Patent Application
20230329592
The present invention relates to the use of electrochemical, aptamer-based (E-AB) sensors.
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.
Interstitial fluid contains many of the same analytes as blood and often at comparable concentrations. As a result, interstitial fluid presents an alternative biofluid to blood for detection of analytes such as glucose for diabetes monitoring. Commonly employed practices for continuous monitoring of glucose in interstitial fluid include (1) in-dwelling sensors, where a needle is utilized to insert the sensor into the dermis of the skin, and (2) ex-vivo sensors, where micro-needles penetrate the surface of the skin and the analyte is coupled from interstitial fluid to the sensor by diffusion to the sensor. In products, and in research, the biosensing of analytes in interstitial fluid monitoring has been dominated by metabolite analytes because electrochemical enzymatic sensors are readily available and well developed for these compounds, and because metabolites are found at generally high concentrations (mM) which simplifies their detection. Even so, use of an enzymatic sensor with a microneedle device has not yet seen commercial success, at least in part because microneedles have difficulty in keeping continuous contact with the dermis. For an enzymatic sensor, which relies on a diffusive flux of analyte to the sensor, any change in coupling between the microneedles and the dermis of the skin would result in a false-change in measured glucose signal. Even in-dwelling sensors can suffer from motion artifacts as an enzymatic sensor’s position in the dermis can impact diffusive flux of glucose to the sensor.
Affinity-based sensors, such as electrochemical or optical aptamers, are known to be inherently reversible (and thus truly continuous) and known to provide ranges of detections in the µM or lower ranges in biofluids such as whole blood. These sensors, however, are quite different than enzymatic sensors, which metabolize and therefore consume the analyte. This is because affinity sensors require equilibration of analyte concentration with the sensor itself, and have a known binding affinity for the target analyte. However, such an approach is ill-suited to a continuous wearable format, as unlike enzymatic sensors, electrochemical aptamer sensors are extremely sensitive to abrasion or pressure against the sensor surface.
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.
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 sample fluids containing at least one analyte of interest to be measured.
Aspects of the disclosed invention are directed to electrochemical aptamer-based sensors that have resistance to the confounding effects of abrasion and pressure. One particular aspect of the present invention is directed to a continuous sensing device for measuring at least one analyte in interstitial fluid. The device includes at least one feature configured to be inserted into a body, and specifically, the at least one feature may be configured to be inserted into a skin of the body. The at least one feature is at least partially coated with at least one electrode functionalized with an aptamer sensing monolayer layer, and the aptamer sensing monolayer layer includes an aptamer with attached redox couples and passivating material. The at least one feature is configured to provide at least one of a resistance to abrasion effect or a pressure effect for the aptamer sensing monolayer when the feature is placed into the body.
In certain embodiments, the feature is a porous or grooved surface of a microneedle. In addition, in certain embodiments including a microneedle, only the inside of the electrode is coated.
In another embodiment, the feature includes a membrane covering the electrode. In another embodiment, the feature includes the aptamer sensing monolayer that is added onto the electrode.
Another aspect of the present invention is directed to a method of fabricating a continuous sensing device for measuring at least one analyte in interstitial fluid. The device contains at least one feature that is coated at least in part with at least one electrode that is functionalized with aptamers and attached redox couples to electrochemically measure the analyte. The method involves fabricating the at least one feature that provides at least one of abrasion resistance or pressure resistance when placed into the dermis of the skin. In one embodiment, the method also involves coating an electrode, an aptamer sensing layer, or both on the feature, and then removing the electrode and/or the aptamer sensing layer from all regions of the feature where variable pressure or abrasion could be problematic. In another embodiment, an aptamer sensing layer, blocking layers, or both are applied after partial removal of the microneedle feature. In one embodiment, an aptamer sensing layer, blocking layers, or both are applied before partial removal of the microneedle feature. In another embodiment, an electrode is coated with a porous protected material, after which an aptamer sensing layer, blocking layers, or both are applied to the remaining electrode surface.
The objects and advantages of the disclosed invention will be further appreciated in light of the following detailed descriptions and drawings in which:
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.
As used herein, the term “aptamer” means a molecule that undergoes a conformation 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. 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.
As used herein, the term “sensing monolayer” means aptamers that are functionalized with a redox tag, such as methylene blue or other redox tag, and attached onto an electrode such as gold by thiol linkage or other suitable chemistry, and the space in between the aptamers on the electrode passivated by a passivating material such as mercaptohexanol or other suitable passivating material.
