer, on a surface of the detecting face of the electrode thereby providing an electrically conductive surface, and vi) optionally immobilizing a quantity of peptidic probe to a surface of the electrically conductive surface, optionally via a thiolate gold bond, the peptidic probe comprising an electroactive moiety and a cleavable peptide thereby providing the working electrode 108. Some embodiments include one or both optional steps.

[0128] In some embodiments, the mechanical polishing comprises polishing with a disc embedded with diamond particles, previously wet, prior to polishing with the alumina slurry.

[0129] In some embodiments, the electrochemically active surface area (ECSA) is about or at least 10'3cm2, about or at least 10'2cm2, or about or at least 10'1cm2. In some embodiments, the roughness factor is about or at least 101, or about or at least 1, or about or at least 10.

[0130] In some embodiments, the peptidic probe is immobilized to the surface of the electrically conductive surface via a linker comprising Cys and / or PEG units. The linker can have a N terminal or C terminal cysteine residue and comprise one or more other amino acids or PEG units depending on the length of the peptide. Other molecules having a thiol group other than cysteine can also be used. For example, hexanethiol, mercaptoethanol, mercaptopropionic acid or dithiols (1 , 2-Ethaned ithiol (EDT) or 1 ,3-Propanedithiol) or PEG-thiol, can also be used.

[0131] If the electrically conductive surface is a metal or non-metal such as platinum, silver, carbon, graphene, or carbon nanotubes, the peptidic probes can be attached via covalent or non-covalent bonding mechanisms using electrochemical oxidation to introduce reactive groups, carbodiimide coupling for amide bond formation, diazonium chemistry for covalent attachment, and TT-TT stacking interactions for aromatic group interactions.

[0132] One or more of the steps described in the Examples can be employed to prepare the needle apparatus 100 or the microelectrode apparatus 114.

[0133] The needle apparatus 100 can also be used without a peptidic probe. As shown herein, functionalizing the needle apparatus 100 with nanospikes increases sensitivity dramatically. An example is shown in FIG. 7C. Such an apparatus was used to detect pyocyanin as described in Example 8 and shown in FIGs. 11A and 11 B.

[0134] Accordingly, in another aspect of the disclosure, is provided a needle apparatus 100 comprising i) a detecting end 102 and a connecting end, ii) a plurality of workingelectrodes, each working electrode 108 of the plurality having a detecting face and extending from the detecting end 102 to the connecting end and comprising: an electrically conductive surface on a surface of the detecting face of each working electrode 108, iii) a reference electrode 106, iv) an electrode spacer 110 configured to support and align the plurality of working electrodes and the reference electrode 106 each inserted or insertable therein, v) a syringe hub counter-electrode 104, optionally configured to mate with and receive the electrode spacer 110; wherein the plurality of working electrodes are substantially parallel at least at and approaching the detecting end 102.

[0135] The counter electrode 104 can be made of steel. The counter electrode 104 is polished mechanically and chemically as for example described herein.

[0136] The counter electrode 104 can include as shown in FIGs. 13B, 14A and 15A, a connecting wire which is connectable to a potentiostat. The connecting wire can be externally affixed or affixable to the counter electrode 104 as shown in FIGs. 13B, 14A and 15A or it can be internally affixed in the syringe hub, for example encapsulated in epoxy. In some embodiments, the connecting wire is externally and removably affixed by way of a clasp or clamp that is detachable from the counter electrode. The connecting wire can be attached for example during use.

[0137] In yet another aspect of the disclosure, is provided a microelectrode apparatus 114 for detection of bacteria, wherein the apparatus is i) the needle apparatus 100 and ii) a plurality of connectors configured to connect to the plurality of working electrodes and the reference electrode 106, wherein detection of the presence of bacteria is determined by the change of an electrochemical signal such as the voltage or current, at one or more of each working electrode 108 of the plurality of working electrodes.

[0138] In some embodiments, the apparatus is for use in detecting Pseudomonas Aeruginosa.

[0139] Pseudomonas Aeuroginosa can be detected without peptides as the bacteria secreted redox virulent factors that can be detected directly on the electrode surface. It is demonstrated herein that the use of nanostructure can increase signal detected.

[0140] Pyocyanin produced by Pseudomonas Aeruginosa can be detected without the addition of the peptidic probes. Inclusion of nanostructures for example, increases selectivity.

[0141] In another aspect of the disclosure, is provided a method for detecting bacteria, the method comprising i) inserting the detecting end 102 of the needle or microelectrode apparatus 114 described herein into a test sample or tissue, optionally a wound, and ii) measuring an electrochemical signal, wherein the electrochemical signal comprises one ormore of current, voltage, charge, impedance, capacitance, or a change therein produced in the presence of bacteria.

[0142] In some embodiments, the bacteria is Pseudomonas Aeruginosa.

[0143] In another aspect of the disclosure, is provided a method of diagnosing a subject, comprising i) performing a method described herein and ii) diagnosing said subject with the presence of bacteria infection based upon the pattern and quantum of peptidic probe cleavage.

[0144] In another aspect of the disclosure, is provided a method of treating a person, comprising i) performing a method described herein and ii) treating the subject with a suitable treatment according to the bacterial infection detected.

[0145] In another aspect of the disclosure, is provided an isolated cleavable peptide comprising any of the peptidic probes and / or cleavable peptides described herein including those in Tables 3, 4, 5, 6, 7 or 8 described herein.

[0146] In an embodiment, the isolated cleavable peptide comprises or consists of one or more of the following:

[0147] Phe-Phe-Arg-Arg (SEQ ID NO:1), Asp-Phe-Phe-Arg-Arg (SEQ ID NO:2), Gly-Asp-Phe-Phe-Arg-Arg (SEQ ID NO:3), Gly-Leu-Leu-Gly-Asp-Phe-Phe-Arg-Arg (SEQ ID NO:4), Arg-Trp-Ala-Arg (SEQ ID NO:5), Gly-Arg-Trp-Ala-Arg (SEQ ID NO:6), Gly-Gly-Arg-Trp-Ala-Arg (SEQ ID NO:7), Ala-Arg-Arg-Leu (SEQ ID NO:8);

[0148] Ala-Thr-Leu-Thr (SEQ ID NO:9), Thr-Leu-Thr-Tyr (SEQ ID NO: 10), Ala-Thr-Leu-Thr-Tyr (SEQ ID NO:11), Gly-Ala-Thr-Leu-Thr (SEQ ID NO:12), Gly-Ala-Thr-Leu-Thr-Tyr (SEQ ID NO:13), Ala-Thr-Leu-Thr-Tyr-Thr (SEQ ID NO:14), Gly-Ala-Thr-Leu-Thr-Tyr-Thr (SEQ ID NO:15), Met-Ser-Leu-Leu (SEQ ID NO:16), Ser-Leu-Leu-Ser (SEQ ID NO:17), Phe-Met-Ser-Leu-Leu (SEQ ID NO:18), Met-Ser-Leu-Leu-Ser (SEQ ID NO:19), Phe-Met-Ser-Leu-Leu-Ser (SEQ ID NQ:20), Met-Ser-Leu-Leu-Ser-Met (SEQ ID NO:21), Phe-Met-Ser-Leu-Leu-Ser-Met (SEQ ID NO:22), Phe-Met-Thr-Leu-Leu-Ser-Met (SEQ ID NO:23), Met-Thr-Leu-Leu (SEQ ID NO:24), Trp-Met-Thr-Leu-Leu (SEQ ID NO:25), Trp-Met-Thr-Leu-Leu-Cys (SEQ ID NO:26), Phe-Met-Gly-Leu-Leu-Ser-Met (SEQ ID NO:27), Met- Gly -Leu-Leu-Ser-Met (SEQ ID NO:28), Trp-Met-Gly-Leu-Leu-Ser-Met (SEQ ID NO:29), Met-Gly-Leu-Leu-Cys-Met (SEQ ID NQ:30), Phe-Met-Gly-Leu-Leu-Cys-Met (SEQ ID NO:31), Phe-Met-Gly-Leu-Leu-Thr-Met (SEQ ID NO:32), Met-Gly-Leu-Leu-Thr-Met (SEQ ID NO:33), Met-Ala-Leu-Leu-Thr-Met (SEQ ID NO:34), Tyr-Met-Ser-Leu-Leu-Ser-Met (SEQ ID NO:35), Phe-Leu-Ser-Leu-Leu-Ser-Met (SEQ ID NO: 56), Phe-lle-Ser-Leu-Leu-Ser-Met (SEQ ID NO: 57), Phe-Met-Ser-Leu-Leu-Ala-Met (SEQ ID NO: 58), Phe-Met-Ser-Leu-Leu-Ser-Leu (SEQ ID NO: 59), or Phe-Met-Ser-Leu-Leu-Ser-lle (SEQ ID NO: 60), X1-X2-X3-Leu-Thr-X4-X5, X1-X2-X3-Leu-Leu-X4-X5;

[0149] Asn-Trp-Val-GIn-Arg (SEQ ID NO:36), Asn-Trp-Val-GIn-Arg-Val (SEQ ID NO:37), Trp-Val-GIn-Arg-Val (SEQ ID NO:38), Asn-Trp-Val-GIn-Arg-Val-Val (SEQ ID NO:39), Gln-Trp-Val-Gln-Arg-Val-Val (SEQ ID NQ:40), Ser-Trp-Val-Gln-Arg-Val-Val(SEQ ID NO:41), Asp-Trp-Val-GIn-Arg-Val-Val (SEQ ID NO:42), Asn-Tyr-Val-GIn-Arg-Val-Val (SEQ ID al-Val (SEQ ID NO:44), Asn-Trp-lle-GIn-Arg-Val-Valn-Arg-Val-Val (SEQ ID NO:46), Asn-Trp-Val-Gln-Arg-lle-Leu (SEQ ID NO:47), Pro-lie- Val-Lys-Lys (SEQ ID NO:48), Pro-lle-Val-Lys-Lys-Leu (SEQ ID NO:49), Pro-lle-Val-Lys-Lys-Val (SEQ ID NQ:50), Pro-Val-Leu-Lys-Lys— lie— lie (SEQ ID NO:51), Pro-lle-Val-Lys-Lys-lle-lle (SEQ ID NO:52), Pro-lle-Val-Lys-Lys-lle-lle-Lys (SEQ ID NO:53), Pro-lle-u-Val (SEQ ID NO:54), Pro-Val-lle-Lys-Lys-Val-Val (SEQ ID NO:55), A-Arg-Val-Val (SEQ ID NO: 61), Asn-Trp-Val-GIn-Arg-Val-lle (SEQ ID N u-lle-Lys-Lys-Val-Leu (SEQ ID NO: 63), X1-X2-X3-Gln-Arg-X4-X5, P-X2-5 .

[0150] In some embodiments, the isolated cleavable peptide comprises one or more linkers. For example, the isolated cleavable peptide can comprise for example an N-terminal or C-terminal Cys residue and optionally two or more units, e.g. PEGn with n=2, 4, 6, 12 or other amino acids bridging the Cys residue and any of SEQ ID NO: 1-74. The Cys residue is optionally an N-terminal ultimate residue.

[0151] In some embodiments, the isolated cleavable peptide can comprise for example an N-terminal or C-terminal Lys or Phe residue. The Lys or Phe residue is optionally a C-terminal ultimate residue or a N-terminal end residue.

[0152] Peptidic probes and isolated peptides can be used to prepare apparatuses described herein. They can also be used in other methods.

[0153] Accordingly, in another aspect of the disclosure, is provided a peptidic probe comprising a cleavable peptide described herein and an electroactive moiety described herein.

[0154] In yet another aspect, is provided a composition comprising the isolated cleavable peptide or the peptidic probes described herein and optionally a carrier, optionally a stabilizer. The stabilizer can for example be a counterion such as TFA, albumin, a salt solution.

[0155] In some embodiments, the composition is lyophilized or frozen.

[0156] In some embodiments, the composition is a coating for preparing a working electrode or apparatus described herein. The composition can include one or more components described in the Examples or herein.

