Patent Application
20230048049
The present invention relates generally to techniques for detecting drugs and drug metabolites and relates more particularly to techniques for detecting drugs and/or drug metabolites of interest in wastewater and other liquid samples.
The misuse/abuse of drugs including opioids is an epidemic crisis and represents a major threat to public health and safety. The consumption of opioids on a routine basis can cause a variety of adverse health effects, including damage to the central nervous system, and often leads to death. According to a report by the United Nations Office on Drugs and Crime (UNODC), there are almost 300 million people of ages 15 to 64 who use illicit drugs like heroin and pharmaceutical opioids. In 2019 alone, there were more than 70,000 opioid-related deaths in the United States, which corresponds to more than 190 opioid-related deaths per day in the United States. To understand the depth of this crisis, a group of U.S. scientists recently surveyed approximately 3,300 students in ten high schools in the Los Angeles area to learn about the misuse of prescription opioids by high school students. The outcome of this four-year study suggests that 2.1% to 13.1% of students misuse opioids, putting such students at a significant risk for later developing a heroin addiction. Additionally, a recent spike in the presence of highly potent synthetic opioids, such as fentanyl, which is commonly found in street heroin, presents significant concerns and challenges to law enforcement. In order to control illicit drug use and to secure public health and safety, law enforcement needs real-time data on the type and usage frequency of illicit drugs. Current sources for this type of data tend to rely on anonymous surveys or on numbers of cases reported by hospitals or emergency rooms. However, as can be appreciated, such an approach is inadequate as it only reflects a small portion of illicit drug users and does not provide the level of information needed to optimize drug surveillance.
Wastewater-based epidemiology (WBE) (sometimes alternatively referred to as wastewater-based surveillance or wastewater monitoring) is a relatively new approach to drug surveillance and is based on the chemical analysis of target drug analytes in raw wastewater. Wastewater monitoring was first implemented in the U.S. in 2000s and has been utilized primarily in Europe, with several multi-city studies, to monitor illicit drug use. The premise of wastewater monitoring is that, after drug use, drugs are excreted and released into wastewater, mostly in metabolite forms. Wastewater monitoring possesses several advantages over existing surveillance techniques, such as near real-time feedback on usage, the ability to detect changes in daily usage, and information on the introduction of new psychoactive substances. Wastewater monitoring is also able to deliver more rapid, comprehensive, and objective measurement of drug use without stigmatizing individuals or communities.
In order to be successful, however, wastewater monitoring must be very sensitive and specific since such metabolites are typically present in wastewater only at very low concentrations (low pg/mL to ng/mL levels) due to dilution. Unfortunately, at present, only a few methods are available for wastewater monitoring, and none are sensitive and reliable enough to detect low concentrations of opioids in environmental wastewater. The currently preferred technique for wastewater monitoring is high-performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS); however, HPLC-MS/MS often cannot detect opioid metabolites unless the sample is highly processed and the drugs are pre-concentrated. HPLC-MS/MS also needs complex instrumentation and is not amenable for use in wastewater plants, manholes, and catch basins. Field-friendly immunoassays based on antibodies, such as lateral flow immunoassays (LFIs), exist, but such immunoassays exhibit similar degrees of sensitivities to HPLC-MS/MS (1-10 ng/mL), and they are either not quantitative or need complex detectors for analysis. The availability and shelf life of the antibodies used in such immunoassays are also of concern.
It is an object of the present invention to provide a novel technique for detecting one or more drugs and/or drug metabolites in wastewater and other liquid samples.
It is another object of the present invention to provide a technique as described above that overcomes at least some of the shortcomings associated with existing techniques.
It is still another object of the present invention to provide a technique that is highly sensitive and specific for detecting one or more drugs and/or drug metabolites of interest and that can be used to analyze a wastewater sample rapidly and accurately. In one embodiment, such a technique may be implemented using a portable, hand-held instrument that may include data storage and wireless communications for real-time dissemination of actionable data.
Therefore, according to one aspect of the invention, there is provided a method for detecting one or more drugs and/or drug metabolites of interest in a liquid sample, the method comprising the steps of (a) providing a device, the device comprising a graphene field effect transistor and a first aptamer coupled to the graphene field effect transistor, the first aptamer being selective for a first drug or drug metabolite of interest; (b) exposing a liquid sample to the first aptamer of the device; (c) then, applying a liquid gate voltage to the device and measuring the resultant resistance; and (d) comparing the resultant resistance to appropriate standards to determine the presence and/or quantity of the first drug or drug metabolite of interest.
