EP4677095A2

EP4677095A2 - Aptamer biosensors with mixed-conductivity monolayers

Info

Publication number: EP4677095A2
Application number: EP24767955.8A
Authority: EP
Inventors: Jason Heikenfeld
Original assignee: University of Cincinnati
Current assignee: University of Cincinnati
Priority date: 2023-03-09
Filing date: 2024-03-08
Publication date: 2026-01-14
Also published as: WO2024187171A2; WO2024187171A3; AU2024232938A1

Abstract

A device for continually sensing at least one analyte in a sample fluid via measurement of the analyte is provided. The device includes at least one sensor with surface having a plurality of aptamers that bind to the analyte. The aptamers carry at least one tag that changes in at least one electrical parameter as analyte binds to the aptamers. The surface also has a protective layer that protects the surface from fouling in between the aptamers. The protective layer includes a monolayer of molecules that form a boundary with the sample fluid and the monolayer is a mixed conductivity monolayer.

Description

APTAMER BIOSENSORS WITH MIXED-CONDUCTIVITY MONOLAYERS

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Application Serial No. 63/451,011, filed March 09, 2023, and U.S. Provisional Application Serial No. 63/456,605, filed April 3, 2023, which applications are hereby incorporated by reference in their entirety.

FIELD OF THE INVENTION

[0002] This invention relates generally to placement of biosensors with improved stability, but which maintain robust electron transfer.

BACKGROUND OF THE INVENTION

[0003] This section is intended to introduce the reader to various aspects of art that may be related to various aspects of the present invention, which are described and/or claimed below. This discussion is believed to be helpful in providing the reader with background information to facilitate a better understanding of various aspects of the present invention. Accordingly, it should be understood that these statements are to be read in this light, and not as admissions of prior art.

[0004] Electrochemical aptamer sensors can identify the presence and/or concentration of an analyte of interest via the use of an aptamer sequence that specifically binds to the analyte of interest. These sensors include aptamers attached to an electrode, wherein each of the aptamers has a redox active molecule (redox tag) attached thereto. The redox couple can transfer electrical charge to or from the electrode. When an analyte binds to the aptamer, the aptamer changes shape, bringing the redox couple closer to or further from, on average, the electrode. This results in a measurable change in electrical current that can be translated to a measure of presence or concentration of the analyte. When used in this manner, then, aptamers are an example of an affinity-based biosensor.

[0005] A major unresolved challenge for aptamer sensors and other affinity -based biosensors (particularly those where the aptamers are bonded to the working electrode) is the lifetime of the sensors, especially for applications where continuous operation is required (“continuous” referring to multiple measurements over time by the same device). Such aptamer sensors are susceptible to degradation due to, among other things, desorption of the aptamers themselves from the electrode, and/or desorption of the protective layer molecules (such as mercaptohexanol) from the electrode. The aptamers and the protective molecules together form a monolayer which can be referred to as a sensing monolayer. The protective layer portion of the sensing monolayer (1) ensures that the aptamer conformation change when binding to an analyte is not physically hindered by foulants, and (2) reduces electrical background current (including oxygen reduction current), which would otherwise wash-out the measured signal from the aptamer and redox tag.

[0006] Current methods of fabrication of these devices uses a very simple and convenient approach of forming a partial monolayer of aptamer by thiol bonding to a gold electrode via incubation of the electrode in solution including aptamer(s), followed by forming a more complete monolayer including the protective molecule such as mercaptohexanol (via incubation of the electrode in mercaptohexanol solution). This process is quite fortuitous for researchers because not only does a monolayer of mercaptohexanol reduce background current, but mercaptohexanol monolayers as-typically-formed have at least one feature such as defects, for example, which allow for electron transfer between the redox tag and the electrode, these defects being few and/or small enough to minimize oxygen reduction current and other major sources of background current. Furthermore, mercaptohexanol monolayers have adequate defects for electron transfer to support a zero-gain frequency that allows two frequency or comparable self-calibration techniques. Lastly, mercaptohexanol has enough surface fouling resistance to allow for short-term in-lab experiments in biofluids such as blood or serum.