[0157] In another aspect of the disclosure, is provided a kit for detecting bacteria comprising an apparatus described herein and optionally one or more standards or reagents for standardizing, such as known quantities of isolated cleavable peptides, peptidic probesand / or composition described herein or a package, comprising an apparatus, isolated cleavable peptide, peptidic probe or composition described herein. The package can be a component of the kit. In some embodiments, the kit further comprises one or more of instructions for use, or a support assembly 112.

[0158] The needle apparatus 100 can be comprised in a single needle or in two or more separate needles. The needle apparatuses and / or the microelectrode apparatus 114 can be disposable. In some embodiments, the needle apparatus 100 comprises one or more working electrodes, the reference electrode 106, the syringe hub counter-electrode 104, and electrode spacer 110 in a single disposable needle.

[0159] The following non-limiting examples indicate preferred embodiments of the invention and are merely illustrative of the presently disclosed invention.ExamplesExample 1 : Peptidic Probe Synthesis

[0160] An exemplary synthesis of one embodiment of a peptidic probe is as follows. First, 4-[(2,4-dimethoxyphenyl)-(Fmoc-amino)methyl]phenoxyacetic acid (Fmoc-Rink-amide linker) is attached on NH2 of resin via peptide link. After dissolution in DMF, Fmoc-Rink-amide linker is mixed with ethyl(hydroxyimino)cyanoacetate (Oxyma, 3 mmol, 3 eq). N,N’ Diisopropylcarbodiimide is then added and stirred. The solution is added to aminomethyl polystyrene resin to react. The mixture is then washed and attachment to resin can be confirmed with Kaiser test.

[0161] Next, Fmoc deprotection is performed to expose NH2 on the Phenyl group linker attached to the resin. Deprotection is carried out on the resin in 20% piperidine in DMF (10 ml_) and the reaction mixture is shaken for 10 min followed by washing.

[0162] Then, to incorporate an electroactive moiety, for example, ferrocenylcarbonyl, a solution of Fmoc-amino acid (Fmoc-aa-OH), or Fmoc-PEG(2, 4, 6, 12)-OH, or Fmoc-Cys(Trt)-OH, or Fc-carboxylic acid (e.g. CAS RN: 115223-09-1 | Product Number: S0820 / V-Succinimidyl Ferrocenecarboxylate), is stirred with N,N,N',N'-tetramethyl-O-(1H-benzotriazol-1-yl)uranium hexafluorophosphate (HBTU) and DIPEA in DMF. The solution is then added to the resin comprising the peptide and the reaction mixture is shaken. The solution is drained and the resin washed.

[0163] Finally, a cleavage mixture of TFA / EDT / water / TIS (94 / 2.5 / 2.5 / 1) (1 mL / 100 mg resin) is added to the modified resin and the mixture shaken for 3 hours at room temperature. The resin is removed by filtration and washed with the cleavage mixture once. Cold diethyl ether is added dropwise to the filtrate to precipitate the crude peptidic probe. The probe is collected by centrifugation and the diethyl ether is decanted. The solid is washed with diethylether and the procedure is repeated three times. All peptidic probes comprising the electroactive moiety were purified on a RP-HPLC equipped with UV detection (collection at 650 nm).

[0164] A schematic of the Fc amino acid residue reaction is provided below.5

[0165] A list of exemplary cleavable peptides and / or peptidic probes is shown in Tables 3, 4, 5, 6, 7 or 8. A list of exemplary electroactive moieties is shown in FIG. 1B.Example 2: Preparation of a gold working electrode with gold nanospike-modified surface

[0166] First, gold electrodes were mechanically polished using an alumina slurry (0.05 pm) for 5 minutes to remove surface impurities. The electrodes were then thoroughly rinsed with Milli-Q water, followed by ultrasonic cleaning in Milli-Q water for 5-10 minutes to further eliminate any residual particulates. A final rinse with Milli-Q water and dry with nitrogen gas (N2) was performed.

[0167] Then, the gold electrodes were chemically polished by immersing in a Piranha solution, composed of a 3:1 volumetric ratio of concentrated sulfuric acid (H2SO4) to hydrogen peroxide (H2O2). This highly oxidative solution effectively etches the electrode surface, removing any remaining organic contaminants and surface impurities. The electrodes were let to soak in the Piranha solution for 5-10 minutes.

[0168] Subsequently, the electrodes were thoroughly rinsed with Milli-Q water to ensure complete removal of residual Piranha solution, and they were then dried under a nitrogen gas (N2) stream. To further cleanse the electrode surface, the electrodes were subjected to ultrasonic agitation in Milli-Q water for an additional 5-10 minutes.

[0169] Then, the electrodes were immersed in reagent-grade ethanol (CH3CH2OH) for 1 hour to eliminate surface oxidation and achieve further surface refinement. Upon completion, the electrodes were rinsed once more with Milli-Q water and dried under nitrogen gas (N2) to prepare for subsequent procedures.

[0170] Next, electrochemical polishing was performed via cyclic voltammetry in a 0.5 M sulfuric acid (H2SO4) solution. A potential range from 0.1 V to 1.4 V was performed for a totalof 25 complete cycles. The scan rate was set to 0.5 V / s to ensure controlled and uniform polishing across the electrode surface. This process enhances the electrode’s electrochemical activity by removing surface oxides and microscopic irregularities, creating a clean and reproducible surface for subsequent analyses. The reproducibility is shown in FIG. 12B, where cyclic voltammetry plots for a microelectrode apparatus with four working electrodes were recorded in 10 mM Fe(CsHs)2 and 10 mM KCI, at 0.5 V / s from 1 to -1V using an apparatus having an electrode spacer (e.g. as shown in FIG. 10C) and support assembly (e.g. as shown in FIG. 10D). Each line represents a different electrode, showing the reproducibility of fabrication.

[0171] Then, gold nanospikes were electrodeposited onto the electrode surface using an electrolyte solution containing 6.9 mM HAuCk and 0.5 mM Pb(CH3COO)2. The deposition process was conducted at a constant potential of 0.05 V for 600 seconds. Upon completion, the electrodes were thoroughly rinsed with Milli-Q water and allowed to dry naturally at room temperature, ensuring they were shielded from light to prevent photodegradation.

[0172] Following electrodeposition, cyclic voltammetry (GV) was performed in a 0.5 M H2SO4solution to characterize the electrode surface. The CV was conducted over a potential range from 0.1 V to 1.4 V at a scan rate of 0.5 V / s for 10 cycles. From these measurements, the electrochemically active surface area (ECSA) and roughness factor (Rf) were subsequently calculated to evaluate the effectiveness of the electrodeposition process.

[0173] The formula used for calculating ECSA was as follows:ECSA (cm2) = Q / (390 pC / cm2)where Q is the electric charge in pC.

[0174] The area of the reductive signal of the cyclic voltammetry graph was used to calculate Q to be 10.194 pC before electrodeposition of gold nanospikes and 36.868 pC after electrodeposition of gold nanospikes. Using the formula provided above, ECSA was calculated to be 0.026 cm2before electrodeposition of gold nanospikes and 0.0945 cm2after electrodeposition of gold nanospikes.

[0175] The formula used for calculating Rf was as follows:Rf = ECSA / Awhere ECSA is the electrochemically active surface area in cm2and A is the geometric surface area of the electrode in cm2.

[0176] The ECSA after electrodeposition of gold nanospikes as calculated above to be 0.0945 cm2as well as the geometric surface area of the electrode, calculated to be A = (rr)r2= 0.02 cm2(where r = 0.08 cm), were used to calculate an Rf of 4.725 using the formula above.

[0177] The gold nanospike-modified surface was also characterized by electron microscopy as shown in FIGS. 2A and 2B.Example 3: Preparation of a gold working electrode with gold nanospike-functionalized surface with peptidic probe immobilized on the surface of the gold nanospikes

[0178] Peptidic probes were immobilized onto gold nanospike-modified gold electrodes as prepared in Example 2. Peptide solutions (stock solution with 1 mg / mL) were thawed at room temperature and mixed thoroughly through vortex agitation followed by pipette aspiration to ensure homogeneity. The Au nanostructures (Au-NS)-modified gold electrodes were then immersed in the peptide solution for two different amounts of time to compare peptide immobilization incubation time; one aliquot for 2 hours at room temperature and one aliquot overnight at room temperature, taking care to avoid bubble formation on the electrode surface, which can interfere with immobilization. Upon completion of the immobilization period, the electrodes were gently rinsed with phosphate-buffered saline (PBS) to remove unbound peptides and prepare the electrodes for subsequent experimental procedures.

[0179] A cyclic voltammetry graph comparing the peptidic probe immobilization incubation time of 2h and overnight of Cys-PEG2-Gly-Ala-Thr-Leu-Thr-Tyr-Thr-Lys-(Ferrocenylcarbonyl) (SEQ ID NO: 69) solution on gold nanospike-modified gold electrodes was plotted and is shown in FIG. 3.

[0180] A cyclic voltammetry graph comparing different scan rates (0.05-1.0 V / s) in 1X PBS (pH 7.4) after peptidic probe immobilization on gold nanospike-modified gold electrodes is shown in FIG. 4A. The relationship between anodic peak current and the square root of the scan rate was also plotted, with an R2of 0.986 (FIG. 4B).Example 4: Cleavage Study using metalloprotease SepA and gold working electrode with gold nanospike-functionalized modified surface with peptidic probe immobilized on the surface of the gold nanospikes

[0181] Using the gold working electrode with gold nanospike-functionalized modified surface with peptidic probe immobilized on the surface of the gold nanospikes as prepared in Example 2, baseline signals were established by performing cyclic voltammetry (GV) in a 1X PBS solution. The potential range was set from -0.05 V to 0.8 V, with a scan rate of 0.5 V / s over 5 cycles. Following this, square wave voltammetry (SWV) was conducted in the same 1X PBS solution, with a potential range of 0.1 V to 0.8 V, to obtain initial reference measurements. Measurements were conducted at 37°C.

[0182] For the cleavage study, a series of SepA enzyme concentrations, the enzyme secreted by Staphylococcus Epidermidis, was prepared in 1X PBS. The peptide-modified AuNSgold electrodes were immersed in the SepA solutions to investigate the relationship between SepA concentration and the percentage change in peak current. This relationship was characterized by conducting CV and SWV as previously outlined, allowing for the assessment of enzymatic activity on the electrode surface based on peak current variations.

[0183] A cyclic voltammetry graph comparing gold working electrodes with gold nanospike-functionalized modified surface with peptidic probe immobilized on the surface of the gold nanospikes both before and after the addition of metalloprotease SepA in 1X PBS solution with a scan rate of 0.5 V / s was plotted and is shown in FIG. 5A. A square wave voltammetry plot comparing gold working electrodes with gold nanospike-functionalized modified surface with peptidic probe immobilized on the surface of the gold nanospikes both before and after the addition of metalloprotease SepA in 1X PBS solution with a pulse height of 25 mV, step height of 10 mV, and frequency of 100 Hz was plotted and is shown in FIG. 5B.

[0184] The relationship between the logarithm of SepA concentration (log[SepA (ng / mL)]) and the percentage in peak current (lo-l / lo) at 37°C, with a linear range between 0.01 and 1 ng / mL, and an R2of 0.98 was plotted and is shown in FIG. 6.Example 5: Comparison study of gold working electrode with surface immobilized peptide versus gold nanostructure-modified working electrode surface immobilized peptide

[0185] Before use, the gold working electrodes were gently polished using a disc embedded with diamond particles, previously wet, for five minutes. After that, they were polished again for five minutes in an alumina powder solution (0.05pm). They were then sonicated for another five minutes to remove any surface contaminants. The electrode surface was rinsed with Milli-Q water and treated with electrochemical polishing with an H2SO4 0.5M solution as electrolyte. The range used for the electrochemical polishing was from 0.1 V to 1.4 V, with a 0.5 V / s scan rate, during 25 cycles.