In a more detailed feature of the invention, step (c) may comprise sweeping the liquid gate voltage to obtain a resistance versus liquid gate voltage plot for the device.
In a more detailed feature of the invention, step (d) may comprise comparing a Dirac voltage shift for the device to appropriate standards.
In a more detailed feature of the invention, the first drug or drug metabolite may be selected from the group consisting of oxycodone, noroxycodone, fentanyl, norfentanyl, morphine, and 2-ethylidene-1,5-dimethyl-3,3-diphenyl-pyrrolidine.
In a more detailed feature of the invention, the liquid sample may be a wastewater sample.
In a more detailed feature of the invention, the one or more drugs and/or drug metabolites of interest may be exactly one drug or drug metabolite.
According to another aspect of the invention, there is provided a method for detecting one or more drug and/or drug metabolites of interest in a liquid sample, the method comprising the steps of (a) providing a device, the device comprising a graphene field effect transistor, the graphene field effect transistor having a first well and a second well, the device further comprising a first aptamer and a second aptamer, the first aptamer being selective for a first drug or drug metabolite of interest and being coupled to the graphene field effect transistor in a first well, the second aptamer being selective for a second drug or drug metabolite of interest and being coupled to the graphene field effect transistor in a second well, the second drug or drug metabolite of interest being different than the first drug or drug metabolite of interest; (b) exposing a liquid sample to the first aptamer and the second aptamer of the device; (c) then, applying a liquid gate voltage to each of the first well and the second well of the device and measuring the resultant resistance; and (d) comparing the resultant resistance from each of the first well and the second well to appropriate standards to determine the presence and/or quantity of the first drug or drug metabolite of interest and the second drug or drug metabolite of interest.
In a more detailed feature of the invention, step (c) may comprise sweeping the liquid gate voltage in each of the first well and the second well to obtain first and second resistance versus liquid gate voltage plots, respectively, for the device.
In a more detailed feature of the invention, step (d) may comprise comparing a Dirac voltage shift for each of the first and second wells to appropriate standards.
In a more detailed feature of the invention, the one or more drugs and/or drug metabolites of interest may be selected from the group consisting of oxycodone, noroxycodone, fentanyl, norfentanyl, morphine, and 2-ethylidene-1,5-dimethyl-3,3-diphenyl-pyrrolidine.
In a more detailed feature of the invention, the liquid sample may be a wastewater sample.
In a more detailed feature of the invention, the device may further comprise a third well and a third aptamer, the third aptamer may be selective for a third drug or drug metabolite of interest and may be coupled to the graphene field effect transistor in the third well, the third drug or drug metabolite of interest may be different than the first and second drugs or drug metabolites of interest, and the method may further comprise exposing the liquid sample to the third aptamer of the device; then, applying a liquid gate voltage to the third well of the device and measuring the resultant resistance; and comparing the resultant resistance from the third well to appropriate standards to determine the presence and/or quantity of the third drug or drug metabolite of interest.
In a more detailed feature of the invention, the device may further comprise a fourth well and a fourth aptamer, the fourth aptamer may be selective for a fourth drug or drug metabolite of interest and may be coupled to the graphene field effect transistor in the fourth well, the fourth drug or drug metabolite of interest may be different than the first, second and third drugs or drug metabolites of interest, and the method may further comprise exposing the liquid sample to the fourth aptamer of the device; then, applying a liquid gate voltage to the fourth well of the device and measuring the resultant resistance; and comparing the resultant resistance from the fourth well to appropriate standards to determine the presence and/or quantity of the fourth drug or drug metabolite of interest.
According to yet another aspect of the invention, there is provided a device for use in detecting one or more drugs and/or drug metabolites of interest in a liquid sample, the device comprising (a) a graphene field effect transistor, the graphene field effect transistor comprising a first well; and (b) a first aptamer, the first aptamer being coupled to the graphene field effect transistor in the first well, the first aptamer being selective for a first drug or drug metabolite of interest.
In a more detailed feature of the invention, the first aptamer may be selective for a drug or drug metabolite of interest selected from the group consisting of oxycodone, noroxycodone, fentanyl, norfentanyl, morphine, and 2-ethylidene-1,5-dimethyl-3,3-diphenyl-pyrrolidine.
