Patent Application
20220341901
The present invention relates to the use of aptamers as recognition elements in the development of electrochemical sensors.
Aptamers are nucleic acid or peptide molecules that bind to a target molecule with high specificity. After selection and enrichment, aptamers possess similar affinities to antibody-antigen pairs, but have the advantage of being able to be synthesized using standard methods. As synthetic molecules, aptamers also have unique advantages in the control of their size and their amenability for chemical modification, and as such have been widely developed and applied in the development of sensors. Electrochemical, aptamer-based (E-AB) sensors have emerged in recent years as a platform to detect proteins, small molecules, and inorganic ions, relying on the induced conformational change of oligonucleotide aptamers in the presence of specific analyte. When a target molecule binds to an aptamer, which is tethered to the electrode surface, changes in the aptamer structure are measured by changes in the electrochemical signal of an attached redox label on the aptamer. E-AB sensor response is typically interrogated using a voltammetric technique such as cyclic voltammetry (CV), alternating current voltammetry (ACV), square wave voltammetry (SWV) or chronoamperometry. However, these techniques require additional equipment and are typically performed in a lab. E-AB sensors would be more useful if they had a simple indicator to show a user that a target material has been detected without the need for additional lab equipment.
The present invention meets that need by providing a simple and reversible colorimetric sensor that is easy to use and produce. One embodiment of the present invention is a sensor comprising a bipolar electrode combined with a redox indicator aptamer-based sensor and an electrocatalyst. In on embodiment, the electrochemical aptamer-based (E-AB) sensor comprises a closed bipolar electrode having a first end and a second end, wherein the first end comprises an electrochromic material and the second end comprises an electrocatalyst and an oligonucleotide aptamer tethered to the second end; wherein the oligonucleotide aptamer is labelled with a redox indicator. In another embodiment, the electrochromic material is selected from the group consisting of prussian white, polymeric viologens, tungsten oxide and N,N′-bis(n-heptyl)-4,4′-bipyridylium (heptyl viologen). In one embodiment, the electrochromic material is prussian white.
In another embodiment, the redox indicator is selected from the group consisting of methylene blue, ferrocene, anthraquinone, and nile blue. In one embodiment, the redox indicator is methylene blue. In another embodiment, the electrocatalyst is selected from the group consisting of ferricyanide, hexaamineruthenium chloride and ferrocyanonide. In one embodiment, the electrocatalyst is ferricyanide. In another embodiment, the electrocatalyst is potassium ferricyanide. In another embodiment, the second end of the bipolar electrode is cathodic.
In another embodiment, a method of detecting an analyte is provided. The method comprises applying a solution containing the analyte to an electrochemical aptamer-based sensor comprising a closed bipolar electrode and detecting a color change; wherein the bipolar electrode has a first end and a second end, the first end comprising an electrochromic material and the second end comprising an electrocatalyst and an oligonucleotide aptamer tethered to the second end; wherein the oligonucleotide aptamer is labelled with a redox indicator.
In one embodiment, the method also involves applying a solution wash to the bipolar electrode after the bipolar electrode has been exposed to the analyte-containing solution. In another embodiment, the solution wash comprises sodium dodecyl sulfate.
In one embodiment, the electrochromic material is selected from the group consisting of prussian white, polymeric viologens, tungsten oxide and N,N′-bis(n-heptyl)-4,4′-bipyridylium (heptyl viologen). In one embodiment, the electrochromic material is prussian white.
In another embodiment, the redox indicator is selected from the group consisting of methylene blue, ferrocene, anthraquinone, and nile blue. In one embodiment, the redox indicator is methylene blue. In another embodiment, the electrocatalyst is selected from the group consisting of ferricyanide, hexaamineruthenium chloride and ferrocyanonide. In one embodiment, the electrocatalyst is ferricyanide. In another embodiment, the electrocatalyst is potassium ferricyanide.