[0007] Therefore, researchers have had at their disposal a very ‘convenient’ way to make aptamer sensors for research applications. However, most aptamer researchers have not historically been motivated to address longevity of aptamer sensors, and the same monolayer approach that is so convenient is also inherently fragile as the monolayer is able to desorb over time. Part of this cause for desorption is that each portion of the monolayer is a single molecule that has a single bond to the electrode, and statistically or energetically breaking one of these bonds with the electrode is not that difficult with conventional monolayer chemistries, especially at elevated temperatures such as body temperature. Multiple bonds to the gold could alleviate this challenge, but also may lack the tight packing density required for a low background current during measurement. According to leading experts in the 2022 review article, see Shaver, et al., “The challenge of long-term stability for nucleic acid-based electrochemical sensors,” Current Opinion in Electrochemistry (2022), 32: 100902 (https://doi.Org/10.1016/j.coelec.2021.100902), “Unfortunately, these chemistries desorb over time when exposed to environmental or experimental factors like, for example, dry air, high temperature, voltage pulsing, and biological fluids. This desorption process simultaneously removes sensing moieties and passivating thiols from the electrode surface, prohibiting their deployment for more than a few hours.” Clearly, even to the experts in the field, aptamer sensor longevity remains an unresolved problem with no obvious solutions for achieving sensor longevity for multiple days or weeks. Even as alternate methods are developed for extending longevity of protective layers, these methods must also support proper electron-transfer for sensor signaling, ideally allow use of one or more calibration-free methods of operation, and prevent over-fouling that otherwise would inhibit movement of the aptamer and therefore proper signaling of the sensor. Inventors Young and co-inventor Heikenfeld recently published a breakthrough on aptamer sensor longevity by stabilizing an alkythiolate based protective layer system for aptamer sensors with greater than one week of operational longevity, see Watkins, et al., “Watkins Z, Karajic A, Young T, White R, Heikenfeld J. Week-Long Operation of Electrochemical Aptamer Sensors: New Insights into Self- Assembled Monolayer Degradation Mechanisms and Solutions for Stability in Serum at Body Temperature. ACS Sensors, 2023 Mar 8; Available from: https://doi.org/10.1021/acssensors.2c02403.” The paper describes in detail degradation mechanisms for aptamer sensors and shows the utility of membrane protection and longer monolayer molecules and other efforts to preserve aptamer sensor functionality over multiple days. However, the techniques to increase monolayer stability also reduce redox current for the sensors which is a drawback. Novel approaches for electrochemical aptamer sensors are needed which reduce or eliminate these drawbacks such the sensors are more broadly attractive for long-lasting biosensing applications.

SUMMARY OF THE INVENTION

[0008] Certain exemplary aspects of the invention are set forth below. It should be understood that these aspects are presented merely to provide the reader with a brief summary of certain forms the invention might take and that these aspects are not intended to limit the scope of the invention. Indeed, the invention may encompass a variety of aspects that may not be explicitly set forth below.

[0009] Many of the drawbacks and limitations stated above can be resolved by creating novel and advanced interplays of chemicals, materials, sensors, electronics, microfluidics, algorithms, computing, software, systems, and other features or designs, in a manner that affordably, effectively, conveniently, intelligently, or reliably brings sensing technology into proximity with biofluid and analytes.

[0010] One aspect of the present invention is directed to a device for continually sensing at least one analyte in a sample fluid via measurement of the analyte. The device includes at least one sensor. The sensor’s surface has a plurality of aptamers that bind to the analyte. The aptamers carry at least one tag that changes in at least one parameter as analyte binds to the aptamers. The sensor’ s surface also has a protective layer that protects the surface from fouling in between the aptamers. The protective layer further comprises a monolayer of molecules that form a boundary with the sample fluid. The monolayer of molecules is a mixed conductivity monolayer of at least a first molecule and a second molecule which have different electrical conductivities.

[0011] The present invention involves a device for continually sensing at least one analyte in a sample fluid via measurement of the analyte. The device includes at least one sensor having a surface. The surface has a plurality of aptamers that bind to the analyte, and the aptamers carry at least one tag that changes in at least one electrical parameter as analyte binds to the aptamers. The surface also has a protective layer that protects the surface from fouling in between the aptamers. The protective layer further includes a monolayer of molecules that form a boundary with the sample fluid. In addition, the monolayer of molecules is a mixed conductivity monolayer.

[0012] In one embodiment, the mixed conductivity monolayer is a horizontally mixed conductivity monolayer, and further, wherein the monolayer comprises a plurality of first molecules and a plurality of second molecules, wherein the first molecules and the second molecules are mixed in the monolayer. In another embodiment, the first molecules and the second molecules each have an electrical conductivity and the electrical conductivity of the first molecules differs by at least 2X from the electrical conductivity of the second molecules under operating conditions of the sensor. In one embodiment, the first molecules and the second molecules are present in the monolayer at percentages that enable lower electrical resistance when compared to a standard aptamer sensor at a value selected from the group consisting of at least 2X, at least 10X, and at least 100X.

[0013] In another embodiment, the at least one tag is a redox tag and further, wherein the first molecules and the second molecules are present in the monolayer at percentages that enable higher redox tag current when compared to a standard aptamer sensor at a value selected from the group consisting of at least 2X, at least 10X, and at least 100X. In one embodiment, the at least one tag is a redox tag and further, wherein the first molecules and the second molecules are present in the monolayer at percentages that enable higher redox transfer rates when compared to a standard aptamer sensor at a value selected from the group consisting of at least 2X, at least 10X, and at least 100X. In another embodiment, the second molecules comprise a material selected from the group consisting of anthracene, benzylmercaptane, 4- biphenylmethanethiol, [r,r :4’;l”-terphenyl]-4-methanethiol, and combinations thereof.