In a more detailed feature of the invention, the first aptamer may be coupled to the graphene field effect transistor using a linker molecule.
In a more detailed feature of the invention, the linker molecule may be pyrenebutyric acid N-hydroxysuccunumide ester.
In a more detailed feature of the invention, the graphene field effect transistor may further comprise a second well, the device may further comprise a second aptamer, the second aptamer may be coupled to the graphene field effect transistor in the second well, the second aptamer may be selective for a second drug or drug metabolite of interest, and the second drug or drug metabolite of interest may be different than the first drug or drug metabolite of interest.
According to a further aspect of the invention, there is provided a system for detecting one or more drugs or drug metabolites in a liquid sample, the system comprising the above-described device, a voltage sweep generator for applying a voltage sweep to the graphene field effect transistor, and a reader/analyzer for measuring the resultant resistance and comparing the resultant resistance to appropriate standards.
Additional objects, as well as aspects, features and advantages, of the present invention will be set forth in part in the description which follows, and in part will be obvious from the description or may be learned by practice of the invention. In the description, reference is made to the accompanying drawings which form a part thereof and in which is shown by way of illustration various embodiments for practicing the invention. The embodiments will be described in sufficient detail to enable those skilled in the art to practice the invention, and it is to be understood that other embodiments may be utilized and that structural changes may be made without departing from the scope of the invention. The following detailed description is, therefore, not to be taken in a limiting sense, and the scope of the present invention is best defined by the appended claims.
The accompanying drawings, which are hereby incorporated into and constitute a part of this specification, illustrate various embodiments of the invention and, together with the description, serve to explain the principles of the invention. These drawings are not necessarily drawn to scale, and certain components may have undersized and/or oversized dimensions for purposes of explication or may omit certain features for purposes of clarity. In the drawings wherein like reference numerals represent like parts:
The present invention is directed at a novel technique for detecting one or more drugs and/or drug metabolites of interest in a liquid sample, such as, but not limited to, a wastewater sample. According to one embodiment, the detection technique of the present invention may be achieved using a novel device that comprises (i) a graphene field effect transistor (G-FET) and (ii) one or more aptamers that are coupled to the G-FET, the one or more aptamers being selective or specific for one or more drugs and/or drug metabolites of interest. As will be discussed below, this combination of a graphene field effect transistor and one or more aptamers that are selective or specific for the one or more drugs and/or drug metabolites of interest enables a detection technique that possesses many advantages over existing detection techniques.
Referring now to
Method 11 may begin with a step 13 of collecting a liquid sample. In the present embodiment, step 13 may comprise obtaining a wastewater sample.
Method 11 may continue with a step 15 of introducing the collected sample to a device, the device comprising a graphene field effect transistor (G-FET) to which one or more aptamers that are selective or specific for the one or more drugs and/or drug metabolites of interest have been coupled. If the liquid sample contains the drugs and/or drug metabolites of interest, such metabolites will tend to bind to the aptamers.
Method 11 may continue with a step 17 of detecting the presence of a metabolite that is bound to one of the foregoing aptamers. This may comprise applying a voltage to the G-FET, measuring the resultant resistance, and comparing the resultant resistance to appropriate standards. The resultant resistance may be indicative of the presence of a drug or drug metabolite bound to the aptamer because, when a drug or drug metabolite binds to the aptamer, the additional charge induced on the graphene coupled to the aptamer causes a change in the Dirac point (the Dirac point representing the peak in resistance at charge neutrality). Consequently, by measuring the change in the Dirac point, one not only can determine whether or not the drug or drug metabolite of interest is present but also can determine the concentration of the drug or drug metabolite of interest in the sample.
Referring now to
Aptamer-based graphene field effect transistor 51 may comprise a graphene field effect transistor that is similar or identical to one of more of the graphene field effect transistors that are disclosed in the following documents, all of which are incorporated herein by reference: Kumar et al., “Rapid, Multianalyte Detection of Opioid Metabolites in Wastewater,” ACS Nano, 16(3): 3704-3714 (Feb. 24, 2022); Kumar et al., “Detection of a multi-disease biomarker in saliva with graphene field effect transistors,” Med. Devices Sens., 3:e101021 (2020); Kumar et al., “Dielectrophoresis assisted rapid, selective and single cell detection of antibiotic resistant bacteria with G-FETs,” Biosensors and Bioelectronics, 156:112123 (2020); Gray et al., “A Cleanroom in a Glovebox,” Rev. Sci. Instrum., 91(7):073909 (2020).
