System and method for electrochemical sensors with reduced signal loss

Abstract

Electrochemical aptamer-based (EAB) sensors assist in high-frequency, real-time monitoring of analytes in vivo. Methods for reducing sensor drift during in vivo deployment are described. Sensor signal drift can be reduced by using EAB sensors containing non-natural nucleic acids. EAB sensors exhibit improved stability and reduced signal loss.

Description

[Technical Field]

[0001] Cross-reference to related applications This application claims priority to U.S. Provisional Patent Application No. 63 / 506,560, filed June 6, 2023, entitled "Electrochemical Sensor Having Reduced Signal Loss." The disclosures of U.S. Provisional Patent Application No. 63 / 506,560 are incorporated herein by reference in their entirety for all purposes.

[0002] Description of federally funded research This invention was made with the assistance of the U.S. Government under AI164483 and R01AI145206, awarded by the National Institutes of Health. The U.S. Government has certain rights in this invention.

[0003] Sequence List This application is filed electronically in XML format and includes a sequence listing which is incorporated herein by reference in its entirety. The above XML copy, generated on May 20, 2024, is named 08507PCT.xml and is 7 kilobytes in size.

[0004] Field of Invention The present invention relates to an electrochemical sensor having nucleic acids, configured to specifically detect analytes in biological fluids. The signal output of the sensor is configured to withstand undesirable signal loss and is therefore useful for monitoring analytes over extended periods. [Background technology]

[0005] Electrochemical aptamer-based (EAB) sensors are known for detecting target analytes in biological fluids. The aptamer portion of the sensor is an oligonucleotide of a defined base sequence that can selectively interact with the target analyte. In one version, the aptamer is coupled to the working electrode surface, and the redox reporter is coupled to the free end of the aptamer. Binding of the analyte to the aptamer causes a conformational change in the aptamer, thereby moving the redox reporter more proximal to the electrode. This movement then affects the electron transfer rate (k) between the redox reporter and the electrode. et This causes an increase in k et The changes provide real-time information about the target analyte concentration, without the addition of exogenous reagents.

[0006] EAB sensors can perform in situ measurements of numerous drugs and metabolites with second- and / or sub-second resolution in vivo, and can assist in closed-loop feedback controlled drug delivery. For in vivo applications, the sensor may include an aptamer-coated microneedle or wire as the working electrode. The microneedle or wire may be inserted through the skin surface so that the aptamer-coated portion is in contact with the biological fluids of the subcutaneous tissue. Alternatively, the sensor may be placed in another body compartment, such as a vein. The sensor may also include a counter electrode and a reference electrode. These electrodes may also be in the form of microneedles or wires, similarly inserted subcutaneously.

[0007] Sensor electrodes may remain in situ for minutes, hours, or even days, and during that period, they can provide clinically valuable information regarding the amount of analytes in body fluids. Reasonable extrapolation can be performed to estimate the amount of analytes in systemic circulation. In this way, EAB sensors can provide clinically relevant information regarding the amount of exogenous analytes (e.g., drugs) or endogenous analytes (e.g., hormones) in a subject. This information can be used for the diagnosis, treatment, and / or monitoring of disease. For example, if the analyte is a drug, the information provided by the sensor can be used to optimize drug therapy. For example, the amount of drug may be monitored to ensure that it is above the minimum effective concentration but remains below the concentration at which toxic effects could occur. The drug dosage may be adjusted to ensure that appropriate levels are maintained.

[0008] Interrogation for an EAB sensor to detect a target analyte requires the application of a potential waveform, resulting in a current output from a working electrode used to determine the amount of the target analyte in the solution. Square wave voltammetry (SWV) may be used for in vivo detection of an analyte if it has the ability to compensate for losses in the current output (also called "drift"). To explain, the amount of analyte reported by an EAB sensor tends to drift downward over time. Measuring square wave voltammograms at two different frequencies allows for drift compensation in an approach called kinetic differential measurement (KDM). KDM subtractively compensates for drift by utilizing the difference between relative SWV measurements performed at two frequencies.

[0009] While drift correction methods are effective, after a certain period of time, the current output from the sensor will inevitably decrease to a level below the noise floor. At that point, the system has no detectable signal above the level of inherent noise, and the sensor is no longer functional in detecting the target analyte. In this art, the potential delivered to the sensor during interrogation generally plays a role in causing aptamer loss from the working electrode surface. Fouling of the working electrode in biological substances (e.g., proteins, lipids, and whole cells) in the biological fluid under analysis is also considered a contributing factor.

[0010] The problem of signal drift, ultimately resulting in signal loss, negatively impacts the usefulness of EAB sensors, particularly in in vivo clinical applications where accurate determination of analyte amounts over long periods is critical. As an example of such applications, a sensor may be configured to detect antibiotics in situ in a subject via an electrode in contact with plasma. Long-term monitoring of antibiotics may be necessary to ensure that drug administration maintains drug concentrations above the minimum inhibitory concentration in vivo, while ensuring they do not reach toxic concentrations. Typically, antibiotics are monitored for at least 24 hours, but prior art sensors cannot maintain a signal for such long periods without significant signal loss and a simultaneous decrease in the signal-to-noise ratio. While signal loss can be addressed by continuous sensor replacement, this approach has a cost penalty. Repeated sensor replacement requires continuous disturbance of the subject. Furthermore, replacing a new sensor (and, furthermore, calibrating it) is time-consuming and can result in data loss over the associated changeover period.

[0011] Considerations of documents, actions, materials, apparatus, articles, etc., are included herein solely for the purpose of providing background to the present invention. Because these issues existed prior to the priority date of each provisional claim of this application, it is not implied or shown that any or all of these issues form part of the prior art base or were common general knowledge in the art related to the present invention. [Overview of the Initiative]

[0012] This specification summarizes, and is described in detail below, EAB sensors with improved performance, such as (but not limited to) reduced signal loss.

[0013] Some embodiments include a working electrode for an electrochemical sensor, which comprises a conductive element and an analyte sensing element associated with the conductive element, configured to specifically interact with a target analyte, the sensing element comprising a non-natural nucleic acid coupled with a redox reporter.

[0014] In some embodiments, non-natural nucleic acids are unreadable and / or replicated by any natural mammalian cell.

[0015] In some embodiments, non-natural nucleic acids exhibit greater resistance to degradation by components of biological fluids compared to similar natural nucleic acids.

[0016] In some embodiments, the similarity relates to one or more of the following: length, base sequence, secondary structure, tertiary structure, and the ability to interact with the target analyte.

[0017] In some embodiments, the biological fluid contains a nuclease.

[0018] In some embodiments, non-natural nucleic acids are formed from single strands.

[0019] In some embodiments, the non-natural nucleic acids are polymers.

[0020] In some embodiments, the unnatural nucleic acid comprises 10 to 100 subunits.

[0021] In some embodiments, the unnatural nucleic acid is a chemical variant of natural deoxyribonucleic acid or natural ribonucleic acid.

[0022] In some embodiments, the chemical variant is to the sugar backbone and / or one or more bases.

[0023] In some embodiments, the unnatural nucleic acid is made by humans or with human assistance.

[0024] In some embodiments, the unnatural nucleic acid is xeno nucleic acid (XNA) or peptide nucleic acid (PNA).

[0025] In some embodiments, the unnatural nucleic acid is a DNA or RNA aptamer having a modified chemical structure.

[0026] In some embodiments, the unnatural nucleic acid is a modified form of a natural nucleic acid that can specifically interact with a target analyte, and the unnatural nucleic acid has an interaction ability of at least 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, or 150% of that of the natural nucleic acid.

[0027] In some embodiments, the unnatural nucleic acid is a modified form of a natural nucleic acid that can specifically interact with a target analyte, and the unnatural nucleic acid has a sensitivity, accuracy, or specificity regarding the recognition of the target analyte of at least 50%, 60%, 70%, 80%, 90%, or 100% of that of the natural nucleic acid.

[0028] In some embodiments, the unnatural nucleic acid is a modified form of a natural nucleic acid that can specifically interact with a target analyte, and the unnatural nucleic acid has an uncorrected signal loss of less than 90%, 80%, 70%, 60%, or 50% of that of the natural nucleic acid.

[0029] In some embodiments, the working electrode is a portion of an electrochemical sensor with an uncorrected signal drift rate, which is determined or averaged over a period of at least 10 minutes, 20 minutes, 30 minutes, 40 minutes, 50 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 24 hours, 36 hours, 48 ​​hours, or 72 hours.

[0030] In some embodiments, non-natural nucleic acids bind to a conductive element at a first end.

[0031] In some embodiments, the redox reporter binds to the second end of the non-natural nucleic acid.

[0032] In some embodiments, the conductive element includes a skin penetration portion to which a non-natural nucleic acid is bound.

[0033] In some embodiments, the conductive element is a needle, a microneedle, or a wire.

[0034] Some embodiments include an electrochemical sensor device comprising a working electrode and a counter electrode according to any one of claims 1 to 21.

[0035] Some embodiments include a reference electrode.

[0036] Some embodiments involve a retainer configured to hold a working electrode that comes into contact with the subject's bodily fluids.

[0037] In some embodiments, the retainer is configured to hold a working electrode in contact with the subject's bodily fluids for a period of at least 10 minutes, 20 minutes, 30 minutes, 40 minutes, 50 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 24 hours, 36 hours, 48 ​​hours, or 72 hours.

[0038] Some embodiments involve a housing configured to enclose a power supply and / or electronics for the function of the sensor.

[0039] Some embodiments include a method for monitoring a target analyte in a biological fluid of a subject, the method comprising the step of bringing a working electrode according to any one of claims 1 to 21 into contact with the biological fluid for a period of time.

[0040] In some embodiments, the working electrode comes into contact with a biological fluid that remains in situ within the subject for the duration of the method.

[0041] In some embodiments, the biological fluid is blood or interstitial fluid.

[0042] In some embodiments, the biological fluid is not removed from the subject.

[0043] In some embodiments, the duration is at least 10 minutes, 20 minutes, 30 minutes, 40 minutes, 50 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 24 hours, 36 hours, 48 ​​hours, or 72 hours.

[0044] Further embodiments and features are partially shown in the following description and will be partially apparent to those skilled in the art in a closer examination of this specification or can be learned through the practice of this disclosure. A further understanding of the nature and merits of this disclosure may be achieved with respect to the remaining parts of the specification and drawings that form part of this disclosure. [Brief explanation of the drawing]

[0045] This specification will be better understood with reference to the following figures. The figures are presented as embodiments of the invention and should not be considered a complete enumeration of the scope of the invention.

[0046] [Figure 1] Figure 1A is a schematic representation of the response of the EAB sensor to the presence of a target analyte, according to prior art.

[0047] Figure 1B illustrates the immersion of an EAB sensor using DNA sequences in whole blood (in vitro) at 37°C, in accordance with prior art.

[0048] Figure 1C illustrates the placement of an EAB sensor using DNA sequences in the jugular vein (in vivo) of a rat, in accordance with prior art.