The foregoing summary, as well as the following detailed description of preferred embodiments of the application, will be better understood when read in conjunction with the appended drawings.
The details of one or more embodiments of the disclosed subject matter are set forth in this document. Modifications to embodiments described in this document, and other embodiments, will be evident to those of ordinary skill in the art after a study of the information provided herein.
The present disclosure may be understood more readily by reference to the following detailed description of the embodiments taken in connection with the accompanying drawing figures, which form a part of this disclosure. It is to be understood that this application is not limited to the specific devices, methods, conditions or parameters described and/or shown herein, and that the terminology used herein is for the purpose of describing particular embodiments by way of example only and is not intended to be limiting. Also, in some embodiments, as used in the specification and including the appended claims, the singular forms “a,” “an,” and “the” include the plural, and reference to a particular numerical value includes at least that particular value, unless the context clearly dictates otherwise. Ranges may be expressed herein as from “about” or “approximately” one particular value and/or to “about” or “approximately” another particular value. When such a range is expressed, another embodiment includes from the one particular value and/or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent “about,” it will be understood that the particular value forms another embodiment.
While the following terms are believed to be well understood by one of ordinary skill in the art, definitions are set forth to facilitate explanation of the disclosed subject matter. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the disclosed subject matter belongs.
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 in some embodiments ±20%, in some embodiments ±10%, in some embodiments ±5%, in some embodiments ±1%, in some embodiments ±0.5%, and in some embodiments ±0.1% from the specified amount, as such variations are appropriate to perform the disclosed method.
As used herein, the term “analyte” means an oligonucleotide or polynucleotide having a sequence to which a particular electrode-bound oligonucleoride is designed to hybridize. It can also refer to a small molecule of the like to which a particular aptamer is designed to hybridize.
As used herein, the term “aptamer” means any polynucleotide molecule (for example, DNA or RNA molecule containing natural or synthetic nucleotides) that has the ability to bind other molecules. For example, aptamers have been selected which bind nucleic acids, proteins, small organic components and even entire organisms.
The particular use of terms “oligonucleotide” and “polynucleotide” should in no way be considered limiting. “Oligonucleotide” is used when the relevant nucleic acid molecules typically comprise less than about 100 bases. “Polynucleotide” is used when the relevant nucleic acid molecules typically comprise more than about 100 bases. Both terms are used to denote DNA, RNA, modified or synthetic DNA or RNA (including but not limited to nucleic acids comprising synthetic and naturally-occurring base analogs, dideoxy or other sugars, and thiols), and PNA or other nucleobase containing polymers. However, probes and/or targets may comprise fewer than or more than 100 bases (inclusive). Accordingly, the terms “oligonucleotide” and “polynucleotide” are used to describe particular embodiments of the invention. The terms in no way define or limit the length of the nucleic acids that may be used to practice the invention.
As used herein, the term “labelled” refers to another molecule being linked, whether covalent or otherwise intercalated to an aptamer.
It should be understood that every maximum numerical limitation given throughout this specification includes every lower numerical limitation, as if such lower numerical limitations were expressly written herein. Every minimum numerical limitation given throughout this specification will include every higher numerical limitation, as if such higher numerical limitations were expressly written herein. Every numerical range given throughout this specification will include every narrower numerical range that falls within such broader numerical range, as if such narrower numerical ranges were all expressly written herein.
