Patent Application
20230050906
The present invention is directed to the fabrication and use of stretchable wrinkled electrodes for electrochemical detection of biomarkers in body fluids, more particularly to a device to be used in portable/wearable point-of-care health monitoring devices.
It is frequently possible to delay the onset or progression of an illness by identifying the cause and acting against it. Unfortunately, access to early detection is an issue; hospital visits and laboratory tests are expensive. Wearable sensors and point-of-care (POC) devices bring personalized healthcare to more people at a lower cost. Detailed insight into the user's health state can be obtained at the molecular level with analysis of biomarkers present in sweat, such as glucose, ethanol, lactic acid, ions (Na+, K+, Cl), and cortisol.
For example, cortisol is a widely accepted biomarker for physiological and emotional stress and exists in two forms in human bodies: bound to another molecule, or not bound (free). Monitoring fluctuations in the concentration of free cortisol can help identify and reduce harmful responses to stress, but invasive sample collection confounds data because of the difficulty of distinguishing the origin of the stress response. Through the non-invasive collection of saliva, the inherent stress response to blood collection is avoided. Electrochemical detection of these biomarkers is attractive because it can achieve clinically relevant sensitivity and accuracy and is easily integrated into electronic systems.
Biosensors that detect analytes in sweat face the challenge of maintaining sensitivity upon miniaturization. Various materials and processes have been developed to create nanostructured electrodes with high surface areas to mitigate this issue. The need remains, however, for biocompatible materials that can be scalably integrated into wearable devices. Integrating electrochemical transducers into wearable devices necessitates miniaturization of the electrodes. This negatively impacts the signal-to-noise ratio of the electrical response, as current is directly related to the sensing electrode surface area. To overcome this issue, nanostructured electrodes with small geometric footprints have been fabricated to achieve a higher surface area than their planar counterparts. Wearable sensors in particular should be biocompatible and withstand physiological strains due to motion and deformation of human skin, which is approximately 30%.
Stretchable gold electrodes have been evaluated as glucose detection platforms. It was previously demonstrated that the stability at strains up to 230% with a solution-processed wrinkled gold glucose sensor, with a limit of detection (LOD) of 1×10−3 M in artificial sweat. Additionally, it was reported that gold nanowire-based electrodes functionalized with glucose oxidase that were stretched to 20% strain to detect glucose concentrations of 1×10−5 M in a solution of NaOH, while it was demonstrated that a gold-fiber based sensor functionalized with glucose oxidase capable of detecting 6×10−6 M glucose in phosphate-buffered solution (PBS) under strains up to 200%. It is critical to have low detection limits and high sensitivity due to a lower concentration of glucose in sweat in comparison with other biological fluids. In this work, a highly stretchable electrode is introduced with the lowest limit of detection to the best of our knowledge for flexible enzyme-free glucose sensing at physiological pH.
It is an objective of the present invention to provide electrodes and fabrication methods for said electrodes that allow for electrochemical, impedimetric, capacitive, and colorimetric detection of biomarkers in body fluids, as specified in the independent claims. Embodiments of the invention are given in the dependent claims. Embodiments of the present invention can be freely combined with each other if they are not mutually exclusive.
In some aspects, a gold thin-film electrode is fabricated using a thermoplastic shape memory polymer to create hierarchical wrinkled structures via a miniaturization process, followed by transfer onto a soft, wearable substrate. In some embodiments, the original electrochemically active surface area (EASA) of the unshrunk electrode is retained in the final flexible electrode, thereby allowing for more reaction sites than on a planar electrode of the same geometric area.
In some embodiments, the flexible electrode sensitively detects glucose without enzymes or additional labels at physiological pH in the range of 1×10−7-1×10−4M with a calculated limit of detection (LOD) of 2.22×108−8M, the lowest ever reported for an enzyme-free sensor on a stretchable substrate. This concentration range is relevant for glucose detection in saliva, sweat, or tears. Diabetic patients seeking to monitor their blood glucose can do so without having to draw blood, and without enzymes, the sensor can have a longer shelf life and be commercialized for in-home use.
The preliminary data for glucose detection indicate that other biomarkers can similarly be detected in very low concentrations, which is important for detecting relevant concentration changes in body fluids in response to stress stimuli, disease progression, and response to medication, among other diagnostic applications.
Although biosensors detect analytes portably and noninvasively, they face the challenge of maintaining sensitivity upon miniaturization. Various materials and processes have been developed to create nanostructured electrodes with high surface areas to mitigate this issue; however, the need remains for biocompatible materials that can be integrated into wearable or point-of-care (POC) devices.
The present invention features methods for a stretchable wrinkled electrode and the fabrication and use of the electrode for electrochemical detection of biomarkers in body fluids, to be used in portable/wearable point-of-care health monitoring devices.
One of the unique and inventive technical features of the present invention is the transfer of a shrunken wrinkled metallic film to a flexible substrate through the use of an oxygen plasma treatment. Without wishing to limit the invention to any theory or mechanism, it is believed that the technical feature of the present invention advantageously provides for even additional electroactive surface area (EASA) of the electrode, allowing for improved signal detection. None of the presently known prior references or work has the unique inventive technical feature of the present invention.
Furthermore, the prior references teach away from the present invention. For example, prior systems teach the transfer of a wrinkled electrode to a substrate through the use of a super wetting polymer in place of an oxygen plasma treatment of the metallic film. Thus, the prior art teaches away from the inventive feature of the present invention. Surprisingly, the oxygen plasma treatment provides better surface wetting while retaining almost all of the electrochemically active surface area of the non-shrunken metallic film.
Any feature or combination of features described herein are included within the scope of the present invention provided that the features included in any such combination are not mutually inconsistent as will be apparent from the context, this specification, and the knowledge of one of ordinary skill in the art. Additional advantages and aspects of the present invention are apparent in the following detailed description and claims.