[0186] For the deposition of gold nanostructures, an electrolyte solution containing 6.9 mM HAUCI4and 0.5 mM Pb(CH3COO)2was used. The deposition process was carried out for 600 seconds at a potential of 0.05 V.

[0187] According to fabricant instructions, the peptide was dissolved in DMF (1mg / mL). Before dissolution, the peptides were allowed to reach room temperature. Since the peptide contained cysteine, thiol chemistry was employed by immersing the gold surface of the 3D electrode in 0.2mL of peptide solution for two hours. After the fabrication, the sensor was rinsed with MilliQ ultrapure water and stored in 10mM PBS, pH 6.8, at -4 °C until use.

[0188] The electrochemical measurements were performed using a PARSTAT4000A standard potentiostat unit manufactured by Princeton Applied Research, TN, USA, with a Pt wire as a counter electrode and Ag|AgCI as a reference electrode.

[0189] A cyclic voltammetry graph comparing different scan rates ranging from 0.1 V / s to 2 V / s in 10 mM PBS applied to the gold working electrode with Ferrocenylcarbonyl-Phe-Gly-p-(2-furyl)-Ala-PEG2-Cys immobilized on the surface was plotted and is shown in FIG.7 A. A linear calibration curve of the redox peaks against the scan rates, with an adjusted R2of 0.9915, was plotted and is shown in FIG. 7B.

[0190] A cyclic voltammetry graph comparing the gold working electrode with Ferrocenylcarbonyl-Phe-Gly-p-(2-furyl)-Ala-PEG2-Cys immobilized on the bare gold surface of working electrode (dashed line, compact detection tracing) or on the Au nanostructure (AuNS or AuNP)-modified gold working electrode (black line, expanded detection tracing) was plotted and is shown in FIG. 7C.

[0191] A cyclic voltammetry graph showing the stability of Ferrocenylcarbonyl-Phe-Gly-p-(2-furyl)-Ala-PEG2-Cys immobilized on the nanostructure-modified gold working electrode from 2 to 8h, recorded in PBS 10 mM, pH 6.8 at 1 V / s, potential ranging from 0.1 to 0.8 V, was plotted and is shown in FIG. 7D.

[0192] A square wave voltammetry graph of the gold nanostructure-modified gold working electrode with Ferrocenylcarbonyl-Phe-Gly-p-(2-furyl)-Ala-PEG2-Cys immobilized on the surface in 10 mM PBS at pH 6.8 over a potential range of 0.15 to 0.75 V that was completed in triplicate was plotted and is shown in FIG. 8A.

[0193] The average percent decrease of the current of the gold nanostructure-modified gold working electrode with Ferrocenylcarbonyl-Phe-Gly-p-(2-furyl)-Ala-PEG2-Cysimmobilized on the surface contacted with 10 mM PBS buffer after 15, 30, 40, and 60 minutes as measured with square wave voltammetry in 10 mM PBS at pH 6.8 was plotted (FIG.8B).

[0194] A cyclic voltammetry graph of the electrode in contact with 0.0003 mg / mL of aureolysin after 0, 15, 30, and 45 minutes over a potential range of 0.12 to 0.8 V at 1 V / s in 10 mM PBS at pH 6.8 was plotted and is shown in FIG. 9A.

[0195] A square wave voltammetry graph showing the average current decrease versus the logarithm of different concentrations of aureolysin for the gold nanostructure-modified gold working electrode with Ferrocenylcarbonyl-Phe-Gly-p-(2-furyl)-Ala-PEG2-Cys immobilized on the surface in 10 mM PBS at pH 6.8, with an R2of 0.9941, was plotted and is shown in FIG. 9B.Example 6: Calculation of density of peptidic probe immobilized on a gold electrode

[0196] The surface probe density of peptidic probe immobilized on a gold electrode was calculated using the formula:Surface Probe Density (mol / cm2) = Q / nFAWhere Q is the electric charge in C, n is the number of electrons per redox event, F is Faraday’s constant in C / mol, and A is the area of the electrode in cm2.

[0197] Cyclic voltammetry was performed on the peptidic probe immobilized on a gold electrode before and after adding SepA with the cyclic voltammetry graph shown in FIG. 5A. The area of the reductive signal of the cyclic voltammetry graph was used to calculate Q to be 1.47 x 10-6 C. Also substituting the values n = 1 (for ferrocene), F = 96485 C / mol, and A = (rr)r2 = 0.020 cm2 (where r = 0.08 cm), surface probe density was calculated to be 7.58 x 10-10 mol / cm2. This was then converted to a molecular density using Avagadro’s number, to yield 4.56 x 1014 molecules / cm2.Example 7: Construction and characterization of a needle apparatus

[0198] A needle apparatus was constructed according to the following procedure, with a schematic of said needle apparatus as shown in FIG. 10A. A schematic of the detecting endof the needle is shown in FIG. 10B. The detecting end is shown “bare” prior to functionalizing with nanostructures and peptidic probes.

[0199] A silver wire (0 0.075mm) was used as the reference electrode, the working electrodes consisted of four gold wires (00.125mm), and a syringe hub’s stainless steel outer layer was used as the counter electrode. To account for potential variations in electrode positioning and surface area, a 3D-printed electrode spacer was used to ensure that the wires remain straight and evenly spaced. The electrode spacer had a diameter of 1 ,6mm and a length of 2mm. It included five 0.3mm wide holes: one in the middle for the silver wire (reference electrode) and the others evenly distributed around it for the gold wires (working electrodes). A schematic of the 3D-printed electrode spacer with 4 holes for working electrodes is shown in FIG. 10C. An electrode spacer with holes for up to 8 working electrodes was also made.

[0200] The electrode spacer was placed inside the syringe hub, and the wires were passed through the holes and fixed with epoxy (EPO-TEK medical grade). The epoxy was allowed to dry for 48 hours, and afterward, the sensor was polished with sandpaper to remove the excess epoxy from the sides. The hub was then inserted in a support assembly e.g. a 3D-printed plastic support assembly, and the connector wires were soldered with standard soldering tin in a printed circuit board also fixed to the support assembly, then the electrode wires were attached to the other side of the board using silver conductive epoxy. A schematic of an embodiment of a 3D-printed support assembly is shown in FIG. 10D.

[0201] Following the epoxy application, the needle apparatus was left to dry for at least six hours. After that, the sensing surfaces were polished first with a diamond pad, wet with MilliQ water, for five minutes, and then with alumina 0.05 pm for another five minutes. The device was sonicated in an ultrasonic bath to remove any impurities or alumina excess for ten minutes.

[0202] The reference electrode, was functionalized by depositing a layer of silver chloride through electrochemical chlorination in a 3 M KCI solution. Alternating currents of - 20 pA for 1s and 20 pA for 9s were applied cyclically for a total duration of one minute. Electrochemical cleaning was performed using H2SO4 0.5 M solution as an electrolyte. The range was from 0.1 V to 1.4 V, with a 0.5 V / s scan rate, during 15 cycles.

[0203] Cyclic voltammetry was carried out in a 10 mM ferrocene solution to investigate the electrochemical behavior of the fabricated electrodes within the device. The four voltammograms were recorded both individually and simultaneously, showing consistent currents across the sensors and indicating stable and reproducible performance as shown in FIG. 10E. The voltammograms of individual electrodes (dotted lines) and simultaneous tests (full lines) overlap, with no significant shifts in peak potential or current. Comparison of the oxidation current measured sequentially and simultaneously showed a relative standard deviation of 1.04 %, indicating that the electrodes operate independently, with no significant interference. A smaller feature around 0.75 V was occasionally observed on some electrodes, likely due to surface oxidation or chlorination of residual silver species from slight contamination near the silver components. Importantly, this minor signal did not affect overall device functionality.

[0204] Table 1 shows the current and potential responses for both measurement types, showing similar outcomes for individual and simultaneous measurements, with a halfwave potential of approximately Ex / 2= 0.23V.Table 1: Current and potential responses for individual and simultaneous CV dataExample 8: Method of Using the Needle Apparatus to identify pyocyanin in a cadaver pig leg

[0205] The needle apparatus of Example 7 was evaluated in an ex vivo model for wound infection. A leg from a cadaver pig was infected and the needle was inserted near the injection site and measurements were run using the parameters identified in vitro. Images of the method of using the needle apparatus are shown in FIG. 11 A. Voltammograms were then run from 0.8 to -0.8V at 5mV / s and plotted, as shown in FIG. 11 B.Example 9: Method of Using the Needle / Microelectrode Apparatus to Detect Bacteria in a Wound

[0206] The needle apparatus or system comprising the needle apparatus can be used to detect bacteria in a wound by inserting the detecting end in the wound. It can also be used during surgical debridement, to detect the boundary of infected versus noninfected tissue. The needle apparatus or system can also be used to detect if specific bacteria are present in an area suspected of bacterial infection for example by piercing the skin of a visibly inflamed area with the detecting end of the needle apparatus or inserting the detecting end of the needle apparatus into a joint etc. The detecting end of the needle apparatus or system can also be gently rinsed prior to measuring electrochemical signal in PBS or other washing solution. When a bacterial infection is detected by one or more of the working electrodes, a suitable treatment option can be selected and / or administered.Example 10: Calibration of the Needle / Microelectrode Apparatus for in vivo Bioanalysis

[0207] For in vivo bioanalysis, calibration to correlate the sensor's output signal (e.g. , current, voltage, capacitance, charge, impedance or a combination thereof) with the target analyte’s concentration can be done via the following method. Standard solutions of known concentrations (for example, 5-7 standard solutions) can be prepared in a biological matrix that mimics the in vivo environment (for example blood plasma or simulated body fluids). The sensor's response to these solutions under controlled conditions, at various pH, temperature, and ionic strength can be recorded. A calibration curve can be created by plotting the sensor's response against known concentrations; where the curve's slope represents sensitivity and its intercept adjusts for baseline drift. Key parameters like the limit of detection (LOD) and the concentration range can also be determined. In vivo, calibration must account for matrix effects, time-dependent drift, temperature, pH variations, and potential interference from other substances. Additionally, calibration may need periodic updates or cross-validation with other techniques, for example PCR or ELISA. A minimum of 10 tests should be performed across a range of concentrations, environmental conditions, and over time to account for variability and maintain accuracy.Example 11 : Comparison of microelectrode apparatus with and without electrode spacer

[0208] A needle apparatus having 8 electrodes without an electrode spacer was made using a method similar to that described in Example 7 (i.e. without the electrode spacer portion) and was characterized using cyclic voltammetry from 0.0 to 0.6 V vs Ag|AgCI (3 M NaCI), 0.05 V / s, in 1 mM ferrocyanide solution in 1 M KCI. The cyclic voltammetry graphs produced are shown in FIG. 12A. Each voltammogram demonstrates the redox reaction of ferrocyanide for each of the 8 working electrodes simultaneously.

[0209] A needle apparatus similar to that shown in FIG. 10A (with 4 working electrodes and with an electrode spacer and nanostructures) was tested for its ability to detectFc in solution. As shown in FIG. 12B, the addition of the electrode spacer improved reproducibility of the working electrode.Example 12: Comparison of various linker lengths in PEG-linked peptides

[0210] Peptides containing PEGs 2, 4, and 6 were dissolved in DMF, and the peptide containing PEG12 was dissolved in 1% acetic acid. Before dissolution, the peptides were allowed to reach room temperature. Since the peptide contained cysteine, thiol chemistry was employed by immersing the Au surface of the 3D electrode in 0.2 ml_ of peptide solution (1 mg / mL) for two hours, generating self-assembled monolayers (SAMs). After the fabrication, the sensor was carefully rinsed with MilliQ ultrapure water and stored in 10 mM PBS, pH 6.8, at -4 °C until use.

[0211] For the comparison study, Ferrocenylcarbonyl-Phe-Gly-p-(2-furyl)-Ala-PEG(2, 4, 6, 12)-Cys were used. The sensors were named AuNP-Cys-PEGx-Pep-Fc.

