Patent Publication Number: US-2022213528-A1

Title: Methods and devices for detection of pathogens

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS 
     This application is a continuation of U.S. patent application Ser. No. 16/260,773, filed on Jan. 29, 2019, entitled “Methods And Devices For Detection Of Pathogens,” which claims the benefit of U.S. Provisional Application No. 62/623,038, entitled “Methods and Devices for Detection for  Listeria Monocytogenes  Bacterium,” and filed on Jan. 29, 2018; U.S. Provisional Application No. 62/676,079, entitled “A Graphene Functionalized Sensor,” and filed on May 24, 2018; and U.S. Provisional Application No. 62/703,702, entitled “Methods and Devices for Detection of Pathogens,” and filed on Jul. 26, 2018, all of which are herein fully incorporated by reference in its respective entirety. 
    
    
     BACKGROUND 
     The present invention relates generally to methods and devices for the detection of pathogens, such as bacteria and viruses, in a sample, e.g., a food sample. 
     Pathogens can cause a variety of diseases and can infect subjects through a myriad of modalities, e.g., food supply or contact with an infected individual. By way of example,  Listeria monocytogenes  is a Gram-positive pathogenic bacterium that causes the infection listeriosis. It is a facultative anaerobic bacterium, which is capable of surviving in the presence or absence of oxygen. It is one of the most virulent foodborne pathogens and can lead to fatality, especially in vulnerable populations. Its ability to grow at typical refrigeration temperatures greatly enhances its ability to evade control in human foodstuffs. Conventional methods for detecting  Listeria  in food samples can be time-consuming and cumbersome. Similar problems are presented in detection of other pathogens, such as  Chlamydia  and gonorrhea. 
       Chlamydia  is a genus of pathogenic bacteria that are obligate intracellular parasites. For example,  Chlamydia trachomatis  ( C. trachomatis ) is responsible for causing blindness of millions of infected patients worldwide. Gonorrhea is a sexually transmitted infection (STI) caused by the bacterium  Neisseria gonorrhoeae.  Other pathogenic agents, such as viruses, can also cause a variety of diseases. For example, human papillomavirus infection is an infection caused by human papillomavirus (HPV). In some patients, an HPV infection can result in warts or precancerous lesions. 
     Accordingly, there is a need for improved methods and systems for detecting pathogenic agents. 
     SUMMARY 
     In one aspect, a method of detecting a pathogen, e.g.,  Listeria  bacterium,  Chlamydia  bacteria, gonorrhea bacteria and/or HPV, in a sample is disclosed, which comprises bringing a sample into contact with a graphene layer functionalized with an antibody exhibiting specific binding affinity to the pathogen, monitoring electrical resistance of said antibody-functionalized graphene layer in response to interaction with said sample, and detecting presence of the pathogen in said sample by detecting a change in said electrical resistance indicative of interaction of the pathogen with said antibody-functionalized graphene layer. By way of example, a change in the electrical resistance of the antibody-functionalized graphene layer that exceeds a predefined threshold can indicate the presence of a pathogen of interest in a sample under study. For example, a decrease of the electrical resistance of the graphene layer can indicate the presence of the pathogen in the sample under study. In some embodiments, a temporal pattern of change in the resistance of the underlying antibody-functionalized graphene layer in response to its exposure to a sample under study can be correlated with the presence of a particular pathogen in the sample under study. By way of example, in some such embodiments, the detected temporal pattern of change in the resistance of the underlying antibody-functionalized graphene layer can be compared with a calibration pattern to determine whether a pathogen of interest is present in the sample under study. 
     In some embodiments, a method according to the present teachings is capable of detecting pathogens, such as  Listeria  bacteria,  Chlamydia  bacteria, gonorrhea bacteria and HPV in a sample at a concentration as low as 4 cfu (colony forming units) per 100 grams of a sample. 
     In some embodiments, the graphene layer is incorporated in a device that comprises a substrate, e.g., a glass or a semiconductor substrate, on which the graphene layer is disposed. A pair of electrically conductive pads can be in electrical contact with the graphene layer to facilitate the measurement of the electrical resistance of the graphene layer in response to interaction with a sample under study. In some embodiments, such a device can include a microfluidic structure that is coupled to said semiconductor substrate, where the microfluidic structure has at least one reservoir and a fluidic channel fluidly coupled to the reservoir. The fluid channel can be in fluid communication with at least a portion of the graphene layer, and the reservoir can be configured for receiving a sample. 
     In a related aspect, a sensor for detecting a pathogen in a sample is disclosed, which comprises a substrate, a graphene layer deposited on a surface of the substrate, where the graphene layer is functionalized with a plurality of antibodies exhibiting specific binding affinity to the pathogen. A reference electrode is disposed in proximity of the antibody-functionalized graphene layer. The sensor can further comprise a microfluidic delivery device coupled to the functionalized graphene layer for delivery of a fluid sample thereto. In some embodiments, the microfluidic device can comprise two fluid reservoirs and a fluid channel connecting said two reservoirs. An AC voltage source can be utilized to apply an AC voltage to the reference electrode to facilitate the detection of a change in at least one electrical property of the antibody-functionalized graphene layer, e.g., its DC resistance, in response to the interaction of the pathogen with that layer. By way of example, the applied AC voltage can have a frequency in a range of about 1 kHz to about 1 MHz and an amplitude in a range of about 1 millivolt to about 3 volts. 
