Patent Publication Number: US-2020300845-A1

Title: Methods and devices for detection of thc

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS 
     The present disclosure claims priority to U.S. Provisional Application No. 62/832,264, filed Apr. 10, 2019 and titled “Methods and Devices for Detection of THC.” The present disclosure is also a continuation-in-part of U.S. application Ser. No. 16/422,743, filed May 24, 2019 and titled “A Functionalized Sensor for Detection of Biomarkers.” U.S. application Ser. No. 16/422,743 claims priority to U.S. Provisional Application No. 62/676,079, filed May 24, 2018 and titled “Graphene-Functionalized Sensor.” The entire contents of each of these applications are incorporated herein by reference. 
    
    
     FIELD 
     The present invention relates generally to methods and devices for the detection of tetrahydrocannabinol (THC) and cannabidiol (CBD). 
     BACKGROUND 
       Cannabis , which is also known as marijuana, is a psychoactive drug from the  Cannabis  plant. It is used increasingly for medical and recreational purposes. The main psychoactive component of  cannabis  is tetrahydrocannabinol (THC). 
     With the increase use of  cannabis , including in food products, methods and systems are needed for easy detection of THC in a variety of different products. 
     SUMMARY 
     In one aspect, a method of detecting tetrahydrocannabinol (THC) in a sample is disclosed, which comprises bringing the sample into contact with a graphene layer functionalized with an antibody exhibiting specific binding to THC, applying a time-varying electric field to said antibody-functionalized graphene layer, monitoring at least one electrical property, e.g., electrical resistance, of said graphene layer in response to interaction with said sample, and detecting presence of THC in the sample by detecting a change in said electrical resistance indicative of interaction of THC with said anti-body functionalized graphene layer. 
     In some embodiments, the time-varying electric field has a frequency in a range of about 1 kHz to about 2 MHz, e.g., in a range of 100 kHz to about 1 MHz, such as 500 kHz. 
     In some embodiments, the graphene layer can be disposed on an underlying substrate. A variety of substrates can be employed. Some suitable examples include, without limitation, a semiconductor substrate and glass. 
     The graphene layer can be electrically coupled to a pair of electrically conductive pads for facilitating the measurement of the electrical conductivity of the antibody-functionalized graphene layer, and specifically, the measurement of a change, if any, in the electrical conductivity of the antibody-functionalized graphene layer in response to interaction with a sample under study. 
     The methods and systems according to the present teachings can be employed to detect the presence of THC in a variety of different samples, including, food samples, medications, biological samples, such as blood, urine and saliva. 
     In some embodiments, the method is employed to detect Δ-9-THC. In some embodiments, the method is employed to detect one or more metabolites of Δ-9-THC, such as 11-OH-THC and 11-COOH-THC. In some embodiments, the method can be employed for concurrent detection of Δ-9-THC and any, or both, of 11-OH-THC and 11-COOH-THC. 
     In some embodiments, the method can exhibit a limit-of-detection of better than 20 ng/ml to 100 ng/ml. 
     In a related aspect, a system for detecting THC in a sample is disclosed, which comprises a sensor having a substrate, and a graphene layer deposited on a surface of said substrate, said graphene layer being functionalized with a plurality of antibodies exhibiting specific binding to THC. The sensor can further include at least a pair of electrically conductive pads coupled to the graphene layer for measuring an electrical property, e.g., an electrical resistance, thereof. A reference electrode can be disposed on the substrate in proximity of the antibody-functionalized graphene layer to allow application of a reference AC voltage thereto, via an AC voltage source. In some embodiments, such a reference electrode can be positioned above the graphene layer. In some embodiments, the distance between the reference electrode and the antibody-functionalized graphene layer can be, for example, in a range of 100 microns to about 3 mm, e.g., about 1 to about 2 mm. 
     The applied AC voltage can have a frequency in a range of about 1 kHz to about 2 MHz, e.g., 1 MHz, and an amplitude in a range of about 1 millivolt to about 3 volts. In some embodiments, in addition to the AC voltage, a ramp voltage (e.g., in a range of about −10 V to about 10 V, e.g., about −1 V to 1 V) can applied to the reference electrode during data acquisition. In other words, a dc offset of the AC voltage is ramped, e.g., in a range of about −10 V to about 10 V, e.g., in a range of about −1 V to about 1 V. 
     In some embodiments, THC includes Δ-9-THC. In some embodiments, THC includes a hydroxylated, or carboxylated metabolite of Δ-9-THC. Some examples of such metabolites include, without limitation, 11-COOH-THC. In some embodiments, THC includes 11-COOH-THC. In some embodiments, the sensor is configured to detect Δ-9-THC as well as one or more metabolites of 11-0H-THC and 11-COOH-THC. In some embodiments, THC includes Δ-8-THC and/or one of its metabolites. 
     In a related aspect, a method of detecting cannabidiol (CBD) in a sample is disclosed, which includes bringing the sample into contact with a graphene layer functionalized with an antibody exhibiting specific binding to CBD, applying a time-varying electric field to said antibody-functionalized graphene layer, monitoring at least one electrical property of the graphene layer in response to interaction with said sample, and detecting presence of CBD in the sample by detecting a change in said electrical property indicative of interaction of CBD with the anti-body functionalized graphene layer. 
     In a related aspect, a system for detecting CBD in a sample is disclosed, which includes a sensor, comprising a substrate and a graphene layer deposited on a surface of said substrate, said graphene layer being functionalized with a plurality of antibodies exhibiting specific binding to CBD, and at least one pair of electrically conductive pads coupled to said graphene layer for measuring an electrical property of the antibody-functionalized graphene layer in response to exposure thereof to a sample under study. In some embodiments, the measured electrical property of the antibody-functionalized graphene layer can be its electrical resistance, e.g., its DC electrical resistance. 
     Further understanding of various aspects of the invention can be obtained by reference to the following detailed description in conjunction with the associated drawings, which are briefly described below. 
    