[0212] Plots of peak current density versus the scan rate of nanospiked sensors functionalized with PEG-linked peptides (PEG2, PEG4, PEG6, and PEG12) is shown in FIG.17A. They were derived from cyclic voltammetry recorded at different scan rates (0.1 to 2 V / s) to assess the electrochemical behavior and determine the reaction type (surface-confined vs. diffusion-controlled) for each peptide-modified electrode. Error bars representing the standard deviation of three independent measurements (n = 3). The resulting plots revealed a linear relationship for all the developed sensors, consistent with a surface-confined redox process rather than a diffusion-controlled one.

[0213] The stability of the Fc modified peptide electrodes were evaluated using SWV recorded after 15, 30, 45, and 60 minutes of incubation at room temperature in PBS 1X pH 7 (FIG. 17B). The sensors containing PEGs 2, 4, and 6 maintained consistent baseline signals during incubation in PBS, with less than 5% signal decrease observed over 1 hour. This high signal retention indicates that the immobilized peptide layers are chemically stable and do not suffer from significant desorption in simple buffered environments.

[0214] Square wave voltammetry graphs and the corresponding calibration curves of nanospiked sensors functionalized with PEG-peptides in 1X PBS are shown in FIG. 17C.

[0215] The sensitivity of the sensors was evaluated to quantify the response of the sensors to variations in analyte concentration (Table 2). The LOD values followed the order PEG2 (1.12 ng / mL) < PEG6 (1.24 ng / mL) < PEG4 (2.34ng / mL) < PEG12 (2.65ng / mL). This non-linear behavior suggests that spacer length influences performance through multiple mechanisms and not just by controlling the distance between the ferrocene probe and electrode. Similar trends were also observed in other studies with different sensors containing PEGs. A potential explanation is that PEG4 shows conformations that reduce access orinterfere with electron transfer, whereas PEG2 has a better balance of accessibility and coupling, and PEG6 provides sufficient flexibility to restore accessibility.

[0216] The shift that can be observed in the SWV peaks with larger PEG chains (6 and 12) is attributed to the ferrocene label being farther from the electrode surface. At the same time, the longer PEG chains create a more hydrated and flexible environment, changing the way the ferrocene interacts with the solvent and the electric field near the electrode.Table 2: Sensitivity of nanospiked sensors functionalized with PEG-peptides in 1 x PBSExample 13: Selectivity of AuNP-Cys-PEG2-Pep-Fc towards staphylococcus aureus

[0217] An interference study using AuNP-Cys-PEG2-Pep-Fc from Example 12, showing the SWV responses of the sensor in different bacterial culture supernatants (pure LB broth, staphylococcus epidermidis, pseudomonas aeruginosa, and staphylococcus aureus) is shown in FIG. 18A. The detection of staphylococcus aureus was carried out using SWV, with a 100-mV pulse height, a 5 mV step height, and a 50 Hz frequency, using 10 mM PBS (pH 6.8) as the electrolyte for all the fabricated sensors.

[0218] A calibration curve obtained from SWV responses in staphylococcus aureus culture supernatant, recorded in PBS 1X pH 7, is shown in FIG. 18B and demonstrates sensor response and analytical performance in complex biological media. The x-axis is displayed on a logarithmic scale, but the points themselves are plotted at their original concentrations. The linear regression trendline was fitted using Iog10-transformed concentrations, giving a coefficient of correlation of 0.98, demonstrating high linearity and reproducibility even in heterogeneous matrices. Obtained LOD was 1.05 CFU / mL, and LOQ was 3.34 CFU / mL, suggesting the applicability of the sensor in real-world applications. Furthermore, the sensor had a variability of only 1.59% of its initial response after storage in PBS at 4 °C for 10 days, indicating strong operational stability.Example 14: Comparison of square wave voltammetry conditions

[0219] SWV conditions were tested with a primary focus on signal stability and reproducibility. Varying condition time and step height did not show high variabilities. A condition time of 5s and a step height of 1 mV were chosen.

[0220] Changing pulse height showed that peak height increased strongly going from low amplitude toward the mid / high range, with the biggest peaks around the higher amplitudes (FIG. 19A). A pulse height of 50mV was chosen because of the stable baseline, and symmetrical and high peak (faradaic current).

[0221] Varying frequency showed that at low frequencies, the signal amplitude is limited and more susceptible to noise, whereas excessively high frequencies may exceed the kinetic response capability of the electrochemical system, leading to peak distortion and reduced reproducibility (FIG. 19B). A frequency of 100 Hz was chosen because peak current is significantly enhanced, the shape remains well-defined and symmetric and there is no observed kinetic mismatch.Example 15: Study of peptide-2 modified AuNP electrode and its selectivity towards staphylococcus epidermidis

[0222] GV scans were recorded at a scan rate of 0.5 V s’1. SWV measurements were obtained over the potential range of 0.1 -0.8 V using a pulse height of 50 mV, a step height of 1 mV, and a frequency of 100 Hz. Following each SWV scan, the electrode was held at 0.1 V for 15 s to stabilize the interface. All experiments were performed in 1* PBS.

[0223] The gold working electrode with gold nanospike-functionalized modified surface with peptidic probe immobilized on the surface of the gold nanospikes was prepared as in Example 2, using Cys-PEG2-Phe-Met-Ser-Leu-Leu-Ser-Met-Lys-(Ferrocenylcarbonyl) (SEQ ID NO: 68) (peptide-2 modified AuNP electrode).

[0224] The modified electrodes were then incubated in SepA solution for 15 mins prior to measurement, unless noted otherwise. Cyclic voltammetry (CV) and square-wave voltammetry characterization of the peptide-2 modified AuNP electrodes before and after exposure to SepA (25 pg / ml_) are shown in FIGs. 20A and 20B respectively. A cyclic voltammogram graph of peptide-2 modified AuNP electrodes recorded at different scan rates (0.2-1.0 Vs’1) is shown in FIG. 20C. A plot of peak current density versus the scan rate, with error bars representing the standard deviation of three independent measurements (n = 3) with R2of 0.971 is shown in FIG. 20D, confirming the successful immobilization of the Fc-modified peptide on the Au electrode.

[0225] A calibration curve obtained from standard SepA solutions based on the SWV responses of peptide-2-modified AuNP electrodes in PBS with commercial enzyme is shown in FIG. 20E. Error bars represent the standard deviation of three independent measurements (n = 3). Excellent linearity (R2= 0.9988) was obtained. This concentration-dependent signal attenuation arises from enzymatic cleavage of the surface-immobilized Fc-modified peptide by SepA, resulting in progressive loss of electroactive Fc from the electrode surface. The highlinearity and reproducibility demonstrate that the sensor enables reliable quantitative detection of SepA.

[0226] The analytical performance of the peptide-2 modified AuNP electrode was evaluated using supernatants from staphylococcus epidermidis cultures of different concentrations. A linear calibration model and a polynomial calibration model are shown in FIGs. 20F and 20G respectively, where the percentage signal change (Al / 10) (%) was plotted against the bacterial concentration. Supernatants were incubated with peptide-2 modified AuNP electrode for 30 mins before measurement. Error bars represent the standard deviation (n = 3). Both figures show that the normalized SWV signal change (y-axis) increased with bacterial concentration. A linear relationship was observed between ( / _0- / ) / / _0 and the logarithm of bacterial concentration over the range tested (R2= 0.946), enabling quantitative analysis under non-saturating conditions. From the linear calibration model, a LOD of 11.1 CFU / mL and a LOQ of 3.0 x 103CFU / mL were obtained.

[0227] An interference test assessing the SWV responses of peptide-1 modified AuNP electrodes in supernatants obtained from the target bacterium, staphylococcus epidermidis, and three non-target species, including staphylococcus aureus, pseudomonas aeruginosa, and escherichia coli (tryptic soy broth (TSB) or LB is media only) is shown in FIG.20H. All bacterial cultures were grown for 24 h at 37 °C with shaking at 200 rpm under identical conditions before bacteria removal. The resulting supernatants were processed and incubated with peptide-2 modified AuNP electrode prior to SWV analysis. Error bars represent the standard deviation of three independent measurements (n = 3).

[0228] Staphylococcus epidermidis induced a pronounced decrease in the Fc signal ((Io - l) / lo “ 0.25), demonstrating high specificity of the peptide-modified electrode towards staphylococcus epidermidis, with minimal interference from non-target species or the growth medium.Example 16: Study of peptide-1 modified AuNP electrode and its selectivity towards staphylococcus epidermidis

[0229] GV scans and SWV measurements followed the same procedure as set out in Example 15.

[0230] The gold working electrode with gold nanospike-functionalized modified surface with peptidic probe immobilized on the surface of the gold nanospikes was prepared as in Example 2, using Cys-PEG2-Gly-Ala-Thr-Leu-Thr-Tyr-Thr-Lys-(Ferrocenylcarbonyl) (SEQ ID NO: 69) (peptide-1 modified AuNP electrode).

[0231] Then the modified electrodes were incubated in SepA solution for 15 mins prior to measurement unless noted otherwise. Cyclic voltammetry and square-wave voltammetry characterization of the peptide-1 modified AuNP electrodes before and after exposure to SepA(10 pg / mL) are shown in FIGs. 21 A and 21 B respectively. A cyclic voltammogram graph of peptide-1 modified AuNP electrodes recorded at different scan rates (0.2-1.0 V s“1) is shown in FIG. 21C A plot of peak current density versus the scan rate, with error bars representing the standard deviation of three independent measurements (n = 3) with R2of 0.9987 is shown in FIG. 21 D confirming successful attachment of peptide-1 to the AuNP modified electrode.

[0232] A calibration curve obtained from standard SepA solutions based on the SWV responses of peptide-1 modified AuNP electrodes is shown in FIG. 21 E. Error bars represent the standard deviation of three independent measurements (n = 3). Good linearity (R2= 0.98) is obtained, demonstrating that peptide-1 enables quantitative detection of SepA with good reproducibility

[0233] The analytical performance of the peptide-1 modified AuNP electrodes was evaluated using supernatants obtained from staphylococcus epidermidis cultures of different concentrations. After bacteria removal, the resulting samples were processed and incubated with peptide-1 modified AuNP electrodes for 15 mins prior to SWV measurement. A linear calibration model from the SWV responses of the peptide-1 modified AuNP electrode is shown in FIG. 21 F. Error bars represent the standard deviation of three independent measurements (n = 3). The signal change ( lo-l ) / lo increased linearly with the logarithm of bacterial concentration, yielding a regression equation of y = -0.54705 + 0.06314x with excellent linearity (R2=0.99), demonstrating that peptide-1 enables reliable, quantitative detection of staphylococcus epidermidis in complex biological matrices. A LOD of 3.0 CFU / mL and a LOQ of 38.5 CFU / ml were obtained.

[0234] An interference test assessing the SWV responses of peptide-1 modified AuNP electrodes in sterile supernatants obtained from the target bacterium, staphylococcus epidermidis, and three non-target species, including staphylococcus aureus, pseudomonas aeruginosa, and escherichia coli is shown in FIG. 21 G. All bacterial cultures were grown for 24 h at 37 °C with shaking at 200 rpm under identical conditions before bacteria removal. The resulting supernatants were processed and incubated with peptide-1 modified AuNP electrode prior to SWV analysis. Error bars represent the standard deviation of three independent measurements (n = 3). Peptide-1 exhibits selectivity toward staphylococcus epidermidis, with minimal interference from non-target bacteria or culture media.Example 17: Medium Comparison between peptide-1 and peptide-2 modified AuNP electrodes to demonstrate alternative method of detecting bacteria

[0235] Bacteria in a wound can be detected following the method described in Example 9, except the detecting end of the needle apparatus can be removed from the wound after incubation and rinsed prior to measuring electrochemical signal in phosphate-buffered saline (PBS) or tryptic soy broth (TSB).

