WO2019055985A1

WO2019055985A1 - Click chemistry aptamer tagging for eab biosensors

Info

Publication number: WO2019055985A1
Application number: PCT/US2018/051516
Authority: WO
Inventors: Jacob A. BERTRAND; Brian Hanley; Haghighi Leila SAFAZADEH
Original assignee: Eccrine Systems, Inc.
Priority date: 2017-09-18
Filing date: 2018-09-18
Publication date: 2019-03-21

Abstract

Methods are disclosed for using click chemistry techniques to functionalize aptamer sequences for use in electrochemical aptamer-based biosensors, and in particular such sensors for use in sweat sensing environments. Specifically, such techniques include preparing a portion of the aptamer sequence to interact with a functionalization component through a bio-orthogonal chemistry reaction, preparing the functionalization component to interact with the aptamer sequence through the click chemistry reaction, and attaching the aptamer sequence to the functionalization component through the click chemistry reaction. The functionalization component may be one or more of an oligonucleotide primer, an anchor molecule, a redox moiety, an oligonucleotide dock, or an electrode surface. Disclosed bio-orthogonal chemistry reactions include an azide-alkyne cycloaddition reaction, and a thiol-maleimide reaction.

Description

CLICK CHEMISTRY APTAMER TAGGING FOR EAB BIOSENSORS

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] The present application claims priority to U.S. Provisional Application No. 62/559,857, filed September 18, 2017, and has specification that builds upon PCT/US17/23399, filed March 21, 2017, the disclosures of which are hereby incorporated herein by reference in their entirety.

BACKGROUND OF THE INVENTION

[0002] Despite the many ergonomic advantages of sweat compared to other biofluids for use in wearable devices, sweat remains an underutilized source of analytes compared to the established biofluids: blood, urine, and saliva. Upon closer comparison to other non-invasive biofluids, the advantages extend beyond ergonomics: sweat can provide superior analyte information. Several challenges, however, historically have kept sweat from becoming a preferred clinical biofluid. These challenges include very low sample volumes (nL to μί), unknown concentration due to evaporation, filtration and dilution of large analytes, mixing of old and new sweat, and the potential for contamination from the skin surface. Rapid progress has been made in the development of wearable sweat sensing devices, resolving several of these challenges. Recent progress has nevertheless been limited to high concentration analytes (μΜ to mM) sampled at high sweat rates (>1 nL/min/gland) found in, for example, athletic applications. Progress will be much more challenging as wearable biosensing moves towards accurate detection of small proteins, and large, low concentration analytes (nM to pM and lower).

[0003] In particular, many sensor technologies known in the art for detecting such molecules are ill-suited for use in wearable biofluid sensing, which requires robust, inexpensive, and miniaturized sensors that permit continuous use on a wearer's skin. These requirements preclude the use of sensor modalities that require complex microfluidic mampulation, the addition of reagents, the use of limited shelf-life components, such as antibodies, or sensors that are designed for a single use will not be sufficient for many biofluid sensing applications. Electrochemical aptamer-based ("EAB") sensor technology, such as is disclosed in U.S. Patent Nos. 7,803,542 and 8,003,374, presents a stable, reliable bioelectric sensor that is sensitive to the target analyte in a sample, while being capable of multiple analyte capture events during the sensor lifespan.

[0004] However, a chief obstacle to the development of EAB sensors is the ability to efficiently and effectively functionalize selected aptamer sequences for use in such sensors, which requires tagging of the aptamer with a redox moiety, one or more primers, and anchoring the aptamer complex to an electrode surface. All of which are needed to allow the sensor to detect target analytes. The current method used to tag aptamer sequences for use in EAB sensors relies on conventional chemistries to achieve the desired bonds. However, conventional chemistries often include low-yield steps, require reagents or solvents with hazardous properties for wearable applications, excessive reaction temperatures or times, or process steps that may need to be empirically derived and which are subject to trial and error. Therefore, what is needed are novel ways to functionalize analyte capture sequences to reduce the uncertainty, cost, and undesirable substances involved in some conventional chemistry methods. Such devices and methods are the subject of the present disclosure.