    
     
       BRIEF DESCRIPTION OF DRAWINGS 
         FIG. 1  schematically depicts a sensor system according to an embodiment of the present teachings for detecting THC in a sample; 
         FIG. 2  is another schematic view of the sensor depicted in  FIG. 1  as well as an AC and DC source for applying AC and/or DC voltage/current to the antibody-functionalized graphene layer and a controller for controlling the AC and DC source; 
         FIG. 3  schematically depicts a combination of a ramp voltage and an AC voltage applied to the reference electrode of the sensor shown in  FIG. 1 ; 
         FIG. 4  schematically depicts the coupling of a plurality of antibodies to the graphene layer via a plurality of linker molecules; 
         FIG. 5A  schematically depicts a hydroxyl-functionalized graphene layer; 
         FIG. 5B  schematically depicts a hydroxyl-functionalized graphene layer to which antibodies are attached; 
         FIG. 6  schematically depicts a voltage-measuring device that can be employed in some embodiments for measuring the electrical resistance of the underlying graphene layer of a sensor according to the present teachings; 
         FIG. 7  schematically depicts an analyzer in communication with the voltage-measuring device depicted in  FIG. 6 ; 
         FIG. 8  schematically depicts an example of implementation of the analyzer depicted in  FIG. 7 ; 
         FIG. 9  schematically depicts an embodiment a system according to the present teachings; 
         FIG. 10  depicts a sensor according to the present teachings having a plurality of sensing elements; and 
         FIG. 11  presents data obtained using a prototype device according to an embodiment of the present teachings. 
     
    
    