[0236] To evaluate the effect of different media on the sensing performance of the modified peptide electrodes from Examples 15 and 16, PBS, staphylococcus epidermidis 24 h culture supernatant, and TSB were selected as comparison media. Peptide-modified electrodes were first tested in each medium to record the initial electrochemical signal. The electrodes were immersed in the supernatant culture for 15 minutes, the supernatant electrode was measured directly and the other two electrodes were rinsed and then measured again under the same conditions. Changes in the electrochemical signal before and after immersion were compared to assess the influence of different media on sensor performance (FIG. 22A for peptide-2; FIG. 22B for peptide-1).

[0237] The slope (sensitivity) of peptide-1 was found to be 0.06314, with a LOD of 3 CFU / mL and a LOQ 38 CFU / ml. In comparison, the slope (sensitivity) of the peptide-2 modified electrodes was determined to be 0.02873, with a LOD of 11 CFU / mL and a LOQ of 3,000 CFU / ml.Example 18: Demonstration of the individual functionality of electrodes

[0238] Gold nanostructures were electrodeposited on one of the four working electrodes (electrode 1), leaving the other three electrodes unmodified. Cyclic voltammetry of the bare gold electrodes recorded in 0.5 M H2SO4from 0.1 to 1.4 V at 0.5 V s'1, showing the characteristic gold oxidation / reduction peaks used for ECSA estimation is shown in FIG. 23A. Cyclic voltammetry of the electrodes after modification with Au nanostructures in electrode 1, exhibiting an increased peak current corresponding to a larger ECSA only for electrode 1 is shown in FIG. 23B.

[0239] This selective modification confirmed that each electrode can be independently functionalized within the array without affecting neighbouring electrodes, highlighting the minimal cross-talk and modularity of the system. Before the modification calculated ECSA of the gold electrodes was approximately 0.04 mm2± 0.01 mm2. This value was determined by averaging the charge obtained from the gold oxide reduction peaks of the four integrated sensors and comparing across devices.Example 19: Method of Using the Needle Apparatus to identify pyocyanin in chicken samples

[0240] Raw SWV data obtained from three independent devices (n = 3) measured in PBS with pyocyanin concentrations ranging from 10 pM to 600 pM in PBS 1X is shown in FIG.24A. A calibration curve showing the average response ± standard deviation (n = 3 devices) is shown in FIG. 24B. The sensor achieved a limit of detection of 2.23 pM, calculated using the baseline response and the lowest calibration concentration (10 pM). The baseline standard deviation was 2.47% (n = 3), demonstrating a reproducible zero-concentration response. The sensitivity was found to be 2.21 *10-1 OA / pM.

[0241] Proof-of-concept detection of electroactive metabolites (pyocyanin) directly in solid tissue was done with the needle apparatus of Example 7. Chicken samples were cut into uniform pieces and inoculated with bacterial culture (in LB broth). The samples were incubated at room temperature overnight to allow interaction between the bacterial metabolites and the chicken tissue. The excess fluid was removed, and the sensor was inserted into the tissue as shown in FIG. 24C. Subsequently, SWV measurement was performed directly in the tissue.

[0242] The detection of pyocyanin was performed using square wave voltammetry as shown in FIG. 24D, within a potential range of -0.6 V to -0.2 V. The measurement parameters were set to a pulse amplitude of 25 mV, a step potential of 10 mV, and a frequency of 25 Hz. The average peak current across three chicken samples was 0.155 ± 0.049 A.Example 20: Comparison study of gold electrode, nanospiked gold electrode, gold electrode with PEG2 immobilized peptide, and nanospiked gold electrode with PEG2 immobilized peptide

[0243] A cyclic voltammogram recorded in 0.5 M H2SO4from 0.1 to 1.4 V at a scan rate of 0.5 V s'1, comparing the nanospiked gold electrode and the bare gold electrode is shown in FIG. 25A, highlighting the characteristic gold oxidation / reduction peak used for ECSA calculation. The nanospiked electrode exhibits substantially enhanced capacitive and faradaic currents, which is consistent with an increased electrochemically active surface area (ECSA) relative to the bare electrode.

[0244] A cyclic voltammetry graph recorded from 0.1 to 0.8 V at 0.5 Vs1, comparing the bare gold electrode (dotted and dashed line), the nanospiked gold electrode with no peptide immobilized (dashed line), and the gold electrode with Ferrocenylcarbonyl-Phe-Gly-p-(2-furyl)-Ala-PEG2-Cys functionalized in 1X PBS either on the bare gold electrode (dotted line) or on the nanospiked gold electrode (black line) is shown in FIG. 25B.

[0245] The CV of the Fc-modified peptide in PBS reveals a well-defined redox couple centered at -0.45 V vs Ag / AgCI, corresponding to the surface-confined Fc / Fc+redox process. The nanostructured Au electrode exhibits a substantially larger Fc peak current compared to the bare Au electrode. This enhancement is attributed to the increased ECSA of the nanospiked (nanostructured) Au, as independently evidenced by the higher double-layer capacitive current observed over a wide potential window.

[0246] As expected, the nanospikes modified electrode presented a higher ECSA (normalized relative difference -69.2%), as well as a higher roughness factor (from -0.9 to -5.6), due to the nature of the morphology formed on the surface, clearly demonstrating that the electrodeposition process increased the active area and a more electrochemically accessible surface.

[0247] To confirm that the peptides were successfully attached, CV was carried out in 10 mM PBS by scanning the potential from 0.1 to 0.7 V at a rate of 0.5 V / s. No redox peaks appeared on the bare electrode, showing that the signal came only from the immobilized peptide. The presence of well-defined redox peaks after modification confirmed the stable attachment and electrochemical activity of the ferrocene moiety. Electrodes modified with nanospikes and functionalized by the peptide with PEG2 spacer exhibited a current increase of -75% compared to the bare electrode.Example 21 : Study of peptide concentration

[0248] Using the peptide modified AuNP electrodes in Examples 15 and 16, different concentrations of the SepA solution used for incubation prior to measurement was studied. All experiments were performed in 1X PBS.

[0249] Square-wave voltammograms of peptide-2 modified AuNP electrodes recorded at concentrations ranging from 31.25 to 1000 pg / mL are shown in FIG. 26A. The curves show the current density (pA cm-2) as a function of the applied potential, measured under identical electrochemical conditions. At relatively high enzyme concentrations (e.g., 1000 pg / mL and 500 pg / mL), the peptide substrate immobilized on the electrode surface is likely cleaved rapidly or nearly completely within a short time. As a result, the electrode interface reaches a stable state quickly, and the electrochemical signal shows little observable change after the addition of either the enzyme solution or bacterial culture supernatant. In contrast, at an intermediate enzyme concentration (e.g., 250 pg / mL), peptide cleavage occurs on a time scale that is compatible with electrochemical measurement. This allows the signal to change measurably in response to enzymatic activity, providing a resolvable difference before and after enzyme exposure.

[0250] Square-wave voltammograms of peptide-1 modified AuNP electrodes recorded at concentrations ranging from 20 to 1000 pg / mL is shown in FIG. 26B. The curves show the current density (pA cm-2) as a function of the applied potential, measured under same electrochemical conditions. At concentrations of 50 pg / mL and higher, the electrochemical responses showed similar current changes and peak characteristics, indicating that the electrode surface was sufficiently covered by the peptide layer and that further increases in peptide concentration did not significantly alter the interfacial electrochemical behavior.Example 22: Study of enzyme SpyCEP and peptide-modified bare gold working electrode

[0251] A bare gold electrode was immobilized with peptide Cys-PEG2-Asn-Trp-Val-Gln-Arg-Val-Val-Lys-(Ferrocenylcarbonyl) (SEQ ID NO: 70). SpyCEP is an enzyme secreted by GAS (group A strep.) which can cleave CXCL-8. The bare gold electrodes were immersed in SpyCEP solutions (12.90 pg / mL) to investigate the relationship between adding SpyCEP and the percentage change in peak current.

[0252] Square-wave voltammetry characterization of peptide-modified bare electrodes before and after exposure to SpyCEP is shown in FIG. 27. SWV measurements were obtained over the potential range of 0.1-0.8 V using a pulse height of 50 mV, a step height of 1 mV, and a frequency of 100 Hz. Experiments were performed in 1* PBS. Current density is higher before the addition of SpyCEP. The addition of SpyCEP lowered current density but did not differ between after 15 mins or 30 mins after addition of SpyCEP.Example 23: List of exemplary cleavable peptides and / or peptidic probesTable 3: List of exemplary cleavable peptides and / or peptidic probesExample 24: Sequences

[0253] In testing for staphylococcus aureus, the following peptide sequences were chosen and tested as described in the Examples: Ferrocenylcarbonyl-Phe-Gly-p-(2-furyl)-Ala-PEG2-Cys, Ferrocenylcarbonyl-Phe-Gly-p-(2-furyl)-Ala-PEG4-Cys, Ferrocenylcarbonyl-Phe-Gly-p-(2-furyl)-Ala-PEG6-Cys, and Ferrocenylcarbonyl-Phe-Gly-P-(2-furyl)-Ala-PEG12-Cys.

[0254] In testing for staphylococcus epidermidis, the following peptide sequences were chosen and tested as described in the Examples: Cys-AEEAc-Gly-Ala-Thr-Leu-Thr-Tyr-Thr-Lys-(Ferrocenylcarbonyl) (SEQ ID NO: 15) and Cys-AEEAc-Phe-Met-Ser-Leu-Leu-Ser-Met-Lys-(Ferrocenylcarbonyl) (SEQ ID NO: 22). The Lys-(Ferrocenylcarbonyl) enables the redox signal and the Cys-AEEAc facilitates the immobilization on the gold electrodes due to the SH of Cys, and the AEEAc acts as an electrode linker.

[0255] In testing streptococcus pyogenes, the following peptide sequences were chosen and tested as described in the Examples: Cys-AEEAc-Asn-Trp-Val-GIn-Arg-Val-Val-Lys-(Ferrocenylcarbonyl) (SEQ ID NO: 39) and Cys-AEEAc-Pro-lle-Val-Lys-Lys-lle-lle-Lys-(Ferrocenylcarbonyl) (SEQ ID NO: 53). The Lys-(Ferrocenylcarbonyl) enables the redox signal and the Cys-AEEAc facilitates immobilization on the gold electrodes due to the SH of Cys, and the AEEAc acts as an electrode linker.Example 25: Sequences that can be Used

[0256] Using PyMOL molecular docking simulations, togetherwith rational amino-acid substitutions through size and physicochemical similarity, including polarity, charge, and sidechain characteristics, the inventors identified other sequences for use.