[0005] Click chemistry techniques have previously been disclosed for the functionalization of electrochemical sensors, see, e.g., US Patent 10,034,625B1 (discussing use of click chemistry for assembling aptamer sensors); US Publication US2011/0210017A1 (disclosing use of click chemistry to attach a redox moiety to a ligand, as well as to attach the ligand to an electrode surface); US Publication US2014/0349005A1 (mentioning use of click chemistry to attach functional groups to RNA aptamer sensors); International Publication WO2017/164982A1 (mentioning use of click chemistry to attach aptamers to an electrode surface). The disclosed invention adds specific click chemistry reactions for functionalizing EAB sensors tailored for use in wearable sweat sensing devices and other biofluid sensing environments, and provides click chemistry-facilitated electrode surface treatments for use with such sensors.

SUMMARY OF THE INVENTION

[0006] Methods are disclosed for using click chemistry techniques to functionalize aptamer sequences for use in electrochemical aptamer-based biosensors. Specifically, such techniques include preparing a portion of the aptamer sequence to interact with a functionalization component through a bio-orthogonal chemistry reaction, preparing the functionalization component to interact with the aptamer sequence through the click chemistry reaction, and attaching the aptamer sequence to the functionalization component through the click chemistry reaction. The functionalization component may be one or more of an oligonucleotide primer, an anchor molecule, a redox moiety, an oligonucleotide dock, or an electrode surface. Disclosed bio-orthogonal chemistry reactions include an azide-alkyne cycloaddition reaction, and a thiol-maleimide reaction.

BRIEF DESCRIPTION OF THE DRAWINGS

[0007] The objects and advantages of the present invention will be further appreciated in light of the following detailed descriptions and drawings in which:

[0008] Figs. 1 A and IB depict an embodiment of a previously disclosed EAB sensing element.

[0009] Figs. 2A and 2B depict an embodiment of a previously disclosed EAB sensing element.

[0010] Fig. 3 depicts an embodiment of the disclosed invention.

[0011] Figs. 4A and 4B depict embodiments of the disclosed invention.

[0012] Figs. 5A and 5B depict embodiments of the disclosed invention.

[0013] Figs. 6A and 6B depict embodiments of the disclosed invention.

[0014] Fig. 7 depicts an embodiment of the disclosed invention.

[0015] Figs. 8A and 8B depict embodiments of the disclosed invention.

[0016] Figs. 9A and 9B depict embodiments of the disclosed invention.

[0017] Fig. 10 depicts an embodiment of the disclosed invention.

[0018] Fig. 11 depicts an embodiment of the disclosed invention.

DEFINITIONS

[0019] Before continuing with the background, a variety of definitions should be made, these definitions gaining further appreciation and scope in the detailed description and embodiments of the present disclosure.

[0020] As used herein, "sweat" means a biofluid that is primarily sweat, such as eccrine or apocrine sweat, and may also include mixtures of biofluids such as sweat and blood, or sweat and interstitial fluid, so long as advective transport of the biofluid mixtures (e.g., flow) is primarily driven by sweat. [0021] As used herein, "biofluid" may mean any human biofluid, including, without limitation, sweat, interstitial fluid, blood, plasma, serum, tears, and saliva.

[0022] "Biosensor" means any type of sensor that measures a state, presence, flow rate, solute concentration, solute presence, in absolute, relative, trending, or other ways in a biofluid. Biosensors can include, for example, potentiometric, amperometric, impedance, optical, mechanical, antibody, peptide, aptamer, or other means known by those skilled in the art of sensing or biosensing.

[0023] "Analyte" means a substance, molecule, ion, or other material that is measured by a fluid sensing device.

[0024] "Measured" can imply an exact or precise quantitative measurement and can include broader meanings such as, for example, measuring a relative amount of change of something. Measured can also imply a binary or qualitative measurement, such as 'y^es' ^or '^ηο' typ^e measurements.

[0025] "Chronological assurance" means the sampling rate or sampling interval that assures measurement(s) of analytes in sample in terms of the rate at which measurements can be made of new fluid analytes as they enter the sample. Chronological assurance may also include a determination of the effect of sensor function, potential contamination with previously generated analytes, other fluids, or other measurement contamination sources for the measurement(s). Chronological assurance may have an offset for time delays in the body (e.g., a well-known 5- to 30-minute lag time between analytes in blood emerging in interstitial fluid), but the resulting sampling interval is independent of lag time, and furthermore, this lag time is inside the body, and therefore, for chronological assurance as defined above and interpreted herein, this lag time does not apply.