[0049] Figures 1D and 1E show graphs of normalized currents in vitro and in vivo as a function of time, obtained by SWV collected at two frequencies, following prior art.

[0050] Figures 1F and 1G show graphs of in vitro and in vivo KDM signals as a function of time, following prior art.

[0051] [Figure 2] Figure 2A schematically and chemically illustrates an EAB sensor using a natural DNA molecule according to one embodiment, and its destruction by an agent in a biological fluid.

[0052] Figure 2B schematically and chemically illustrates an EAB sensor having a non-natural nucleic acid that functions similarly to the aptamer in Figure 2A in its interaction with a target analyte, but is resistant to degradation by the agent in Figure 2A, according to one embodiment.

[0053] Figure 2C shows a graph of the relative sensor output signals as a function of time, comparing the output of sensors having native versus non-native nucleic acids as sensing elements in the presence of nucleases, according to one embodiment.

[0054] [Figure 3] Figure 3A shows the KDM signal as a function of tobramycin, using natural and non-natural nucleic acids as sensing elements, according to one embodiment.

[0055] Figure 3B shows a graph of the normalized current as a function of time for a sensor having native and non-native nucleic acids separately as sensing elements, placed in the left and right jugular veins of a single rat exposed to an aminoglycoside antibiotic, according to one embodiment.

[0056] Figure 3C shows the KDM signal as a function of time for a sensor like the one used in Figure 3B, according to one embodiment.

[0057] Figure 3D shows the concentration of aminoglycosides as a function of time for a sensor like the one used in Figure 3B, according to one embodiment.

[0058] Figure 3E illustrates the signals of DNA and Ome RNA aptamer sensors according to one embodiment.

[0059] Figures 3F and 3G illustrate the charge transfer dynamics of a DNA aptamer sensor and an OMe RNA aptamer sensor, respectively, according to one embodiment.

[0060] Figure 3H illustrates the normalized signals of a DNA aptamer sensor and an OMe RNA aptamer sensor according to one embodiment.

[0061] [Figure 4] Figure 4A schematically shows in vitro whole blood immersed in an EAB sensor containing either a natural or non-natural nucleic acid as a sensing element, according to one embodiment. The graph on the right shows the relative sensor signal intensity as a function of time for each sensor type.

[0062] Figure 4B schematically illustrates an EAB sensor, according to one embodiment, placed in the jugular vein of a rat, having either a native or non-native nucleic acid as a sensing element. The non-native nucleic acid sensor was inserted before the native nucleic acid sensor. The graph on the right shows the relative sensor signal intensity as a function of time for each sensor type and for each of the two treated rats.

[0063] Figure 4C schematically illustrates an EAB sensor, according to one embodiment, placed in the jugular vein of a rat, having either a native or non-native nucleic acid as a sensing element. The native nucleic acid sensor was inserted before the non-native nucleic acid sensor. The graph on the right shows the relative sensor signal intensity as a function of time for each sensor type and for each of the two treated rats.

[0064] [Figure 5]Figures 5A–5F show a series of charge-transfer-versus-frequency plots (Lovric plots) over 5 hours, illustrating the time evolution of electron transfer dynamics for EAB sensors deployed under various conditions according to one embodiment. Figure 5A shows a Lovric plot for a sensor made from natural nucleic acids exposed to DNase in vitro. Figure 5B shows a Lovric plot for a sensor made from non-natural nucleic acids exposed to DNase in vitro in PBS buffer. Figure 5C shows a Lovric plot for a sensor made from natural nucleic acids exposed to DNase in whole blood in vitro. Figure 5D shows a Lovric plot for a sensor made from non-natural nucleic acids exposed to DNase in whole blood in vitro. Figure 5E shows a Lovric plot for a sensor made from natural nucleic acids in whole blood in vivo. Figure 5F shows a Lovric plot for a sensor made from non-natural nucleic acids in whole blood in vivo.

[0065] [Figure 6] Figure 6 illustrates a top perspective view of a microneedle implantation device according to one embodiment. This embodiment relies on providing power for the user to insert microneedles into the skin. The arm is shown in a first position, as presented to the user, before the microneedles are implanted into the skin.

[0066] [Figure 7] Figure 7A illustrates a lower perspective view of Figure 6 according to one embodiment.

[0067] Figure 7B illustrates an upper perspective view of Figure 6 according to one embodiment.

[0068] [Figure 8] Figure 8 illustrates a lower perspective view of Figure 6 according to one embodiment, more clearly showing the removable flexible layer that is removed to expose a dermatologically acceptable adhesive.

[0069] [Figure 9] Figure 9 illustrates, in a lower perspective view, the microneedle implantation device of Figure 6, according to one embodiment, having a removable flexible layer that is removed to expose a dermatologically acceptable adhesive.

[0070] [Figure 10] Figure 10 illustrates, in a lower perspective view, the microneedle implantation device of Figure 9, which includes microneedles in an extended position for implantation in the skin of a subject, according to one embodiment.

[0071] Unless otherwise specified herein, features in figures labeled with the same number are to be interpreted as being the same feature, or at least functionally similar, when used across different figures.

[0072] The figures are not provided to any particular scale or dimension, nor are they presented as a completely accurate representation of various embodiments. [Modes for carrying out the invention]

[0073] After considering this description, it will be apparent to those skilled in the art how to carry out the present invention in various alternative embodiments and applications. However, while various embodiments of the present invention will be described herein, it will be understood that these embodiments are presented only as examples and not as limiting. Thus, this description of various alternative embodiments should not be considered as limiting the scope or breadth of the present invention. Furthermore, references to advantages or other aspects apply to specific exemplary embodiments and not necessarily to all embodiments, or in fact to any embodiments included by the claims.

[0074] Throughout this description and claims, the word “comprise” and its variations, such as “comprising” and “comprises,” are not intended to exclude other additions, components, integers, or steps.

[0075] Throughout this specification, any reference to “one embodiment” or “an embodiment” means that certain features, structures, or characteristics described in relation to that embodiment are included in at least one embodiment of the present invention. Therefore, the phrases “in one embodiment” or “in one embodiment” in various places throughout this specification do not necessarily all refer to the same embodiment, although they may refer to the same one.

[0076] The term "subject" is used to refer to animals (including humans and non-human animals) to which the present invention may be applied.

[0077] As used herein, “body fluids” include, but are not limited to, interstitial fluid (ISF), blood, saliva, tear secretions, lactation secretions, nasal secretions, tracheal secretions, bronchial secretions, alveolar secretions, gastric secretions, gastric contents, glandular secretions, vaginal secretions, uterine secretions, prostatic secretions, semen, urine, sweat, cerebrospinal fluid, glomerular filtrate, hepatic secretions, bile, or exudates, any of which come into contact with the electrode of the present invention during use.

[0078] Based at least in part on our findings, the present invention predicts that non-natural nucleic acids, which have similar functionality to natural nucleic acid aptamers (e.g., DNA and RNA) but exhibit significantly lower signal loss rates when used in in vivo EAB sensors, can be used as detection elements. The result is a sensor that can provide a signal output higher than the noise floor over extended periods.

[0079] Many embodiments demonstrate that the degradation of aptamers in EAB sensors by agents present in biological fluids is a significant, and perhaps primary, cause of signal loss observed in vivo. In some embodiments, the agent is a nuclease. These findings represent a new fact about the previously unknown underlying cause of the signal drift problem. In light of this new fact, we have discovered that, in the background of EAB sensors, non-natural nucleic acids resist signal drift while functioning similarly to (and in some situations better than) natural nucleic acid aptamers in detecting analytes.

[0080] These findings are consistent with the accepted dogma in the art that signal loss is due to interrogate current and aptamer loss caused by fouling in biological materials.

[0081] Significant advantages are offered in applications where the degradation of sensing aptamers in EAB sensors is a concern. Apart from the advantages in in vivo applications discussed above, certain ex vivo applications may benefit. In some embodiments, electrochemical sensors used to monitor the presence of analytes in industrial processes involving biological materials may be resistant to degradation over the course of process execution. Some embodiments provide in vitro testing of clinical samples in pathology facilities. Even brief contact with a clinical sample can lead to significant degradation of the sensor, resulting in unstable output signals. Furthermore, sensors may be cleaned and used with several samples; in such situations, resistance to degradation would extend the sensor's service life.

[0082] In this specification, the term “non-natural nucleic acid” is intended to include polymers that are biosimilars to natural nucleic acid polymers, such as DNA or RNA, but have been modified and possess chemical structures not found in nature. As a result of structural modification, non-natural nucleic acids may be more resistant than natural nucleic acids to degradation (e.g., cleavage of chemical bonds) caused by agents found in biological fluids, such as blood and ISF. These agents may be nucleases, such as DNases or RNases.

[0083] Non-natural nucleic acids may be derived from naturally occurring nucleic acids, but they have been modified in their chemical structure so that their chemical structure is considered non-natural. More typically, non-natural nucleic acids will be synthesized de novo in their modified form.

[0084] The term "non-natural nucleic acids" is intended to include nucleic acids that are synthesized by humans, or with human assistance, but nevertheless possess a natural chemical structure. These molecules are not natural products, but nevertheless have the same chemical structure as naturally occurring nucleic acids.

[0085] In the context of the present invention, useful non-natural nucleic acid molecules (i.e., useful as sensing elements associated with the working electrode of an EAB sensor) may be modified aptamers. For example, a DNA aptamer known to detect an analyte (e.g., an antibiotic) may be chemically modified to retain its ability to detect the analyte while also being resistant to degradation by agents in biological fluids. Non-natural nucleic acids may be oligomers having a non-natural backbone and being molecular analogs of DNA or RNA. Examples of non-natural backbone oligomers include, without limitation, 2'-fluoroarabinoside nucleic acids (FANA), 2'-O-methylRNA, loct nucleic acids (LNA), and threose nucleic acids (TNA). Collectively, these non-natural backbone oligomers are referred to as xeno nucleic acids (XNA). Because they are not produced naturally, XNAs are generally highly resistant to enzymatic degradation. In some embodiments, the non-natural nucleic acid may be a peptide nucleic acid (PNA).

[0086] Apart from modified chemical structures whose stability in biological fluids has been confirmed, the non-natural nucleic acids of the present invention may share one or more features with prior art aptamers. These features may include, but are not limited to, length, base sequence (primary structure), secondary structure, and tertiary structure. Aptamers are small (typically 20-60 nucleotides) RNA or DNA oligonucleotides formed from a single strand that can bind to a target analyte with high affinity and specificity. Aptamers may be considered nucleotide analogs of antibodies, but aptamer production is an in vitro, cell-free process, which is easier and less expensive than antibody production by cell culture or in vivo methods.

[0087] One method for identifying useful aptamers in the background of the present invention is to use a method for identifying native DNA or RNA aptamers (e.g., SELEX), and then modify the identified aptamers to have a non-native chemical structure. Alternatively, a method such as SELEX may be adapted by using an enzyme configured to synthesize and amplify the non-native nucleic acid of the first example.