     DETAILED DESCRIPTION 
     It has been discovered that the change in the conductivity of an antibody-functionalized graphene layer can be employed to detect the presence of tetrahydrocannabinol (THC) in a sample. The present teachings are generally related to graphene-based sensors that can be employed for detection of THC in a sample, including metabolites of  cannabis  consumption. 
     An “antibody,” as that term is used herein, refers to a polypeptide that exhibit specific binding affinity, e.g., an immunoglobulin chain or fragment thereof, comprising at least one functional immunoglobulin variable domain sequence. An antibody encompasses full length antibodies and antibody fragments. In some embodiments, an antibody comprises an antigen binding or functional fragment of a full-length antibody, or a full-length immunoglobulin chain. For example, a full-length antibody is an immunoglobulin (Ig) molecule (e.g., an IgG antibody) that is naturally occurring or formed by normal immunoglobulin gene fragment recombinatorial processes. In embodiments, an antibody refers to an immunologically active, antigen-binding portion of an immunoglobulin molecule, such as an antibody fragment. An antibody fragment, e.g., functional fragment, comprises a portion of an antibody, e.g., Fab, Fab′, F(ab′)2, F(ab)2, variable fragment (Fv), domain antibody (dAb), or single chain variable fragment (scFv). A functional antibody fragment binds to the same antigen as that recognized by the intact (e.g., full-length) antibody. 
     The term “antibody” also encompasses whole or antigen binding fragments of domain, or signal domain, antibodies, which can also be referred to as “sdAb” or “VHH.” Domain antibodies comprise either V H  or V L  that can act as stand-alone, antibody fragments. Additionally, domain antibodies include heavy-chain-only antibodies (HCAbs). Antibody molecules can be monospecific (e.g., monovalent or bivalent), bispecific (e.g., bivalent, trivalent, tetravalent, pentavalent, or hexavalent), trispecific (e.g., trivalent, tetravalent, pentavalent, hexavalent), or with higher orders of specificity (e.g., tetraspecific) and/or higher orders of valency beyond hexavalency. An antibody molecule can comprise a functional fragment of a light chain variable region and a functional fragment of a heavy chain variable region, or heavy and light chains may be fused together into a single polypeptide. 
     The term “immunogen” as used herein refers to a substance that is capable of inducing a humoral antibody response. 
     Various terms are used herein in accordance with their ordinary meanings in the art. The term “about” as used herein to modify a numerical value is intended to denote a variation of at most 10% of a numerical value. 
       FIGS. 1 and 2  schematically depict an example of a device  1000  (herein also referred to as sensor  1000 ) according to an embodiment of the present teachings for detecting THC in a sample. The device  1000  includes a substrate  1002  on a top surface of which a layer of graphene  1004  is deposited. A variety of different substrates can be employed. By way of example, the substrate  1002  can be any of a semiconductor, such as silicon, glass, or plastic. In this embodiment, a silicon oxide layer  1003  separates the underlying silicon layer from the graphene layer. 
     In this embodiment, the graphene layer is functionalized with an antibody  1004   a  that can specifically bind to THC. By way of example, in some embodiments, the graphene layer can be functionalized with a commercially available antibody, such as an antibody marketed by Fitzgerald Industries (#10-T43B). Two metallic pads  1005 / 1007  in electrical contact with the graphene layer allow measuring the electrical resistance of the antibody-functionalized graphene layer, and particularly, a change in the electrical resistance of the graphene layer in response to exposure thereof to a sample containing THC. In some embodiments, the electrically conductive pads can be formed of silver high conductive paste, though other electrically conductive materials can also be employed. The conductive pads can be electrically connected to a measurement device, e.g., a voltmeter, via a plurality of conductive wires for measuring the Ohmic electrical resistance of the graphene layer. 
     In this embodiment, the sensor  1000  includes a reference electrode  3001  disposed in proximity of the antibody-functionalized graphene layer, e.g., at a distance in a range of about 50 micrometers to about a few millimeters (e.g., 1-2 millimeters) on the silicon oxide layer  1003 , or alternatively, above the functionalized graphene layer. The reference electrode can be utilized to generate a time-varying electric field at the interface of the functionalized graphene layer and the sample in contact with that layer. For example, in this embodiment, an AC voltage source  3002  can be employed to apply an AC voltage to the reference electrode, which can in turn result in the generation of a time-varying electric field in the space between the reference electrode and the functionalized graphene layer. As discussed in more detail below, the AC voltage source  3002  can also apply a DC offset voltage to the reference electrode. In other embodiments, a sensor according to the present teachings may not include a reference electrode. 
     Further, a power supply  3005  is provided for applying a DC voltage across, or a DC current to, the antibody-functionalized graphene layer to measure a response thereof (e.g., a change in a voltage across the antibody-functionalized layer when a constant current is applied to that layer) upon exposure of the antibody-functionalized layer to a sample under study. 
     More specifically, a controller  3007  (see  FIG. 2 ) is programmed to control the AC and the DC sources. Although in this embodiment the AC voltage source  3002  and the power supply  3005  are shown as two independent units, in other embodiments the functionalities of the AC voltage source for applying an AC voltage and a DC offset voltage to the reference electrode and that of the power supply  3003  can be combined in a single unit. 