[0014] In one embodiment, the monolayer further comprises water. In another embodiment, the at least one tag is a redox tag and further, wherein redox tag current is increased by electron transfer through the water. In one embodiment, the monolayer further comprises methylene. In another embodiment, the second molecules comprise a percentage of the total molecules in the monolayer selected from the group consisting of at least 0.1, at least 1, and at least 10%.

[0015] In one embodiment, at least a portion of the first molecules, the second molecules, or both, are terminated with a hydrophilic group. In another embodiment, at least a portion of the first molecules, the second molecules, or both, are terminated with a charged group. In one embodiment, at least a portion of the first molecules, the second molecules, or both, comprise zwitterionic groups. In another embodiment, the second molecules form domains of more than one molecule in isolation. In one embodiment, the aptamers are attached to conductive linker chemistry. In another embodiment, the second molecules have conductive portions which are separated from the sample fluid by at least one group. In one embodiment, all the molecules in the monolayer of molecules are at least in part conductive and form a vertically mixed conductivity monolayer.

[0016] In another embodiment, the second molecules have conductive portions, and further, wherein the second molecules are shorter than the first molecules. In one embodiment, the device also includes water which forms part of a conductive pathway for redox electron transfer, wherein the conductive pathway is located above the second molecules in the monolayer. In another embodiment, at least part of the conductive pathway is located between adjacent first molecules in the monolayer. In one embodiment, the second molecules have conductive portions, and further, wherein the second molecules are internally charged. In another embodiment, the first molecules comprise a number of methyl groups selected from the group consisting of 8, 9, 10, 11, and 12. In one embodiment, the second molecules comprise a number of phenyl groups selected from the group consisting of 1, 2, and 3.

[0017] In another aspect, the present invention involves a method of fabricating a sensor for sensing at least one analyte in a sample fluid. The sensor has a surface with a plurality of aptamers that bind to the analyte. The aptamers carry at least one tag that changes in at least one parameter as analyte binds to the aptamers. The surface also has a protective layer that protects the surface from fouling in between the aptamers. The protective layer further includes a monolayer of molecules that form a boundary with the sample fluid. The monolayer of molecules is a mixed conductivity monolayer. The method involves first incubating the sensor surface with aptamer followed by incubating the sensor surface with a mixed conductivity monolayer.

[0018] In one embodiment, the mixed conductivity monolayer is incubated on the sensor surface by a method involving incubating the sensor surface with first molecules to form a first monolayer. Then, forming defects in the first monolayer, and after defects are formed, incubating the sensor with second molecules to fill in part or all of the defects with conductive second molecules. In another embodiment, the mixed conductivity monolayer is incubated on the sensor surface by a method involving incubating the sensor surface with first molecules to form a first monolayer. At least 0.1% of the first molecules are inverted in orientation such that they are not bound chemically to the electrode. The method further involves desorbing the first monolayer to form defects or vacancies, and then incubating with second molecules to fill in part or all of the defects or vacancies with conductive second molecules.

BRIEF DESCRIPTION OF THE DRAWINGS

[0010] The objects and advantages of the disclosed invention will be further appreciated in light of the following detailed descriptions and drawings in which:

[0011] FIG. 1 A is a schematic of one embodiment of a conventional prior art sensor device.

[0012] FIG. IB is a schematic of one embodiment of a conventional prior art sensor device.

[0013] FIG. 2A is a schematic of an embodiment of a conventional prior art sensor device.

[0014] FIG. 2B is a schematic of another embodiment of a conventional prior art sensor device.

[0015] FIG. 3 A is an embodiment of the present invention.

[0016] FIG. 3B is an embodiment of the present invention.

[0017] FIG. 4 is an embodiment of the present invention.

[0018] FIG. 5 is an embodiment of the present invention.

[0019] FIG. 6 is an embodiment of the present invention

DEFINITIONS

[0020] As used herein, “continuous sensing” with a “continuous sensor” means a sensor that changes in response to changing concentration of at least one solute in a solution such as an analyte. Similarly, as used herein, “continuous monitoring” means the capability of a device to provide multiple measurements of an analyte over time. [0021 ] As used herein, the term “about,” when referring to a value or to an amount of mass, weight, time, volume, pH, size, concentration or percentage is meant to encompass variations of ±20% in some embodiments, ±10% in some embodiments, ±5% in some embodiments, ±1% in some embodiments, ±0.5% in some embodiments, and ±0.1% in some embodiments from the specified amount, as such variations are appropriate to perform the disclosed method.

[0022] As used herein, the term “electrode” means any material that is electrically conductive such as gold, platinum, nickel, silicon, conductive liquid infused materials such as ionic liquids, PEDOT:PSS, conductive oxides, carbon, boron-doped diamond, nanotubes or nanowire meshes, or other suitable electrically conducting materials.

[0023] As used herein, the term “protective layer” means a monolayer protective layer or a non-monolayer protective layer, or a combination of both.