Accordingly, aptamer-based graphene field effect transistor 51 may comprise a SiO2/Si substrate 53, a graphene layer 55, a drain electrode 57 (which may be a Pt/Ti electrode), a source electrode 59 (which may be a Pt/Ti electrode), a side gate 61 (which may be Pt), and an A1Ox layer 63.
Aptamer-based graphene field effect transistor 51 may further comprise an aptamer 71. Although aptamer-based graphene field effect transistor 51 is shown in
Aptamers are small, single-stranded nucleic acids (DNA or RNA) that are usually about 20-100 bases in length. Aptamers tend to adopt conformational structures that enable selective binding to a target of interest. To find an aptamer that selectively binds to a target of interest, one may use a technique commonly known as “SELEX,” which typically involves the following steps: (i) start with a large library of aptamers; (ii) immobilize the target; (iii) expose the library of aptamers to the immobilized target; (iv) wash away non-binding aptamers; (v) elute bound aptamers; (vi) amplify the eluted aptamers; (vii) expose the amplified library to the immobilized target; and (viii) repeat steps (iv)-(vii) under conditions of increasing stringency. Additional information relating to aptamers and the SELEX technique for identifying aptamers that are selective or specific for a target of interest may be found in the following documents, all of which are incorporated herein by reference: Zhuo et al., “Recent Advances in SELEX Technology and Aptamer Applications in Biomedicine,” Int. J. Mol. Sci., 18:2142 (2017); Gold, “SELEX: How It Happened and Where It will Go,” J Mol Evol, 81:140-143 (2015); and Ellington et al., “In vitro selection of RNA molecules that bind specific ligands,” Nature, 346:818-822 (1990).
In view of the above, where, for example, the drug or drug metabolite of interest is noroxycodone, one may apply the SELEX technique to a library of aptamers and immobilized noroxycodone to find an aptamer that is selective for noroxycodone. A similar approach may be used to find aptamers that are selective for norfentanyl and EDDP, respectively. In fact, aptamers that are selective for noroxycodone, norfentanyl and EDDP are already commercially available, for example, from Base Pair Biotechnologies, Inc., Pearland, TX, such aptamers including CFA0079-GP5-25, AKA-H4LFD (for binding to noroxycodone); CFA0661-GP5-25 (for binding to EDDP); and CFA0071-GP5-25, AKA-H6AAZ (for binding to norfentanyl). A similar approach may be used to find aptamers selective for other drug metabolites or drugs.
Aptamers 71 may be coupled to graphene layer 55 via a linker molecule 73, such as pyrenebutyric acid N-hydroxysuccunumide ester (PBASE).
Referring now to
Microfluidics cartridge 81, which may be designed for multiplexed analysis, may include a port 83 that may be connected to a syringe to receive a liquid sample. Microfluidic cartridge 81 may further include a microfluidic sensor chip (not shown) with a plurality of (e.g., four) independent wells (one drug for each well for simultaneous detection) and a plurality of (e.g., five) G-FET devices per well for signal robustness and reproducibility. Microfluidic cartridge 81 may further include with control interfaces (not shown) which may be operated by a portable analyzer/reader shown in
As can be appreciated, the present technique possesses many advantages over existing techniques. Current cutting-edge sensing in wastewater typically relies on high-performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS) or optical techniques, such as enzyme-linked immunosorbent assay (ELISA). These methods achieve high levels of sensitivity and permit the processing of numerous tests in parallel on multiple bioanalytes. However, these methods also require trained personnel and expensive laboratory equipment. As such, collection and testing are rarely done at the same location, with only a limited number of analytes and potential collection sites. Electrical sensors are significantly cheaper, more comfortable to operate, easier for multiplexed analysis, and readily scalable.
Graphene-based sensors offer ultrasensitive, rapid, and accurate detection of targets. The electrical resistance of graphene is highly sensitive to the attached targets (or the probes conformal changes), enabling direct and rapid readout, while the Dirac-point (peak in resistance at charge neutrality) is measured by a gate, providing a quantitative determination of the target concentration. Graphene is also attractive, given its ease of functionalization with an array of biological probes and its ability to be implemented on a wide variety of substrates. Wafer-scale graphene can be commercially purchased from multiple sources in the U.S., Europe, and Asia.