The features and advantages of the present invention will become apparent from a consideration of the following detailed description presented in connection with the accompanying drawings in which:
Following is a list of elements corresponding to a particular element referred to herein:
The electrode of the present invention is stretchable and unobtrusive, making it an excellent candidate for wearable health monitoring. The geometric area of the flexible electrode is approximately 30 times smaller than that of the unshrunk electrode. Remarkably, a further enhancement to signal is achieved upon stretching the electrode to 210% of its original length then relaxing it overnight. Stretching the electrode also aids diffusion limited reactions by “unraveling” some of the wrinkles, allowing diffusion to all of the electrochemically active surface area (EASA) and producing a higher signal than its unstretched counterpart.
The electrode has the lowest limit of detection (LOD) of glucose of any reported non-enzymatic, stretchable sensor thus far, which is crucial to detecting small changes in low concentrations of glucose. This suggests that similarly low detection limits can be achieved for other biomarkers, like ethanol, lactic acid, ions (Na+, K+, Cl—), and cortisol. Additionally, the lack of enzymes ensures no degradation of sensor components and promotes scalable manufacturing for commercialization.
Referring now to
In some embodiments, body fluids may refer to but are not limited to, saliva, sweat, tears, Cerebrospinal fluid, serum, or urine.
As used herein “biomarkers” or “analytes” may be used interchangeably and may refer to a substance whose chemical constituents are being identified and measured. In some embodiments, the measured amount of a substance may be indicative of some phenomenon such as disease, infection, or environmental exposure.
In some embodiments, non-limiting examples of biomarkers may include but are not limited to, glucose, ethanol, lactic acid, cortisol, or ions such as but not limited to sodium, potassium, or chloride.
In some embodiments, non-limiting examples of analytes may include but are not limited to cortisol or viral proteins.
The present invention may feature a method for producing stretchable wrinkled electrodes (200) for electrochemical, impedimetric, capacitive, and colorimetric sensing. In some embodiments, the method may comprise mounting a polymer layer (20) onto a conductive substrate (10). The polymer layer (20) may then be coated with a sacrificial layer (30). After allowing the sacrificial layer (30) to dry, an electrode shape template (60) may be cut out of an adhesive and applied on top of the sacrificial layer (30). The electrode shape template (60) may act as a stencil for the deposition of a metallic film (40). In some embodiments, the method may further comprise depositing the metallic film (40) on top of the sacrificial layer (30) by the electrode shape template (60). After depositing the metallic film (40), the electrode shape tem on the sacrificial layer (30). The polymer layer (20), sacrificial layer (30), and metallic film (40) may then be removed from the conductive substrate (10) and placed in an oven to create a shrunken polymer layer (120), a shrunken sacrificial layer (130) and a shrunken metallic film (140). In further embodiments, the method comprises treating the shrunken metallic film (140) with a first solution. In some embodiments, treating the shrunken metallic film (140) promotes bonding between the shrunken metallic film (140) and an elastomer (150). The elastomer (150) may then be drop-casted onto the shrunken metallic film (140) such that the shrunken metallic film (140) bonds to the elastomer (150). The shrunken sacrificial layer (130) between the shrunken metallic film (140) and the shrunken polymer layer (120) may be dissolved using a second solution, such that the shrunken metallic film (140) detaches from the shrunken polymer layer (120). In some embodiments, the method comprises placing the shrunken metallic film (140) and the elastomer (150) in a chemical bath and drying the shrunken metallic film (140) and the elastomer (150) to produce the stretchable wrinkled electrode (200).
The present invention may also feature a method for producing stretchable wrinkled electrodes (200) for electrochemical, impedimetric, capacitive, and colorimetric sensing. In some embodiments, the method may comprise mounting a polymer layer (20) onto a conductive substrate (10). The polymer layer (20) may then be coated with a sacrificial layer (30). In some embodiments, the method comprises applying the electrode shape template (60) on top of the sacrificial layer (30) and depositing a metallic film (40) on top of the sacrificial layer (30) by the electrode shape template (60). In further embodiments, the method comprises removing the polymer layer (20), sacrificial layer (30), and metallic film (40) from the conductive substrate (10). In some embodiments, the method comprises shrinking the polymer layer (20), sacrificial layer (30), and metallic film (40) to create a shrunken polymer layer (120), a shrunken sacrificial layer (130), and a shrunken metallic film (140), treating the shrunken metallic film (140) with a first solution, wherein the first solution (140) promotes bonding between the shrunken metallic film (140) and an elastomer (150), and dissolving the shrunken sacrificial layer (130) between the shrunken metallic film (140) and the shrunken polymer layer (120) using a second solution. In some embodiments, the method further comprises producing the stretchable wrinkled electrode (200) comprising a shrunken metallic film (140) and the elastomer (150).
In some embodiments, the polymer layer (20) or the shrunken polymer layer (120) may comprise polyolefin (PO). In some embodiments the polymer layer (20) or the shrunken polymer layer (120) may comprise polystyrene. In some embodiments the polymer layer (20) or the shrunken polymer layer (120) may comprise any thermoplastic shape-memory polymer.
In some embodiments, the conductive substrate (10) may comprise a silicon wafer. In some embodiments, the conductive substrate (10) is glass. In other embodiments, the conductive substrate (10) is not conductive. In some embodiments, the conductive substrate (10) is any rigid support.
In some embodiments, the sacrificial layer (30) or the shrunken sacrificial layer (130) may comprise poly(methyl methacrylate) (PMMA) dissolved in toluene. In other embodiments, the sacrificial layer (30) is applied to the polymer layer (20) via spin coating. In some embodiment, an adhesive may comprise frisket film.
In some embodiments, the metallic film (40) or the shrunken metallic film (140) may comprise gold. In some embodiments, the metallic film (40) or the shrunken metallic film (140) may comprise platinum. In some embodiments, the metallic film (40) or the shrunken metallic film (140) may comprise silver. In some embodiments, the metallic film (40) or the shrunken metallic film (140) may comprise copper. In some embodiments, the metallic film (40) or the shrunken metallic film (140) may comprise carbon. In some embodiments, the metallic film (40) or the shrunken metallic film (140) may comprise any conductive material. In other embodiments, the metallic film may be deposited onto the sacrificial layer (30) via sputtering.