[0024] As used herein, the term “protective monolayer” means a homogeneous or heterogeneous layer of material or of one or more types of molecules which enables electron transfer and at least one of: reduced electrochemical background current and/or current due to electrochemical interference; reduced fouling in a sample fluid; and which may promote proper freedom of movement for the aptamer which is required for creating a measurable response to analyte concentration.

[0025] As used herein, the term “non-monolayer protective layer” means a homogeneous or heterogeneous layer of material or of one or more types of molecules on an electrode which do not represent a monolayer configuration, and which enables electron transfer and at least one of: reduced electrochemical background current and/or current due to electrochemical interference; reduced fouling in a sample fluid; and which may promote proper freedom of movement for the aptamer which is required for creating a measurable response to analyte concentration. For example, a metal or semiconductor oxide can be a non-monolayer protective layer, or a thin polymer film may be a non-monolayer protective layer, because they are comprised of multiple layers of atoms or molecules. A single atomic monolayer of SiCh for example would be a monolayer, whereas 2 nm of SiCh is a non-monolayer.

[0026] As used herein, the term “mixed charge monolayer” may be a protective monolayer and means a monolayer of at least partially vertically oriented molecules on a surface, comprising at least a first plurality of molecules with a first polarity of charge at or near their terminus facing the sample fluid, and at least a second plurality of molecules with a second polarity of charge at or near their terminus facing the sampling fluid, where the first polarity and second polarity are oppositely charged. [0027] As used herein, the term “mixed conductivity monolayer” means a monolayer with a plurality of first molecules and a plurality of second molecules, where the first molecules and second molecules are mixed in the monolayer and where the electrical conductivity of the first molecules vs. the second molecules differs by at least 2X at the potentials required for redox electron transfer from the redox tagged aptamer. Practically, if the more conductive molecules of the first or second molecules comprises <10% of the total number of first and second molecules, then the more conductive molecules would preferably be at least 10X more conductive than the other molecules. A “mixed conductivity monolayer” may also encompass the antifouling properties of a mixed charged monolayer. The mixed conductivity (1) the monolayer may be primarily “horizontally mixed” meaning two or more molecule types are interspersed throughout the monolayer, and in at least one of the specific embodiments of present invention one of those molecules must be electrically conductive at the potentials used for redox electron transfer in the present invention; (2) the monolayer may be primarily “vertically mixed” meaning the monolayer has at least one molecule type which is along the inner length of the molecule electrically conductive at the potentials used for redox electron transfer in the present invention. This vertically mixed conductivity monolayer may include the linker chemistry used to attach aptamers to the electrode.

[0028] As used herein, the term “aptamer” means a molecule that undergoes a conformation or binding change as an analyte binds to the molecule, and which satisfies the general operating principles of the sensing method as described herein. Such molecules are, e.g., natural or modified DNA, RNA, or XNA oligonucleotide sequences, spiegelmers, peptide aptamers, and affimers and other affinity-based probes. Modifications may include substituting unnatural nucleic acid bases for natural bases within the aptamer sequence, replacing natural sequences with unnatural sequences, or other suitable modifications that improve sensor function, but which behave analogous to traditional aptamers. Two or more aptamers bound together can also be referred to as an aptamer (i.e., not separated in solution). Aptamers can have molecular weights of at least 1 kDa, 10 kDa, or 100 kDa.

[0029] As used herein, the term “tag” is a molecule carried on an aptamer that has a measurable response as analyte binds to the aptamer, such as a redox tag, or for example a fluorescent tag or quencher tag like that used in molecular beacons, or some other suitable tag that is measurable.

[0030] As used herein, the term “redox tag” or “redox molecule” means any species such as small or large molecules with a redox active portion that when brought adjacent to an electrode can reversibly transfer at least one electron with the electrode. Redox tag or molecule examples include methylene blue, ferrocene, quinones, or other suitable species that satisfy the definition of a redox tag or molecule. In some cases, a redox tag or molecule is referred to as a redox mediator. Redox tags or molecules may also exchange electrons or change in behavior when brought into proximity with other redox tags or molecules. Exogenous redox molecules are those added to a device, e.g., they are not endogeneous and provided by the sample fluid to be tested.

[0031] As used herein, the term “change in electron transfer” means a redox molecule whose electron transfer with an electrode has changed in a measurable manner. This change in electron transfer can, for example, originate from availability for electron transfer, distance from an electrode, diffusion rate to or from an electrode, a shift or increase or decrease in electrochemical activity of the redox molecule, or any other embodiment as taught herein that results in a measurable change in electron transfer between the redox molecule and the electrode.

[0032] As used herein, the term “sensing monolayer” means at least a plurality of aptamers on a working electrode, which may also include a plurality of molecules or mixtures of molecules that form a non-monolayer protective layer or monolayer protective layer.

[0033] As used herein, the term “analyte” means any solute in a solution or fluid or sample fluid which can be measured using a sensor. Analytes can be small molecules, proteins, peptides, electrolytes, acids, bases, antibodies, molecules with small molecules bound to them, DNA, RNA, drugs, chemicals, pollutants, or other solutes in a solution or fluid.