[0026] "Click chemistry" or "bio-orthogonal chemistry" describes high yield reactions that are wide in scope, create only byproducts that can be removed without chromatography, are stereospecific, simple to perform, and can be conducted in benign solvents.

[0027] "NHS chemistry" means the attachment of an N-IIydroxySiicciiiiniide (NHS) ester to an amine.

[0028] "EAB sensor" means an electrochemical aptamer-based biosensor that is configured with multiple aptamer sensing elements that, in the presence of a target analyte in a fluid sample, produce a signal indicating analyte capture, and which signal can be added to the signals of other such sensing elements, so that a signal threshold may be reached that indicates the presence of the target analyte.

[0029] "Docked aptamer EAB sensor" means an EAB sensor that employs docking strategies to connect analyte capture complexes with the sensor electrode, as disclosed in PCT/US 18/39274, filed June 25, 2018, which is hereby incorporated by reference in its entirety.

[0030] "Aptamer sensing element" means an individual aptamer that is functionalized to operate in conjunction with an electrode to detect the presence of a target analyte. Such functionalization may include tagging the aptamer with a redox moiety, such as methylene blue, adding nucleotide bases or primers, thiol binding molecules, or other components to the aptamer. Multiple aptamer sensing elements functionalized on an electrode comprise an EAB sensor.

[0031] "Analyte capture complex" means an aptamer, or other suitable molecules or complexes, such as proteins, polymers, molecularly imprinted polymers, polypeptides, and glycans, that experience a conformation change in the presence of a target analyte, and are capable of being used in an EAB sensor. Such molecules or complexes can be modified by the addition of one or more primers comprised of nucleotide bases.

[0032] "Fluid sensor data" means all the information collected by fluid sensing device sensor(s) and communicated to a user or a data aggregation location.

[0033] "Correlated aggregated fluid sensor data" means fluid sensor data that has been collected in a data aggregation location and correlated with outside information such as time, temperature, weather, location, user profile, other biofluid sensor data, or any other relevant data.

DETAILED DESCRIPTION OF THE INVENTION

[0034] One skilled in the art will recognize that the various embodiments may be practiced without one or more of the specific details described herein, or with other replacement and/or additional methods, materials, or components. In other instances, well-known structures, materials, or operations are not shown or described in detail herein to avoid obscuring aspects of various embodiments of the invention. Similarly, for purposes of explanation, specific numbers, materials, and configurations are set forth herein in order to provide a thorough understanding of the invention. Furthermore, it is understood that the various embodiments shown in the figures are illustrative representations and are not necessarily drawn to scale.

[0035] Reference throughout this specification to "one embodiment" or "an embodiment" means that a particular feature, structure, material, or characteristic described in connection with the embodiment is included in at least one embodiment of the invention, but does not denote that they are present in every embodiment. Thus, the appearances of the phrases "in an embodiment" or "in another embodiment" in various places throughout this specification are not necessarily referring to the same embodiment of the invention. Further, "a component" may be representative of one or more components and, thus, may be used herein to mean "at least one."

[0036] Certain embodiments of the disclosed invention show sensors as simple individual elements. It is understood that many sensors require two or more electrodes, reference electrodes, or additional supporting technology or features which are not captured in the description herein. Sensors are preferably electrical in nature, but may also include optical, chemical, mechanical, or other known biosensing mechanisms. Sensors can be in duplicate, triplicate, or more, to provide improved data and readings. Sensors may be referred to by what the sensor is sensing, for example: a biofluid sensor; an impedance sensor; a sample volume sensor; a sample generation rate sensor; and a solute generation rate sensor. Certain embodiments of the disclosed invention show sub-components of what would be sensing devices with more sub-components needed for use of the device in various applications, which are obvious (such as a battery), and for purposes of brevity and focus on inventive aspects, such components are not explicitly shown in the diagrams or described in the embodiments of the disclosed invention. As a further example, many embodiments of the disclosed invention could benefit from mechanical or other means known to those skilled in wearable devices, patches, bandages, and other technologies or materials affixed to skin, to keep the devices or sub-components of the skin firmly affixed to skin or with pressure favoring constant contact with skin or conformal contact with even ridges or grooves in skin, and are included within the scope of the disclosed invention.