[0088] Aptamers may be selected from a combinatorial library containing a very large number (up to 10¹⁸) different oligonucleotides. RNA aptamers offer greater structural diversity compared to DNA aptamers, but their application is complicated by stability issues such as the presence of RNases, high temperatures, and unfavorable pH levels.

[0089] The selection of aptamers that are selective for a given analyte can be facilitated by a process known as SELEX (systematic evolution of ligands by exponential enrichment). This process can be considered as two alternating steps. In the first step, library oligonucleotides are amplified to the desired concentration by polymerase chain reaction (PCR). For RNA aptamer selection, single-stranded oligonucleotides are generated by in vitro transcription of double-stranded DNA with T7 RNA polymerase. For DNA aptamers, a pool of oligodeoxyribonucleotides is generated by strand separation of the double-stranded PCR product. In the second step, the amplified product is incubated with the target analyte, and the oligonucleotides that bind to the analyte are used in the next SELEX round.

[0090] The isolation of oligonucleotides with higher affinity for the target drug, and the removal of unbound oligonucleotides, are achieved through strong competition for binding sites. Selective pressure occurs in every SELEX round. Sufficient enrichment of the oligonucleotide pool with the aptamer having the strongest affinity for the target analyte can be achieved after approximately 5–15 rounds.

[0091] Once identified as useful, non-natural nucleic acids may be associated with the working electrode of the EAB sensor. The working electrode may have at least one associated counter electrode and at least one associated reference electrode. Each working electrode may have a dedicated counter electrode, but in some embodiments, the counter electrodes are shared among some or all of the set of working electrodes. Each working electrode may have a dedicated reference electrode, but in some embodiments, the reference electrodes are shared among some or all of the set of working electrodes.

[0092] In many embodiments, the EAB sensor useful in the context of this disclosure may be voltametric, chronoamperometric, or impedimetric. In a voltametric sensor, a potential waveform is applied to the sensor interface and the resulting current response is recorded. In a chronometric approach, a step potential is applied and the resulting time-evolving current response is recorded. In impedimetric sensing, a sinusoidal potential waveform is applied and the resulting sinusoidal current response is recorded.

[0093] EAB sensors are typically voltametric, and an aptamer (or, according to many embodiments, a non-natural nucleic acid) binds to a working electrode. Gold may be used as the probe surface of the working electrode. The aptamer has a relevant redox active species that acts as a reporter. The redox reporter may be (but not limited to) methylene blue. Upon target (e.g., drug) binding, the aptamer undergoes a conformational change to bring the redox reporter more proximal to the working electrode surface. This increased proximal positioning increases electron transfer from the redox reporter to the electrode. The increased electron transfer rate contributes to a change in the Faraday current detected by a potentiostat. The EAB sensor may be incorporated into a circuit having a reference electrode. The reference electrode is a site of a known chemical reaction with a known redox potential. For example, a reference electrode based on a silver-silver chloride (Ag / AgCl) redox pair has a fixed, known potential, which forms a point against which the redox potential of the working electrode is measured. Typically, the circuit includes a counter electrode that functions as either a cathode or anode relative to the working electrode. Since the current does not pass through the reference electrode (due to the impedance of the potentiostat), any generated current originates from the working electrode and the counter electrode. The current is measured as a function of the potential difference between the interrogating electrode and the reference electrode. The potential difference generates a current in the circuit, which in turn produces an output signal. The signal, in accordance with electron transfer, quantifies the target bond, which is ideally stoichiometrically proportional to the target bond.

[0094] The device, when assembled, is particularly suitable for use as a wearable device, allowing measurements to be performed while the subject is going about their normal activities and / or over a long period of time. In some embodiments, the wearable device may be a necklace, bracelet or other suitable piece of jewelry, a watch, clothing, a strap, adhesive, or a patch. Those skilled in the art will recognize that, during use, means to assist in attaching and / or holding the wearable device to the subject, such as micro-anchors, may be provided.

[0095] In some embodiments, the wearable device may include a housing structure comprising one or more other components, such as (but not limited to) electronic processing units. The electronic processing units are configured to have direct or indirect electrical communication with at least one conductive element (e.g., an electrode) and will generally include any one or more of a power supply, a data processing unit, an analog front end, and a wireless transmitter.

[0096] In some embodiments, the housing structure may be configured to at least partially enclose the device such that electrodes (e.g., microneedles) are exposed from the plane of the housing structure. The electrodes may be protected by a protective cover, which may be removed before use to expose the protruding electrodes.

[0097] In many embodiments, if the reliability of the output depends on the temperature or pH of the body fluid, or if the operation or output is adjustable, the device may further include means for monitoring the temperature or pH of the body fluid.

[0098] In some embodiments, the housing structure may be configured to enclose or couple with a device by any suitable mechanism. Examples of mechanisms include (but are not limited to) electromagnetic couplings, mechanical couplings, adhesive couplings, magnetic couplings, and the like. In some embodiments, the coupling mechanism allows the device and the housing structure to be attached and detached, which allows the housing structure and other components to be reused, while also allowing the device to be discarded and replaced with another device if necessary.

[0099] In many embodiments, the wearable device may further include a computer program product that can be run as a software application on a mobile communication device that communicates with an electronic processing unit, the computer program product being capable of controlling one or more of the following: (i) detection of electrochemical measurements performed on an electrode-based platform, (ii) data analysis, (iii) data transmission, (iv) device configuration, and (v) device power management. Suitable mobile communication devices include, but are not limited to, smartphones, smartwatches, tablets, smart glasses, laptops, or other personal computers.

[0100] In some embodiments, the device itself includes a processor that includes program instructions configured to drive mounted functions, such as voltammetry, and transmit output to a remote device via a wireless module, such as a Bluetooth® module.

[0101] In some embodiments, the working electrode or any other electrode may be a wire, needle, microneedle, electrode array, or microneedle array, which comes into contact with the subject's ISF, blood, or any other suitable somatic substance. For transdermal applications where skin penetration is required to come into contact with the ISF, microneedles and / or microneedle arrays are preferred.

[0102] Electrodes according to many embodiments can be manufactured in a range of diverse shapes and arrangements, but for reliable skin penetration, their specific arrangement for transdermal application is optimized to disrupt the stratum corneum. In some embodiments, the device may be configured to facilitate entry into the subject's skin, promoting stratum corneum disruption of the electrode and penetration through the skin layer. For non-human applications, the stratum corneum may be replaced by a similar or even dissimilar layer on the subject's surface.

[0103] Generally, each electrode will have a protruding, pointed structure that extends from the mount. Typically, the electrodes will generally extend perpendicularly from the mount.

[0104] The protruding structures of each electrode may be of any needle shape. In some embodiments, the protruding structures may taper smoothly from a base to form a pointed tip (e.g., conical), have multiple sides extending from a base which converge to form a pointed tip (e.g., pyramidal or triangular prism), taper in only one direction, or have a base with curved sides of a relatively constant diameter which are divided to form a pointed tip (e.g., tubular segments). Typically, the pointed tip will be sharp. The electrodes may or may not include shape changes along their length. Furthermore, any edges or sides of the shape may be oblique, curved, or rounded.

[0105] In some embodiments, the electrode shape is conical or pyramidal, such as triangular, square, or hexagonal. In other embodiments, the electrode shape is tetrahedron or triangular prism. In further embodiments, it may take the shape of a rocket, turret, arrowhead, spike, or spear.

[0106] It will be recognized that other shapes within a certain range may be used. In certain embodiments, the shape may be a truncated circular or elliptical cylinder. Any of the other shapes described herein may or may not be truncated. The term “truncated,” when used in this context, may refer to a shape cut by a plane parallel to the base, which may be called a parallel truncated shape, or more specifically a frustum, or a shape cut at an angle to the axis of the electrode, which may be called an angular-truncated shape. With respect to an angular-truncated shape, the angle of the truncation with respect to the axis of the shape is at least about 50° and not exceeding about 75°. It will be recognized that the same or different shapes may be provided on a single EAB sensor.

[0107] The outer wall of the microneedle may have a smooth or rough surface and may include (but not limited to) surface features such as raised areas, etching, serrations, anchors, or barbs, which may assist in engaging with biological tissue to keep the electrode in the subject once it has broken through the stratum corneum. The ability of the EAB sensor to remain in situ would be particularly beneficial, as it ensures that continuous measurements over extended periods are performed at the same site within the subject. Furthermore, restricting the location where measurements are performed ensures more accurate long-term monitoring. In some embodiments, the EAB sensor is configured to remain in situ for at least 1 minute, or at least 1 hour, or at least about 8 hours, or at least about 18 hours, or at least 1 day (about 24 hours), or at least about 3 days, or at least about 4 days, or at least 1 week. In some applications, it may be necessary or desirable for the sensor to remain in situ for more than one month.

[0108] It will be recognized that the size of the electrodes and their placement on the mount may vary depending on the intended application.

[0109] In many embodiments, the electrodes may be at least longer than the thickness of the stratum corneum, penetrate the skin layer to a depth of at least 100 μm, and be positioned in biological tissue to be in contact with the subject's bodily fluids. In some embodiments, the length is at least about 10% greater than the thickness of the stratum corneum, or at least about 20% greater than the thickness of the stratum corneum, or at least about 50% greater than the thickness of the stratum corneum, or at least about 75% greater than the thickness of the stratum corneum, or at least about 100% greater than the thickness of the stratum corneum. In some embodiments, the length is less than about 1500 μm, or less than about 1000 μm, or less than about 500 μm, or less than about 50 μm, or less than about 20 μm, or less than about 10 μm. In some embodiments, the length is about 100 μm to about 1000 μm.

[0110] In some embodiments, the electrodes have a stepped arrangement and therefore will not all be the same length.

[0111] The base width of the electrode may be less than at least about 50% of the length, or less than about 25% of the length, or less than about 20% of the length, or less than about 15% of the length, or less than about 10% of the length, or less than about 5% of the length. In some embodiments, the base width is at least about 100 μm, but not exceeding about 400 μm. In some embodiments, the diameter is about 200 μm, or about 300 μm.

[0112] The electrode diameter is less than at least about 50% of the length, or less than about 25% of the length, or less than about 20% of the length, or less than about 15% of the length, or less than about 10% of the length, or less than about 5% of the length. In some embodiments, the diameter is at least about 0.1 mm to about 5 mm. In some embodiments, the diameter is at least about 0.5 mm to about 1 mm.

[0113] It may be desirable for one microneedle to penetrate deeper into the skin than the other. The two microneedles may therefore terminate at different distances from the skin surface or at different distances from the electrode mounting portion. In some embodiments, the two microneedles are of different lengths. In some embodiments, the microneedles are of the same length, and the mounting portion is set to move one microneedle axially compared to others. In certain embodiments, the mounting portion may be multi-level, including a first electrode extending from a first level and a second electrode extending from a second level.