In some embodiments, the electrode (200) is placed in a convection oven. In other embodiments, the electrode (200) is placed in a conventional oven. In preferred embodiments, the electrode (200) is placed in an oven at 140° C. In other embodiments, the electrode (200) is placed in an oven ranging in temperature from about 125° C. to 145° C. In preferred embodiments, the electrode (200) is placed in an oven for 13 minutes. In other embodiments, the electrode (200) is placed in an oven for about 10 to 15 minutes.
In some embodiments, the first solution comprises silane. In other embodiments, the first solution promotes bonding. In other embodiments the second solution comprises acetone. In further embodiments, the second solution may cause the shrunken metallic film (140) detaches from the shrunken polymer layer (120). In some embodiments, the chemical bath comprises isopropanol. In other embodiments, the chemical bath comprises toluene.
As used herein, “elastomer” may refer to a polymer with viscoelasticity and has very weak intermolecular forces, generally low Young's modulus and high failure strain compared with other materials. In some embodiments, the elastomer (150) is silicon-based. In some embodiments, the elastomer (150) is any material which fits the aforementioned definition of an elastomer.
In some embodiments, the stretchable wrinkled electrode (200) has an average geometrical area of 0.2 cm2 to 0.4 cm2. In some embodiments, the electrode (200) has an area of about 0.1 cm2 to 0.15 cm2. In some embodiments, the electrode (200) has an area of about 0.2 cm2 to 0.25 cm2. In some embodiments, the electrode (200) has an area of about 0.3 cm2 to 0.35 cm2. In some embodiments, the electrode (200) has an area of about 0.4 cm2 to 0.45 cm2. In some embodiments, the electrode (200) has an area of about 0.5 cm2 to 0.55 cm2. In some embodiments, the electrode (200) has an area of 0.6 cm2. In some embodiments, the electrode (200) has an area of about 0.1 cm2, or about 0.15 cm2, or about 0.2 cm2, or about 0.25 cm2, or about 0.3 cm2, or about 0.35 cm2, or about 0.4 cm2, or about 0.45 cm2, or about 0.5 cm2, or about 0.55 cm2, or about 6.0 cm2. In other embodiments, the electrode has an area greater than 6.0 cm2. In some embodiments, the final size of the stretchable wrinkled electrode (200) depends on the initial working area of the electrode (i.e., before the electrode is shrunk). In other embodiments, the amount the initial electrode (i.e., before the electrode is shrunk) shrinks depends on the type of polymer and/or the amount of time spent shrinking.
In some embodiments, the stretchable wrinkled electrode comprises hierarchical, wrinkled structures. In some embodiments, a “hierarchical wrinkled structure” may refer to a larger wrinkle on the electrode having wrinkles (
Referring to
In some embodiments, the elastomer (150) may be silicon-based. In some embodiments, the metallic film (40) and the shrunken metallic film (140) may comprise gold. Non-limiting examples of materials that the metallic film (40) or the shrunken metallic film (140) may comprise may include but are not limited to platinum, silver, copper, carbon, or any other conductive material.
In some embodiments, the polymer layer (20) and the shrunken polymer layer (120) may comprise polyolefin. In some embodiments the polymer layer (20) or the shrunken polymer layer (120) may comprise polystyrene. In some embodiments the polymer layer (20) or the shrunken polymer layer (120) may comprise any thermoplastic shape-memory polymer.
In some embodiments, the sacrificial layer (30) and the shrunken sacrificial layer (130) may comprise poly(methyl methacrylate) (PMMA) dissolved in toluene. In other embodiments, the shrunken sacrificial layer (130) is dissolved using a second solution comprising acetone
In some embodiments, the first solution may comprise a silane. In other embodiments, the first solution promotes bonding. In other embodiments, the second solution may comprise acetone. In some embodiments, the chemical bath may comprise isopropanol or toluene.
An exemplary embodiment of the present invention features a method for fabricating a wrinkled electrode, as follows. PO is mounted onto a silicon wafer to be coated with poly(methyl methacrylate) (PMMA) (Sigma Aldrich) dissolved in toluene (22.5 mg mL-1) to comprise the sacrificial layer. The surface tension of the solvent in which the PMMA is dissolved is taken into consideration to achieve good wetting with the surface energy of the PO, which is in the range of 30 dynes cm2. The surface energy for polyolefins is relatively low, and favors wetting by the lower-surface energy solution. Toluene (28.40 dyne cm−1 at 20° C.) has a lower surface tension than the previously used solvent, anisole (35.00 dyne cm−1 at 20° C.). The PMMA is spin coated at 3200 rpm to ensure uniform spreading and reduce the thickness of the sacrificial layer and subsequently the time necessary to dissolve it. After drying the PMMA film overnight, masks for the electrode design are laser-cut from adhesive and applied to the films as stencils for gold deposition. The initial working area of the electrode was 10.5 cm2. An 80 nm gold film is deposited on top of the PMMA-coated PO via sputtering (Quorum 150R). The PO-PMMA-Au is then removed from the silicon wafer substrate and prepared for thermal treatment. The films are shrunk in a convection oven at 140° C. for 13 min. The average geometrical area of shrunk electrode is 0.48 cm2.
An exemplary embodiment of the present invention features a method for transferring a wrinkled electrode to an elastomer, as follows. The wrinkled gold film is treated with a silane solution to promote bonding to the flexible silicon-based elastomer prior to the liftoff procedure. The elastomer is drop casted onto the silane-treated gold film and allowed to cure overnight at 60° C. The wrinkled films are transferred to the Ecoflex by dissolving the PMMA between the gold and polyolefin in acetone. Once the films are fully transferred, they are placed in an isopropanol bath for an hour and dried overnight. The average geometrical area of the transferred, working electrode is 0.31 cm2. SEM images of samples prepared with sputtered iridium (4 nm) are taken using the GAIA Tescan system (5 kV, 0.20 pA). The average geometric area of the shrunk and transferred electrodes is 0.48 and 0.31 cm2, respectively. Samples are prepared for SEM imaging with sputtered iridium (6 nm) and are taken using the GAIA Tescan system (8 kV, 0.20 pA).