[0037] The detailed description of the present invention will be primarily, but not entirely, limited to devices, methods and sub-methods using wearable biofluid sensing devices. Therefore, although not described in detail here, other essential steps which are readily interpreted from or incorporated along with the present invention shall be included as part of the disclosed invention. The disclosure provides specific examples to portray inventive steps, but which will not necessarily cover all possible embodiments known to those skilled in the art. For example, the specific invention will not necessarily include all obvious features needed for operation. Several specific, but non-limiting, examples can be provided as follows. The invention includes reference to the article in press for publication in the journal IEEE Transactions on Biomedical Engineering, titled "Adhesive RFHD Sensor Patch for Monitoring of Sweat Electrolytes"; the article published in the journal AIP Biomicrofluidics, 9 031301 (2015), titled "The Microfluidics of the Eccrine Sweat Gland, Including Biomarker Partitioning, Transport, and Biosensing Implications."

[0038] EAB sensor technology is a developing field that faces several challenges, one of which is efficiently and effectively functionalizing aptamer sequences for use in EAB sensors, i.e., attaching various components to the aptamer and anchoring the entire complex to an electrode base. To construct an EAB sensor, a randomized aptamer sequence is identified (through a process such as a version of systematic evolution of ligands by exponential enrichment (SELEX)) for its ability to interact with a target analyte. Copies of the selected aptamer are then functionalized into aptamer sensing elements that are arranged on an electrode surface. Each EAB sensor can have thousands, millions, or billions of individual sensing elements, with an upper limit of 10¹⁴/cm². As an example of such functionalization, Fig.lA depicts a single aptamer sensing element 110 for a multiple-capture EAB sensor ("MCAS"). The MCAS sensing element comprises an analyte capture complex 112, which includes a randomized aptamer sequence 140 selected to bind a target analyte 160, a redox moiety 150, such as methylene blue, bonded to a first end of the aptamer, and a first nucleotide primer 142 that is bonded to the other end of the aptamer. In other embodiments (not shown), the first end of the aptamer may be bonded to a second nucleotide primer, which is then bonded to the redox moiety. The analyte capture complex 112 is covalently bonded via the first primer 142 to a thiol 120 or other suitable anchoring molecule or complex, which is in turn covalently bonded to an electrode 130. The electrode 130 maybe comprised of gold or another suitable conductive material. [0039] Another EAB sensor modality used with some embodiments of the disclosed invention is a docked aptamer EAB sensor (DAS). With reference to Fig. 2 A, a DAS aptamer sensing element is depicted. The aptamer sensing element 210 includes an analyte capture complex 212 and a molecular docking structure 220 immobilized on an electrode 230. The docking structure 220 may be attached to the electrode 230 by covalently bonding a first end to a thiol, which is then in turn covalently bonded to the electrode. The docking structure 220 includes a 9 to 12 base nucleotide sequence that is selected to be complementary with a nucleotide sequence on the analyte capture complex 212, specifically, the dock is configured to pair with a first primer section 242. A redox chemical moiety 250 is immobilized on the unattached end of the dock 220, on the opposite end of the dock from the electrode 230. The dock 220 further includes two complementary nucleotide sequences 222, 224. In the initial arrangement, the analyte capture complex 212 is attached to a dock 220 that is, in turn, attached to the electrode 230.

[0040] Such aptamer functionalization has previously been accomplished through traditional chemistry methods, such as thiol-iodoacetamide chemistry, or N-HydroxySuecimmide (NHS) ester- amine chemistry, which include developing a series of reactions which should theoretically result in the desired product, then enacting the steps to empirically determine if the desired molecule can be created. An exemplary NHS-type reaction used to tag the aptamer with a redox moiety comprises first anchoring the aptamer to a gold electrode via a thiol bond. Then, the electrode is covered by a self-assembled monolayer, and placed into a solution containing the NHS -bound redox moiety. The solution is buffered to potential of hydrogen (pH) of 9 with a bicarbonate/carbonate buffer, and is kept at room temperature in the dark on an orbital laboratory shaker for one hour. The NHS redox tag reacts with the free amine on the aptamer, attaching the redox moiety to the aptamer. Then the redox - tagged aptamer solution is purged with argon, and the electrode is rinsed in a neutral buffer solution to de-salt the electrode.