[0114] In many embodiments, electrodes may be arranged in pairs, groups, or as a matrix. A paired arrangement would include an even number of electrodes. A grouped arrangement may include 1 to about 5 groups, each group containing about 4 to about 8 electrodes. A matrixed arrangement may include either an even or odd number of electrodes, and as such, the arrangement may or may not have the same number of columns and / or rows. In some embodiments, electrodes are arranged in a matrix selected from the group consisting of 2x2, 2x3, 2x4, 2x5, 2x6, 3x2, 3x3, 3x4, 3x5, 3x6, 4x2, 4x3, 4x4, 4x5, 4x6, 5x2, 5x3, 5x4, 5x5, 5x6, 6x2, 6x3, 6x4, 6x5, and 6x6. In any configuration, electrodes may be spaced less than approximately 5 mm, or less than approximately 4 mm, or less than approximately 3 mm, or less than approximately 2 mm, or less than approximately 1 mm, or less than approximately 0.5 mm, and with a gap of more than approximately 0.1 mm between them. The gap may be measured from midpoint to midpoint of each electrode.

[0115] The present invention will be described more fully hereby by reference to the following non-limiting embodiments. [Examples]

[0116] The following examples are provided to those skilled in the art to offer a complete disclosure and explanation of how the present invention is constructed and used, and are not intended to limit the scope of what the inventors consider to be their invention, nor to indicate that the following experiments are all or only experiments performed. While efforts have been made to ensure accuracy with respect to the numerical values ​​used (e.g., quantities, temperatures, etc.), some degree of experimental error and deviation should be taken into consideration.

[0117] Example 1: Materials and Sources The following materials were used in Examples 2-5.

[0118] In vitro sensors were fabricated from 0.2 mm diameter gold wire (99.99%) insulated with polyolefin heat shrink tubing (0.05”, 0.017”, 0.007”). For in vitro testing, commercially available Ag|AgCl(or) reference electrodes and commercially available platinum reference electrodes were used. Sensors used for in vivo measurements consisted of 0.2 mm diameter gold wire, 0.005 inch diameter platinum wire (99.99% purity), and 0.005 inch diameter silver wire (99.99% purity). The insulation used for these sensors was polytetrafluoroethylene heat shrink tubing (PTFE, 0.02”, 0.005”, 0.003”, black).

[0119] Sodium hydroxide, 6-mercapto-1-hexanol, tris(2-carboxyethyl)phosphine, sulfate, and DNase were used. Phosphate-buffered saline (PBS) was diluted from 20x stock. Heparinized bovine whole blood was used. Tobramycin sulfate was used. Other methylene blue- and HO-C6S-S-C6-modified DNA sequences, including T37 methylene blue- and HO-C6S-S-C6-modified sequences and 2'-O-methyl sequences, were used.

[0120] Example 2: Manufacturing of an EAB sensor In vitro sensors were fabricated by exposing a 3 mm length of wire and insulating it with polyolefin (see, e.g., Leung et al; ACS Sensors 2021, 6 (9), 3340-3347; this disclosure is incorporated by reference). After fabrication, these sensors were then electrochemically cleaned. In vivo sensors were fabricated by bundling gold (working electrode), platinum (counter electrode), and silver (reference electrode) wires parallel to each other. These wires were insulated from each other and bundled together in a staggered manner using polytetrafluoroethylene heat shrink tubing. The gold wire was positioned at the bottom with an exposed length of 3 mm, followed by 6 mm of exposed platinum, and finally 1 cm of exposed silver. Once bundled together, this three-electrode sensor was immersed overnight in household bleach to chlorine-treat the silver electrode. The three electrodes were then rinsed with Millipore water before electrochemical cleaning.

[0121] After electrochemically cleaning the gold portions of both the in vitro and in vivo sensors in NaOH, they were roughened in H2SO4 using a CH1040C potentiostat. Cleaning involved 1000 cycling cycles at a potential between -1.0V and -2V at 2V / s in 0.5M NaOH. Following this, the gold wire was roughened in 0.5M H2SO4 by applying 20ms pulses 32000 times at 0V and 2.2V to increase the microscopic surface area of ​​the electrode. Subsequently, the gold electrode was analyzed in 0.5M H2SO4 by cyclic voltammetry (1.5 to -0.35V at 1V / s) to determine its electroactive surface area. The electrode was then thoroughly rinsed with Millipore water before DNA deposition. At this point, and then in vivo, the sensor was inserted into a 20G catheter.

[0122] Before depositing DNA onto the gold, the HO-C6S-S-C6 modified aptamer was first deprotected by combining 14 μL of 10 mM tris(2-carboxyethyl)phosphine with 2 μL of 100 μM DNA in the dark for 1 hour. After deprotection, the DNA was diluted to 500 nM in PBS. The electrochemically cleaned sensor was then rinsed by immersion in the DNA solution in the dark. After a 1-hour incubation period, the sensor was transferred to a 10 mM 6-mercapto-1-hexanol solution prepared in PBS and stored overnight before use. [Table 1]

[0123] Example 3: Electrochemical Measurement In vitro electrochemical measurements were generally performed using SWV (see, e.g., Leung et al; ACS Sensors 2021, 6 (9), 3340-3347; this disclosure is incorporated by reference). As a quality check, all sensors were first tested at room temperature in a shot glass containing 1x PBS before use. These were then transferred at 37°C to a shot glass containing whole blood or PBS containing 2.5 mM CaCl2 and 1.5 mM MgCl2, and electrochemical measurements were immediately started. For DNase experiments, after obtaining a stable baseline, measurements were paused, sensors were quickly removed, 5 μg / mL DNase was added, the solution was mixed, and then measurements were resumed. The sensor was repeatedly interrogated at multiple square wave frequencies (5, 7, 10, 15, 30, 50, 70, 100, 200, 250, 300, 600, and 1000 Hz), and sensor resolution was monitored using a square wave frequency close to the methylene blue electron transfer velocity.

[0124] In vivo electrochemical sensors were placed in the jugular veins of living rats. For experiments involving monitoring the degradation of DNA and 2'-O-methylRNA sensor analogs (using T37 and OMe37 sequences), the sensors were repeatedly interrogated at numerous square wave frequencies, using a square wave frequency close to the methylene blue migration velocity, for analysis. For experiments involving tobramycin detection sensors, SWV was performed alternately at frequencies of 100 Hz and 200 Hz. Signal drift was corrected by calculating the KDM signal using these two frequencies as follows:

number

[0125] Example 4: In vivo surgery and drug administration All in vivo experiments were performed in male Sprague-Dawley rats (4-5 months old). The rats weighed 350-500g and were housed in pairs in a room with a standard light cycle (12:12 periodic light cycle, light turned on at 8 AM). The rats had free access to food and water.

[0126] Prior to measurement, rats were anesthetized in a Plexiglas anesthesia chamber using 4% isofluorane, and then anesthesia was maintained through the nose cone for the entire duration of the experiment using a 2–2.5% isofluorane / oxygen mixture. The necks were shaved, and the left and right jugular veins were incised to surgically isolate them. Using spring-loaded microscises, small incisions were made into each vein to allow insertion of a catheter (for heparin delivery) with an in vivo sensor and infusion line. Both the sensor and infusion line were secured using two sterile 6-0 silk sutures. Prior to any recording, 30 units of heparin were infused through the infusion line (jugular vein delivery for tobramycin experiments and femoral vein for aminoglycosides). To administer tobramycin intravenously to the rats (30 mg / kg), 0.1 M tobramycin sulfate diluted in a pre-calculated volume of PBS was injected through the femoral vein using a syringe pump.

[0127] Example 5: Experiment and Discussion of Results EAB sensors assist in high-frequency, real-time measurements of specific molecules in vivo, in situ. Signaling in this type of sensor occurs when a binding-inducible conformational change in the target analyte-recognizing aptamer alters the electron transfer rate from the attached redox reporter. This signaling mechanism allows for the generation of aptamers to bind to a wide range of small molecule analytes, thus making EAB sensors independent of specific chemical structures or the reactivity of their targets. Therefore, EAB sensors represent the first in vivo molecular monitoring approach that can be generated across numerous types of analytes. In line with this, EAB sensors can perform in situ measurements of numerous drugs and metabolites with second- and / or sub-second resolution in the veins, brain, and subcutaneous ISF of living animal subjects, potentially assisting closed-loop feedback-controlled drug delivery.

[0128] Figures 1A–1G illustrate how an electrochemical aptamer-based (EAB) sensor, following prior art, can assist in continuous real-time molecular monitoring in unprocessed biological fluids, both in vivo and in situ. Figure 1A shows an EAB sensor, each having a gold electrode to which a redox reporter-modified aptamer is attached via the formation of an alkane-thiol self-assembly monolayer on gold. Upon exposure to the target, the aptamer undergoes a conformational change that alters the electrochemical behavior of the redox reporter (methylene blue in this case), producing an easily measurable electrochemical signal. The EAB sensor was exposed in vitro in whole blood (commercially available bovine blood) (Figure 1B) or in vivo (in rat jugular vein, in situ) at 37°C (Figure 1C). Figures 1D and 1E show the electrochemical peak current, which exhibits a downward drift over time (data shown are collected at the square wave frequency pairs shown). Figures 1F and 1G demonstrate that this drift can be compensated for by taking the difference between relative currents observed in square wave frequency pairs where the signals drift simultaneously; this is a drift-compensating approach called dynamical differential measurement (KDM). Excellent return to baseline when the sensor is returned to drug-free blood in vitro, or when the drug is eliminated from the body in vivo, demonstrates the accuracy of this approach in compensating for drift. However, while KDM drift compensation ensures that the EAB sensor remains accurate, the loss of peak current associated with the drift reduces its signal-to-noise ratio and ultimately diminishes its accuracy (e.g., the standard deviation of the signal in Figure 1G increases from 0.008 before drug injection to 0.020 4 hours later in vivo). The data presented herein pertain to vancomycin-detecting EAB sensors exposed to vancomycin over the indicated time. The return to baseline when the sensor is returned to drug-free blood (in vitro) or when the drug is eliminated from the body (in vivo) demonstrates the accuracy of KDM's drift correction.

[0129] A limitation of EAB sensors is that when they are placed in bodily fluids, they suffer time-dependent signal loss, an effect that occurs both in vitro (Figure 1D) and in vivo (Figure 1E). This "drift" can be accurately compensated for using dual-frequency square wave approaches (Figures 1F, 1G), such as KDM, or it can be avoided by using interrogation methods such as chronoamperometry or electrochemical impedance spectrometers, which do not affect measurement accuracy. Nevertheless, the loss in the signaling current reduces the signal-to-noise ratio (regardless of the approach used to compensate for or avoid it), and this effect ultimately harms the accuracy of the measurement (see, for example, the noise increase on the right side of Figure 1G).

[0130] Prior art engineers have characterized the underlying mechanisms of in vitro EAB sensor drift in undiluted whole blood maintained at 37°C. Under these conditions, the drift is predominantly due to the electrochemically induced loss of its target recognition aptamer (presumably due to the loss of an attached thiol monolayer on the metal surface) as well as fouling of the electrode surface by proteins and cells, the former of which can be easily avoided by careful selection of the potential window used for interrogation. However, subsequent experiments have shown that the in vivo drift behavior of EAB sensors differs from that observed in in vitro whole blood. By elucidating the origin of this difference, many embodiments provide hardware and methods that can significantly reduce the in vivo drift of EAB sensors.