     The controller  3007  can be implemented in hardware, software, and/or firmware in a manner known in the art as informed by the present teachings. For example, the controller  3007  can have the components illustrated in  FIG. 6  for the analyzer. 
     In other embodiments, a sensor according to the present teachings can be implemented without the reference AC electrode. 
     By way of illustration,  FIG. 3  schematically depicts a combination of an AC voltage  3010  and a DC offset voltage  3012  applied to the reference electrode. By way of example, the DC offset voltage can extend from about −10 V to about 10 V (e.g., from −1 V to about 1 V), and the applied AC voltage can have the frequencies and amplitudes disclosed above. 
     Without being limited to any particular theory, in some embodiments, it is expected that the application of such an AC voltage to the reference electrode can minimize, and preferably eliminate, an effective capacitance associated with a sample, e.g., a liquid sample, with which the functionalized graphene layer is brought into contact as the sample is being tested, thereby facilitating the detection of a change in the resistance of the underlying graphene layer in response to the interaction of the antibodies with THC present in the sample. In some cases, the effective capacitance of the sample can be due to ions present in the sample. 
     The application of a such a time-varying electric field to the interface between the graphene layer and the liquid in contact with the graphene layer can advantageously facilitate the detection of one or more electrical properties of the antibody-functionalized graphene layer, e.g., a change in its resistance in response to its interaction with a THC present in a sample under investigation. In particular, it has been discovered that the application of an AC voltage having a frequency in a range of about 1 kHz to about 2 MHz, e.g., in a range of about 100 kHz to about 1 MHz, or 10 kHz to about 500 kHz or in a range of about 20 kHz to about 400 kHz, or in a range of about 30 kHz to about 300 kHz, or in a range of about 40 kHz to about 200 kHz, can be especially advantageous in this regard. By way of example, the amplitude of the AC voltage applied to the reference electrode can be in a range of about 1 millivolt to about 3 volts, e.g., in a range of about 100 millivolts to about 2 volts, or in range of about 200 millivolts to about 1 volt, or in range of about 300 millivolts to about 1 volt, e.g., in a range of about 0.5 volts to 1 volt. Further, in some cases, the voltage applied to the reference electrode can have an AC component and a DC offset, where the DC offset can be in a range of about −40 volts to about +40 volts, e.g., −1 volt to about +1 volt. 
     In some embodiments, in use, a sample suspected of containing THC can be introduced onto the sensor  1000 . Without being limited to any particular theory, the interaction of THC in the sample, if any, with the antibodies that are coupled to the underlying graphene layer can cause a change in the electrical conductivity of the graphene layer. This change in the electrical conductivity of the graphene layer can be in turn measured to detect the presence of THC in the sample under study. 
     In some embodiments, a linker can be employed to couple the antibodies to the graphene layer. For example, in such embodiments, a plurality of linker molecules can be bound to the graphene layer, e.g., via π-π interactions, and the antibody molecules can be in turn covalently coupled to the linker molecules. By way of example, as shown schematically in  FIG. 4 , a variety of linker molecules  4000  can be employed for coupling the antibodies to the underlying graphene layer  1004 . By way of example, in some embodiments, 1-pyrenebutonic acid succinimidyl ester is employed as a linker to facilitate the coupling of the antibodies to the underlying graphene layer. 
     With reference to  FIG. 6 , in some embodiments, a voltage-measuring device  701  can be employed to measure the resistance of the underlying graphene layer. In this embodiment, such a voltage-measuring device  701  can be electrically coupled to the antibody-functionalized graphene layer via the electrical pads  1005 / 1007 , shown in  FIG. 1 . The voltage-measuring module can be implemented using routine methods in the art. Specifically,  FIG. 6  illustrates a sensor  702  according to the present invention as an equivalent circuit corresponding to an antibody-functionalized graphene layer. A fixed voltage V (e.g., 1.2 V) is generated at the output of a buffer operational amplifier  703 . This voltage is applied to one input (A) of a downstream operational amplifier  704  whose other input B is coupled to VRI ground via a resistor R 1 . The output of the operational amplifier  704  (Vout 1 ) is coupled to the other end of the sensor  702  (in this schematic diagram, resistor R 2  denotes the resistance between two electrode pads at one end of the equivalent sensor  702 , resistor R 3  denotes the resistance of the graphene layer extending between two inner electrodes of the sensor, and resistor R 4  denotes the resistance between two electrode pads at the other end of the sensor). As the operational amplifier maintains the voltage at the end of the resistor R 1  that is not connected to VR 1  ground at the fixed voltage applied to its input (A), e.g., 1.2 V, a constant current source is generated that provides a constant current flow through the sensor  702  and returns to ground via the resistor R 1  and VR 1 . 
     The voltage generated across the antibody-functionalized graphene layer is measured via the two inner electrodes of the sensor. Specifically, one pair of the inner electrode pads is coupled to a buffer operational amplifier  706  and the other pair is coupled to the other buffer operational amplifier  708 . The outputs of the buffer operational amplifiers are applied to the input ports of a differential amplifier  710  whose output port provides the voltage difference across the antibody-functionalized graphene layer. This voltage difference (V out1 −GLO) can then be used to measure the resistance exhibited by the antibody-functionalized graphene layer. The current forced through R 3  is set by I=(Vref−VR 1 )/R 1 , where the value of VR 1  is digitally controlled. For each value of current I, the corresponding voltage (Vout 1 _GLO) is measured and stored. The resistance of the antibody-functionalized graphene layer can be calculated as the derivative of the voltage, Vout 1 _GLO, with respect to current I, i.e., R=dV/dI. 