[0034] As used herein, the term “sample fluid” is the fluid containing the analyte.

[0035] As used herein, a “device” comprises at least one sensor based on at least one aptamer, and at least one sample solution. Devices can sense multiple samples and be in multiple configurations such as a device to measure a pin-prick of blood, or a microneedle or in-dwelling sensor needle to measure interstitial fluid, or a device to measure saliva, tears, sweat, or urine sensor, or a device to measure water pollutants or food processing solutes, or other devices which measure at least one analyte found in a sample solution.

[0036] As used herein, the term “redox tag current” means the amplitude of the faradaic redox tag peak current (such as that collected in voltammogram) minus the background current amplitude outside the redox peak in a given voltammetric scan; “normalized redox-tag current” is the redox-tag current normalized to the first measurement taken; “background current” is the current that would be measured if the aptamer molecules were not tagged with a redox reporter including, for example, capacitive currents and competing redox processes such as oxygen reduction; “adjusted current” is the combined redox tag current and background current of a square-wave voltammogram adjusted such that the minimum current is set to 0 A in the presentation of the voltammogram such that voltammograms can be plotted side by side and compared with greater ease; “sensor response” is the change in redox tag current due to binding of the target analyte to the aptamer, also known as signal gain, which can either increase or decrease based on the aptamer and the voltammetric time scale (such as operating frequency for square wave votammetry). The above terms can be used for representation or interpretation in other types of sensor scans such as chronoamperometric and should not be narrowly interpreted in the specifically to only represent scanning methods such as square wave voltammetry.

[0037] As used herein, the term “2X” means a difference of two times in magnitude. Similarly, the term “10X” means a difference of ten times in magnitude. The term “100X” means a difference of one hundred times in magnitude.

DETAILED DESCRIPTION OF THE INVENTION

[0038] One or more specific embodiments of the present invention will be described below. In an effort to provide a concise description of these embodiments, all features of an actual implementation may not be described in the specification. It should be appreciated that in the development of any such actual implementation, as in any engineering or design project, numerous implementation-specific decisions must be made to achieve the developers’ specific goals, such as compliance with system-related and business-related constraints, which may vary from one implementation to another. Moreover, it should be appreciated that such a development effort might be complex and time consuming, but would nevertheless be a routine undertaking of design, fabrication, and manufacture for those of ordinary skill having the benefit of this disclosure.

[0039] 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 can be in duplicate, triplicate, or more, to provide improved data and readings. Sensors may provide continuous or discrete data and/or readings. 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 known (e.g., a reference or counter electrode, a battery, antenna, adhesive), and for purposes of brevity and focus on inventive aspects, such components may not be explicitly shown in the diagrams or described in the embodiments of the disclosed invention. All ranges of parameters disclosed herein include the endpoints of the ranges.

[0040] With reference to FIGS. 1A and IB, a conventional prior art sensor device 100 as placed initially in a sample fluid, such as interstitial fluid, is shown. The device 100 includes at least one working electrode 120 such as gold, carbon, or other suitable electrode material; at least one protective layer that is a monolayer protective layer 126 such as a plurality of molecules such as mercaptohexanol that are thiol bonded to the electrode; at least one aptamer 124 that is responsive to binding to an analyte 180; and a redox tag 170, such as methylene blue, associated with the at least one aptamer, such as by being bound thereto. In the generic example taught for FIGS. 1 A and IB, the aptamer 124 is a simple stem loop (hairpin) aptamer where analyte 180 binding causes the stem loop to form and the redox tag current measured from the redox tag 170 to increase, as measured using square wave voltammetry, chronoamperometry, or other suitable technique. In absence of analyte 180 binding to the aptamer 124 the stem loop conformation does not form and the redox current thus does not increase. Alternately, analyte 180 binding to different example aptamer can cause a decrease in redox tag current. Thus, with aptamer sensors changes in a measurement of electrical redox tag current can be used as a signal to interpret changes in concentration of the analyte 180.

[0041] With further reference to FIGS 2A, and 2B, where like numerals refer to like features, a challenge with aptamer sensors is that when based on monolayers the monolayers can desorb over time and the sensor degrades, which can at least in part be alleviated by increasing monolayer molecule length and resulting increased Van Der Waals and other forces to keep the layer intact, as taught in detail by Watkins et al. in Watkins Z, Karajic A, Young T, White R, Heikenfeld J. Week-Long Operation of Electrochemical Aptamer Sensors: New Insights into Self-Assembled Monolayer Degradation Mechanisms and Solutions for Stability in Serum at Body Temperature. ACS Sensors, 2023 Mar 8; Available from: https://doi.org/10.1021/acssensors.2c02403. However, increased monolayer thickness due to increased molecule length also decreases the rate of electron transfer and the measurable current, and for Watkins et al moving from C6-OH to C8-OH decreases the current by approximately 5X. As illustrated FIG. 2B where even with analyte 280 binding to aptamer 224 there is no or reduced electron transfer between redox tag 270 and electrode 220. The amount of reduction in electron transfer can be substantial, for example in a comparison of mercaptohexanol (C6-OH) to mercaptoundecanol (Cl l-OH) for molecules 226 the redox current decreased non-linearly by approximately 50X, as taught m Lai RY, Seferos DS, Heeger AJ, Bazan GC, Plaxco KW. Comparison of the signaling and stability of electrochemical DNA sensors fabricated from 6- or 11-carbon self-assembled monolayers. Langmuir. 2006 Dec 5 ;22(25): 10796-800. doi: 10.1021/la0611817. PMID: 17129062. The reduction in redox current is due to multiple factors including reduced monolayer defectivity, reduced tunneling, and increased electrical resistance through defects due to increased thickness.