In some embodiments, overnight may refer to a timeframe between 4 and 18 hours.
An exemplary embodiment of the present invention is tested using electrochemical characterization, as follows. Electrochemical characterization is performed on a Reference 600 potentiostat (Gamry Instruments) with the unshrunk, shrunk and transferred electrodes as working electrodes, platinum (Pt) wire as the counter electrode and silver/silver chloride (Ag/AgCl) as the reference electrode. Cyclic voltammetry is firstly performed in a H2SO4 0.5 M solution from 0 to 1.5 V at 10, 25, 50, 100, 250, 500 and 1000 mV s-1. The process is repeated in 0.005 M [Fe(CN)6]3-/4- solution in 0.05 M PBS solution at pH 7.4 from −0.2 to 0.7 V.
An exemplary embodiment of the present invention is tested using electrochemical characterization on transferred electrodes under strain, as follows. A stretching system was developed to maintain the flexible electrodes under strain while an electrochemical measurement was performed. Cyclic voltammetry is performed at every 30% strain of electrode length in H2SO4 and [Fe(CN)6]3-/4- solutions until it reaches 210% of its original length. The electrode is allowed to relax overnight before repeating the stretching procedure.
An exemplary embodiment of the present invention is tested by calculating surface area by charge associated with gold oxide reductions, as follows. The electroactive surface area (EASA) of the nanostructured electrode is evaluated by gas sorption and by several electrochemical measurements, such as underpotential deposition of metal, capacitance measurement by electrochemical impedance spectroscopy (EIS) or cyclic voltammetry, and, most commonly used cyclic voltammetry in H2SO4. The latter technique is widely applied to characterize nanostructured gold surfaces because it can be easily performed and provides a good estimate of the surface area. The surface area estimated by gold oxide reduction was reported to agree with the areas measured by underpotential deposition (UPD) and nitrogen adsorption/desorption isotherms. In fact, it was previously demonstrated that the gold oxide reduction in H2SO4 was proven as the most accurate and reliable method to determine the real area when compared to several other methods such as assessment of monolayer (copper, 6(ferrocenyl)hexanethiol and cytochrome c), redox reactions ([Fe(CN)6]3-/4-, [Ru(NH3)6]3-/4- and O2) and double layer capacitance. The measurement of gold electrode by cyclic voltammetry in H2SO4 consists in the formation and successive reduction of gold oxide and the charge associated with this reaction at a relatively slow scan rate is directly proportional to the surface area by the following relation:
EASA=Q/c
where Q is charge which is calculated by integrating the area under the reduction peak and c is the specific charge equivalent, which equals to 390 ρC cm−2 for polycrystalline gold.
An exemplary embodiment of the present invention is tested by calculating the dependency of signal enhancement with scan rate, as follows. Diffusion-limited redox reactions, such as for the redox par [Fe(CN)6]-3/-4, is affected by the scan rate of the cyclic voltammetry. At fast scan rates, the redox species are forced to move more rapidly, which results in the decrease of the diffusion layer. Consequently, higher currents are observed since more redox species are able to access the surface area. The dependency of the Faradaic current from diffusion-limited reactions on scan rate is described by the Randles-Sevcik equation:
where n is the number of electrons involved in the electrochemical reaction, F is the Faraday constant, C0 is the concentration of the redox species, v is the scan rate, D0 is the diffusion coefficient of the oxidized analyte, R is the gas constant, and T is the temperature. As shown in
An exemplary embodiment of the present invention is tested by calculating the sheet resistance of transferred electrodes before and after strain, as follows. A Signatone Pro4 Resistivity Test System with an SP4 inline probe is used to measure the sheet resistance of transferred electrodes before, during and after strain. The sheet resistances are first measured on the unstrained electrodes. The same electrodes are stretched up to 200% of the length and the sheet resistance is measured again. The preconditioning step is applied to the electrode, which consisted of straining the sensors up to 200% 10 times. After 5 minutes of relaxation, the sheet resistance of the electrodes is once again measured.
An exemplary embodiment of the present invention features glucose detection with a stretchable electrode, as follows. The glucose oxidation potential was measured in a 5×10−3 M solution of glucose in PBS (pH 7) by cyclic voltammetry. Chronoamperometry is performed at 0.3 V and glucose was titrated into 20 mL of PBS (pH 7.4) every 30 s. The titration is performed for non-strained electrodes (n=3) and pre-strained electrodes (n=3). An analytical calibration curve is plotted using current values corresponding to sequential additions of glucose. The sensitivity is obtained from the slope and the limit of detection was calculated according to IUPAC
In some embodiments, the present invention features a method for a three-electrode electrochemical mini-cell fabrication. In some embodiments, the method comprises sputtering gold onto polystyrene using a sputtering machine (e.g., QuorumDesign Q150R Plus) and shrinking the gold-sputtered polystyrene in a conventional oven at 130 C. In some embodiments, the method comprises securing the shrunken gold sputtered polystyrene on a laser cutter (e.g., Universal Laser System Ultra R5000) stage and adjusting the power setting to remove all gold from the shrunken gold-sputtered polystyrene sample surface except that which will form the final electrode design. In other embodiments, the method comprises removing the shrunken gold-sputtered polystyrene sample for the laser cutter stage and sonicating in a bath of isopropanol for 5 minutes to remove residues from the surface of the electrodes. In some embodiments, the method results in a shrunken polystyrene substrate with wrinkled gold electrodes in the desired formation. In some embodiments, the three-electrode electrochemical mini-cell is finished when the Ag/AgCl ink is applied to create the third and final electrode.