[0041] Unfortunately, these conventional chemistry processes often must be developed anew for each type of molecule to be bonded to a new aptamer sequence. Such development can be time consuming and expensive, and the resulting processes may be low yield (e.g., NHS attachment chemistry can have conjugation efficiency as low as 2%), may require significant energy inputs, or may involve toxic catalysts. Functionalization of aptamers by conventional means therefore injects an element of uncertainty into the process of developing EAB sensors. Rather than rely on such uncertain processes, the disclosed invention seeks to standardize the process of functionalizing aptamer sequences through the use of click chemistry and other suitable bio-orthogonal chemistry techniques.

[0042] In addition, EAB sensors used in sweat sensing devices face a very challenging environment. Sweat is a highly variable excretory fluid, with pH values that can vary as much as 300X, from about 4.5 to about 7. Salinity levels in sweat can also vary significantly with sweat generation rates, ranging from roughly 10 mMol to 60 mMol. In addition, sweat contains large solutes, e.g., proteins (measured in 1000's of Daltons), which can foul EAB electrode surfaces after only a few hours immersion in sweat. Further, sweat contains enzymes, DNases, RNases, and other solutes that can cause aptamer breakdown, and hence EAB sensor failure. Finally, aptamer detachment from electrode surfaces is a substantial problem for EAB sensors having to operate for extended times in sweat biofluid. For these reasons, click chemistry reactions, functionalizations, and electrode surface treatments tailored for use in sweat biofluid may prove particularly useful for functionalizing EAB sensors for use in such environments.

[0043] With reference to Fig. 3 A, at least a portion of an aptamer sensing element 310 is immobilized on a gold electrode surface 330. The selected aptamer sequence 340 is then tagged with a redox moiety 350 through click chemistry. The steps for this click-chemistry tagging include labelling the redox moiety 350 with an azide molecule 352, and labelling the free end of the aptamer sequence with an alkyne 354. The aptamer 340 can be labelled with the alkyne during solid-phase synthesis, or post synthesis. When placed together in solution, the redox moiety attaches to the aptamer sequence through an azide-alkyne cycloaddition reaction. The click chemistry reaction described benefits from orthogonality, simplicity, and fast reaction time, i.e., less than one minute, and provides higher conjugation efficiency compared to traditional chemistries, and even other click chemistry techniques, such as thiol-maleimide click chemistry.

[0044] The disclosed click chemistry method enables the surface tagging of aptamers with a wide range of functional groups, including a variety of redox moieties, oligonucleotide primers, anchor molecules, oligonucleotide docking structures, and electrode surface materials. Further, the disclosed method lends itself to sweat and other biofluid sensing applications because it is adaptable for use in different solvents, such as water, sweat, or other biofluids, and does not require toxic catalysts, making it suitable for use in vi^'vo/on-body. Click chemistry is preferred over N~ HydfoxySuccinimide (NHS) ester-amine chemistry in certain situations, such as when using non- native base aptamers in conjunction with free amines, where NHS chemistry produces undesired side reactions.

[0045] In another embodiment of the disclosed invention, click chemistry is used to attach the analyte capture complex to electrodes made from gold, or from a variety of alternate materials. EAB sensors are typically constructed on a gold electrode surface because gold is resistant to corrosion, and the gold-thiol bond is well understood and robust enough to keep a quorum of aptamer sensing elements attached to the electrode for more than two hours when immersed in biofluid. However, if an efficient, reliably robust bond were available, the aptamer sensing element could be attached to electrodes made from other metals, alloys, or materials, such as carbon traces, conductive metal oxides, or conductive metal nitrides. These alternate electrode materials could allow the EAB sensor to display desirable operational properties, such as increased electrode adhesion time in biofluid (reduced sensor drift), the use of more inexpensive materials, cheaper or easier assembly, allowing standardized aptamer attachment procedures, increasing device flexibility, decreasing weight, or other advantage.

[0046] Another advantage of constructing EAB sensors on alternate electrode materials is the tendency of the gold electrode to degrade in the presence of NaCl and electrical current, a particular problem for sweat biosensing. During sweat sensing, electrical potential is applied to the electrode to interrogate the aptamer sensing elements as to the presence or concentration of target analyte molecules. The use of a direct current in the presence of chloride containing salt results in the formation of gold chloride, a soluble gold salt. Gold chloride fouls the electrode surface and decreases sensor lifetime. Similarly, an alternating current interrogation waveform can also form gold chloride, depending on frequency and voltages used. [0047] While electrodes constructed from alternate substances may present advantages for use in biofluid sensing device technologies, reliably attaching aptamer sensing elements to the electrode is a significant problem. Attachment chemistries can vary widely with the properties of the prospective electrode material, and the materials may display additional chemical properties that interfere with the function of EAB sensing elements. Therefore, what is needed are reliable methods of attaching aptamer sensing elements and blockers (or monolayers) to the electrode surface, regardless of the material's chemical properties. Disclosed herein are various methods for solving this problem, to include means of attaching layers of molecules that enable click chemistry or NHS chemistry attachments.