The devices and methods described herein encompass the use of sensors. A “sensor,” as used herein, is a device that is capable of measuring the concentration of a target analyte in solution. As used herein, an “analyte” may be any inorganic or organic molecule, for example: a small molecule drug, a metabolite, a hormone, a peptide, a protein, a carbohydrate, a nucleic acid, or any other composition of matter. The target analyte may comprise a drug. The drug may be of any type, for example, including drugs for the treatment of cardiac system, the treatment of the central nervous system, that modulate the immune system, that modulate the endocrine system, an antibiotic agent, a chemotherapeutic drug, or an illicit drug. The target analyte may comprise a naturally-occurring factor, for example a hormone, metabolite, growth factor, neurotransmitter, etc. The target analyte may comprise any other species of interest, for example, species such as pathogens (including pathogen induced or derived factors), nutrients, and pollutants, etc.
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.
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 measure a characteristic of an analyte. 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 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 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.
With reference to
As has been noted above, aspects of the present invention include the use of aptamers (such as in electrochemical aptamer-based sensors). However, if a feature 114 were to be coated with an aptamer on its surface, there are several challenges presented by insertion into the body such as the dermis 12b. Firstly, tissue can abrade the surface and remove the monolayer of aptamer and blocking layer such as mercaptohexanol which are both typically thiol bonded to a gold surface. Secondly, and in some instances even more potentially challenging, pressure of tissue such as collagen against the sensor can cause signal changes from the aptamer, for example by physical pressure or by the negatively charged membrane proteins on surface of many types of tissue that can cause electronic or steric repulsion and could change the distance of the redox tag on the aptamer (e.g., methylene blue) with the electrode that transfers charge to/from the redox tag. Cellular or other materials in the dermis can also interfere with aptamers. Drawbacks such as these were discussed above in the “Background” section.
Aspects of the present invention eliminate these drawbacks. With reference now to
Alternately, as shown in
With further reference to the present invention, an alternate method of fabrication (not shown) is additive instead of subtractive like the previous taught method. In an embodiment, a surface is coated with a gold or other electrode, and a porous or non-continuous material is deposited onto the gold electrode, such as electrodeposited or spray-coated polymer such as electrodeposited photoresist or spray coated acrylic in a solvent such as acetone. This polymer then forms a porous yet protected network on the electrode, and the electrode can then be functionalized with aptamer and blocking layer on the remaining exposed electrode areas. In this method, an electrode is coated with a porous protecting material, after which the aptamer and blocking layers can be applied to the remaining electrode surface.
With further reference to the present invention, any of the embodiments as taught herein can be coated with a biocompatible dissolvable material such as sucrose, for example, the aptamer sensing monolayer 122 may be coated with a bio-compatible dissolvable material, denatured serum that is 5 kDa filtered, or other suitable material, to protect any surfaces until the device is inserted into the skin. The biocompatible dissolvable material can then be naturally removed (dissolved) by the body to reveal the sensor surface.
With reference to
With further reference to the present invention, as illustrated in
With further reference to the present invention, any of the embodiments as taught herein can have various porosities for the electrode 120, 220. For example, porous gold can be formed by a simple procedure which involves an acidic treatment of a commercially available complex white-gold or gold-silver alloy. 24 hour HNO3 treatment can provide up to 12,400 times surface enlargement and resulted in a surface area of 14.2 m2/g. With use with aptamers, pores can typically be as small as allows freedom of movement for the aptamer (approximately 10 nm or larger). In an embodiment, the feature 114 includes an exposed area configured to be exposed to tissue or cellular content in the body after the feature is inserted into the body, and an unexposed area that is configured to be unexposed to tissue or cellular content in the body after the feature 114 is inserted into the body, and which carriers the aptamer sensing monolayer 122, 222. The ratio of protected and unexposed porous surface with the aptamer sensing monolayer 122, 222 to the surface of the electrode 120, 220 that is exposed and unprotected to abrasion can therefore be at least one of >1.3X, >3X, >10X, >30X, >100X. Alternately, porous electrodes can be electrodeposited, including for example for gold by adding a sufficient amount of ammonium chloride is added to the electrolyte as a hydrogen source during electrodeposition. Porous electrodes can also have such small porosity that they also, in effect, act as a protecting membrane by excluding larger solutes such as cells, albumin, enzymes, nucleases, etc. The ratio of protected and unexposed porous surface also applies to other embodiments, such as
With further reference to the present invention, too great of a porosity could disable proper functionalization of the electrode. The first step in electrochemical aptamer electrode functionalization is to bond the redox-tagged aptamer to the electrode, often using chemistry such as thiol-linkage if the electrode is gold. Typically, aptamer densities range from ~1010 to 1012/cm2. An ultra-porous surface would suffer from low aptamer densities in its deepest crevices and too high aptamer densities near the surface, because during wet functionalization of the electrode with aptamer the aptamers will link to nearby gold surfaces as they slowly diffuse into the porous electrode. This will often appear as a noisier signal or a sensor that does not properly respond to changes and analyte signal. Also, too high of electrode porosity can cause a long lag time for the sensor response as greater amount of analyte must diffuse to the aptamers and also diffuse through a more tortuous network of pores. Therefore, the present invention may utilize a ratio of protected porous surface to the surface of the electrode 120, 220 that is exposed and unprotected to abrasion that is at least >1.3X but at least one of less than <3X, <10X, <30X, <100X, <300X. As a result, the coverage density of aptamer across the sensing monolayer will vary by at least one of <30%, <100%, <300%, <1000%. Pore sizes also matter, because if the pore size is too small, aptamers bound on opposite sides of a pore can interfere with each other’s free motion in solution and/or the pores can be too small to allow proper diffusive transport of analyte such as large proteins to the aptamers. Therefore, average pore size may be at least one of >5 nm, >15 nm, >45 nm, >100 nm.