     As shown schematically in  FIG. 7 , in some embodiments, an analyzer  600  can be in communication with the voltage measuring circuitry  701  to receive the applied current and the measured voltage value and use these values to calculate the resistance of the antibody-functionalized graphene layer. The analyzer can then employ the calculated resistance, e.g., a change in the resistance in response to exposure of the antibody-functionalized graphene layer to a sample under investigation, to determine, in accordance with the present teachings, whether the sample contains an immunogen of interest. 
     By way of example, as shown schematically in  FIG. 8 , in this embodiment, the analyzer  600  can include a processor  602 , an analysis module  604 , a random access memory (RAM)  606 , a permanent memory  608 , a database  610 , a communication module  612 , and a graphical user interface (GUI)  614 . The analyzer  600  can employ the communication module  612  to communicate with the voltage measuring circuitry  701  to receive the values of the applied current and the measured voltage. The communication module  612  can be a wired or a wireless communication module. The analyzer  600  further includes a graphical user interface (GUI)  614  that allows a user to interact with the analyzer  600 . 
     The analysis module  604  can employ the values of a current applied to the antibody-functionalized graphene layer as well as the voltage induced across the graphene layer to calculate a change in the resistance of the antibody-functionalized graphene layer in response to exposure thereof to a sample under investigation. The instructions for such calculation can be stored in the permanent memory  608  and can be transferred at runtime to RAM  606  via processor  602  for use by the analysis module  604 . In some embodiments, the database  610  can store calibration data that can be employed for determining whether a pathogen of interest is present in a sample under study. By way of example, the database  610  can store calibration data indicative of a temporal change in the electrical resistance of an antibody-functionalized graphene layer in response to exposure to a particular pathogen. A comparison of a measured temporal variation of a similar antibody-functionalized graphene exposed to a sample suspected of containing the pathogen with the calibrated response can be used to determine whether the pathogen is present in the sample. The GUI  614  can allow a user to interact with the analyzer  600 . 
     By way of example, a suitable voltage-measuring module is disclosed in U.S. Pat. No. 9,664,674, which is herein incorporated by reference in its entirety. 
     In another embodiment, a constant DC voltage can be applied across the functionalized graphene layer via a DC voltage source and a change in the current flowing through the functionalized graphene layer can be measured to determine whether THC is present in a sample under test. The DC voltage source and the circuitry for measuring the current can be implemented using known methods as informed by the present teachings. 
     With reference to  FIGS. 5A and 5B , in some embodiments, a sensor  5000  according to the present teachings can include a hydroxyl-functionalized graphene layer  5002  that is further functionalized with anti-THC antibodies via a molecule containing an aldehyde moiety. 
     More specifically, with reference to  FIG. 5B , in this embodiment, the hydroxyl-functionalized graphene layer  5002  can be incubated with 2% 3-Aminopropyl triethoxysilane (APTES) in 95% ethanol for 1 hour to allow for aqueous silanization of the surface. The graphene layer can then be incubated in 2.5% glutaraldehyde in milli-Q water for a few hours (e.g., for 2 hours). This incubation can create aldehyde groups (—COH), which can react with amine groups (—NH 2 ) of the antibody, e.g., via a covalent bond, thus coupling the antibody to the hydroxyl-functionalized graphene layer. 
     Similar to the previous embodiment, in this embodiment, the graphene layer can be initially deposited on an underlying substrate  5004 . The underlying substrate  5004  can be, for example, a semiconductor, such as silicon, or a polymeric substrate, e.g., plastic. 
     Though not shown in  FIG. 5B , similar to the above sensor  1000 , the sensor  5000  includes metallic pads that can allow application of an electrical signal (e.g., a current or a voltage) to the antibody-functionalized graphene layer and monitor at least one electrical property of the antibody-functionalized graphene layer, e.g., its DC electrical resistance. 
     In some embodiments, a sensor according to the present teachings can be employed by law enforcement officials as an on-site testing device against illicit use of  cannabis . In particular, in some such embodiments, a sensor according to the present teachings can detect not only Δ-9-THC but also one or more of its metabolites. 
     By way of example, with reference to  FIG. 1 , in some embodiments, the graphene layer  1004  can be functionalized with an antibody that exhibits binding not only to Δ-9-THC, or Δ-8-THC but also to one or more of its metabolites, such as hydroxylated and/or carboxylated metabolites. By way of example, a monoclonal antibody marketed by MybioSource of San Diego, Calif. under catalogue number MBS310888 can be used. The specification of this antibody indicates that it recognizes THC and its metabolites and does not cross-react with Opiates, Cocaine Metabolite, Amphetamines and Phencyclidine. Table 1 below provides examples of other antibodies that can be employed in the practice of the present teachings for detecting THC, e.g., Δ-9-THC, or Δ-8-THC and their metabolites. 
     