Traditional back-gated FETs require substantial voltages (>60 V) with special electronics. By contrast, the “solution” gated FET sensors of the present invention may employ a side gate directly incorporated onto the chip that operates with up to twenty devices in parallel, offering additional sensitivity, redundancy, and multiplexed detection. Thus, lower voltages (1-2 V) are sufficient for device operation. This is a reliable approach with less complex electronics than are required for other FET approaches. For clean graphene, one observes a maximum in the resistivity at zero applied voltage, indicated as the Dirac Point.
The devices of the present invention can be made by a process such that their intrinsic doping is nearly zero, and the devices are protected by an A12O3 layer, exposing only the active area of graphene and the on-chip electrical gate to the biological targets. This results from a fabrication process that significantly reduces the cost of production and fabrication time. Furthermore, a mask-less lithography system of the type used allows numerous device configurations, and the single-layer graphene on rigid silicon provides a sturdy surface for contact electrodes.
By contrast, recent experimental approaches for drug detection, such as ELISA assays and laminar flow immunoassays, mostly rely on antibodies. Current attempts to multiplex assays using antibodies suffer from inconsistencies between vendors and product lots. The use of aptamers rather than antibodies, coupled with graphene FETs, offer several advantages that enhance their development potential: (1) Aptamers are tolerant to temperatures that cause denaturation of antibodies (no need for refrigeration); (2) Antibody development can take months in animals compared to hours for in vitro aptamer development; (3) Material scales up for aptamers are straightforward via standard in vitro nucleic acid synthesis methods; (4) Functionalization of surfaces with antibodies is not trivial and compromises their binding activity; (5) The strong aptamer binding affinity over antibodies coupled with the high sensitivity of G-FET generates rapid, sensitive, and highly specific biosensors. To turn graphene into a sensor, it may be functionalized with an aptamer specific to the target drug or drug metabolite. When the target binds to the aptamer, the additional charge induced on the graphene is measured by the change in the Dirac point and plotted as a function of the drug or drug metabolite concentration. This enables in-situ optimization of the attachment process. The flat micro-scale, single-layer graphene on a rigid silicon substrate provides a sturdy surface for depositing the contact electrodes by conventional masking techniques.
In summary, there is disclosed herein, according to one embodiment, a compact and label-free graphene field effect transistor sensor utilizing high-specificity aptamers for rapid, sensitive, and multiplexed detection of drugs and/or drug metabolites in wastewater to accurately assess drug misuse and abuse in municipal communities. In particular, in a preferred embodiment, two, three, four or more drugs and/or drug metabolites may be targeted for detection in wastewater samples. For example, such drugs or drug metabolites could include one or more of the following opioids, opioid metabolites or related compounds: oxycodone, noroxycodone (a metabolite of oxycodone), fentanyl, norfentanyl (a metabolite of fentanyl), morphine, and EDDP (a metabolite of methadone, fully synthetic opioid. The strong opioid metabolite/aptamer binding complex at the graphene surface provides a rapid, highly selective change in the G-FET source-drain current and the Dirac point. The instrument may be label-free since there is no requirement to modify or label the target. The instrument may be a portable hand-held instrument capable of detecting drug and/or drug metabolites at actionable levels in wastewater in less than 30 minutes. The instrument may be used for easy and frequent field sampling further upstream in community sewer lines which can be accessed by removing manhole covers. The instrument may include data storage and wireless communications for the real-time dissemination of actionable data.
Some desirable features, attributes and/or advantages of one or more embodiments of the present invention may include one or more of the following:
The following examples are given for illustrative purposes only and are not meant to be a limitation on the invention described herein or on the claims appended hereto.
Three different opioid metabolites were selected, namely, (i) noroxycodone (NX), which is a metabolite of oxycodone (a semisynthetic opioid); (ii) 2-ethylidene-1,5-dimethyl-3,3-diphenyl-pyrrolidine (EDDP), which is a metabolite of methadone (a fully synthetic opioid), and (iii) norfentanyl (NF), which is a metabolite of fentanyl (a synthetic opioid). The following 5’-amine-aptamer-3’ aptamers were obtained from Base Pair Biotechnologies, Inc., Pearland, TX, for selective binding to the aforementioned opioid metabolites: (i) CFA0079-GP5-25, AKA-H4LFD (for binding to noroxycodone); (ii) CFA0661-GP5-25 (for binding to EDDP); and (iii) CFA0071-GP5-25, AKA-H6AAZ (for binding to norfentanyl). All selected aptamers were believed to have strong binding affinities to their respective opioid metabolites. Nevertheless, to verify, the nanoplasmonic assay described below was performed.