In one embodiment, the present invention incorporates a redox indicator such as methylene blue (MB) in an electrochemical sensor. Further, the present invention uses an electrocatalyst, such as a solution-based redox molecule, to electrocatalytically interact with the redox indicator. In one embodiment, the reduced form of MB, leucomethylene blue (LB), is combined with potassium ferricyanide ([Fe(CN)6]3−), as shown in equation 1 and equation 2. The catalytic cycle starts with the rapid, two-electron reduction of MB to LB, which occurs when the electrode is held at a suitably negative potential (Equation 1):
MB+2e−+H+→LB (Equation 1)
Two equivalents of [Fe(CN)6]3− then rapidly oxidize LB back to MB (Equation 2):
LB+2[Fe(CN)6]3−→MB+2[Fe(CN)6]4−+H+ (Equation 2)
In the present invention, we take advantage of the catalytic cycle described above by using a redox indicator-tethered aptamer-based sensor with an electrocatalyst and combine it with a closed bipolar electrode (BPE) setup. BPEs are conductors that are polarized in an electric field within an electrolyte solution, such that one end of the electrode becomes cathodic while the opposite end becomes anodic. Such devices have applications in motion control and monitoring, biosensing, the screening of electrocatalysts, analyte separation and enrichment and electrochemical synthesis. BPEs can be classified as either a conventional open-bipolar electrode (O-BPE) system or a closed-bipolar electrode (C-BPE) system. A C-BPE system is preferred for use with the E-AB sensor platform of the present invention, since one end of the BPE needs to be aptamer-functionalized while the opposite and separate end is in a reduced state (vide infra).
The present invention involves color-changing BPE E-AB sensors, using a C-BPE system. In one embodiment, the BPEs of the present invention may be fabricated using a lithographic process with an indium tin oxide (ITO)-coated glass slide (
PW→PB+e− (Equation 3)
Using the electrocatalytic mechanism described above, these three reactions (1, 2 and 3) will support each other. The performance of the E-AB sensors can be visualized directly by the electrochromic reaction. One advantage of a colorimetric sensor is that it can be readily combined with smartphone algorithms for analysis.
For a visual example of the implementation of these mechanisms, we refer to
The present invention is a simple and reversible colorimetric sensor that combines the concentration-dependent aptamer-target binding phenomenon with a universal C-BPE, in which K3[Fe(CN)6] is used as a catalyst. Compared with the traditional E-AB sensor platform, the C-BPE architecture represents a quantitative colorimetric sensor, which is relatively simple and easy to use and produce. Using this method, sensors for ATP and tobramycin have been fabricated and used to accurately determine target molecule concentration. This phenomenon indicates that an E-AB sensor based on a C-BPE is very useful for easy-to-interpret POC devices that do not require a potentiostat for use. With further characterization and optimization of the operational and fabrication parameters, the color changing C-BPE E-AB sensor strategy of the present invention opens up a new class of color-changing, point-of-use sensors.
The redox indicator of the present invention is a reducible/oxidizable chemical moiety such as ferrocene or methylene blue. More generally, any reducible/oxidizable chemical moiety that is stable under assay conditions can be used. Examples include, but are not limited to, purely organic redox labels, such as viologen, anthraquinone, ethidium bromide, daunomycin, methylene blue, and their derivatives, organo-metallic redox labels, such as ferrocene, ruthenium, bis-pyridine, tris-pyridine, bis-imidizole, and their derivatives, and biological redox labels, such as cytochrome c, plastocyanin, and cytochrome c′. Other examples include methosulfate, p-benzoquinone, 2,6-dichlorophenolindophenol, methylene blue, potassium β-naphthoquinone-4-sulfonate, phenazine etsulfate, vitamin K, viologen, pyrroloquinoline quinone, and the like.
In one embodiment, the electrocatalyst of the present invention is ferricyanide. In another embodiment, the electrocatalyst of the present invention is hexaamineruthenium chloride, ferrocyanonide, or metal nanoparticles.
In one embodiment, the electrochromic material of the present invention can be metal oxides like tungsten oxide or silver oxide, viologens like polymeric viologens or N,N′-bis(n-heptyl)-4,4′-bipyridylium (heptyl viologen), metal coordination complexes, and metal hexacyanometallatesis. In another embodiment, the electrochromic material is prussian white.
In one embodiment, the aptamer of the present invention is an aptamer that binds to aminoglycoside antibiotics like tobramycin. In another embodiment, the aptamer can be sequences that bind to small molecules like cocaine, adenosine triphosphate, methamphetamine, polychlorinated biphenyls, kanamycin, peptides and proteins like 17β-estradiol, progesterone, neuropeptide Y, cells both mammalian and bacterial, and viral particles.