       
         
           
               
               
               
             
               
                   
                 TABLE 1 
               
               
                   
                   
               
               
                   
                 Source 
                 Catalogue # 
               
               
                   
                   
               
             
            
               
                   
                 Abbexa (Cedarlane) 
                 ABX021068-1MG 
               
               
                   
                 Abbexa (Cedarlane) 
                 ABX023622-1MG 
               
               
                   
                 Abbexa (Cedarlane) 
                 ABX120099-1MG 
               
               
                   
                 Abbexa (Cedarlane) 
                 ABX021066-1MG 
               
               
                   
                 Abbexa (Cedarlane) 
                 ABX021067-1MG 
               
               
                   
                   
               
            
           
         
       
     
     It is noted that the antibody under the catalogue number ABX021068-1MG recognizes 8-THC-BSA. Thus, in some embodiment in which the detection of 8-THC-BSA is desired, a sample under study is initially processed so as to conjugate 8-THC to BSA (bovine serum albumin) and then introduced into a sensor according to the present teachings. 
     In some embodiments, a blood or a urine, or a  salvia  sample can be introduced into a sensor according to the present teachings for the detection of Δ-9-THC and its metabolites. By way of example, a saliva sample can be obtained using a swab, or any other suitable device for collecting saliva from an individual, and can be subsequently placed in a suitable liquid, such as a phosphate buffer solution, to prepare a sample for testing by a sensor according to the present teachings. 
     In some embodiments, a sensor according to the present teachings can exhibit a limit of detection of about 20-100 ng/ml. 
     The two primary metabolic products of Δ-9-THC include psychoactive metabolite 11-OH-THC and non-psychoactive metabolite 11-COOH-THC. First-pass metabolism (in the liver or Phase I metabolism) also generates to a lesser degree 8β-0H-THC (epoxy-hexahydrocannabinol) and 8α-OH-THC (8-keto-THC) as part of the process. The liver (cytochrome P450 enzymes) breaks down Δ-9-THC into these hydroxylated and carboxylated metabolites: 
     
       
         
         
             
             
         
       
     