The present method features a method for producing wrinkled electrodes (200). In some embodiments, the method comprises depositing a metallic film (40) on top of a polymer layer (20) and shrinking the polymer layer (20) and the metallic film (40) to create a shrunken polymer layer (120) and a shrunken metallic film (140). The method further comprises adding Ag/AgCl ink to the electrode. The method further comprises mounting the electrode to an elastomer. In some embodiments the electrodes are functionalized.
As used herein, a “mini cell” may refer to an electrode that has all three critical components of a 3-electrode electrochemical cell (see
In some embodiments, the mini cell comprises one or more working electrodes sequentially measured against a reference electrode and a counter electrode. In other embodiments, multiple mini cells each comprising at least one reference electrode, at least one counter electrode and at least one working electrode may be put together to form an array.
In some embodiments, the counter electrode and/or working electrode may be comprised of a wrinkled gold (Au) electrode, or a wrinkled platinum (Pt) electrode, or an electrode made from other suitable materials. In some embodiments, the reference electrode may comprise silver/silver chloride (Ag/AgCl).
As used herein, a “reference electrode” refers to an electrode which has a stable and well-known electrode potential. As used herein, a “counter electrode” refers to an electrode used in two or three electrode systems to complete the circuit so that electrochemical measurements can be performed. As used herein, a “working electrode” refers to the electrode in an electrochemical system on which the reaction of interest is occurring.
In some embodiments, the mini cell may comprise one working electrode. In some embodiments, the mini cell may comprise working two electrodes. In some embodiments, the mini cell may comprise three working electrodes. In some embodiments, the mini cell may comprise four working electrodes. In some embodiments, the mini cell may comprise five working electrodes. In some embodiments, the mini cell may comprise six working electrodes. In some embodiments, the mini cell may comprise seven electrodes. In some embodiments, the mini cell may comprise eight working electrodes. In some embodiments, the mini cell may comprise nine working electrodes. In some embodiments, the mini cell may comprise working ten electrodes. In some embodiments, the mini cell may comprise greater than ten working electrodes.
In some embodiments, the resulting electrode may be flexible. In other embodiments, the resulting electrode may be non-flexible. In some embodiments, the resulting electrode may be functionalized with materials. Non limiting examples of functionalized materials may include but are not limited to aptamers, antibodies, fragments of antibodies, oligonucleotides, or hydrogels containing functionalized material (such as enzymes). In other embodiments, the resulting electrodes may not be functionalized with materials.
Without wishing to limit the present invention to any theory or mechanisms it is thought that stretching the wrinkled surface reveals “hidden” surface area or wrinkled pockets.
The present invention may also feature a method of detecting severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). In some embodiments, the method comprises obtaining a biological sample from a subject and placing the biological sample on a mini cell. In some embodiments, the mini cell comprises at least one reference electrode (Ag/AgCl), at least one counter electrode (i.e., wrinkled Au) and at least one working electrode (i.e., wrinkled Au). In some embodiments, the working electrode is functionalized with methylene blue (MB)-modified aptamers to detect SARS-CoV-2. In some embodiments, the method comprises detecting square wave voltammograms. In some embodiments, the detection of SARS-CoV-2 causes a decrease in the square wave voltammogram as compared to a reference sample with no SARS-CoV-2.
In some embodiments, the mini cell is connected via electrical leads to the potentiostat, which is connected to a computer which allows for the detection of the square wave voltammograms. In some embodiments, the biological sample or reference sample is incubated on the mini cell for about 1 hour. In some embodiments, the biological sample or reference sample is incubated on the mini cell for about 30 minutes. In some embodiments, the biological sample or reference sample is incubated on the mini cell for about 10 minutes. In some embodiments, the biological sample or reference sample is incubated on the mini cell for over 1 hour.
The following is a non-limiting example of the present invention. It is to be understood that said example is not intended to limit the present invention in any way. Equivalents or substitutes are within the scope of the present invention.
Fabrication and transfer of wrinkled electrodes: Detailed protocol is described below. The average geometric area of the shrunken and transferred electrode was 0.48 and 0.31 cm2, respectively. Samples were prepared for SEM imaging with sputtered iridium (6 nm) and were taken using the GAIA Tescan system (8 kV, 0.20 pA).
Fabrication of wrinkled electrodes: PO is mounted onto a silicon wafer to be coated with poly(methyl methacrylate) (PMMA) (Sigma Aldrich) dissolved in toluene (22.5 mg mL-1) to comprise the sacrificial layer. The surface tension of the solvent in which the PMMA was dissolved was taken into consideration to achieve good wetting with the surface energy of the PO, which is in the range of 30 dynes cm2. The surface energy for polyolefins is relatively low, and favors wetting by the lower-surface energy solution. Toluene (28.40 dyne cm-1 at 20° C.) has a lower surface tension than the previously used solvent, anisole (35.00 dyne cm-1 at 20° C.). The PMMA was spin coated at 3200 rpm to ensure uniform spreading and reduce the thickness of the sacrificial layer and subsequently the time necessary to dissolve it. After drying the PMMA film overnight, masks for the electrode design were laser-cut from adhesive and applied to the films as stencils for gold deposition. The initial working area of the electrode was 10.5 cm2. An 80 nm gold film was deposited on top of the PMMA-coated PO via sputtering (Quorum 150R). The PO-PMMA-Au is then removed from the silicon wafer substrate and prepared for thermal treatment. The films were shrunk in a convection oven at 140° C. for 13 min. The average geometrical area of the shrunk electrode was 0.48 cm2.
Transfer of wrinkled electrodes to elastomer: The wrinkled gold film was treated with a silane solution to promote bonding to the flexible silicon-based elastomer prior to the liftoff procedure. The elastomer was drop cast onto the silane-treated gold film and allowed to cure overnight at 60° C. The wrinkled films were transferred to the Ecoflex by dissolving the PMMA between the gold and polyolefin in acetone. Once the films were fully transferred, they were placed in an isopropanol bath for an hour and dried overnight. The average geometrical area of the transferred, working electrode was 0.31 cm2. SEM images of samples prepared with sputtered iridium (4 nm) were taken using the GAIA Tescan system (5 kV, 0.20 pA).