C-BPEs that incorporate an aptamer-based sensor on an Au nanoparticle-decorated substrate were fabricated and tested under a range of conditions. To obtain a colorimetric sensor with optimal performance, parameters such as applied potential, buffer composition, and aptamer concentration were varied. The sensors were then tested using a range of target molecule concentrations, using ATP and tobramycin as model systems.
Chemicals and Materials
Ethanol, hydrochloric acid (HCl), sodium chloride (NaCl), potassium chloride (KCl), magnesium chloride (MgCl2), ferric chloride (FeCl3), sodium bicarbonate (NaHCO3), potassium ferricyanide (K3[Fe(CN)6]), hydrochloric acid (HCl), 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 6-mercapto-1-hexanol (C6-OH), adenosine triphosphate (ATP), tobramycin, chlorauric acid, tris-(2-carboxyethyl) phosphine hydrochloride (TCEP), 2-amino-2-(hydroxymethyl)-1,3-propanediol (Tris base), sulfuric acid (H2SO4), 10% sodium dodecyl sulfate (SDS), polydimethylsiloxane (PDMS) and calcium chloride (CaCl2)) were used as received (Sigma-Aldrich, St. Louis, Mo.). The interrogation buffer was a Tris buffer (50 mM Tris base, 10 mM KCl, 100 mM NaCl, 50 mM MgCl2, pH=7.4). Aptamers were selected that bind specifically to ATP and tobramycin, with a thiol group on the 5′ end and with or without a MB label on the 3′ end. Three DNA aptamer sequences were used: an ATP-specific aptamer with MB-label (5′-HSC6-CTGGGGGAGTATTGCGGAGGAAA-MB-3′), the same ATP-specific aptamer without the MB-label (5′-HSC6-CTGGGGGAGTATTGCGGAGGAAA-3′) and a tobramycin-specific aptamer (5′-HSC6-GGGACTTGGTTTAGGTAATGAGTCCC-MB-3′), all purchased from Biosearch Technologies, Inc. (Novato, Calif.) and used as received. ITO-coated glass substrates (resistance: ˜6 Ω/square) were obtained from CSG Holding Co., Ltd. (Shenzhen, China). A photosensitive dry photoresist sheet (MonkeyJack 30 cm×1 m), and a transparency film for laser printers (Trulam, 4 mil thickness) were purchased from Amazon. All other chemicals were used as received (analytical reagent grade), without any further purification. All solutions were made with deionized (DI) water, purified through a water purification system (18.2 MΩ/cm, Milli-Q Advantage A10, Millipore, Billerica, Mass.).
The photosensitive dry film was employed to photolithographically fabricate patterned ITO electrodes. The photosensitive dry film was applied directly onto the clean ITO substrate. A photomask with the designed patterned template was fabricated by printing the BPE patterns using a laser printer (MFC-9330CDW, Brother), and was positioned on the coated ITO substrate. This was exposed to direct sunlight for 30 seconds, which hardened the film for the desired pattern (dry film is negative). The sample was submerged in 0.1 M NaOH to remove the exposed photoresist layer. The exposed ITO was etched by the FeCl3—HCl solution and the patterned ITO electrodes were obtained by removing the remaining photoresist with 1 M NaOH. Finally, the C-BPE system was achieved by direct bonding of a PDMS membrane (by physical contact, no adhesive) to create the two reservoirs on the patterned ITO substrate, thus separating the opposing ends of the BPE.