[0048] For example, a number of potential carbon-based electrode materials, including graphite, and carbon nanotubes, have a graphene surface characterization. Graphene' s near uniform two- dimensional hexagonal lattice structure can be exfoliated, e.g., through treatment with NaOH, to dislodge carbon atoms, thereby creating attachment points for anchor molecules. With reference to Fig. 4A, the exfoliated graphene surface can be exposed to hydroxyl (also referred to as primary alcohol) groups 410, which can serve as attachment points, or these hydroxyls can be oxidized with CrC>3, aqueous H2SO4, and acetone to yield carboxylic acid attachment points 412. With reference to Fig. 4B, a similar transformation can occur with more complex hydroxyls 414 and resulting carboxylic acid groups 416. In both Figs. 4A and 4B, the R groups represent various functional groups that can be attached to the surface via the carboxylic acid structures. Example functional groups are depicted in Figs. 5A and 5B, and include tert-butyl cyclopentane carboxylate 510, methyl benzoate 512, ethyl acetate 514, methyl pivalate 516, and allyl benzoate 518, among others.

[0049] Figures 6A and 6B depict additional alcohol reactions that can create different types of attachment points for anchoring EAB components to a surface. For example, Fig. 6A depicts the first two steps depicted in Fig. 4 A, i.e., 410, 412, in which a hydroxyl 610 is reacted to form a carboxylic acid group 612. The carboxylic acid can be further reacted to form an acid chloride 614, reacted again to form an amide 616, and again to form a free amine 618. These four products 612, 614, 616, 618 are standard in the art for attaching molecules to a surface, with each offering different properties depending on the needs of the application, e.g., the amide 616 facilitates click chemistry techniques, and the amine 618 allows NHS chemistry. To provide another example, Fig. 6B depicts a transformation beginning with the hydroxyl of Fig. 4B, 414. In this example, the hydroxyl 620 is tosylated to protect the alcohol group 622. Then the tosylated molecule 622 can be reacted to form two different products: an azide 624, which is useful for click chemistry attachments, or a free amine 626, which is used for NHS-type attachments. Additionally, the azide 624 can also be reacted again to form the amine 626. The use of similar techniques to those illustrated in Figs. 4 through 6 for use with carbon-based surfaces can also be used to attach organic functional groups to metal oxide surfaces. By the disclosed means therefore, EABs can include various electrode materials.

[0050] Another set of click chemistry and NHS chemistry reactions can be used with vacuum deposition to functionalize electrode surfaces for EAB sensors. A standard vacuum deposition technique used in the art for many purposes is trimethyl-aluminum (TMA) water deposition, which will reliably attach to almost any surface. With reference to Fig. 7, the electrode surface 730 is placed in a vacuum, and is exposed to gaseous TMA 710 in a first step, and water 714 in a second step. The reaction produces a hydroxyl-terminated surface 720, upon which additional organic functional groups, or additional layers 722, can be deposited. The functional groups, such as depicted in Figs. 5 A and 5B, in turn enable the use of click chemistry or standardized chemistry attachment methods on the surface. Because the binding reactions are self-limiting, i.e., each open surface molecule will react with only one introduced gaseous molecule, individual layers can be deposited with great precision. The allows several layers 722, e.g., 3 to 50 layers, each of which is about 1 A thick, to be built up on the electrode to form a blocking surface similar to a self-assembled monolayer. Blocking surfaces constructed by this technique may be used to tune the electrode properties for use in a particular EAB sensor biofluid medium or application, can shield the aptamer sensing elements from undesirable physical or chemical properties of the electrode, or may tune the interaction of EAB redox moieties with the electrode to adjust the performance of the EAB sensor.