Electrochemical characterization: Electrochemical characterization was performed on a Reference 600 potentiostat (Gamry Instruments) with the unshrunk, shrunk, and transferred electrodes as working electrodes, Pt wire as the counter electrode, and Ag/AgCl as the reference electrode. Cyclic voltammetry was firstly performed in a H2SO4 0.5 M solution from 0 to 1.5 Vat 10, 25, 50, 100, 250, 500 and 1000 mV s-1. The process was repeated in 0.005 M [Fe(CN)6]3-/4- solution in 0.05 M PBS solution at pH 7.4 from −0.2 to 0.7 V.
Electrochemical characterization of transferred electrodes under strain: A stretching system was developed to maintain the flexible electrodes under strain while an electrochemical measurement was performed. Cyclic voltammetry was performed at every 30% strain of electrode length in H2SO4 and [Fe(CN)6]3-/4- solutions until it reached 210% of its original length. The electrode was allowed to relax overnight before repeating the stretching procedure.
Glucose detection with stretchable electrode: The glucose oxidation potential was measured in a 5×10-3 M solution of glucose in PBS (pH 7) by cyclic voltammetry. Chronoamperometry was performed at 0.3 V and glucose was titrated into 20 mL of PBS (pH 7.4) every 30 s. The titration was performed for non-strained electrodes (n=3) and pre-strained electrodes (n=3). An analytical calibration curve was plotted using current values corresponding to sequential additions of glucose. The sensitivity was obtained from the slope and the limit of detection was calculated according to IUPAC.
The present invention is directed to stretchable wrinkled electrodes for electrochemical sensing by depositing thin gold metal thin film on polyolefin (PO), which shrinks to 5% of its original area. During the shrinking process, the stiffness mismatch between the polymer and the gold thin film leads to buckling of the gold and results in hierarchical, wrinkled structures. The shrinking factor (the ratio of the average unshrunk electrode's geometric area to that of the processed electrode) of the pre-stressed thermoplastic polyolefin (PO) is 21:8. The wrinkled thin film is then transferred to an elastomer. The transfer of the electrode from shape-memory polymer to elastomer results in additional shrinking due to the lift-off process, with a total shrinking factor of 33:4 (
Electrochemical properties of the wrinkled electrodes were characterized by measuring the electrochemical active surface area (EASA) in different solutions. The wrinkled structures contribute to the high surface area, which directly correlates with the Faradaic current. The electrodes were measured by cyclic voltammetry in H2SO4 and [Fe(CN)6]3-/4- solutions (
In H2SO4, signal enhancements (the ratio of current densities of processed electrodes to unshrunk electrodes) of 21- and 32-fold were achieved for shrunk and transferred electrodes, respectively (
Interestingly, the signal enhancements are different depending on the solution in which the measurement was performed. The signal enhancements in the [Fe(CN)6]3-/4- solution are approximately 14- and 13-fold for shrunk and transferred electrodes, respectively. The lower signal enhancement in this solution is due to the [Fe(CN)6]3-/4- reaction being a diffusion limited process with relatively fast electron transfer kinetics. As the potential is applied, the concentration of [Fe(CN)6]3-/4- at the outermost surface of the electrode approaches zero, and a concentration gradient forms in the solution. Diffusion takes place at the surface of the electrodes as the reaction proceeds; however, the diffusion of electroactive species is impeded by the morphology of the wrinkles. A significant portion of the inner surface of the gold wrinkles is therefore not accessed by the [Fe(CN)6]3-/4- ions because the time for diffusion deep into the wrinkles is not sufficient, leading to the disagreement between signal enhancement and shrinking factor. Similarly, the electrochemical behavior of nanoporous gold in high-surface-area, porous electrodes were shown to be biased against redox couples like [Fe(CN)6]3-/4-. A high dependence of signal enhancement on scan rate was more evident for the diffusion-limited reaction in [Fe(CN)6]3-/4- than for H2SO4; higher signal enhancement can be achieved at greater scan rates (
The electrochemical behavior of the transferred, stretchable electrodes with applied strain was characterized by stretching the electrodes in H2SO4 and [Fe(CN)6]3-/4- up to 210% of its length in increments of 30% (
Importantly, comparing the voltammograms of unstrained electrodes before and after stretching, a gain in signal of 16 and 13% in H2SO4 and [Fe(CN)6]3-/4- respectively was observed (
The transferred electrodes were thus evaluated for label- and enzyme-free electrochemical detection of glucose in the clinically relevant concentration range found in sweat, saliva, and tears. Glucose is a commonly studied biomarker, and as a proof of concept, the measurements were taken at physiological pH. The transferred electrode detects glucose without the use of enzymes through its oxidation mechanism at 0.3 V (
Chronoamperometry was used to measure sequential addition of glucose to PBS pH 7.2 (
The achieved LOD is lower than those reported for electrodes utilizing structures with nanoparticles of gold, platinum, or other alloy combinations with carbon, as well as for nanoporous substrates and electrodes functionalized with glucose oxidase. Table 1 (below) compares the figures of merit obtained in the present invention with other published works investigating flexible, glucose sensors. The range of detection achieved using the flexible, pre-strained electrodes is relevant for monitoring blood sugar via sweat in diabetic patients, as well as for noninvasive methods utilizing saliva, tears, or any application that requires detection of small changes in glucose concentration.
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In summary, the present invention is directed to high surface area electrodes on soft, stretchable substrates for electrochemical, impedimetric, capacitive, and colorimetric detection of nanomolar quantities of glucose at physiological pH. The method for fabricating the enhanced EASA electrodes requires neither a cleanroom, nor specialty materials or equipment. The wrinkled, transferred gold electrode shows a 32-fold increase in current density compared to a planar electrode of equal geometric size. Stretching the electrode introduces cracks to the wrinkled film and provides additional surface area. Stretching the electrode also resulted in the “unfolding” of some of the wrinkles, which facilitated electroactive species to reach areas not accessed otherwise in reactions limited by diffusion, such as in the redox reaction of [Fe(CN)6]3-/4-.