     In a related aspect, a system for detecting pathogenic agents in a sample is disclosed, which comprises a sensor configured for detecting at least two pathogenic agents. The system comprises a substrate, and a plurality of sensing units disposed on said substrate, where each of said sensing units comprises an antibody-functionalized graphene layer disposed on said substrate. At least two of said sensing units comprise antibodies configured for specific binding to at least two different pathogens. 
     In some embodiments, the above system includes at least one reference electrode for application of an AC signal to at least one of the sensing units. In some such embodiments, a plurality of reference electrodes are provided, each of which is configured for application of an AC signal to one of the sensing units. By way of example, the AC signal can have a frequency in a range of about 1 kHz to about 1 MHz and an amplitude in a range of about 1 millivolt to about 3 volts. 
     In some embodiments, the system can further include a device for measuring the resistance of the underlying graphene layer for detecting a change, if any, in the resistance of the underlying graphene layer in response to its exposure to a sample under investigation. By way of example, the system can include at least one voltage measuring device for measuring a voltage developed across one or more of said sensing units in response to application of a predefined current thereto. An analyzer in communication with the voltage measuring device can receive the measured voltage and utilize the measured voltage together with the predefined current applied to a sensing unit to determine the resistance of the graphene layer associated with that sensing unit. The analyzer can further determine whether the calculated resistance of the sensing unit is indicative of the presence of a pathogen of interest in a sample that was brought into contact with the sensing unit, for example, in a manner discussed above. 
     In some embodiments, a multiplexer can be disposed between the voltage measuring device and the sensing units to allow sequential measurement of the voltages developed across the sensing units of the sensor in response to application of a predefined current thereto. 
     In some embodiments, a sensor according to the present teachings includes a plurality of sensing units, each of which is functionalized with a different antibody exhibiting specific binding affinity to a different pathogen. A microfluidic device is coupled to the sensing units for dividing a sample among the sensing units. The microfluidic device can include an input port for receiving a fluid sample, an input manifold for dividing the sample among a plurality of channels each of which is in communication with one of the sensing units. A return manifold can receive the fluid exiting the sensing units and can return the fluid to the input manifold to provide a closed-loop circulation path. In some cases, rather than utilizing a single return path, each sensing unit can include its dedicated fluid return path. In some embodiments, one or more pumps can be employed to facilitate the circulation of the sample through the microfluidic channels. 
     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 THE DRAWINGS 
         FIG. 1A  is a schematic top view of a sensor system according to an embodiment of the present teachings for detecting a pathogen in a sample, 
         FIG. 1B  is a schematic cross-sectional view of the system depicted in  FIG. 1A , 
         FIG. 2  is a schematic depiction of an antibody-functionalized graphene layer employed in a device according to the present teachings, 
         FIG. 3  schematically depicts a sensor according to an embodiment for detecting pathogens in a sample, where the sensor includes a reference electrode to which an AC voltage can be applied, 
         FIG. 4A  schematically depicts a functionalized graphene layer in which a plurality of linker molecules are employed for coupling antibodies to the graphene layer, 
         FIG. 4B  schematically depicts the use of a passivation layer to cover the portions of the graphene layer that are not coupled to an antibody molecule, 
         FIG. 5  depicts a circuit diagram of an example of a voltage-measuring device that can be employed for measuring a voltage induced across an antibody-functionalized graphene layer in response to application of a current thereto, 
         FIG. 6A  schematically depicts an analyzer in communication with the voltage-measuring device shown in  FIG. 5  for receiving the voltage measured by the voltage-measuring device as well as the current applied to the antibody-functionalized graphene layer, 
         FIG. 6B  depicts an example of implementation of the analyzer shown in  FIG. 6A , 
         FIG. 7  schematically depicts a sensor according to an embodiment, which includes a plurality of sensing units configured to detect different types of pathogens, 
         FIG. 8  schematically depicts a microfluidic device that is coupled to the sensor shown in  FIG. 7  for delivering a liquid sample thereto, 
         FIG. 9  schematically depicts a sensor according to an embodiment of the present teachings, which includes a plurality of sensing unit having graphene layers functionalized with different antibodies and a microfluidic device for introducing a sample onto the sensing units, and 
         FIG. 10  presents data indicative of detection of  Listeria  using an embodiment of the present teachings. 
     