[0257] For use in testing for Staphylococcus Aureus, based on Phe-Gly- -(2-furyl)-Ala-Cys (SEQ ID NO: 71), sequences have been identified (Table 4) which follow the generic sequence:X-Gly-R-Lswherein X is selected from Phe, Leu, lie, Vai, Met, Trp, or Tyr. R is a furyl modified amino acid, for example p-(2-furyl)-Ala, (2-(furan-2-y l)-Gly), (p-(furan-2-yl)-Val), or (y-(furan-2-y I)-Leu). LE is an electrode linker, for example PEGn, where n = 2, 4, 6, 12, or small amino acids like Gly, Ala, p-Ala, or Ser.Table 4: Sequences for use in testing for Staphylococcus Aureus

[0258] For use in testing for Staphylococcus epidermidis, based on Gly-Ala-Thr-Leu-Thr-Tyr-Thr (SEQ ID NO: 15), sequences have been identified (Table 5) which follow the equation:X1-X2-X3-Leu-Thr-X4-X5wherein when X2 is present, X1 is either not present or is selected from Gly, Ser, or Ala, X2 is either not present or is selected from Ala, Vai, Ser, or Gly, X3 is selected from Thr, Ser, or Vai, X4 is either not present or is selected from Tyr, Trp, or Phe, and when X4 is present, X5 is either not present or selected from Thr, Ser, or Vai.Table 5: Sequences for use in testing for Staphylococcus epidermidis

[0259] For use in testing for Staphylococcus epidermidis, based on Phe-Met-Ser-Leu-Leu-Ser-Met (SEQ ID NO: 22), sequences have been identified (Table 6) which follow the generic sequence:X1 -X2-X3-Leu-Leu-X4-X5wherein when X2 is present, X1 is either not present or is selected from Phe, Trp, or Tyr, X2 is either not present or is selected from Met, Leu, or lie, X3 is selected from Ser, Thr, or Gly, or Ala, X4 is either not present or is selected from Ser, Thr, or Gly, or Ala, and when X4 is present, X5 is either not present or selected from Met, Leu, or lie.Table 6: Sequences for use in testing for Staphylococcus epidermidis

[0260] For use in testing for Streptococcus pyogenes, based on Asn-Trp-Val-Gln-Arg-Val-Val (SEQ ID NO: 39), sequences have been identified (Table 7) which follow the generic sequence:X1-X2-X3-Gln-Arg-X4-X5wherein X1 is either not present or is selected from Asn, Gin, Ser, or Asp, X2 is selected from Trp, Tyr, or Phe, X3 is selected from Vai, lie, or Leu, X4 is either not present or is selected fromVai, lie, or Leu, and when X4 is present, X5 is either not present or is selected from Vai, lie, or Leu.Table 7: Sequences for use in testing for Streptococcus pyogenes

[0261] For use in testing for Streptococcus pyogenes, based on Pro-lle-Val-Lys-Lys-lle-lle (SEQ ID NO: 52) and Pro-lle-Val-Lys-Lys-lle-lle (SEQ ID NO: 53), sequences have been identified (T able 8) which follow the generic sequence:P-X2-X3-Lys-Lys-X4-X5wherein X2 and X3 are independently selected from Vai, lie, or Leu and X4 and X5 are independently either not present or selected from Vai, lie, or Leu.Table 8: Sequences for use in testing for Streptococcus pyogenesExample 26: Attachment of ferrocene carbonyl to lysine

[0262] Ferrocenylcarbonyl is added to the peptidic probe which may be N terminus or C terminus. If positioned in the C terminus, a Lys group may be added whereas if positioned inN terminus, no functional amino acid is needed. Ferrocene Carboxylic N-hydroxysuccinimide Ester is reacted with the primary amine in the (CH2)4-NH2 side chain of Lys. Lys was chosen because it is a simple way to attach redox Ferrocene carbonyl tag.

[0263] All publications, patents, and patent applications referred to 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.

[0264] While the applicant's teachings described herein are in conjunction with various embodiments for illustrative purposes, it is not intended that the applicant's teachings be limited to such embodiments. On the contrary, the applicant's teachings described and illustrated herein encompass various alternatives, modifications, and equivalents, without departing from the embodiments, the general scope of which is defined in the appended claims

[0265] The scope of the claims should not be limited by the preferred embodiments and examples but should be given the broadest interpretation consistent with the description as a whole.

Claims

1. CLAIMS1. A working electrode for detecting a bacteria comprisinga body having a detecting face and an electrically conductive surface on at least the detecting face, anda peptidic probe immobilized on at least a portion of the electrically conductive surface, the peptidic probe comprising an electroactive moiety and a cleavable peptide, the cleavable peptide optionally comprising at least one linker.

2. A needle apparatus for detecting a bacteria comprisinga detecting end and a connecting end,a plurality of working electrodes, comprising:a body having a detecting face and an electrically conductive surface on at least the detecting face of each working electrode, a peptidic probe immobilized on at least a portion of a surface of the electrically conductive surface, the peptidic probe comprising an electroactive moiety and a cleavable peptide, the cleavable peptide optionally comprising at least one linker, optionally wherein the plurality of working electrodes is a plurality of the working electrode of claim 1;a reference electrode;an electrode spacer configured to support and align the plurality of working electrodes and the reference electrode each inserted or insertable therein;a syringe hub counter-electrode, optionally configured to mate with and receive the electrode spacer;wherein each working electrode of the plurality extends from the detecting end to the connecting end; andwherein the plurality of working electrodes is substantially parallel at least at and approaching the detecting end.

3. The needle apparatus of claim 2 wherein the needle apparatus further comprises a non-functionalized working electrode comprising an electrically conductive surface lacking peptidic probe.

4. The needle apparatus of claim 2 or 3 wherein the reference electrode is centrally located and the working electrodes are regularly spaced around the reference electrode.

5. The needle apparatus of any one of claims 2 to 4, wherein each of the working electrodes are approximately equidistant from the reference electrode.

6. The needle apparatus of any one of claims 2 to 5, wherein the detecting face is disc shaped or elliptically shaped, optionally and independently wherein each working electrode is flush, recessed or protruding from the detecting end.

7. A microelectrode apparatus comprising:the needle apparatus of any one of claims 2 to 6; anda plurality of connectors configured to connect to the connecting end of the plurality of working electrodes and the reference electrode;wherein detection of the presence of bacteria is determined by an electrochemical signal at one or more of each working electrode of the plurality of working electrodes following cleavage of the cleavable peptide of the peptidic probe by a bacterial enzyme which releases the electroactive moiety.

8. The working electrode of claim 1 or the apparatus of any one of claims 2 to 7, wherein the working electrode or one or more of each of the plurality of working electrodes is or comprises gold, carbon, platinum, silver or a combination thereof.

9. The working electrode of claim 1 or 8 or the apparatus of any one of claims 2 to 8, wherein the working electrode or one or more of each of the plurality of working electrodes is or comprises gold.

10. The working electrode of any one of claims 1 , 8 or 9 or the apparatus of any one of claims 2 to 9, wherein the electrically conductive surface comprises a carbon material, gold, platinum, silver or a combination thereof.

11. The working electrode of any one of claims 1 , 8, 9 or 10, or the apparatus of any one of claims 2 to 9, wherein the electrically conductive surface comprises nanostructures.

12. The working electrode or the apparatus of claim 11 wherein the nanostructures are nanospikes.

13. The working electrode of any one of claims 1, 8-12 or the apparatus of any one of claims 2-12, wherein the surface of the electrically conductive surface has a peptidicprobe density of about or at least 1013molecules / cm2, of about or at least 1014molecules / cm2, or of about or at least 1015molecules / cm2.

14. The working electrode of any one of claims 1, 8-13 or the apparatus of any one of claims 2-13, wherein the electroactive moiety comprises ferrocene, methylene blue, anthraquinone, tetrathiafulvalene, Ruthenium complexes, Osmium complexes, quinones such as hydroquinone or benzoquinone, phenothiazines such as toluidine blue.

15. The working electrode of any one of claims 1, 8-14 or the apparatus of any one of claims 2-14, wherein the electroactive moiety comprises ferrocene.

16. The micro electrode apparatus of any one of claims 7-15, further comprising one or more of:a support assembly configured to mate with the electrode spacer, wherein the support assembly is configured to provide support and alignment of the plurality of connectors; anda binder configured to immobilize the plurality of working electrodes and the reference electrode enclosed in the syringe hub counter-electrode.

17. The working electrode of any one of claims 1 , 8-15 or the apparatus of any one of claims 2-16, wherein the at least one linker is an electrode linker, optionally comprising an ultimate Cys- and / or at least 2 PEG subunits, optionally 2, 4, 6, 8 10 or 12 PEG subunits , and / or 2, 4, 6, 8 10 or 12 amino acid residues, such as glycine, alanine, isoleucine, leucine, methionine or valine, optionally wherein the electrode linker comprises Cys and 2 PEG units or Cys and 4 PEG units.

18. The working electrode of any one of claims 1, 8-16 or the apparatus of any one of claims 2-17, wherein the bacteria is selected from Staphylococcus Aureus, Staphylococcus epidermidis, Escherichia Coli, Streptococcus pyogenes or Pseudomonas Aeruginosa or combinations thereof.

19. The working electrode or the apparatus of claim 18, wherein the bacteria is or comprises Staphylococcus Aureus.

20. The working electrode or the apparatus of claim 19, wherein the peptidic probe comprises Phe-Gly-P-(2-furyl)-Ala-Cys (SEQ ID NO: 71), Phe-Gly-P-(2-furyl)-Ala, Tyr-Gly-P-(2-furyl)-Ala, Trp-Gly-P-(2-furyl)-Ala, Val-Gly-P-(2-furyl)-Ala, Met-Gly-P-(2-f uryl)-Ala, lle-Gly-£-(2-furyl)-Ala, Leu-Gly-P-(2-furyl)-Ala, Phe-Gly-3-(furan-2- yl)-P-Ala, Ala-Gly-3-(furan-2-yl)-P-Ala, Gly-Gly-3-(furan-2-yl)-P-Ala, Vai -Gly-3- (furan-2-yl)-P-Ala Met- Gly-3-(furan-2-yl)-P-Ala, lie- Gly-3-(furan-2-yl)-P-Ala, Leu- Gly-3-(furan-2-yl)-P-Ala, Phe-Gly-P-(furan-2-yl)-Val, Ala-Gly-P-(furan-2-yl)-Val, Gly-Gly-P-(furan-2-yl)-Val, Val-Gly-P-(furan-2-yl)-Val, Met-Gly-P-(furan-2-yl)-Val, lle-Gly-P-(furan-2-yl)-Val, Leu-Gly-P-(furan-2-yl)-Val, Phe-Gly-y-(furan-2-yl)-Leu, Ala- Gly-y-(furan-2-yl)-Leu, Gly-Gly-y-(furan-2-yl)-Leu, Val-Gly-y-(furan-2-yl)-Leu, Met- Gly-y-(furan-2-yl)-Leu, lie- Gly-y-(furan-2-yl)-Leu, or Leu- Gly-y-(furan-2-yl)- Leu or an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 71 where the Gly-P-(2-furyl)-Ala or Gly-3-(furan-2-yl)-P-Ala or Gly-P-(furan- 2-yl)-Val or Gly-y-(furan-2-yl)-Leu cleavage site residues are maintained and / or any N terminal or C terminal Cys, Phe or Lys residues are maintained.

21. The working electrode of any one of claims 1 , 8-19 or the apparatus of any one of claims 2-19, wherein the peptidic probe is or comprises Ferrocenylcarbonyl-Phe- Gly-p-(2-furyl)-Ala-PEG2-Cys, Ferrocenylcarbonyl-Phe-Gly-p-(2-furyl)-Ala-PEG4- Cys, Ferrocenylcarbonyl-Phe-Gly-p-(2-furyl)-Ala-PEG6-Cys, or Ferrocenylcarbonyl-Phe-Gly-p-(2-furyl)-Ala-PEG12-Cys.