Au was electrodeposited on the cathodic end of the ITO bipolar electrode (3 mm diameter circle geometry,
The Au acted as a substrate to which aptamer could be tethered. 1 μL aptamer solution (200 μM) and 1 μL TCEP (10 mM) were added together, and allowed to stand for 1 hour (to reduce any 5′ disulfide bonds). The solution was diluted to 2 μM using 98 μL of buffer (20 mM Tris and 100 mM NaCl, pH=7.4). The 100 μL solution was placed on the Au nanoparticle-coated electrode of ITO for 1 hour to allow for self-assembled monolayer formation, in a covered petri dish to minimize evaporation. The surface was rinsed with DI water, followed by incubation in 2 mM C6—OH for 12 hours, to remove non-specifically adsorbed DNA and passivate the surface, in a covered petri dish.35 Probe aptamer surface density was determined by integrating the MB reduction peak in CV, at 20, 50 and 100 mV s−1 scan rates.
The electrochemical measurements were performed at room temperature using a CHI 660D (CH Instruments, Austin, Tex.) Electrochemical Workstation. Chronoamperometry was used for investigating optimal holding potentials for the PB to PW sensor initialization and the PW to PB sensor interrogation. The C-BPE was positioned between two Pt sheet electrodes (Surepure Chemetals, L.L.C., 2.5 cm×6.5 cm, 50 μm thickness), which were held at a potential to polarize the C-BPE sensor. The Pt electrode at the Au-deposited end was the working electrode and the electrode at the electrochromic end was the quasi-reference counter electrode (QRCE). Unless otherwise stated, all potentials refer to the potential applied at the Pt working electrode.
Prior to testing the C-BPE E-AB sensor device, the PB-coated anode was reduced to PW, so that it could be oxidized back to PB anodically. To ensure an even distribution, a potential of 1.2 V was applied directly to the center contact of the bipolar electrode (not the Pt working electrode) to reduce PB to PW, for at least 10 s (
Images of the C-BPEs were taken using a smartphone (iPhone 7 Plus, 12 MP camera) and semi-quantitative analysis was performed in Adobe Photoshop. Analysis involved selecting an area from the image of the PW/PB anodic end of the device (free from artifacts) and tracking the blue color intensity of these pixels over time. Color changes were determined as a percentage using:
% Color change=(η/ηfinal)×100% (Equation 4)
where η is the blue color intensity, and ηfinal is the final color of PB (fully oxidized), taken from a single reference device that had been fully converted from PW to PB. Note that a light screening box was not used, and some images contain light reflections in the droplet and artifact shadows, but the camera was in a fixed position, so the same area of the image could be monitored and compared for the effective color change over time in this proof of principle setup.
Table 1 shows buffer composition with % color change with and without 16 mM ATP.
As is common in the performance of E-AB sensors, the surface coverage of the aptamer probe on the electrode surfaces affect signaling of the resulting sensor. We employed concentrations of aptamer ranging from 0.2 μM, 1 μM, 2.0 μM and 5.4 μM, leading to a range of packing densities of aptamer on the electrode surface. Each was tested in the presence and absence of target analyte (0, 8 and 16 mM ATP). At the high target concentration (16 mM ATP) the rate of color change was rapid, such that the end point (100%) was reached in less than 3 mins for sensor surfaces prepared with 2.0 μM and 5.4 μM aptamer. The lower target concentration (8 mM ATP) showed a slower response. Sensor surfaces prepared with 2 μM aptamer during modification (for ATP-specific E-AB sensors) were determined to be optimal as surfaces prepared with 0.2 μM and 1 μM aptamer were slow to reach maximum color change, reaching just 69.7% and 64.3% after 6 minutes, respectively. The concentration of 2 μM ATP-based E-AB sensors reached the endpoint (100%) in 6 minutes with 8 mM ATP. Moreover, after 6 minutes the 5.4 μM ATP-based E-AB sensor reached a value of 84.3% color change, i.e. not faster than the sensor prepared using 2 μM ATP aptamer.