[0010] With further reference to FIGS 3 A, and 3B, where like numerals refer to like features, in an embodiment of the present invention, layer 326 is a horizontally mixed conductivity monolayer with a plurality of first molecules 326a and a plurality of second molecules 326b, where the first molecules and second molecules are mixed in the monolayer and where the electrical conductivity of the second molecules are at least 2X and more preferably 5X, 10X, 20X or even 50X greater in electrical conductivity of the first molecules under the conditions needed to operate the sensor 300. As illustrated in FIG. 3, the second molecules 326b are more conductive. Conductive molecules 326b could be for example conjugated molecules (as illustrated in FIG. 3), redox tags or redox active portions of molecules, or other suitable molecular constructs that have increased electrical conductivity or which promote increased electrical tunneling. For example, molecules 326b could preferably be benzylmercaptane, 4-biphenylmethanethiol, [r,r:4’;l”-terphenyl]-4-methanethiol. The mixed conductively monolayer can therefore have lower electrical resistance or higher redox tag current or redox transfer rates than a layer of unmixed and less-conductive first molecules such as mercaptoundecanol (Cl l-OH), or mercaptooccanol (C8-OH), or mercaptononanol (C9-OH), or mercaptodecanol (C10-OH), or mercaptododecanol (C12-OH). Embodiments of the present invention enable lower electrical resistance or higher redox tag current or higher redox transfer rates of at least 2X, 10X, or 100X depending on the percentages of the first and second molecules 326a and 326b and the assembly and testing conditions. Less preferably molecules 326 could be l,r :4’,l”-terphenyl]-4-ethanethiol which has an extra methyl group between the phenyl groups and electrode which decreases tunneling current between the molecule and the electrode 320. Alternately, molecules 326b could be in part comprised of anthracene or other conductive molecules, and preferably molecules that are linear and/or narrow in geometry such that they arrange in the monolayer without overly disrupting order and stability of the monolayer (for example, PEDOT or Polypyrrole conductive polymer could be less preferred due to less linearity). If the monolayer is formed on gold where Van Der Waals and other forces to keep the layer intact then the conductive molecules are also preferably hydrophobic or at least not highly hydrophilic. Alternately, molecules 326b could induce defects in monolayer 326 and or be hydrophilic which contain at least in part water (water wires), and which also increases redox tag current by electron transfer through the water which when done purposely captures the effect of a ‘horizontally mixed conductivity monolayer’. Alternately, molecules 326b could be formed in part of 1 or less methylene spacers between the conductive portion of the molecule 326b and electrode 320 which is the case for 4-biphenylmethanethiol, benzylmercaptane, and [r,l’ :4’;l”-terphenyl]-4- methanethiol, but not the case for l,r :4’,l”-terphenyl]-4-ethanethiol. Second molecules may comprise at least 0.1, 1, or 10% of the total molecules in monolayer 326.

[0011] With further reference to FIGS 3A, and 3B, where like numerals refer to like features, in an embodiment of the present invention, layer 326 a is mixed conductivity monolayer with a plurality of first molecules 326a and a plurality of second molecules 326b, wherein at least a portion of the molecules are terminated (not shown) with a hydrophilic group such as an OH group or with a charged group, or the molecules themselves include zwitterionic groups, all of which can reduce fouling of the monolayer 326 by increasing the amount of locally bound water near the monolayer surface and at the interface with the sample fluid. Chemistries include mixed charge terminated and in zwitterionic monolayer chemistries, and include for example terminal chemistries using dimethyl-amino-propane-1 -sulfonic acid.

[0012] With further reference to FIG 4, where like numerals refer to like features, in an embodiment of the present invention, layer 426 is a mixed conductivity monolayer with a plurality of first molecules 426a and a plurality of second molecules 426b, where the first molecules and second molecules are mixed in the monolayer and where the second molecules may form domains of more than one molecule in isolation as illustrated for both molecules 426a and 426b. Furthermore, aptamers 424 may also be attached to conductive linker chemistry as illustrated in FIG. 4, the conductive linker chemistry in between non-conductive molecules for example being similar to conductive molecules as taught herein and therefore capturing the embodiment of a ‘horizontally mixed conductivity monolayer’.