22. The working electrode or the apparatus of claim 18, wherein the bacteria is or comprises Staphylococcus epidermidis.

23. The working electrode or the apparatus of claim 22, wherein the peptidic probe comprises Ala-Thr-Leu-Thr (SEQ ID NO:9), Thr-Leu-Thr-Tyr (SEQ ID NO: 10), Ala- Thr-Leu-Thr-Tyr (SEQ ID NO:11), Gly-Ala-Thr-Leu-Thr (SEQ ID NO:12), Gly-Ala- Thr-Leu-Thr-Tyr (SEQ ID NO:13), Ala-Thr-Leu-Thr-Tyr-Thr (SEQ ID NO:14), Gly- Ala-Thr-Leu-Thr-Tyr-Thr (SEQ ID NO:15), Met-Ser-Leu-Leu (SEQ ID NO:16), Ser- Leu-Leu-Ser (SEQ ID NO:17), Phe-Met-Ser-Leu-Leu (SEQ ID NO:18), Met-Ser-Leu- Leu-Ser (SEQ ID NO:19), Phe-Met-Ser-Leu-Leu-Ser (SEQ ID NQ:20), Met-Ser-Leu- Leu-Ser-Met (SEQ ID NO:21), Phe-Met-Ser-Leu-Leu-Ser-Met (SEQ ID NO:22), Phe- Met-Thr-Leu-Leu-Ser-Met (SEQ ID NO:23), Met-Thr-Leu-Leu (SEQ ID NO:24), Trp- Met-Thr-Leu-Leu (SEQ ID NO:25), Trp-Met-Thr-Leu-Leu-Cys (SEQ ID NO:26), Phe- Met-Gly-Leu-Leu-Ser-Met (SEQ ID NO:27), Met- Gly -Leu-Leu-Ser-Met (SEQ ID NO:28), Trp-Met-Gly-Leu-Leu-Ser-Met (SEQ ID NO:29), Met-Gly-Leu-Leu-Cys-Met (SEQ ID NQ:30), Phe-Met-Gly-Leu-Leu-Cys-Met (SEQ ID NO:31), Phe-Met-Gly- Leu-Leu-Thr-Met (SEQ ID NO:32), Met-Gly-Leu-Leu-Thr-Met (SEQ ID NO:33), Met-Ala-Leu-Leu-Thr-Met (SEQ ID NO:34), Tyr-Met-Ser-Leu-Leu-Ser-Met (SEQ ID NO:35), Phe-Leu-Ser-Leu-Leu-Ser-Met (SEQ ID NO: 56), Phe-lle-Ser-Leu-Leu-Ser-Met (SEQ ID NO: 57), Phe-Met-Ser-Leu-Leu-Ala-Met (SEQ ID NO: 58), Phe-Met-Ser-Leu-Leu-Ser-Leu (SEQ ID NO: 59), or Phe-Met-Ser-Leu- Leu-Ser-lle (SEQ ID NO: 60), X1-X2-X3-Leu-Thr-X4-X5, X1-X2-X3-Leu-Leu-X4-X5, Phe-Met-Ser-Leu-Leu-Ser-Met-Lys (SEQ ID NO: 68), Gly-Ala-Thr-Leu-Thr-Tyr-Thr- Lys (SEQ ID NO: 69), or Cys- PEG2-Phe-Met-Ser-Leu-Leu-Ser-Met-Lys(SEQ ID NO: 68) or Cys-PEG2-Gly-Ala-Thr-Leu-Thr-Tyr-Thr-Lys-(Ferrocenylcarbonyl) (SEQ ID NO: 69) or an amino acid sequence having at least 90% sequence identity to any of SEQ ID NO: 9-60 or 68-69 or X1-X2-X3-Leu-Thr-X4-X5 or X1-X2-X3-Leu-Leu-X4-X5 as defined herein where the Leu-Thr or Leu-Leu cleavage site residues are maintained and / or any N terminal or C terminal Cys, Phe or Lys residues are maintained.

24. The working electrode or the apparatus of claim 18, wherein the peptidic probe comprises Phe-Met-Ser-Leu-Leu-Ser-Met (SEQ ID NO: 22) or Gly-Ala-Thr-Leu-Thr- Tyr-Thr (SEQ ID NO: 15), optionally wherein the peptidic probe is or comprises Cys- PEG2-Phe-Met-Ser-Leu-Leu-Ser-Met-Lys-(Ferrocenylcarbonyl) (SEQ ID NO: 68) or Cys-PEG2-Gly-Ala-Thr-Leu-Thr-Tyr-Thr-Lys-(Ferrocenylcarbonyl) (SEQ ID NO: 69).

25. The working electrode or the apparatus of claim 18, wherein the bacteria is or comprises Escherichia Coli.

26. The working electrode or the apparatus of claim 25, wherein the peptidic probe comprises Phe-Phe-Arg-Arg (SEQ ID NO: 1 ), Asp-Phe-Phe-Arg-Arg (SEQ ID NO:2), Gly-Asp-Phe-Phe-Arg-Arg (SEQ ID NO:3), Gly-Leu-Leu-Gly-Asp-Phe-Phe-Arg-Arg (SEQ ID NO:4), Arg-Trp-Ala-Arg (SEQ ID NO:5), Gly-Arg-Trp-Ala-Arg (SEQ ID NO:6), Gly-Gly-Arg-Trp-Ala-Arg (SEQ ID NO:7), Ala-Arg-Arg-Leu (SEQ ID NO:8), Cys-Gly-Leu-Leu-Gly-Asp-Phe-Phe-Arg-Arg-Lys-(Ferrocenylcarbonyl) (SEQ ID NO: 72), Cys-Gly-Gly-Arg-Trp-Ala-Arg-Lys-(Ferrocenylcarbonyl) (SEQ ID NO: 73); or Cys-Ala-Arg-Arg-Leu-Lys-(Ferrocenylcarbonyl) (SEQ ID NO: 74) or an amino acid sequence having at least 90% sequence identity to any of SEQ ID NO: 1 to 8, or 72- 74 where the Arg-Arg, Ala-Arg or Arg-Leu cleavage site residues are maintained and / or any N terminal or C-terminal Cys, Phe or Lys residues are maintained .

27. The working electrode or the apparatus of claim 18, wherein the bacteria is or comprises Streptococcus pyogenes.

28. The working electrode or the apparatus of claim 27, wherein the peptidic probe comprises Asn-Trp-Val-GIn-Arg (SEQ ID NO:36), Asn-Trp-Val-GIn-Arg-Val (SEQ IDNO:37), Trp-Val-GIn-Arg-Val (SEQ ID NO:38), Asn-Trp-Val-GIn-Arg-Val-Val (SEQ ID NO:39), Gln-Trp-Val-Gln-Arg-Val-Val (SEQ ID NQ:40), Val-Val(SEQ ID NO:41), Asp-Trp-Val-GIn-Arg-Val-ValVal-GIn-Arg-Val-Val (SEQ ID NO:43), Asn-Phe-Val-GIn-Arg-Val-Val (SEQ ID NO:44), Asn-Trp-lle-GIn-Arg-Val-Val (SEQ ID NO:45), Asn-Trp _eu-Gln-Arg- Val-Val (SEQ ID NO:46), Asn-Trp^Val-GIn-Arg-lle-Leu (SEQ ID NO:47), , Pro-lle- Val-Lys-Lys (SEQ ID NO:48), Pro-lle-Val-Lys-Lys-Leu (SEQ ID NO:49), Pro-lle- Val-Lys-Lys-Val (SEQ ID NQ:50), Pro-Val-Leu-Lys-Lys-lle-lle (SEQ ID NO:51), Pro-lle-Val-Lys-Lys-lle-lle (SEQ ID NO:52), Pro-lle-Val-Lys-Lys-lle-lle-Lys (SEQ ID NO:53), Pro-lle-Leu-Lys-Lys-Leu-Val (SEQ ID NO:54), Pro-Val-lle-Lys-Lys- Val-Val (SEQ ID NO:55), Asrg-Val-Val (SEQ ID NO: 61), Asn- Trp-Val-GIn-Arg-Val-lle (S Pro-Leu-lle-Lys-Lys-Val-Leu (SEQ ID NO: 63), X1-X2-X3- defined , P-X2-X3-Lys-Lys-X4-X5 as defined herein, Cys-PEG2-Ag-Val-Val-Lys (SEQ ID NO: 70) or Cys-PEG2-Pro-lle-Val-Lys-Lys-lle-lle-Lys (SEQ ID NO: 53) or an amino acid sequence having at least 90% sequence identity to any of SEQ ID NO: 36-55, 61-63, or 70 , or X1-X2-X3-Gln-Arg-X4-X5 as defined herein, or P-X2-X3-Lys-Lys-X4-X5 as defined herein, where the Gin-Arg or Lys-Lys cleavage site residues are maintained and / or any N terminal or C-terminal Cys, Phe or Lys residues are maintained, optionally wherein the peptidic probe is Cys-PEG2-Asn-Trp-Val-Gln-Arg-Val-Val-Lys- (Ferrocenylcarbonyl) (SEQ ID NO: 70) or Cys-PEG2-Pro-lle-Val-Lys-Lys-lle-lle-Lys- (Ferrocenylcarbonyl) (SEQ ID NO: 53).

29. The apparatus of any one of claims 2-28, wherein the reference electrode comprises an Ag / AgCI electrode.

30. The apparatus of any one of claims 2-29, wherein the electrode spacer comprises between 2 to 8 holes, each of the holes for housing one of the plurality of working electrodes.

31. The apparatus of any one of claims 2-30, wherein the syringe hub counter-electrode is a stainless-steel syringe hub.

32. The apparatus of any one of claims 16-31 , wherein the binder comprises medical grade epoxy.

33. A package comprising the working electrode or the apparatus of any one of claims 1 to 32 and inert gas, wherein the package is hermetically sealed.

34. A detection system comprising the working electrode or the apparatus of any one of claims 1-32 or the package of claim 33; and a potentiostat configured to connect to the plurality of connectors,wherein the potentiostat is configured to control a difference of potential between the reference electrode and working electrodes while measuring the currents flowing through the working electrodes.

35. The system of claim 34, further comprising a data processor and / or a display.

36. The system of claim 34 or 35, wherein the system is a unit.

37. The system of claims 34 or 35, wherein the needle apparatus or microelectrode apparatus are a unit and the potentiostat is a separate unit.

38. A method for detecting bacteria, the method comprising:a. inserting the detecting end of the working electrode or the needle or microelectrode apparatus of any one of claims 1 to 32, the package of claim 33 or the system of any one of claims 34 to 37 in a test sample or tissue suspected of bacterial infection, optionally in a wound; andb. measuring an electrochemical signal;wherein an electrochemical signal comprises one or more of current, voltage, charge, impedance, capacitance, or a change therein is produced upon cleavage of a peptidic probe.

39. The method of claim 38, wherein the needle is contacted with the test sample or tissue, optionally wound, for at least 5 minutes or at least 10 minutes.

40. The method of claim 38, wherein the needle is contacted with the test sample or tissue, optionally wound for between about 10 minutes to about 20 minutes.

41. The method of claim 38, wherein the needle is contacted with the test sample or tissue, optionally wound for about 15 minutes.

42. The method of any one of claims 38-41, wherein measuring the electrochemical signal comprises calibrating the microelectrode apparatus.

43. The method of any one of claims 38-42, wherein measuring the voltage or current comprises using square wave voltammetry (SWV).

44. The method of any one of claims 38-43, wherein the bacteria causes necrotizing fasciitis (NF).

45. The method of any one of claims 38-44, wherein the detecting end of the working electrode or the needle or microelectrode apparatus of any one of claims 1 to 32, the package of claim 33 or the system of any one of claims 34 to 37 is rinsed prior to measuring an electrochemical signal optionally in PBS.

46. A method of making a working electrode, comprising:a. obtaining an electrode comprising a detecting face, the electrode being a gold, platinum, silver or carbon electrode;b. mechanically polishing the detecting face of the electrode; the mechanical polishing comprising polishing with an alumina slurry, ultrasonic cleaning, rinsing and drying;c. chemically polishing the detecting face using an oxidative solution, optionally a Piranha solution, rinsing, drying and immersing in ethanol, rinsing and drying; d. electropolishing the detecting face via cyclic voltammetry;e. optionally electrodepositing a gold nanostructure layer on a surface of the detecting face of the electrode wherein the surface, optionally with the god nanostructure layer, comprised an electrically conductive surface; and f. optionally immobilizing a quantity of peptidic probe to the electrically conductive surface, optionally via a thiolate gold bond, the peptidic probe comprising an electroactive moiety and a cleavable peptide,thereby providing the working electrode, optionally the working electrode of claiml.

47. The method of claim 46, wherein the mechanical polishing comprises polishing with a disc embedded with diamond particles, previously wet, prior to polishing with the alumina slurry.

48. The method of claim 46 or 47, wherein the electrochemically active surface area (ECSA) is about or at least 10’3cm2, about or at least 10’2cm2, or about or at least 101cm2and roughness factor is about or at least 101, or about or at least 1, or about or at least 10.

49. The method of claim 48, wherein the peptidic probe is immobilized to the surface of the electrically conductive surface via a linker.

50. A needle apparatus comprisinga detecting end and a connecting end,a plurality of working electrodes, each working electrode of the plurality having a detecting face and extending from the detecting end to the connecting end and comprising:an electrically conductive surface of the detecting face of each working electrode;a reference electrode;an electrode spacer configured to support and align the plurality of working electrodes and the reference electrode each inserted or insertable therein;a syringe hub counter-electrode, optionally configured to mate with and receive the electrode spacer;wherein the plurality of working electrodes is substantially parallel at least at and approaching the detecting end.