Straining the electrode therefore functions as a preconditioning step to improve the SNR and enhance the LOD by an order of magnitude over unstrained electrodes. These improvements are crucial for overcoming the challenge of detecting low concentrations of biomarkers. The pre-strained, wrinkled gold electrodes demonstrate a LOD of 2.22×10−8 M, among the lowest reported for enzyme-free glucose detection.
In conclusion, the present invention features a stretchable, electrochemical electrode that has a high surface area allowing for 32-fold enhancement of signal. Stretching the electrode as a conditioning treatment improves the quality of the signal through crack formation by mechanically stabilizing the metal thin film and its electrical characteristics, as well as further increasing the electrochemically active surface area. Furthermore, the device described herein allows for the ability to detect biomarkers like glucose at concentrations as low as 20 nM in physiological solutions.
The following is a non-limiting example of the present invention. It is to be understood that said example is not intended to limit the present invention in any way. Equivalents or substitutes are within the scope of the present invention.
It has been demonstrated that signal enhancement in the form of greater dynamic range of detection on electrodes made using the '80's children's toy, Shrinky-Dink®, which saturates the color of a drawing made on a thermoplastic polystyrene sheet that shrinks when heated (
To determine the functionality and affinity of the aptamers in the aptamer-based electrochemical (E-AB) format, all initial experiments were first performed on commercial disc (CD) Au electrodes (CH Instruments, 2 mm diameter) in a standard 3-electrode electrochemical cell (
The change in peak current correlates with increasing concentrations of S1 protein, as shown in
The signal change in response to addition of S1 protein on CD electrodes confirmed the viability of the chosen aptamer probes; however, using CD electrodes for screening and diagnostic applications is not feasible. CD electrodes cost $90 each, and given the contagious nature of the disease, reusing them presents significant practical challenges. For a truly deployable and scalable approach, the entire sensor must be disposable. With such considerations of cost of production and scaling in mind, electrodes fabricated with a simple sputtering deposition process on a commercial polystyrene substrate were investigated. Before the shrinking process, the adhesion of sputtered gold on polystyrene is too weak and the gold thin film can delaminate. When shrunk, however, the resulting Shrinky-Dink wrinkled (SDW) electrodes are extremely robust and retain their original surface area, resulting in enhanced current density and dynamic range. Because of the advantages in cost and performance, experiments were initiated on SDW gold electrodes (
The total accessible electrochemically active surface area (EASA) of SDW electrodes was confirmed to be greater than that of commercial disc (CD) electrodes, as calculated by integration of the reduction peak of gold oxide in sulfuric acid for the respective electrode types. The electrodes were of equivalent geometric areas. The wrinkles provide a diffusion-based challenge to surface coverage, as previously described. With longer incubation time, however, this limitation was overcome. The outcome was a greater number of moles of aptamers tethered to the surface of SDW electrodes relative to the same geometric area of CD electrodes. The increased aptamer density resulted in greater absolute MB peak heights on SDW electrodes.
To determine whether the introduction of S1 protein to saliva would produce a differentiable signal change relative to untreated saliva, titration curves were built by measuring square wave voltammograms at 10 Hz with SDW electrodes exposed to varying concentrations of S1 protein. The change in signal with increasing concentrations of S1 protein is shown in
As evidenced from
To improve the portability of the sensor, next an entire miniaturized electrochemical cell was fabricated, abbreviated as “mini cell”, using the same shrinking process used for individual SDW electrodes. The SDW mini cells' working and counter electrodes were created by sputtering gold onto the pre-stressed polymer substrate and heated to induce shrinking. Ag/AgCl ink was applied as a reference electrode on the shrunk substrate; thus, all three electrodes were located on one substrate, allowing electrochemical measurements within a 250 μL droplet instead of the 20 mL used in the beaker cell. The performance of the SDW mini cell was evaluated by characterizing the gold oxidation and reduction in sulfuric acid. Compared to measurements collected using the commercial reference and counter electrodes, the SDW mini cell demonstrated similar performance, with slightly increased peak-to-peak separation (ΔEp) between oxidation and reduction peaks.
It was observed that during the cleaning protocol performed in sulfuric acid, the area under the peak of the SDW mini cell increased more than that of individual SDW electrodes (
The signal gain of 40% at the highest concentration of spike protein was greater in the SDW mini cells than in the individual SDW electrodes, in which the maximum change was approximately 16% (
The SDW mini cell demonstrated detection of as little as 0.1 fg mL-1 S1 protein in 10% saliva, with the full comparison to system noise. In comparison, a recent study utilizing the same aptamer to target the RBD region of the spike protein achieved a LOD six orders of magnitude greater in buffer solution than the lowest concentration presented here. Additionally, the low-complexity process does not involve the use of costly materials and does not rely on enzymatic reactions or biotinylation, which is known to cause interference with clinical samples
SDW Electrode Fabrication: The electrode design was created by applying an adhesive polymer (Grafix Frisket Film, Grafix Arts, OH) shadow mask stencil to the polystyrene (Grafix Shrink Film KSF50-C, Grafix Arts, OH) prior to sputtering. A Q150R Plus—Rotary Pumped Coater was used to sputter 60 nm of gold onto the pre-stressed polystyrene to create the working electrode. The substrate was de-masked and placed into a conventional oven at 130° C. for 13 minutes until fully shrunk. Following this step, the electrode was treated with oxygen plasma for 3 minutes to achieve temporary hydrophilicity to ensure full surface wettability during the following cleaning step.
SDW mini cell Fabrication: Electrodes were created by applying an adhesive mask to the polystyrene prior to sputtering. Following the same protocol as for individual SDW electrodes, 60 nm of gold was sputtered onto the pre-stressed polystyrene to create the working and counter electrode. The substrate was then de-masked and placed into a conventional oven at 130° C. for 13 minutes until fully shrunk. Following this step, the reference electrode was painted onto the shrunk polystyrene using commercially available Ag/AgCl ink (Creative Materials). Working and counter electrodes were treated with oxygen plasma for 3 minutes to achieve temporary hydrophilicity, while the Ag/AgCl electrode was covered to prevent oxidation.