[0131] In body fluids and in vitro, the enzymatic degradation of the aptamers used by EAB sensors for target recognition is only a minor contributing factor to drift. For example, it has been previously shown that significant signal loss observed with EAB sensors exposed to undiluted whole blood at 37°C is approximately 80% recoverable when the sensor is washed with concentrated urea, indicating that physical removal of DNA from the surface, such as that which would occur during enzymatic degradation, contributes relatively little to the drift observed under these conditions. Several prior art engineers have similarly shown that, despite the exceptional DNase resistance of non-natural L-enantiomerized DNA oligonucleotides ("spiegelmer"), the in vitro blood and serum drift characteristics of sensors fabricated using this polymer are similar to those observed with natural D-DNA-based sensors placed under the same conditions. However, it is worth noting that the commercially available bovine blood and plasma used in these studies, several days old, can differ significantly from in situ blood in the veins of living animals. For example, DNases are known to be active in circulating blood. Given the potential for significant differences between these two experimental conditions, many embodiments provide the underlying mechanism for the drift observed with respect to EAB sensors placed in the jugular veins of living rats.

[0132] The blood-driven drift of EAB sensors in vivo differs significantly from that observed in vitro. In some embodiments, sensor-like constructs consisting of unstructured oligonucleotide sequences (i.e., lacking significant internal complementarity) were first used, prepared using either DNA or 2' methoxy-ribonucleic acid (OMeRNA), which is a non-natural xenonucleic acid (XNA) relatively resistant to enzymatic degradation.

[0133] Devices using unstructured oligomers of XNA 2'-methoxyRNA (OMeRNA) are more resistant to DNase degradation than devices using equivalent unstructured DNA oligonucleotides. Figures 2A–2C illustrate the significantly reduced enzymatic degradation of oligonucleotides by introducing non-natural oligonucleotide XNA according to one embodiment. To confirm that this applies to the 2'-methoxy-ribose backbone (OMeRNA) XNA used herein, several embodiments produce EAB sensor analogs using either DNA (Figure 2A) or OMeRNA (Figure 2B), modified with methylene blue on its 3' end and with a thiol for surface adhesion on its 5' end. The sequences used lack any significant self-complementarity to avoid any influence that may arise from the differential population of secondary structures. In Figure 2C, the signal from the DNA-using device decreases monotonically when exposed to DNase in PBS (containing the necessary divalent cations and maintained at 37°C). In contrast, after an initial small decrease, the signal from OMe RNA-based devices stabilizes. The shaded areas indicate the standard deviation of (n=4) independently fabricated devices; no significant difference in reproducibility was observed between devices fabricated using either DNA or XNA.

[0134] Given this observation, and the possibility that DNase activity may be higher in fresh in vivo blood than in aged in vitro blood, some embodiments investigate whether the use of OMe RNA can reduce the in vivo drift observed with respect to the EAB sensor when placed in vivo. However, to test this, it may be necessary to have an XNA aptamer available that binds to a molecular target and is suitable for use in vivo; that is, a target that is nontoxic at concentrations far above the detection limit of the sensor and is cleared from the blood at a rapid pace compared to the several-hour timeframe provided by experiments on anesthetized animals. The development of an aptamer selection scheme accepts the selection of XNA aptamers, but no XNA aptamer can satisfy both needs simultaneously. For example, most XNA aptamers reported to date bind to proteins, which are generally target species that are cleared from plasma fairly slowly. There is one small molecule-binding XNA aptamer that can bind to the mycotoxin orchratoxin, but its toxicity excludes its use in in vivo studies.

[0135] Many embodiments generate novel small molecule-binding XNA aptamers for desired in vivo applications. Some embodiments utilize nucleic acid receptors that bind to aminoglycoside antibiotics. Aminoglycosides, including tobramycin, gentamicin, and kanamycin, bind to prokaryotic ribosomal RNA, interfering with translation and inhibiting bacterial growth. Previous reports have included the observation that 14-nucleotide RNA hairpins excised from prokaryotic ribosomal RNA fold in vitro into a conformation that binds to this type of drug with micromorphic affinity. Motivated by the clinical importance of measuring aminoglycosides (the therapeutic windows for these nephrotoxic and ototoxic drugs are very narrow), the usefulness of adapting and using this RNA in an EAB sensor has been investigated (see, e.g., AA Rowe, et al., Anal. Chem. 2010, 82, 7090-7095; this disclosure is incorporated by reference). Unfortunately, while the resulting sensor achieved clinically adequate limits of detection, accuracy, and specificity, the RNA "aptamer" rapidly degraded when exposed to unprocessed biological fluids. An unexpected solution to this problem emerged during an atomically detailed structural investigation of the RNA / drug complex. In particular, (1) the hairpin forms a structure between the B-type of the DNA helix and the A-type of the RNA helix, and (2) the aminoglycoside binds in the main groove of the hairpin, opposite to the 2' hydroxyl of the double helix that distinguishes RNA from DNA. Given these observations, it may be worthwhile to test whether, in this unusual and specific case, an otherwise naive approach of simply substituting the RNA sequence with DNA would not significantly alter the structure or drug-binding properties of the nucleic acid. Tests showed that while its affinity was significantly reduced (four times compared to that of the equivalent RNA construct), the DNA construct also bound to the aminoglycoside.

[0136] Some embodiments provide that, considering the possibility that methoxy-RNA is more RNA-like than DNA, the same sequence synthesized from OMe RNA can also bind to aminoglycosides. In some embodiments, sensors using equivalent OMe RNA sequences not only bind to the antibiotic tobramycin but also bind with higher affinity than sensors using equivalent DNA aptamers. This increased affinity significantly improves the in vivo detection limit for detection with respect to OMe RNA aptamers. In particular, when placed in vivo, the root mean square noise observed before drug exposure corresponds to detection limits of 1.2 μM and 9.9 μM (with a coefficient of variation of 3) for sensors using OMe RNA and DNA aptamers, respectively.

[0137] Figures 3A–3D illustrate, according to one embodiment, that the use of an aminoglycoside-binding OMe RNA sequence significantly reduces the drift observed with respect to an EAB sensor placed in situ in the jugular vein of a living rat. Figure 3A shows an aminoglycoside detection EAB sensor using either an OMe RNA or DNA sequence that binds to an aminoglycoside antibiotic, and characterizes its binding to the drug tobramycin in vitro in bovine whole blood at 37°C. Error bars indicate the standard deviation of (n=4) independently fabricated sensors. In Figure 3B, the DNA-using and OMe RNA-using sensors are placed in the left and right jugular veins of rats, respectively. After 3 hours, the animals were exposed to a 30 mg / kg dose of tobramycin via a femoral catheter. The signal from the OMe RNA-using sensor drifts relatively little under these conditions (shown are the peak currents at 100 and 200 Hz, normalized to the initial voltammogram). In particular, over the course of this 5-hour in vivo experiment, the drift observed with the OMe RNA sensor was only 7%, nearly seven times less than the 48% signal loss observed with the DNA sensor. Figure 3C shows that while the drift observed with both sensors can be corrected using KDM, the signal loss associated with the DNA construct ultimately reduces accuracy by decreasing the signal-to-noise ratio. Note that the peak KDM signal observed with the OMe RNA sensor (in response to drug exposure) is due to the higher tobramycin binding affinity of this sequence (Figure 3A). Figure 3D shows that the conversion of these KDM signals to concentrations allows for a reproducible approximation of plasma drug levels. The slightly lower C observed with the OMe RNA sensor max C maxSuggests that it may be due to slower rises thereto and restrictions (e.g., blood clot formation or sensor intrusion into the vein wall) that delay the transport of the agent to the sensor. The duration of these experiments is limited by concerns for animal welfare, with restrictions on the time animals can be placed under anesthesia. Figure 3E illustrates the signals of DNA and OMe RNA aptamer sensors according to one embodiment. Figure 3E is an enlarged view of the signal changes before and after a 30 mg / kg intravenous tobramycin injection into the femoral vein of a rat. The injection is delivered to the DNA and OMe RNA aptamer sensors in the left and right jugular veins, respectively, of a single rat. For the OMe RNA sensor, there is a slight delay (about 2.5 minutes) before reaching C max Suggests that there may be a possibility that the transport of the agent to this sensor is restricted due to blood clot or intrusion of the working electrode into the vein wall. Both effects delay the transport of the agent to the sensor and lead to a somewhat lower C max observed.

[0138] To determine whether the in vivo drift performance of the OMe RNA sensor is improved compared to that of the DNA sensor, one of each was placed in the left and right jugular veins of anesthetized rats, respectively. The signal from the DNA device decreased by 48% after 5 hours under these conditions, while the signal from the equivalent OMe RNA device decreased by only 7% (Figure 3B), and the difference was maintained across a range of square wave frequencies. KDM drift correction accurately compensates for the drift observed in both (Figure 3C), but after only a few hours, the signal-to-noise ratio drops significantly due to the greater signal loss from the DNA sensor. Crucially, both sensors remain functional in animals, as demonstrated by intravenous tobramycin (30 mg / kg) exposure. Due to the higher affinity of the OMe RNA aptamer (Figure 3A), the KDM signal change resulting from this exposure is particularly greater for the OMe RNA sensor (Figure 3C). However, when these signals are converted to concentrations, the two datasets produce very similar concentration-time profiles (Figure 3D). Figures 3F and 3G illustrate the charge transfer dynamics of a DNA aptamer sensor and an OMe RNA aptamer sensor according to one embodiment, respectively. When sensors using aminoglycoside-linked DNA (Figure 3F) or OMe RNA (Figure 3G) constructs are exposed in vivo, the drift observed for both is largely independent of the square wave frequency used. The charge transfer dynamics shown here are recorded before tobramycin administration. Figure 3H illustrates the normalized signals of a DNA aptamer sensor and an OMe RNA aptamer sensor according to one embodiment. At a frequency of 70 Hz, which best matches the electron transfer rate, mismatched in vivo drifts are observed between the aminoglycoside-linked constructs.

[0139] The use of OMe RNA significantly improves the in vivo drift characteristics of aminoglycoside detection EAB sensors, in contrast to previous observations that the use of this backbone only slightly reduces drift when the EAB sensor is used in vitro in undiluted whole blood maintained at 37°C (see, e.g., KK Leung, et al., ACS Sensors 2021, 6, 3340-3347; this disclosure is incorporated by reference). To elucidate the origin of this difference, several embodiments characterize the in vivo performance of devices using unstructured constructs of DNA and OMe RNA, eliminating any complexity that may arise from differences in secondary or tertiary structure. Previously, devices using these simpler constructs exhibited significant initial drift when placed in vitro in whole blood at 37°C, even though OMe RNA-using devices showed somewhat less drift. Following this rapid decrease, the signals produced by both devices then "level off," suggesting that they may rise due to some saturable effect, e.g., fouling. Several embodiments built upon this foundation replicated these experiments in vivo in the left and right jugular veins of four living rats. Under these conditions, devices using both DNA and OMe RNA again exhibited a rapid loss of signal. As was the case in vitro, the signal emanating from the OMe RNA-using device then flattened and remained relatively stable. However, the signal emanating from the DNA-using device continued to decline throughout the duration of our experiments, a behavior in contrast to that of the same device in vitro in blood. These observations suggest that, in both in vivo and in vivo drift observed with respect to the OMe RNA device, a mechanism that eventually saturates, such as fouling, is dominant, while the in vivo drift observed with respect to the DNA-using device is primarily caused by some unsaturating process, such as enzymatic degradation.