With further reference to the present invention, if too great of a porosity could disable proper functionalization of the electrode, then instead of or in addition to limiting the electrode porosity and surface area, the aptamer could be deposited in a manner such that it is reaction rate limited or diffusion rate limited inside the pore, not diffusion rate limited between the pore and aptamer-containing solution outside of feature 114. Aptamer deposition (incubation) typically requires 1-2 hours with 100′s nM aptamer concentrations in solution, a diffusion rate limited process (not reaction rate limited by thiol bonding to the gold). A simple illustrative calculation is as follows. Assume a final aptamer density on the gold surface of 1E11 aptamers/cm2. Assume pores in the gold that on average have a thickness of fluid above them that is ~50 nm thick, which per cm2 would be 5E-6 cm * 1 cm2 = 5E-6 mL or 5 nL. 1E11 aptamers/cm2 in that 5 nL represents 1E11 aptamers / 5 nL * 1 mole / 6.02E23 aptamers * 1E9 nL / L = 33 µM of aptamer. Therefore, if aptamer solution were introduced to the pores with standard concentrations of aptamer (100′s nL to µL’s) the deposition would be diffusion rate limited through the porous network of the gold. To have the deposition rate be limited locally by diffusion rate or reaction rate inside the pores, the aptamer concentration would need to be >10 µM, and preferably > 100 µM. The above math for aptamer density, pore size, and other aspects of the aptamer deposition can be arranged such that generally aptamer concentrations in solution during deposition of the aptamer may preferably be at least one of >10, >100, >1000, >10,000 µM of aptamer. With such higher concentrations, the incubation time for deposition will be much shorter, and therefore the porous gold electrode could be dry, placed in vacuum, then dipped into the aptamer solution, and vacuum released, to rapidly introduce aptamer into the pores and avoid diffusion limited aptamer deposition, then incubated, then rapidly placed into a well-stirred buffer solution to remove aptamer as quickly as possible. Wicking into the pores of the gold is also possible if air-trapping is minimized by wicking the fluid primarily horizontally along the plane of the porous gold layer.
With further reference to the present invention, a membrane 140 as taught herein may also be applied to fill the pores 116 or only to the entrance of pores 116 where pores 116 meet area of feature 114 that would interface with tissue in the body. For example, a sensor as shown in
An example is taught with respect to creating a nano-porous electrode on a gold-plated stainless steel acupuncture needle, that can then be coated with an apatamer and be inserted into the skin and used as an electrochemcial working electrode per the present invention.
Deep Eutectic Solvent Preparation: 1:3.5 mole ratio of ZnCl:Urea was prepared in a beaker; the solution was heated to 60° C. on a hotplate while being stirred via a stir-bar until completely liquid and homogenous.
Potentiostat setup: A 3-electrode system was used for deposition. The counter wire was connected to a Zinc wire, the reference wire was connected to a flat, larger piece of zinc, and the working wire was connected to a gold-coated needle. These electrodes were immersed in the deep eutectic solvent in a beaker on a hot plate.
Electrochemical Processes: For cleaning, CV’s from 0-1.6 V were done in 0.5 M H2SO4 at a scan rate of 100 mV/s. For the nanoporous gold deposition, the gold-plated acupuncture needle was placed in the heated deep eutectic solvent. Then a -0.2 V potential was applied via chronocoulometry until a charge of 0.07 C was reached. Immediately afterwards a 1 V potential was applied via chronoamperometry until a steady anodic current of 1 µA was reached. The resulting nanoporous electrode was functionalized and tested with aptamers for cortisol and vancomycin and demonstrated to work in in-vitro with buffer or serum and in real biological tissue.
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.
This application claims priority to, and the benefit of the filing date of, U.S. Provisional Application No. 63/082,810 filed Sep. 24, 2020 and U.S. Provisional Application No. 63/150,634 filed Feb. 18, 2021, the disclosures of which are incorporated by reference herein in their entirety.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2021/051865 | 9/24/2021 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 63150634 | Feb 2021 | US | |
| 63082810 | Sep 2020 | US |