In order to optimize the performance of the C-BPE based sensor, several parameters that affect the rate of color change, and thus signal, of the indicator electrode were explored. The first parameter tested was the driving potential employed during sensing. A range of driving potentials (−0.1, −0.3, −0.8, −1.0, −1.2 and −1.4 V) were applied for 400 s (
Adding ferricyanide to the solution in combination with target analyte at the aptamer-based sensing electrode results in a higher rate of color change at the indicator electrode. To demonstrate this, a series of experiments were performed comprising tests with C-BPEs with or without K3[Fe(CN)6] and with or without target (ATP), which were used to show the electrocatalytic redox cycling effect (
To demonstrate the effect of the MB redox label on the aptamer probes, tests were conducted using sensors fabricated with the ATP-specific aptamer sequence without an appended redox label (
The magnitude and rate of color change of the C-BPE E-AB sensor is quantitatively related to concentration of target analyte and the trend is general for different aptamer-target binding partners. For example, sensors fabricated against ATP (
C-BPE E-AB sensors were also made for tobramycin and tested against different concentrations of this target molecule (
Using a simple DC power supply to polarize the C-BPE, and a smartphone camera to monitor the rate of color change of the sensor, this C-BPE E-AB colorimetric sensor can be used for the rapid detection of specific analytes in POC applications, without the need for a potentiostat and software for interpreting voltammetric data. The use of smartphones in POC colorimetric sensors has been realized for pH, protein content and glucose sensing. Moreover, this type of sensor is cheap to produce and relatively simple to make on a large scale. Furthermore, individual sensors are reusable for multiple times, with a solution wash (10% SDS) and the application of a potential to reset the sensor.
It should be noted here that there are several operational parameters that can be optimized to further improve the sensitivity and reproducibility of the described C-BPE devices. More specifically, the electrochromic film material and thickness, the surface quality of the gold substrate electrode, the size and position of the device with respect to the Pt working and quasi-reference electrodes and the electrocatalyst concentration can all affect sensor-to-sensor variability. PW was selected as the material for the electrochromic film, since the color change is reversible and repeatable. However, under no driving potential PW will convert to PB in −30 mins (c.f. 6 mins using a driving potential of −0.8 V). If this background process was reduced, or eliminated, a higher sensitivity could be achieved, and a lower limit of detection. This may be accomplished using a different electrochromic material that does not change color under the applied driving potential required for the E-AB sensor to function. The sensing electrode employs aptamers with a surface probe coverage, Γ, of 3.5±1.8×1012 molecules cm−2 (n=3). Under identical aptamer modification conditions (1 hour incubation in 2 μM aptamer solution), this coverage is largely governed by the surface roughness and quality of the underlying electrodeposited gold layer, which could be improved to give more reproducible sensor performance. Another consideration for the use of BPEs is the position of the working electrode and QRCE with respect to the BPE. Changes in the positions of these electrodes will result in a difference in induced polarization, which could inhibit sensor performance. Additionally, a higher electrocatalyst concentration may result in faster conversion of LB to MB, thus faster conversion of PW to PB in the presence of target molecule.
Regarding
Regarding
A series of four buffer conditions were investigated before and after adding ATP (Table 1,
All documents cited are incorporated herein by reference; the citation of any document is not to be construed as an admission that it is prior art with respect to the present invention.
It is to be further understood that where descriptions of various embodiments use the term “comprising,” and/or “including” those skilled in the art would understand that in some specific instances, an embodiment can be alternatively described using language “consisting essentially of” or “consisting of.”
While particular embodiments of the present invention have been illustrated and described, it would be obvious to one skilled in the art that various other changes and modifications can be made without departing from the spirit and scope of the invention. It is therefore intended to cover in the appended claims all such changes and modifications that are within the scope of this invention.
This application is a continuation of PCT Application No. PCT/US20/40902 filed Jul. 6, 2020, which claims benefit of U.S. Provisional Application Ser. No. 62/870,392, filed Jul. 3, 2019, which applications are hereby incorporated by reference in their entirety.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US20/40902 | 7/6/2020 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 62870392 | Jul 2019 | US |