51. A microelectrode apparatus for detection of bacteria, wherein the apparatus is:a) the needle apparatus of claim 50; andb) a plurality of connectors configured to connect to the plurality of working electrodes and the reference electrode;wherein detection of the presence of bacteria is determined by the change of electrochemical signal at one or more of each working electrode of the plurality of working electrodes.

52. The apparatus of claim 51, wherein the apparatus is for use in detecting Pseudomonas Aeruginosa.

53. A method for detecting bacteria, the method comprising:a. inserting the detecting end of the needle apparatus of the microelectrode apparatus of claim 51 or 52 into a test sample or tissue, optionally a wound; andb. measuring an electrochemical signal ;wherein an electrochemical signal comprises one or more of current, voltage, charge, impedance, capacitance, or a change therein is produced in the presence of bacteria54. The method of claim 53, wherein the bacteria is Pseudomonas Aeruginosa.

55. A method of diagnosing a subject, comprising:a) performing the method of any of claims claim 38-44; andb) diagnosing said subject with the presence of bacteria infection based upon the pattern and quantum of peptidic probe cleavage.

56. A method of treating a person, comprising:performing the method of claim 55; andtreating the subject with a suitable treatment according to the bacteria infection detected.

57. An isolated cleavable peptide comprising or consisting ofPhe-Phe-Arg-Arg (SEQ ID NO:1), Asp-Phe-Phe-Arg-Arg (SEQ ID NO:2), Gly-Asp- Phe-Phe-Arg-Arg (SEQ ID NO:3), Gly-Leu-Leu-Gly-Asp-Phe-Phe-Arg-Arg (SEQ ID NO:4), Arg-Trp-Ala-Arg (SEQ ID NO:5), Gly-Arg-Trp-Ala-Arg (SEQ ID NO:6), Gly-Gly- Arg-Trp-Ala-Arg (SEQ ID NO:7), Ala-Arg-Arg-Leu (SEQ ID NO:8), Cys-Gly-Leu-Leu- Gly-Asp-Phe-Phe-Arg-Arg-Lys (SEQ ID NO: 72), Cys-Gly-Gly-Arg-Trp-Ala-Arg-Lys- (SEQ ID NO: 73); or Cys-Ala-Arg-Arg-Leu-Lys (SEQ ID NO: 74);Ala-Thr-Leu-Thr (SEQ ID NO:9), Thr-Leu-Thr-Tyr (SEQ ID NO: 10), Ala-Thr-Leu-Thr- Tyr (SEQ ID NO: 11), Gly-Ala-Thr-Leu-Thr (SEQ ID NO: 12), Gly-Ala-Thr-Leu-Thr-Tyr (SEQ ID NO:13), Ala-Thr-Leu-Thr-Tyr-Thr (SEQ ID NO:14), Gly-Ala-Thr-Leu-Thr-Tyr- Thr (SEQ ID NO:15), Met-Ser-Leu-Leu (SEQ ID NO:16), Ser-Leu-Leu-Ser (SEQ ID NO:17), Phe-Met-Ser-Leu-Leu (SEQ ID NO:18), Met-Ser-Leu-Leu-Ser (SEQ ID NO:19), Phe-Met-Ser-Leu-Leu-Ser (SEQ ID NO:20), Met-Ser-Leu-Leu-Ser-Met (SEQ ID NO:21), Phe-Met-Ser-Leu-Leu-Ser-Met (SEQ ID NO:22), Phe-Met-Thr-Leu-Leu- Ser-Met (SEQ ID NO:23), Met-Thr-Leu-Leu (SEQ ID NO:24), Trp-Met-Thr-Leu-Leu (SEQ ID NO:25), Trp-Met-Thr-Leu-Leu-Cys (SEQ ID NO:26), Phe-Met-Gly-Leu-Leu- Ser-Met (SEQ ID NO:27), Met- Gly -Leu-Leu-Ser-Met (SEQ ID NO:28), Trp-Met-Gly- Leu-Leu-Ser-Met (SEQ ID NO:29), Met-Gly-Leu-Leu-Cys-Met (SEQ ID NO:30), Phe- Met-Gly-Leu-Leu-Cys-Met (SEQ ID NO:31), Phe-Met-Gly-Leu-Leu-Thr-Met (SEQ ID NO:32), Met-Gly-Leu-Leu-Thr-Met (SEQ ID NO:33), Met-Ala-Leu-Leu-Thr-Met (SEQ ID NO:34), Tyr-Met-Ser-Leu-Leu-Ser-Met (SEQ ID NO:35), Phe-Leu-Ser- Leu-Leu-Ser-Met (SEQ ID NO: 56), Phe-lle-Ser-Leu-Leu-Ser-Met (SEQ ID NO: 57), Phe-Met-Ser-Leu-Leu-Ala-Met (SEQ ID NO: 58), Phe-Met-Ser-Leu-Leu- Ser-Leu (SEQ ID NO: 59), or Phe-Met-Ser-Leu-Leu-Ser-lle (SEQ ID NO: 60), X1- X2-X3-Leu-Thr-X4-X5, X1-X2-X3-Leu-Leu-X4-X5 , Phe-Met-Ser-Leu-Leu-Ser-Met- Lys (SEQ ID NO: 68), Gly-Ala-Thr-Leu-Thr-Tyr-Thr-Lys (SEQ ID NO: 69), Cys-PEG2- Phe-Met-Ser-Leu-Leu-Ser-Met-Lys (SEQ ID NO: 68) or Cys-PEG2-Gly-Ala-Thr-Leu- Thr-Tyr-Thr-Lys- (SEQ ID NO: 69);Asn-Trp-Val-GIn-Arg (SEQ ID NO:36), Asn-Trp-Val-GIn-Arg-Val (SEQ ID NO:37), Trp-Val-GIn-Arg-Val (SEQ ID NO:38), Asn-Trp-Val-GIn-Arg-Val-Val (SEQ ID NO:39), Gln-Trp-Val-Gln-Arg-Val-Val (SEQ ID NQ:40), Ser-Trp-Val-GIn-Arg-Val- Val(SEQ ID NO:41), Asp-Trp-Val-GIn-Arg-Val-Val (SEQ ID NO:42), Asn-Tyr-Val- Gln-Arg-Val-Val (SEQ ID NO:43), Asn-Phe-Val-GIn-Arg-Val-Val (SEQ ID NO:44), Asn-Trp-lle-GIn-Arg-Val-Val (SEQ ID NO:45), Asn-Trp-Leu-GIn-Arg-Val-Val (SEQ ID NO:46), Asn-Trp-Val-GIn-Arg-lle-Leu (SEQ ID NO:47), Pro-lle-Val-Lys- Lys (SEQ ID NO:48), Pro-lle-Val-Lys-Lys-Leu (SEQ ID NO:49), Pro-lle-Val-Lys- Lys-Val (SEQ ID NQ:50), Pro-Val-Leu-Lys-Lys-lle-lle (SEQ ID NO:51), Pro-lle- Val-Lys-Lys-lle-lle (SEQ ID NO:52), Pro-lle-Val-Lys-Lys-lle-lle-Lys (SEQ ID NO:53), Pro-lle-Leu-Lys-Lys-Leu-Val (SEQ ID NO:54), Pro-Val-lle-Lys-Lys-Val-Val (SEQ ID NO:55), Asn-Trp-Asn-GIn-Arg-Val-Val (SEQ ID NO: 61), Asn-Trp-Val- Gln-Arg-Val-lle (SEQ ID NO: 62), or Pro-Leu-lle-Lys-Lys-Val-Leu (SEQ ID NO: 63), X1-X2-X3-Gln-Arg-X4-X5, P-X2-X3-Lys-Lys-X4-X5, Cys-PEG2-Asn-Trp-Val- Gln-Arg-Val-Val-Lys (SEQ ID NO: 70) or Cys-PEG2-Pro-lle-Val-Lys-Lys-lle-lle-Lys (SEQ ID NO: 53);Phe-Gly-P-(2-furyl)-Ala-Cys (SEQ ID NO: 71), Phe-Gly-P-(2-furyl)-Ala, Tyr-Gly-P-(2- furyl)-Ala, Trp-Gly-P-(2-furyl)-Ala, Val-Gly-P-(2-furyl)-Ala, Met-Gly-P-(2-furyl)-Ala, lle-Gly-P-(2-furyl)-Ala, Leu-Gly-P-(2-furyl)-Ala, Phe-Gly-3-(furan-2-yl)-P-Ala, Ala- Gly-3-(furan-2-yl)-P-Ala, Gly-Gly-3-(furan-2-yl)-P-Ala, Val-Gly-3-(furan-2-yl)-P-Ala, Met- Gly-3-(furan-2-yl)-P-Ala, lie- Gly-3-(furan-2-yl)-P-Ala, Leu-Gly-3-(furan-2-yl)-P- Ala, Phe-Gly-P-(furan-2-yl)-Val, Ala-Gly-P-(furan-2-yl)-Val, Gly-Gly-P-(furan-2-yl)- Val, Val-Gly-P-(furan-2-yl)-Val, Met-Gly-P-(furan-2-yl)-Val, lle-Gly-P-(furan-2-yl)- Val, Leu-Gly-P-(furan-2-yl)-Val, Phe-Gly-y-(furan-2-yl)-Leu, Ala-y Gly-(furan-2-yl)- Leu, Gly-Gly-y-(furan-2-yl)-Leu, Val-Gly-y-(furan-2-yl)-Leu, Met-Gly-y-(furan-2-yl)- Leu, lle-Gly-y-(furan-2-yl)-Leu, or Leu- Gly-y-(furan-2-yl)-Leu.

58. A peptidic probe comprising the isolated peptide of claim 57 and an electroactive moiety, wherein the electroactive moiety is conjugated to a C terminus or N terminus of the isolated peptide, optionally via an electroactive moiety linker, optionally wherein the peptidic probe comprises or is Cys-PEG2-Phe-Met-Ser-Leu-Leu-Ser-Met-Lys- (Ferrocenylcarbonyl) (SEQ ID NO: 68), Cys-PEG2-Gly-Ala-Thr-Leu-Thr-Tyr-Thr-Lys- (Ferrocenylcarbonyl) (SEQ ID NO: 69), Cys-PEG2-Asn-Trp-Val-Gln-Arg-Val-Val-Lys- (Ferrocenylcarbonyl) (SEQ ID NO: 70), Cys-PEG2-Pro-lle-Val-Lys-Lys-lle-lle-Lys- (Ferrocenylcarbonyl) (SEQ ID NO: 53), Cys-Gly-Leu-Leu-Gly-Asp-Phe-Phe-Arg-Arg- Lys-(Ferrocenylcarbonyl) (SEQ ID NO: 72), Cys-Gly-Gly-Arg-Trp-Ala-Arg-Lys- (Ferrocenylcarbonyl) (SEQ ID NO: 73); or Cys-Ala-Arg-Arg-Leu-Lys-(Ferrocenylcarbonyl) (SEQ ID NO: 74)..

59. A composition comprising the isolated cleavable peptide of claim 57 or the peptidic probe of claim 58 and a carrier, optionally a stabilizer.

60. The composition of claim 59, wherein the composition is lyophilized or frozen.

61. A kit for detecting bacteria comprising the working electrode or the apparatus of any one of claims 1-32, the package of claim 33, the isolated cleavable peptide of claims 57, the peptidic probe of claim 58 or the composition of any one of claims 59 and 60 optionally further comprising one or more of instructions for use, or one or more standards or reagents for standardizing, such as known quantities of isolated cleavable peptides, peptidic probes and / or composition described herein or a package, comprising an apparatus, isolated cleavable peptide, peptidic probe or composition described herein.

62. The kit of claim 61, wherein the needle comprises one or more working electrodes, the reference electrode, the syringe hub counter-electrode, and electrode spacer in a single disposable needle.

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