[0013] With further reference to FIG 5, where like numerals refer to like features, in an embodiment of the present invention, layer 526 is a vertically mixed conductivity monolayer with a plurality of first molecules 556a and a plurality of second molecules 526b, where the first molecules and second molecules are mixed in the monolayer and where second molecules 526b have conductive portions (shown as the phenyl rings) which are separated from the sample fluid by at least one group such as a methyl group. This can be advantageous to prevent fouling or aptamer sticking to molecules 526b. If the terminal end of the molecule 526b facing solution has a zwitterion chemistry or charged chemistry, that chemistry binds water and locally adds conductivity such that a larger portion or the entire molecule 526b is effectively conductive. [0014] With further reference to FIG 6, where like numerals refer to like features, in an embodiment of the present invention, all the molecules in layer 626 can be at least in part have vertically mixed conductivity, but where at least a portion of the molecules in layer 626 are electrically insulating adjacent to electrode 620 or the sample fluid. When a molecule is only partially electrically conductive along its length it may require tunneling to allow electron transfer unless water and/or other additional conductivity enables electron transfer (other conductivity being for example defects in the monolayer).

[0015] With further reference to embodiments of the present invention, in a mixed conductivity monolayer with a plurality of first molecules and a plurality of second molecules, where the first molecules and second molecules are mixed in the monolayer and where second molecules have conductive portions the second molecules can be shorter than the first molecules, for example with first molecules that are mercaptoundecanol or mercaptododecanol and second molecules that are 4-biphenylmethanethiol. As a result, water may form part of a conductive pathway for redox electron transfer that is above the second molecules in the monolayer and at least in part between adjacent first molecules in the monolayer. This embodiment, for example can reduce fouling and/or aptamer sticking to the second molecules. [0016] With further reference to embodiments of the present invention, in a mixed conductivity monolayer with a plurality of first molecules and a plurality of second molecules, where the first molecules and second molecules are mixed in the monolayer and where second molecules have conductive portions, and where the second molecules that are internally charged, for example having internal zwitterionic charge surrounded on either end by one more more methylene groups. Here the internal charge causes hydration and local ability to promote redox electron transfer by tunneling or other suitable means.

[0017] With further reference to embodiments of the present invention, embodiments can be fabricated, for example, by conventional methods for aptamer sensors such as first incubation with aptamer followed by mixed conductivity monolayer incubation all the same time in the same solution. Alternately, a device could be made by aptamer incubation followed by first molecules incubation and then defects formed in first monolayer of first molecules by temperature or voltage cycling. After defects are formed the device can then be incubated with second molecules to fill in part or all of defects with conductive second molecules. Alternately, a device could be made by aptamer incubation followed by first molecules incubation at a higher concentration or different solvent conditions for first molecules such that at least 0.1% of first molecules are inverted in orientation such that they are not bound chemically to the electrode, and then desorbed to form defects or vacancies. After defects are formed the device can then be incubated with second molecules to fill in part or all of defects with conductive second molecules. First molecules could also be a mixture of molecules with two or more different charges, hydrophilicities, or other features that promotes desorption and vacancy formation for at least a portion of first molecules. After defects are formed the device can then be incubated with second molecules to fill in part or all of defects with conductive second molecules.

[0018] With further reference to embodiments of the present invention, first molecules may contain at least one of 8, 9, 10, 11, 12, or more methyl groups. With further reference to embodiments of the present invention, second molecules may contain at least one of 1, 2, 3 or more conjugated phenyl groups. Embodiments of the present invention may also include embodiments with less methyl groups but such embodiments will in most cases be less stable and exhibit greater monolayer desorption over time.

[0019] With further reference to embodiments of the present invention a non-monolayer protective layer may be used in conjunction with a covering of a monolayer protective layer using one or more embodiments of the present invention.

[0020] As can be seen, current sensor devices include several drawbacks and limitations, particularly resulting from the protective layers found on such current devices. Various aspects of the present invention, however, resolve such drawbacks and limitations (including the amount of initial or longer-term desorption, instability, and/or fouling of the sensing monolayer). In that regard, one aspect of the present invention is directed to a device for detecting the presence of, or measuring the concentration or amount of, at least one analyte in a sample fluid.

[0021] Although not described in detail herein, other steps which are readily interpreted from or incorporated along with the disclosed embodiments shall be included as part of the invention. The embodiments that have been described herein provide specific examples to portray inventive elements, but will not necessarily cover all possible embodiments commonly known to those skilled in the art.

Claims

WHAT IS CLAIMED IS:

1. A device for continually sensing at least one analyte in a sample fluid via measurement of the analyte, comprising at least one sensor having a surface comprising: a plurality of aptamers that bind to the analyte, wherein the aptamers carry at least one tag that changes in at least one electrical parameter as analyte binds to the aptamers; and a protective layer that protects the surface from fouling in between the aptamers, wherein the protective layer further comprises a monolayer of molecules that form a boundary with the sample fluid; wherein the monolayer of molecules is a mixed conductivity monolayer.