Citrate-reduced gold nanoparticles (AuNP) possess negative charge. The electrostatic repulsive forces between gold nanoparticles give them their characteristic red color (520 nm) when they are dispersed. In the presence of a negatively charged aptamer and 150 mM NaCl, a negative charge cloud protects the gold nanoparticles from any aggregation (
This simple mechanism allows one to obtain quantitative binding information by monitoring the optical density (OD) at 520 nm and the ratio of (OD520/ OD700) (
Using this colorimetric assay, sensitivity and specificity analyses of the subject aptamers for noroxycodone, norfentanyl, and EDDP were performed.
A graphene field effect transistor (G-FET) was fabricated according to a technique like that disclosed in Kumar et al., “Rapid, Multianalyte Detection of Opioid Metabolites in Wastewater,” ACS Nano, 16(3): 3704-3714 (Feb. 24, 2022), which is incorporated herein by reference. The G-FET device included a graphene film on a SiO2/Si substrate, Au/Cr (or Pt/Ti) drain and source electrodes, and a Pt side gate. To reduce unwanted chemical reactions, the graphene device was surrounded by an Al2O3 layer such that only the graphene was able to be in contact with the test solution. The device was fabricated with two wells, each with two active G-FETs, as shown in
The G-FET was initially treated with pyrenebutyric acid N-hydroxysuccunumide ester (PBASE) to act as a heterobifunctional linker molecule, followed by incubation with the amino-modified opioid aptamer in phosphate-buffered saline, PBS (pH 7.4). Fourier-transform infrared (FTIR) spectroscopy was used to confirm the similar PBASE peak is unchanged on different modification steps on the graphene surface (see
Additional G-FETs were designed and fabricated with strips of graphene of various widths. Initial tests of electrical performance and uniformity were performed. These initial devices had PDMS wells attached. Chemical vapor deposition (CVD) graphene was obtained, where the CVD process was optimized for the needed grain size and doping levels. The graphene films were tested for uniformity using micro-Raman spectroscopy. Each device was put through a gate voltage sweep to measure the as-fabricated maximum resistance and to determine the mobility. By dividing the AC voltage by the AC bias current, Dirac points and resistance across the channel contacts were obtained. A number of device designs were attempted, with the final one chosen to maximize the reliability of fabrication and sensing area while minimizing extrinsic contributions. In a final design, a switch was made to using platinum bottom contacts with the graphene transferred on top. This minimized the number of fabrication steps and enhanced the protection of the contacts. In addition, an on-chip gate contact was added to each well. Finally, the chip was scaled up to four wells, each well with 5 G-FET devices, all measured independently and nearly simultaneously. As with the initial design, the entire chip was covered with an Al2O3 protective layer to prevent shorting and biofouling, with small windows opened only on the active region. This methodology ensured that treating with raw wastewater did not adversely affect the G-FETs (see
Noroxycodone, norfentanyl, and EDDP were detected with the above-described aptamer-based G-FETs in buffer and wastewater solutions at pg/mL levels. In
Detection limit of opioids was initially affected in wastewater due to interference caused by the presence of several other contaminants. This issue was resolved by mixing both end amine terminated polyethylene glycol (PEG) with aptamers. PEG helps to minimize the unspecific interaction with the graphene surface and reduces the Debye screening effect, which resulted in significant improvement in LOD. In
The embodiments of the present invention described above are intended to be merely exemplary and those skilled in the art shall be able to make numerous variations and modifications to it without departing from the spirit of the present invention. All such variations and modifications are intended to be within the scope of the present invention as defined in the appended claims.
The present application claims the benefit under 35 U.S.C. 119(e) of U.S. Provisional Pat Application No. 63/232,463, inventors Avni A. Argun et al., filed Aug. 12, 2021, the disclosure of which is incorporated herein by reference in its entirety.
This invention was made with government support under 1R43DA051105-01 awarded by the Department of Health and Human Services, National Institute on Drug Abuse. The government has certain rights in the invention.
| Number | Date | Country | |
|---|---|---|---|
| 63232463 | Aug 2021 | US |