     When  cannabis  is smoked, there is typically a steep increase of Δ-9-THC in the circulating blood after about 10 minutes of consumption while the blood concentration of 11-OH-THC peaks slightly later, at about 15 minutes after consumption. Subsequently, the levels of both Δ-9-THC and 11-OH-THC drop sharply between 20-30 mins and their concentrations typically fall below 0.5 ng/ml (limit of detection) after 1 hr. The blood (plasma) concentration of the non-psychoactive metabolite THC-COOH peaks later between 45-60 min after consumption and circulates in the blood stream for much longer (e.g., on average 7 days, but can be up to 12 days in chronic users), before falling below the detection limits. 
     The bioavailability of inhaled Δ-9-THC is typically in a range of about 10-35%, (actually can be as much as 2-56% due to variability in subject smoking dynamics), but can vary significantly among different individuals. For example, regular users can exhibit a bioavailability that is 50-70% greater than that exhibited by infrequent users. 
     When  cannabis  is consumed orally, Δ-9-THC can enter the blood stream via the stomach and/or intestines due to its high absorption rate (octanol/water coefficient), e.g., 90% to 95%. After the first-pass through the liver, most of Δ-9-THC is either degraded in the stomach or metabolized into the respective hydroxylated and oxidation forms. Although to a lesser degree, Δ-9-THC metabolism has also been reported in other tissues such as brain, intestines, and lungs using redundant physiological processes. 
     The bioavailability of oral Δ-9-THC administration is on average between 4-20%. The timing to peak concentration of Δ-9-THC in (plasma) blood can vary (widely) among different individuals, e.g., from about 1 hour to about 6-7 hours, but usually take 2 to 4 hours. 
     During the secondary phase (Phase II) of THC metabolism, the hydroxylated and carboxylated metabolic products undergo a conjugation reaction with glucuronic acid. This process allows the glucuronide-bound THC, 11-OH-THC, and THC-COOH to become much more water-soluble, which help with distribution and elimination by urine and feces. 
     In some embodiments, to determine the concentrations of THC and its metabolites in blood, urine, feces, and other tissues following in vivo administration, a sample obtained from an individual is processed to release THC from glucuronide-bound THC prior to introduction of the sample onto a sensor according to the present teachings. 
     In many embodiments, the samples can be processed using one (or both) of the following processes: enzymatic-hydrolysis (cleaving) with Beta-glucuronidase or alkaline hydrolysis (cleaving) with sodium hydroxide (NaOH). 
     With reference to  FIG. 9 , in some embodiments, an AC source  9000  can be employed to apply an AC voltage or current, e.g., with a frequency in a range of about 100 Hz to about 2 MHz, e.g., 1 kHz to 1 MHz, across an antibody-functionalized graphene layer  9002 , such as a graphene layer functionalized with antibodies exhibiting specific binding to THC and/or one of its metabolites. The AC voltage or current can also provide a reference signal to a lock-in amplifier  9001  whose input port receives a signal associated with the functionalized graphene layer in response to the application of AC voltage or current (e.g., AC voltage or current depending on the signal applied to the functionalized graphene layer). The output of the lock-in amplifier can be used to determine whether THC and/or one or more of its metabolites are present in a sample under study. 
     In some embodiments, the graphene layer of a sensor according to the present teachings can be functionalized with an antibody fragment that exhibits specific binding to Δ-9-THC and/or one or more of its metabolites. For example, an article entitled “Antibody fragments for on-site testing of cannabinoids generated via in vitro affinity maturation,” published in Bio Pharm Bull, 2017; 40(20: 174-181 by Morita et al., which is herein incorporated by reference, discloses the use of an in vitro affinity maturation technique to generate a single-chain Fv fragment (scFv) that recognizes with high affinity Δ9-tetrahydrocannabinol (THC). 
     In particular, Morita et al. explain that mouse monoclonal antibody against THC, Ab-THC#33, with K a  6.2×107 M-1 (as Fab fragment) was established by the hybridoma technique. Then, a “wild-type” scFv (wt-scFv) with K a , 1.1×107 M-1 was prepared by bacterial expression of a fusion gene combining the V H  and V L  genes for Ab-THC#33. Subsequently, random point mutations in V H  and V L  were generated separately, and the resulting products were assembled into mutant scFv genes, which were then phage-displayed. Morita et al. further explains that repeated panning identified a mutant scFv (scFv#m1-36) with 10-fold enhanced affinity (K a  1.1×108 M-1) for THC, in which only a single conservative substitution (Ser50Thr) was present at the N-terminus of the V H -complementarity-determining region 2 (CDR2) sequence. Morita et al. also indicate that in competitive enzyme-linked immunosorbent assay (ELISA), the mutant scFv generated dose-response curves with midpoint 0.27 ng/assay THC, which was 3-fold lower than that of wt-scFv. Even higher reactivity with a major THC metabolite, 11-nor-9-carboxy-Δ9-tetrahydrocannabinol, indicated that the mutant scFv will be useful for testing not only THC in confiscated materials, but also the metabolite in urine. Indeed, the antibody fragment is potentially suitable for use in advanced on-site testing platforms for cannabinoids. 