[0140] Figures 4A–4C illustrate the behavior of the EAB sensor in in vitro and in vivo studies according to one embodiment. In Figure 4A, when exposed in vitro at 37°C in whole blood, both sensors fabricated using DNA and OMe RNA exhibit a rapid loss of signal, after which these signals remain stable. The shaded areas indicate the standard deviation of (n=4) independently fabricated sensors. In Figures 4B and 4C, the OMe RNA analog behaves similarly when placed in vivo in rat jugular veins. In contrast, the DNA analog continues to lose signal throughout these in vivo experiments. Surgical insertion of each probe into the lumen of the jugular vein takes approximately 3–4 minutes, creating a delay between the time the sensor is exposed to the in vivo environment and the time measurements begin. Therefore, this specification shows an example in Figure 4B where the OMe RNA sensor is inserted before the DNA sensor, and an example in Figure 4C where this order is reversed.

[0141] Several embodiments provide characterizations of the electron transfer dynamics of EAB sensors under diverse in vitro and in vivo conditions. These characterizations support the finding that DNase-driven degradation is dominant in the in vivo drift observed with respect to DNA-using devices. To observe this, DNA-using devices exposed to DNases may first be studied in buffer, by measuring charge transfer from each device as a function of square wave frequency.

[0142] Figures 5A–5F illustrate the time evolution of electron transfer dynamics of an EAB sensor under various conditions according to one embodiment. These measurements provide insight into the origin of signal loss observed when placed in blood, both in vitro and in vivo. Lovric plots of charge transfer as a function of frequency over a 5-hour measurement period are shown in Figures 5A–5F. Arrows indicate the degree of signal loss over time and whether this corresponds to a decrease in electron transfer rate, as indicated by a shift in the peak of the charge transfer distribution. In vitro, when exposed to DNA in phosphate-buffered saline at 37°C, signals from sensors using unstructured (Figure 5A) DNA or (Figure 5B) OMe RNA constructs decrease at all frequencies, and the number of methylene blue molecules on the surface decreases, while the transfer dynamics of the reporter remaining on the surface remain unchanged. In particular, the degree of this decrease is smaller with respect to the OMe RNA construct, reflecting its relative nuclease resistance. In contrast, Figures 5C and 5D show that when immersed in bovine whole blood at 37°C in vitro, both devices exhibit a rapid initial decrease in charge transfer, simultaneously shifting to lower frequencies. After this, the electron transfer properties of both devices stabilize. Figures 5E and 5F show that the behavior of the two devices differs fundamentally when exposed in rat jugular vein in situ. In particular, the DNA-based device shows a dramatic decrease in charge transfer at all frequencies, an effect very similar to that observed during enzymatic degradation in vitro (Figure 5A). This is in contrast to the charge transfer behavior of the OMe RNA sensor, mimicking what is seen with the OMe RNA sensor when exposed in vitro in whole blood (Figure 5D), suggesting that fouling is dominant in the in vivo drift of this sensor.

[0143] Under these conditions, the degree of charge transfer decreases dramatically and continuously throughout the experiment, while the transfer rate remains constant (Figure 5A). This observation is consistent with the loss of methylene blue due to DNA cleavage, where the number of methylene blue redox reporters decreases without alteration of the transfer kinetics of the remaining ones. Under these same conditions, the decrease in charge transfer observed for OMe RNA-using devices is much smaller. As with DNA, the decrease also occurs equally at all frequencies (Figure 5B), which also suggests that this is due to the more limited nuclease-driven degradation of this oligonucleotide. When the two devices were exposed in vitro in whole blood at 37°C, under conditions previously shown to be predominantly fouling-driven in drift, their charge transfer initially decreases and shifts to lower frequencies before finally stabilizing (Figures 5C and 5D). This likely occurs as the protein adsorbs to the surface, inhibiting redox reporter access and delaying electron transfer. In contrast, the behavior of the two devices differed more significantly when exposed in vivo in blood (Figures 5E and 5F). In particular, the behavior of the DNA-using device in vivo was similar to that when exposed in vitro in DNase-containing buffer; the signal drifted downward throughout the experimental period without any significant change in migration speed (compare Figures 5A and 5E). In contrast, the behavior of the OMe RNA-using device in vivo was similar to that when exposed in vitro in whole blood; the degree of charge transfer decreased rapidly at first, and after a simultaneous decrease in migration speed, both effects flattened out (compare Figures 5D and 5F). These observations indicate that the in vivo behavior of the DNA and OMe RNA-using devices differs at a significant mechanical level, suggesting that the in vivo drift observed with the OMe RNA device is predominantly due to fouling that eventually saturates, while the drift observed with the DNA-using device is due to loss driven by oligonucleotide DNase.

[0144] In many embodiments, the use of DNase-resistant OMe RNA aptamers significantly reduces the drift observed when aminoglycoside detection EAB sensors are used intravenously. In some embodiments, the in vivo drift of aminoglycoside sensors using OMe RNA aptamers after 5 hours in the jugular vein of viable rats is seven times less than that of sensors using equivalent DNA aptamers. Tracking mechanism studies following some embodiments using simple model oligonucleotides (i.e., lacking secondary or tertiary structures) suggest that the drift observed with respect to EAB sensors deployed in vivo occurs because enzymatic degradation is the major contributing factor, and this observation contrasts with previous studies suggesting that fouling is dominant in the drift observed in vitro in blood at 37°C. Several embodiments provide the application of XNA aptamers to EAB sensors and other aptamer-based technologies over in vivo duration.

[0145] Example 6: Microneedle-based wearable sensor device The working electrodes (and any other electrodes) described herein may be configured as microneedles and incorporated into a wearable sensor device, exemplary types of which are shown in Figures 6, 7A, 7B, 8, 9, and 10.

[0146] The device includes an upper housing portion (25) and a skin contact portion (30). Also provided is a removable flexible layer (90) graspable by a tab (95), which, when removed, exposes a dermatologically acceptable adhesive on the skin contact surface (35). The adhesive is intended for the purpose of retaining the device on the subject's skin for an extended period. The flexible layer (90) functions to prevent the adhesive from hardening or drying and to prevent contamination of the adhesive layer before use and / or premature adhesion of the adhesive to the packaging or other surfaces. In a particularly preferred embodiment, in addition to covering the adhesive layer, the flexible layer (90) extends over the space (45) to prevent contamination of the microneedles (15) and also helps to prevent unintended needle stick injuries to the user.

[0147] The device may have a retaining portion that functions to hold the device on the skin such that the protruding portion remains in contact with the subject's biological fluids. The retaining portion may be dedicated solely to that function or may perform other functions.

[0148] In many embodiments, a dermatologically acceptable adhesive or a retaining part containing such an adhesive would be useful. The adhesive allows for a simplified application of the device by the user, often requiring only the removal of a protective backing sheet to expose the adhesive, and then the exposed adhesive to contact with the skin. This application method is similar to that of applying a bandage and is therefore a process already familiar to the user.

[0149] As an alternative to the use of adhesive, the retaining portion may be some mechanical means for maintaining the device in the desired position on the skin. For example, the device may include a dedicated strap that is adjustable to engage with the limbs to keep the device firmly attached to the subject. Alternatively, the device may be incorporated into a wearable item that functions to hold the device in place, such as a glove or shirt, or a jewelry item, such as a ring. The device may be configured to associate with a separate wearable item (e.g., by complementary hook-and-loop means) or may have a wearable item that is integrated with it.

[0150] In some embodiments, the device is simply held in place by a wearable item that applies weight to the housing. For example, the holding part may be a snug-fitting, stretchable glove attached to the device.

[0151] In some embodiments, the retaining portion is any surface or part of the device that comes into contact with the subject's skin, and the subject's features are at least partially involved in maintaining the device in place on the subject. For example, the device may be configured to be retained between two parts of the body, typically juxtaposed close together or within existing anatomical structures. The device may be shaped and / or have dimensions to be retained in the toes, between the buttocks, between the groin, in the cheek cavities, in the nostrils, in the external auditory canal, or in the umbilicus.

[0152] In some embodiments, the device housing may be shaped and / or sized to fit snugly to, for example, a finger, toe, or ear. The device housing may be elastically deformable and made of, for example, a rubberized material, and may be set to stretch across any anatomical part (e.g., a finger).

[0153] Each embodiment may be a holding part in the background of the present invention.

[0154] The device further includes a release member (100) having a gripping portion (105) and a wedge-shaped portion (110), the function of which will be described in more detail below.

[0155] Now, referring to the exploded assembly diagrams in Figures 7A and 7B, it will be immediately clear that the components are similar to those in the previous diagram.

[0156] In certain embodiments, the driving force involved in moving the arm (205) and thereby guiding the microneedle (15) into the underlying skin is provided by the user. During use, the user places their finger on the upper housing (25) and presses downward. Furthermore, the arm (205) is movable by a hinged arrangement.

[0157] The hinged arrangement is provided by opposing lugs (115) extending from the skin contact portion (30), each lug containing an aperture. The arm (205) includes opposing laterally extending discs (120), each of which is housed within the aperture of the lugs (115). It will be apparent that the arm (205) is hinged to the skin contact portion (30), allowing movement from a first position to a second position.

[0158] The arm (205) is presented to the user with the arm in a first position. The arm (205) is maintained in the first position by the wedge-shaped portion (110) of the discharge member (100). Before removing the discharge member (100), the wedge-shaped portion is inserted between the skin contact portion (30) and the arm (205) to retain the microneedle within the device.

[0159] When intending to apply the device to the subject's skin, the user removes the flexible layer (90) by pulling the tab (95) and exposes the adhesive layer on the skin contact surface (35). The device is then applied to the skin, and the adhesive is maintained in situ for an extended period.

[0160] Once the device is applied to the skin, the user grasps the gripping portion (105) and pulls it laterally to the left (as shown in the figure) to completely remove the release member (100). The release member (100) has no further function and is discarded at this point. Removal of the release member (100) releases the arm (205) from the first position and allows it to move to the second position (by a downward force exerted by the user), thereby bringing the underside of the arm (205) into contact with the upper surface of the skin contact portion (30). In the second position, the microneedle (15) extends through the space (45) into the underlying skin.