Cleaning electrodes: Commercial electrodes were polished using the 0.5 μm-sized Ag particles included in the kit provided with the electrodes prior to cycling in sulfuric acid. The electrodes were then individually immersed in a 0.5 M solution of H2SO4 and subjected to 120 cycles at 1000 mV s-1 followed by five cycles at 100 mV s-1 in the potential window of 0-1.5 V. Individual SDW electrodes were similarly immersed in a 0.5 M solution of H2SO4 and subjected to 120 cycles at 1000 mV s-1 followed by five cycles at 100 mV s-1 in the potential window of 0-1.5 V. SDW mini cell electrodes were cleaned by drop-casting a 250 μL drop of 0.5 M solution of H2SO4 onto the surface, ensuring coverage of all three electrodes' working areas, and subjected to 120 cycles at 1000 mV s-1 followed by five cycles at 100 mV s-1 in the potential window of 0-1.5 V.
Aptamer preparation: Aptamers were received in liquid form and diluted to a concentration of 100 μM. For each experiment, a small volume of aptamer solution (100 μM) was combined with a reducing buffer (Basepair Technologies) at a 1:2 volume ratio for one hour to reduce the 3′ ends of the aptamers. Following the reduction step, the solution was diluted with a phosphate buffer solution to a final concentration of 1 μM. 1 μM is an excessive concentration to use, but additional studies must be carried out to determine optimal concentration of probes for incubation. Optimal probe density must also be investigated in order to confirm optimal sensor sensitivity. Probe density affects the magnitude of signal change and is assumed to be a function not only of initial concentration of applied aptamers, but also of incubation time and temperature.56 In the case of oligonucleotides greater than 24 bases, such as in this investigation, the probe density is greatly affected by incubation time. From the SDW mini cell experiments, it was evident that a lesser probe density contributed to enhanced signal gain.
Functionalization of Electrodes with aptamers: A 30 μL of the aptamer solution at 1 μM was applied to each electrode and allowed to incubate 4 and 8 hours, respectively, for CD and SDW electrodes, at room temperature. The aptamer incubation time was optimized for SDW electrodes; from previous investigations, it is clear that the wrinkled morphology presents diffusion limitations for molecular access to the surface. Thus, the incubation time of aptamers was increased to ensure adequate surface coverage by the aptamer probes. During this time, the aptamers formed thiol bonds with the gold surface. The presence of the characteristic MB peak at −0.28 V confirmed the attachment of the aptamer to the gold surface. The peak height collected from each type of electrode (CD, SDW individual and mini cell) was normalized to their respective EASA for comparison of current density. The current density was assumed to correlate with the density of MB and the aptamer probes to which it is attached on the electrode surface. The results of a two-sample t-test show a rejection of the null hypothesis at the 5% significance level that the current densities of CD electrodes and individual SDW electrodes have an equal mean, indicating that the individual SDW electrodes may have a greater probe density. We believe this may be attributed to the longer incubation time chosen for SDW electrodes to compensate for diffusion limitations posed by wrinkles. In contrast, due to the greater EASA of the SDW mini cell working electrodes, the probe density was found to be lesser than that of individual SDW electrodes.
Blocking of electrodes with MCH: Blocking the electrode surface avoids nonspecific binding with interfering species in real samples, enhances the stability of aptamers, and passivates any remaining EASA. A six-carbon blocking molecule, MCH, was chosen as the blocking agent because it has been shown to create more stable monolayers than a 2- or 3-carbon molecule, although at the expense of greater conductivity of the SAM achieved using shorter chains. After the incubation with the aptamers, the electrodes were rinsed and incubated with 30 μL of 3 mM MCH in phosphate buffer for 18 and 39 hours, respectively, for CD and SDW electrodes (both individual and mini cell), at 4° C.
Example 3: Using a saliva sample to detect the 51 protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The following example describes a detection strategy for the 51 protein of the SARS-CoV-2 using a saliva sample
A 35-year-old man wakes up one morning with a fever of 102.4° F. and tightness in his chest. He is an essential hospital worker, and so believes he may have contracted COVID-19. Therefore, he calls his primary care physician to determine his next steps. The primary care physician decides that he should be brought in for testing since he is at a higher risk for complications because the patient has severe asthma. The man provides a saliva sample and it is placed on a mini cell. After about an hour incubating the mini cell detects the presence of the 51 protein. The man is told to quarantine for two weeks before returning to work.
As used herein, the term “about” refers to plus or minus 10% of the referenced number.
Although there has been shown and described the preferred embodiment of the present invention, it will be readily apparent to those skilled in the art that modifications may be made thereto which do not exceed the scope of the appended claims. Therefore, the scope of the invention is only to be limited by the following claims. Reference numbers recited in the below claims are solely for ease of examination of this patent application, are exemplary, and are not intended in any way to limit the scope of the claims to the particular features having the corresponding reference numbers in the drawings. In some embodiments, the figures presented in this patent application are drawn to scale, including the angles, ratios of dimensions, etc. In some embodiments, the figures are representative only and the claims are not limited by the dimensions of the figures. In some embodiments, descriptions of the inventions described herein using the phrase “comprising” includes embodiments that could be described as “consisting essentially of” or “consisting of”, and as such the written description requirement for claiming one or more embodiments of the present invention using the phrase “consisting essentially of” or “consisting of” is met.
This application claims benefit of U.S. Provisional Application No. 62/958,092 filed Jan. 7, 2020, and U.S. Provisional Application No. 62/991,956 filed Mar. 19, 2020, the specification(s) of which is/are incorporated herein in their entirety by reference.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US21/12503 | 1/7/2021 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 62991956 | Mar 2020 | US | |
| 62958092 | Jan 2020 | US |