     In some embodiments, a sensor according to the present teachings can be functionalized with anti-cannabidiol (CBD) antibodies. In such embodiments, a graphene layer of the sensor can be functionalized with anti-CBD antibody. An example of an anti-CBD antibody suitable for use in the practice of the present teachings is marketed by Abbiotec of Escondido, Californian, under catalogue #2002223. 
     In some embodiments, a sensor according to the present teachings can include a plurality of graphene-based sensing elements according to the present teachings. By way of example,  FIG. 10  schematically depicts such a sensor  2000  having a plurality of sensing elements  2001 ,  2002 ,  2003 , and  2004 . Each of the sensing elements includes a graphene layer functionalized with an anti-THC or anti-CBD antibody and has a structure similar to that discussed above in connection with sensor shown in  FIG. 1  above. 
     In some embodiments, the signals generated by the sensing elements can be averaged to generate a resultant signal. Further, in some embodiments, at least one of the sensing elements can be configured as a calibration sensing element to allow quantification of THC and/or CBD present in a sample. By way of example, the calibration can be achieved by utilizing a calibrated sample and detecting a change in at least one electrical property of the functionalized graphene layer in response to exposure to the calibration sample. 
     The following example is provided for further elucidation of various aspects of the invention and is not intended to provide necessarily the optimal ways of practicing the present teachings or optical results that can be obtained. 
     Example 
     A sensor based on the design depicted in  FIG. 1  was fabricated. The conductive pads were formed of silver high conductive paste purchased from MG Chemicals of Canada. The high conductive paste was employed to electrically connect the graphene layer to a measurement circuit for detecting the electrical response of the graphene layer to a test sample containing THC. 
     The graphene layer was functionalized with an antibody marketed by Fitzgerald Industries (#10-T43B). The functionalization process included coupling a plurality of linker molecules to the graphene layer at one end thereof and coupling the antibody molecules to the other end of the linker molecules. In this example, the linker molecule was 1-pyrenebutonic acid succinimidyl ester. The procedures for attaching the linker molecules to the graphene layer and coupling antibody molecules to the linker molecules described in U.S. Pat. No. 9,664,674, which is herein incorporated by reference in its entirety, were followed. 
     A sample of THC was extracted from a cartridge of an electrical cigarette available over counter. The sample was observed to be very viscous with dark yellowish color. The sample was completely dissolved in pure ethanol and was agitated in a sonicator for about 2 minutes. The solution was then diluted in TBS (Tris-buffered saline) buffer to 60%. Upon dilution, a cloudy liquid was formed. The cloudy liquid sample was then centrifuged for about 5 minutes to separate the cloudy phase and was diluted in TBS buffer 1 to 10 ratio. The clear liquid was then applied to six sensors in accordance with the following protocol. 
     1) A portion of the THC sample was applied to 3 sensors (Group 1). 
     2) An irrelevant protein, i.e., gliadin, sample prepared in TBS/Ethanol solution was applied to the other 3 sensors (Group 2). 
     3) The electrical resistance of the sensors in both Group 1 and Group 2 was measured. 
     4) The sensors in Group 2 were exposed to the THC sample for 3 minutes and the electrical measurement of the resistance was repeated for Group 2 sensors. 
     The electrical resistance of the sensors was monitored using a four-probe based circuit such as that described in the aforementioned U.S. Pat. No. 9,664,674 and data was recorded on the connected analyzer.  FIG. 11  shows the results of the measurements in terms of percentage change in measured current when the sensors were exposed to THC and irrelevant antigen samples. 
     The above results show the detection of THC in the prepared sample. Further, the sensors in Group 2 exhibited low reaction to the irrelevant antigen (e.g., gliadin molecules soluble in Ethanol). The sensors in Group 2, however, exhibited a response commensurate with that observed for Group 1 sensors when they were exposed to THC samples. 
     Those having ordinary skill in the art will appreciate that various changes can be made to the above embodiments without departing from the scope of the invention.