[0161] As will be recognized, the release member (100) may be configured to prevent the upper housing (25) of the device from closing to the skin contact area (30) when the user does not intend to. The release member (100) is inserted between the upper housing (25) and the skin contact area (30) or otherwise positioned alongside it to prevent the upper housing (25) from closing to the skin contact area (30) sufficiently to allow the tip (i.e., protruding portion) of the microneedle to protrude from the base of the hole in the skin contact area (30). The prevention of closing also prevents the arm (205) from moving from a first position to a second position. Thus, when the release member (100) is in place, the tip of the microneedle is not accessible by accident and is not capable of causing microneedle contamination or injury. When using the device, the user removes the release member (100) as a step in the usage process. In a preferred embodiment of the device's use, the user first adheres the device to the subject's skin, then removes the release member (100), and then pushes in the upper housing (25) to insert the microneedle into the skin.

[0162] Prior to removal by the user, the release member (100) may be held in place by any one of a variety of features. In one example, the release member (100) includes a projection that fits into a recess in either the upper housing (25), the skin contact portion (30), or both the upper housing (25) and the skin contact portion (30) to help hold it in place until it is intentionally removed. In another example, the release member (100) is designed to be slidably assembled to the skin contact portion (30) or the upper housing (25) so that friction between the release member (100) and either the upper housing (25) or the skin contact portion (30) helps hold it in place until the release member is intentionally removed. In yet another example, magnetism may be used to help hold the release member (100) in place. In one embodiment of the present invention, a magnet mounted within the emission member (100) is positioned so as to be close to a Hall effect sensor in either the upper housing (25) or the skin contact portion (30) when the emission member (100) is in place. According to this embodiment, when the emission member (100) is removed by the user, the Hall effect sensor detects the removal of the magnet and causes the device to perform some action, for example, to power on an electrical circuit that is ready for use and to switch it from sleep mode to active mode. The above are examples of possible methods for assisting in keeping the emission member (100) in place before intentional removal, which may be used alone or in combination, and other methods known in the art may also be used alone or in combination with the examples shown.

[0163] In some embodiments of the present invention, the release member (100) may also function as a cover element used to cover the microneedles after the device has been removed from the subject. In a preferred example of this embodiment, the locking element is located on the upper housing (25), extending downward toward the skin contact portion (30). The release member (100) includes a groove that allows the release member (100) to slide past the locking element when withdrawn from the device, while maintaining the surface of the release member (100) continuously facing the upper surface of the skin contact portion (30). During use, after the release member (100) according to this preferred embodiment has been removed by the user, the upper housing (25) is pressed to insert the microneedles into the subject's skin and hold them in place. After the device has been removed from the subject after use, the user is instructed to adhere the release member (100) to the adhesive layer on the lower surface of the skin contact portion (30) to cover the protruding microneedles. In another example of this embodiment, the release member (100) is flexibly attached to the device so that it remains attached to the device after it has been withdrawn by the user and can then be repositioned to cover the protruding microneedles after the device has been removed from the subject after use. In yet another example of this embodiment, the release member (100) and the upper housing (25) may be designed such that the release member (100) is slidably engaged with the upper housing (25) once it has been removed, in which case the release member (100) is intended to be stored while the device is in use and removed after the device has been removed from the subject to be used as a cover element.

[0164] In some embodiments, the device is configured to facilitate the user's removal of the device from the subject. As will be recognized, the use of an adhesive layer can make removal of the device from the skin difficult. An example of such configuration is to leave a portion of the skin contact surface (35) uncoated with adhesive, creating a gap between the subject's skin and the surface (35), where the user can use the gap as a fulcrum to assist in pulling the device away from the skin by breaking the adhesive bond. In another example, a lever mechanism not located on the skin contact surface is incorporated to allow for a larger gap than that created by the absence of adhesive on a portion of the skin contact surface. In yet another example, a tab may be incorporated that protrudes over at least one edge of the skin contact portion (30) and adheres to the adhesive layer, in which case the user can pull the tab with sufficient force to stretch and yield the adhesive layer, further separating the adhesive from the skin contact surface (35) and the skin.

[0165] In some embodiments, the device is designed so that the release member (100) locks in place in its position before use, unless pressure is applied to the upper housing (25). This embodiment is intended to further reduce the risk of the release member (100) being pulled out prematurely. In one example of this embodiment, there are features on the release member (100) and on at least one of the upper housing (25) and the skin contact portion (30) that are lockable when no pressure is applied to the upper housing (25). When the upper housing (25) is pressed, the features on the upper housing (25) and at least one of the skin contact portion (30) twist to release the release member (100) and allow it to be pulled out.

[0166] In yet another embodiment, the release member (100) does not need to be removed from the device by the user. According to these embodiments, the release member (100) includes a flexible element that is sufficiently rigid to not substantially deform when subjected to closing forces that may be present on the device during manufacturing, storage, and in the user's hands before application to a subject, but is flexible enough to deform when applied to the skin of a subject and the user intentionally applies a closing force to the device. When bending in this way, the release member (100) deforms, allowing the upper housing (25) to close toward the skin contact portion (30). In these embodiments, the release member (100) may also function as a locking element, or the release member (100) may be separate from the locking portion. In some of these embodiments, features such as those indicated as (220) in Figures 7A and 7B form the release member (100).

[0167] Each space (45) of the device is sized such that a microneedle can clearly extend through them, and at least the tapered portion of the microneedle does not impact the sides of the hole during insertion. In some embodiments, the hole may have a sufficient cross-section so that no portion of the microneedle contacts the sides of the space during insertion. In some embodiments, the hole will have a cross-section such that at least a portion along its length contacts the sides of the hole during insertion. In this embodiment, the hole functions to help prevent the microneedle from bending when inserted, with a portion of the microneedle's length contacting the sides of the hole.

[0168] In some embodiments, the skin contact portion (30) includes additional spaces or recesses configured to receive projections on the release member, assisting in the retention of the release member until removed by the user. Furthermore, or otherwise, the skin contact portion (30) includes projections designed to be received by recesses in the release member, assisting in the retention of the release member until intentionally removed by the user.

[0169] The device includes a locking portion in the form of a latch (220), which permanently locks the arm (205) in a second position, preventing any hinge movement of the arm (205). In the depicted embodiment, the latch (220) is a simple single member that is capable of deforming in response to movement of the arm (205) toward the closed position, but then returns to its original position when the arm (205) is in the second position (205b), thereby locking the arm (205) in place.

[0170] Rather than acting on the arm (205), the locking portion may act on another component of the device, which then locks the arm in place. For example, the locking portion may act on the upper housing (25), which then holds the arm (205) in a second position. In a further alternative, the locking portion may act on the PCB (65), which then holds the arm (205) in a second position.

[0171] In some embodiments, the locking portion includes a recess into which a projection on the upper housing (25) is inserted to lock the upper housing (25) in a closed position (i.e., with the arm (205) in a second position). In one embodiment, the locking portion includes a flexible element designed to allow the locking portion to move so as to allow the housing (25) to close to the skin contact portion (30) when the locking portion is affected by the upper housing (25), thereby allowing the locking portion to move so as to lock the upper housing (25) in place in the closed position when the upper housing (25) is closed. In one embodiment, the device includes a projection on the upper housing (25) designed to be inserted into a recess in the locking portion, the projection including a flexible element that allows the projection to move so as to allow the upper housing (25) to close to the skin contact portion (30), and then the projection moves so as to be inserted into the recess in the locking portion so as to allow the housing (25) to close to the skin contact portion (30) and lock the upper housing (25) in the closed position. The flexible element includes a shaft that is sufficiently deformable to allow the upper housing (25) to close without yielding of the shaft, and so the flexible element will attempt to return to its original position after the upper housing (25) is closed. In a less preferred but nevertheless functional embodiment, the flexible element includes a coil spring.

[0172] The flexible element of the locking portion may be manufactured from any suitable material having the required rigidity and yield strength. Examples of suitable materials include amorphous plastics, crystalline plastics, spring steel, unsprung steel, stainless steel, or other materials known to have suitable mechanical properties in the art.

[0173] In some embodiments, the locking portion is manufactured from the same material as the skin contact portion (30), facilitating the manufacture of the skin contact portion including the integrated locking portion.

[0174] In certain embodiments, the force required to deform or otherwise move the flexible element is designed to be sufficiently large so that the pressure supplied by the user to deform the flexible element and thus close the upper housing (25) toward the skin contact portion is sufficient to insert the microneedle into the skin. In this embodiment, the flexible element of the locking portion is used to set the force required to close the device (thereby ensuring that the arm is in the second position) and to ensure that the force is sufficient to insert the microneedle into the intended position where it will be embedded in the skin.

[0175] In some embodiments, the locking portion includes at least one adhesive area located on the lower surface of the upper housing (25) and the upper surface of the skin contact surface (35). When the device is closed, one or more adhesive areas bond the upper housing (25) to the skin contact portion (30), locking the device in the closed position.

[0176] In some embodiments, the locking portion may assume three different stable states. In the first state, the locking portion is in a disengaged setting before the upper housing (25) is pushed downward toward the skin contact portion (30) to close the device. In the second state, the locking portion is in the first engaged position. When the locking portion is in the first engaged position, it works to lock the microneedle (15) in the implanted position in the skin (i.e., the arm (205) is in the second position). In the third state, the locking portion is in the second engaged position. In this state, the locking portion locks the device in the open position (i.e., the arm (205) is in the first position), and the microneedle is withdrawn into the device, reducing the possibility of needle stick injuries resulting from the microneedle protruding after use of the device. In some embodiments, the locking portion includes a user-engaged portion, which can be engaged by the user grasping or otherwise engaging, for example, by placing a fingernail under a protruding ledge so that the user can deform the flexible portion of the locking portion. To close the device according to this embodiment, the user presses on the upper housing (25) and locks it in place, as in other embodiments disclosed herein. If it is desirable to remove the device from the subject, the user engages with the locking portion and deforms it in a first direction, thus unlocking the upper housing (25) from the skin contact portion (25), and then deforms the locking portion in a second direction to lock the device in the open position (i.e., the arms are in the first position) and the microneedles in the withdrawn position. In a preferred embodiment of this example, the locking portion moves away from the device body in the first direction and moves towards the device body in the second direction. When sufficiently deformed in the second direction, the locking portion is designed to prevent the device from closing unintentionally, for example, by engaging in stabilization in a recess.

[0177] In some embodiments, a downward force is provided to the microneedle when inserted into the skin through a flexible element of the locking portion that applies a downward force when the device is locked in the closed position (i.e., the movable arm is in the second position). In some embodiments, effective locking of the movable arm in the second position is provided by a dedicated spring or other suitable biasing means. In some embodiments, the spring or other biasing means may not be dedicated to the locking function but may act, for example, as a driving force to move the arm from the first position to the second position. For example, a torsion spring may apply a closing torque at the axis of rotation (if present). In yet another example, a flat disc or coil spring is mounted behind the microneedle so that the spring is deformed or compressed when the device is closed, applying a downward force on the microneedle when the device is in the closed position.

[0178] Although not an essential feature of the present invention, a PCB (65) would be required in many applications if the microneedles are intended to conduct electric current through or into the skin. In this regard, the PCB may contain a microprocessor and / or volatile electronic memory (e.g., RAM) and / or non-volatile electronic memory (e.g., ROM) and / or a wireless network module (e.g., a Bluetooth® module). The device would, of course, include a power source, typically a button battery.