[0051] Figs 8 A and 8B represent additional embodiments of the disclosed invention in which alternate end products of vacuum surface functionalization are illustrated. With reference to Fig. 8A, the first reaction step, where the gaseous TMA 810 reacts with the hydroxyl surface groups, proceeds as was depicted in Figs. 7A and 7B. During the second step, 3-butyn-l-ol 812 reacts with the intermediate product to produce an alkyne-terminated surface 820, which is suitable for click chemistry. With reference to Fig. 8B, during step 2, the intermediate product is reacted with ethanolamine 814 to produce an amine-terminated surface 822, suitable for NHS chemistry.

[0052] Fig. 9A depicts vacuum functionalization to an electrode surface 930 using trimethylsilyl azide 910 to produce an azide terminated surface layer 920, which is suitable for click chemistry attachment reactions. Fig. 9B depicts vacuum functionalization to an electrode 930 using aminopropyl triethoxysilane 912, which produces an amine terminated surface layer 922, which allows NHS attachment reactions.

[0053] Fig. 10 depicts a generalized means of functionalizing an electrode surface 1030 by molecular layer deposition. The functionalization begins with a free amine surface 1020, which may be deposited as depicted in Fig. 8B, or by other suitable means. The first step includes introducing a gaseous form of bi-functional acid chloride 1010, yielding an acid chloride terminated surface 1022. Step two includes introducing a gas of a bi-functional amine molecule 1012, which yields an amine terminated surface 1024 suitable for NHS attachment chemistry. As with vacuum deposition techniques discussed previously, R units represent functional groups, e.g., those depicted in Figs. 5 A and 5B, which allow the deposited layers to have a variety of selected properties. Also similarly, multiple organic layers can be built upon the electrode surface, each having the same or different functional groups in the R positions, allowing the layers to tune the physical or chemical interactions between the electrode and EAB sensor elements. Further, the molecular layer deposition techniques described are not limited to the attachment of EAB sensor components, and can be used to attach other layers or components as required.

[0054] Fig. 11 depicts a specific example of molecular layer deposition to functionalize an electrode surface 1130, in which the first step includes introducing gaseous acid chloride 1110, yielding an acid chloride terminated surface 1122. Step two includes introducing a gaseous bi- functional amine molecule 1112, which yields an amine terminated surface 1124 suitable for NHS attachment chemistry. Because the phenol rings in these layers are electrically conductive, the layers deposited with this molecule can be used to tune the electrical exchange between the electrode surface and the EAB sensor redox moiety. [0055] This has been a description of the disclosed invention along with a preferred method of practicing the disclosed invention, however the invention itself should only be defined by the appended claims.

Claims

CLICK CHEMISTRY APTAMER TAGGING FOR EAB BIOSENSORS WHAT IS CLAIMED IS:

1. A method of functionalizing an aptamer sequence for use in an electrochemical aptamer- based biosensor, comprising:

labelling a portion of the aptamer sequence to interact with a functionalization component through a bio-orthogonal chemistry reaction, wherein said functionalization component is one of the following: an oligonucleotide primer; an anchor molecule; a redox moiety; an oligonucleotide dock; and an electrode surface;

labelling the functionalization component to interact with the portion of the aptamer sequence through the bio-orthogonal chemistry reaction; and

attaching the aptamer sequence to the functionalization component through the bio- orthogonal chemistry reaction.

2. The method of claiml , wherein the electrochemical aptamer based biosensor is configured to measure a characteristic of an analyte in sweat.

3. The method of claim 1, where the bio-orthogonal chemistry reaction is one of the following: an azide-alkyne cycloaddition reaction; and a thiol-maleimide reaction.

4. The method of claim 1, where the electrode surface is one of the following: a carbon trace; a conductive metal oxide; and a conductive metal nitride.

5. The method of claim 1, where preparing the electrode surface further comprises depositing a plurality of layers by a vacuum deposition procedure, where the layers include at least one functional group.

6. The method of claim 4, where the vacuum deposition procedure includes exposing the electrode surface to gaseous trimethyl-aluminum.

7. The method of claim 4, where the vacuum deposition procedure includes exposing the electrode surface to one of the following gaseous molecules: tnmethylsilyl azide; and aminopropyl triethoxysilane.

8. The method of claim 4, where the vacuum deposition procedure includes exposing the electrode surface to a bi- functional acid chloride molecule.

9. The method of claim 4, where the plurality of layers is at least 3 and no more than 50 layers.

10. The method of claim 4, where the functional group is at least one of the following: tert-butyl cyclopentanecarboxylate; methyl benzoate; ethyl acetate; methyl pivalate; and allyl benzoate.