2. The device of claim 1 wherein the mixed conductivity monolayer is a horizontally mixed conductivity monolayer, and further, wherein the monolayer comprises a plurality of first molecules and a plurality of second molecules, wherein the first molecules and the second molecules are mixed in the monolayer.

3. The device of claim 2 wherein the first molecules and the second molecules each have an electrical conductivity and the electrical conductivity of the first molecules differs by at least 2X from the electrical conductivity of the second molecules under operating conditions of the sensor.

4. The device of claim 2 wherein the first molecules and the second molecules are present in the monolayer at percentages that enable lower electrical resistance when compared to a standard aptamer sensor at a value selected from the group consisting of at least 2X, at least 10X, and at least 100X.

5. The device of claim 2 wherein the at least one tag is a redox tag and further, wherein the first molecules and the second molecules are present in the monolayer at percentages that enable higher redox tag current when compared to a standard aptamer sensor at a value selected from the group consisting of at least 2X, at least 10X, and at least 100X.

6. The device of claim 2 wherein the at least one tag is a redox tag and further, wherein the first molecules and the second molecules are present in the monolayer at percentages that enable higher redox transfer rates when compared to a standard aptamer sensor at a value selected from the group consisting of at least 2X, at least 10X, and at least 100X.

7. The device of claim 2 wherein the second molecules comprise a material selected from the group consisting of anthracene, benzylmercaptane, 4-biphenylmethanethiol, [r,r:4’;l”- terphenyl]-4-methanethiol, and combinations thereof.

8. The device of claim 1 wherein the monolayer further comprises water.

9. The device of claim 8 wherein the at least one tag is a redox tag and further, wherein redox tag current is increased by electron transfer through the water.

10. The device of claim 1 wherein the monolayer further comprises methylene.

11. The device of claim 2 wherein the second molecules comprise a percentage of the total molecules in the monolayer selected from the group consisting of at least 0.1, at least 1, and at least 10%.

12. The device of claim 2 wherein at least a portion of the first molecules, the second molecules, or both, are terminated with a hydrophilic group.

13. The device of claim 2 wherein at least a portion of the first molecules, the second molecules, or both, are terminated with a charged group.

14. The device of claim 2 wherein at least a portion of the first molecules, the second molecules, or both, comprise zwitterionic groups.

15. The device of claim 2 wherein the second molecules form domains of more than one molecule in isolation.

16. The device of claim 1 wherein the aptamers are attached to conductive linker chemistry.

17. The device of claim 2 wherein the second molecules have conductive portions which are separated from the sample fluid by at least one group.

18. The device of claim 1 wherein all the molecules in the monolayer of molecules are at least in part conductive and form a vertically mixed conductivity monolayer.

19. The device of claim 2 wherein the second molecules have conductive portions, and further, wherein the second molecules are shorter than the first molecules.

20. The device of claim 2 further comprising water which forms part of a conductive pathway for redox electron transfer, wherein the conductive pathway is located above the second molecules in the monolayer.

21. The device of claim 20 wherein at least part of the conductive pathway is located between adjacent first molecules in the monolayer.

22. The device of claim 2 wherein the second molecules have conductive portions, and further, wherein the second molecules are internally charged.

23. The device of claim 2 wherein the first molecules comprise a number of methyl groups selected from the group consisting of 8, 9, 10, 11, and 12.

24. The device of claim 2 wherein the second molecules comprise a number of phenyl groups selected from the group consisting of 1, 2, and 3.

25. A method of fabricating a sensor for sensing at least one analyte in a sample fluid, the sensor having a surface comprising: a) a plurality of aptamers that bind to the analyte, wherein the aptamers carry at least one tag that changes in at least one parameter as analyte binds to the aptamers; and b) a protective layer that protects the surface from fouling in between the aptamers, wherein the protective layer further comprises a monolayer of molecules that form a boundary with the sample fluid; wherein the monolayer of molecules is a mixed conductivity monolayer; the method comprising first incubating the sensor surface with aptamer followed by incubating the sensor surface with a mixed conductivity monolayer.

26. The method of claim 25 wherein the mixed conductivity monolayer is incubated on the sensor surface by a method comprising: i) incubating the sensor surface with first molecules to form a first monolayer; ii) forming defects in the first monolayer; and iii) after defects are formed, incubating the sensor with second molecules to fill in part or all of the defects with conductive second molecules.

27. The method of claim 25 wherein the mixed conductivity monolayer is incubated on the sensor surface by a method comprising: i) incubating the sensor surface with first molecules to form a first monolayer, wherein at least 0.1% of the first molecules are inverted in orientation such that they are not bound chemically to the electrode; ii) desorbing the first monolayer to form defects or vacancies; and iii) incubating with second molecules to fill in part or all of the defects or vacancies with conductive second molecules.