[0179] Those skilled in the art will recognize that the invention described herein is subject to further modifications and alterations other than those specifically described. It will be understood that the invention includes all such modifications and alterations that fall within the spirit and scope of the invention.

[0180] Therefore, the spirit and scope of the present invention are not limited to the examples described above, but are understood in the broadest sense permitted by law.

[0181] Examples Example 1: A working electrode for an electrochemical sensor, comprising a conductive element and an analyte sensing element associated with the conductive element, configured to specifically interact with a target analyte, wherein the sensing element comprises a non-natural nucleic acid coupled with a redox reporter.

[0182] Example 2: The working electrode according to Example 1, wherein the non-natural nucleic acid is unreadable and / or replicated by any natural mammalian cell.

[0183] Example 3: The working electrode according to Example 1 or 2, wherein the non-natural nucleic acid has greater resistance to degradation by components of a biological fluid compared to a similar natural nucleic acid.

[0184] Example 4: A working electrode according to Example 1, 2, or 3, wherein the similarity relates to one or more of the following: length, base sequence, secondary structure, tertiary structure, and ability to interact with the target analyte.

[0185] Example 5: The working electrode according to any one of Examples 1 to 4, wherein the biological liquid contains a nuclease.

[0186] Example 6: A working electrode according to any one of Examples 1 to 5, wherein the non-natural nucleic acid is formed from a single strand.

[0187] Example 7: The working electrode according to any one of Examples 1 to 6, wherein the non-natural nucleic acid is a polymer.

[0188] Example 8: A working electrode according to any one of Examples 1 to 7, wherein the non-natural nucleic acid comprises 10 to 100 subunits.

[0189] Example 9: The working electrode according to any one of Examples 1 to 8, wherein the non-natural nucleic acid is a natural deoxyribose nucleic acid or a chemical variant of a natural ribose nucleic acid.

[0190] Example 10: The working electrode according to any one of Examples 1 to 9, wherein the chemical mutant is for the sugar backbone and / or one or more bases.

[0191] Example 11: The non-natural nucleic acid is produced by or with human assistance, as in Example 1. A working electrode as described in one of the following ten options.

[0192] Example 12: The working electrode according to any one of Examples 1 to 11, wherein the non-natural nucleic acid is xeno nucleic acid (XNA) or peptide nucleic acid (PNA).

[0193] Example 13: The working electrode according to any one of Examples 1 to 12, wherein the non-natural nucleic acid is a DNA or RNA aptamer having a modified chemical structure.

[0194] Example 14: A working electrode according to any one of Examples 1 to 13, wherein the non-natural nucleic acid is a modified form of a natural nucleic acid that is specifically interactable with the target analyte, and the non-natural nucleic acid has at least 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, or 150% of the interacting ability of the natural nucleic acid.

[0195] Example 15: A working electrode according to any one of Examples 1 to 14, wherein the non-natural nucleic acid is a modified form of a natural nucleic acid that is specifically interactable with the target analyte, and the non-natural nucleic acid has at least 50%, 60%, 70%, 80%, 90%, or 100% of the sensitivity, accuracy, or specificity for recognizing the target analyte of the natural nucleic acid.

[0196] Example 16: A working electrode according to any one of Examples 1 to 15, wherein the non-natural nucleic acid is a modified form of a natural nucleic acid that is specifically interactable with the target analyte, and the non-natural nucleic acid has an uncorrected signal loss of less than 90%, 80%, 70%, 60%, or 50% of that of the natural nucleic acid.

[0197] Example 17: The working electrode according to any one of Examples 1 to 16, wherein the working electrode is a portion of an electrochemical sensor with an uncorrected signal drift rate, and the uncorrected signal drift rate is determined or averaged over a period of at least 10 minutes, 20 minutes, 30 minutes, 40 minutes, 50 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 24 hours, 36 hours, 48 ​​hours, or 72 hours.

[0198] Example 18: A working electrode according to any one of Examples 1 to 17, wherein the non-natural nucleic acid is bound to a conductive element at its first end.

[0199] Example 19: A working electrode according to any one of Examples 1 to 18, wherein the redox reporter is bound to the second end of the non-natural nucleic acid.

[0200] Example 20: The working electrode according to any one of Examples 1 to 19, wherein the conductive element includes a skin penetration portion to which the non-natural nucleic acid is bound.

[0201] Example 21: The working electrode according to any one of Examples 1 to 20, wherein the conductive element is a needle, a microneedle, or a wire.

[0202] Example 22: An electrochemical sensor device comprising one working electrode and a counter electrode from any of Examples 1 to 21.

[0203] Example 23: The apparatus according to Example 22, including a reference electrode.

[0204] Example 24: The apparatus according to Example 22 or 23, relating to a retainer configured to hold the working electrode in contact with the bodily fluids of a subject.

[0205] Example 25: The apparatus according to Example 22, 23, or 24, wherein the retainer is configured to hold the working electrode in contact with the subject's bodily fluids for a period of at least 10 minutes, 20 minutes, 30 minutes, 40 minutes, 50 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 24 hours, 36 hours, 48 ​​hours, or 72 hours.

[0206] Example 26: The apparatus according to any one of Examples 22-25, relating to a housing configured to enclose a power supply and / or electronics for the function of the sensor.

[0207] Example 27: A method for monitoring a target analyte in a biological fluid of a subject, comprising the step of bringing one working electrode from Examples 1 to 21 into contact with the biological fluid for a period of time.

[0208] Example 28: The method of Example 27, wherein the working electrode is in contact with a biological fluid that remains in situ within the subject for the duration of the method.

[0209] The method according to Example 27 or 28, wherein the biological fluid is blood or interstitial fluid.

[0210] Example 30: The method according to Example 27, 28, or 29, wherein the biological fluid is not removed from the subject.

[0211] Example 31: The method according to any one of Examples 27 to 30, wherein the period is at least 10 minutes, 20 minutes, 30 minutes, 40 minutes, 50 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 24 hours, 36 hours, 48 ​​hours, or 72 hours.

Claims

1. A working electrode for an electrochemical sensor, comprising a conductive element and an analyte sensing element associated with the conductive element, configured to specifically interact with a target analyte, wherein the sensing element comprises a non-natural nucleic acid coupled with a redox reporter.

2. The working electrode according to claim 1, wherein the non-natural nucleic acid is unreadable and / or replicated by any natural mammalian cell.

3. The working electrode according to claim 1 or 2, wherein the non-natural nucleic acid has greater resistance to degradation by components of a biological fluid compared to a similar natural nucleic acid.

4. The working electrode according to claim 3, wherein the similarity relates to one or more of the following: length, base sequence, secondary structure, tertiary structure, and ability to interact with the target analyte.

5. The working electrode according to claim 3 or 4, wherein the biological fluid comprises a nuclease.

6. The working electrode according to any one of claims 1 to 5, wherein the non-natural nucleic acid is formed from a single strand.

7. The working electrode according to any one of claims 1 to 6, wherein the non-natural nucleic acid is a polymer.

8. The working electrode according to claim 7, wherein the non-natural nucleic acid comprises 10 to 100 subunits.

9. The working electrode according to any one of claims 1 to 8, wherein the non-natural nucleic acid is a natural deoxyribose nucleic acid or a chemical variant of a natural ribose nucleic acid.

10. The working electrode according to claim 9, wherein the chemical variant is for a sugar backbone and / or one or more bases.

11. The working electrode according to any one of claims 1 to 10, wherein the non-natural nucleic acid is produced by or with human assistance.

12. The working electrode according to any one of claims 1 to 11, wherein the non-natural nucleic acid is xeno nucleic acid (XNA) or peptide nucleic acid (PNA).

13. The working electrode according to any one of claims 1 to 12, wherein the non-natural nucleic acid is a DNA or RNA aptamer having a modified chemical structure.

14. The working electrode according to any one of claims 1 to 13, wherein the non-natural nucleic acid is a modified form of a natural nucleic acid that is specifically interactable with the target analyte, and the non-natural nucleic acid has at least 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, or 150% of the interacting ability of the natural nucleic acid.

15. The working electrode according to any one of claims 1 to 14, wherein the non-natural nucleic acid is a modified form of a natural nucleic acid that is specifically interactable with the target analyte, and the non-natural nucleic acid has at least 50%, 60%, 70%, 80%, 90%, or 100% of the sensitivity, accuracy, or specificity for recognizing the target analyte of the natural nucleic acid.

16. The working electrode according to any one of claims 1 to 15, wherein the non-natural nucleic acid is a modified form of a natural nucleic acid that is specifically interactable with the target analyte, and the non-natural nucleic acid has an uncorrected signal loss of less than 90%, 80%, 70%, 60%, or 50% of that of the natural nucleic acid.

17. The working electrode according to claim 1, wherein the working electrode is a portion of an electrochemical sensor with an uncorrected signal drift rate, the uncorrected signal drift rate is determined or averaged over a period of at least 10 minutes, 20 minutes, 30 minutes, 40 minutes, 50 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 24 hours, 36 hours, 48 ​​hours, or 72 hours.

18. The working electrode according to any one of claims 1 to 17, wherein the non-natural nucleic acid is bound to a conductive element at its first end.

19. The working electrode according to any one of claims 1 to 18, wherein the redox reporter is bound to the second end of the non-natural nucleic acid.

20. The working electrode according to any one of claims 1 to 19, wherein the conductive element includes a skin penetration portion to which the non-natural nucleic acid is bound.

21. The working electrode according to claim 20, wherein the conductive element is a needle, a microneedle, or a wire.

22. An electrochemical sensor device comprising a working electrode and a counter electrode according to any one of claims 1 to 21.

23. The apparatus according to claim 22, comprising a reference electrode.

24. The apparatus according to claim 22 or 23, relating to a retainer configured to hold the working electrode in contact with the bodily fluids of a subject.

25. The apparatus according to claim 24, wherein the retainer is configured to hold the working electrode in contact with the bodily fluids of the subject for a period of at least 10 minutes, 20 minutes, 30 minutes, 40 minutes, 50 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 24 hours, 36 hours, 48 ​​hours, or 72 hours.

26. The apparatus according to any one of claims 22 to 25, relating to a housing configured to enclose a power supply and / or electronics for the function of the sensor.

27. A method for monitoring a target analyte in a biological fluid of a subject, comprising the step of bringing a working electrode according to any one of claims 1 to 21 into contact with the biological fluid for a period of time.

28. The method according to claim 27, wherein the working electrode is in contact with a biological fluid that remains in situ within the subject for the duration of the method.

29. The method according to claim 27 or 28, wherein the biological fluid is blood or interstitial fluid.

30. The method according to any one of claims 27 to 29, wherein the biological fluid is not removed from the subject.

31. The method according to any one of claims 27 to 30, wherein the period is at least 10 minutes, 20 minutes, 30 minutes, 40 minutes, 50 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 24 hours, 36 hours, 48 ​​hours, or 72 hours.

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