    
    
     DETAILED DESCRIPTION 
     It has been discovered that an anti-body functionalized graphene layer can be employed to detect the presence of pathogenic agents, such as  Listeria monocytogene  bacterium (herein referred to as “ Listeria ”) in a sample, for example, a food sample, a biological sample, such as urine and saliva. In particular, the interaction of a pathogen in a sample under investigation with the antibody-functionalized graphene layer can cause a change in at least one electrical property of the underlying graphene layer, e.g., a change in the electrical resistance of the underlying graphene layer. A detection of such a change in the electrical property of the underlying graphene layer can be employed to detect the presence of the pathogen in the sample under study. In some embodiments, the detection of a pathogen via an antibody-functionalized graphene layer can be facilitated via application of an AC signal to a reference electrode disposed in proximity of the graphene layer, e.g., positioned at a distance in a range of about 50 microns to about a few millimeter (e.g., 1-2 mm) from the graphene layer. By way of example, the frequency of the AC signal applied to the reference electrode can be in a range of about 1 kHz to about 1 MHz and the amplitude of the AC signal can be in a range of about 1 millivolt to about 3 volts. 
     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 the numerical value. 
     An “antibody”, as that term is used herein, refers to a polypeptide exhibiting 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 single 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. 
     In many embodiments, an antibody is a glycoprotein produced by B lymphocytes in response to stimulation with an immunogen. An antibody can be composed of 4 polypeptides—2 heavy chains and 2 light chains—bound together by disulfide bonds to form a Y-shaped molecule. 
       FIGS. 1A and 1B  schematically depict an example of a device  100  according to an embodiment of the present teachings for detecting a pathogenic agent in a sample. The device  100  includes a substrate  12  on a top surface of which a layer of graphene  14  is deposited. A variety of different substrates can be employed. By way of example, the substrate  12  can be any of a semiconductor, such as silicon, or glass or plastic. In some embodiments in which the substrate  12  is formed of silicon, a layer of silicon oxide can separate the upper graphene layer from the underlying silicon layer. 
     As shown schematically in  FIG. 2 , in this embodiment, the graphene layer  14  is functionalized with an antibody  16  that can specifically bind to a pathogen of interest. By way of example, in some embodiments, the graphene layer can be functionalized with  Listeria monocytogenes  antibody LZF7 (BGN/0884/67), which is an IgG2a mouse anti- Listeria monocytogenes  monoclonal antibody. In another embodiment, the graphene layer can be functionalized with an anti- Chlamydia  antibody, such as monoclonal or polyclonal antibodies. A number of anti- Chlamydia  antibodies are commercially available. For example, anti- Chlamydia  antibody marketed by Genetex under Cat#GTX40387 can be employed. In another embodiment, the graphene layer can be functionalized with anti-gonorrhea antibodies, e.g., monoclonal or polyclonal antibodies. For example, anti-gonorrhea antibody marketed by Creative Diagnostics under Cat#DMZB9759 can be employed. Yet, in another embodiment, the graphene layer can be functionalized with anti-HPV (human papilloma virus) antibodies. For example, anti-HPV antibody marketed by Abcam under Cat#ab2417 or by Thermo Fisher under Cat#MA5 12446 can be employed. 
     With reference to  FIG. 4A , in some embodiments, the antibodies  16  are coupled to the underlying graphene layer via a linker  18 , where the linker is covalently attached at one end thereof to the graphene layer. The antibodies can be attached to the other end of the linker, e.g., via a covalent bond. By way of example, in some embodiments, 1-pyrenebutonic acid succinimidyl ester can be employed as the linker to facilitate the coupling of the antibody molecules to the underlying graphene layer. It has been discovered that 1-pyrenebutonic acid succininmidyl ester can be used to couple a variety of different antibodies to the underlying graphene layer. As such, this linker can be used for coupling antibodies that specifically bind to a variety of different pathogens to the underlying graphene layer, where the pathogen-antibody interaction can mediate a change in one or more electrical properties of the underlying graphene layer. 
     In some embodiments, the graphene layer can be incubated with the linker molecule (e.g., a 5 mM solution of 1-pyrenebutonic acid succimidyl ester) for a few hours (e.g., 2 hours) at room temperature to ensure covalently coupling of the linker molecules to the underlying graphene layer. The linker modified graphene layer can then be incubated with an antibody of interest in a buffer solution (e.g., NaCO 3 —NaHCO 3  buffer solution (pH 9)) at a selected temperature and for a selected duration (e.g., 7-10 hours at 4° C.), followed by rinsing with deionized (DI) water and phosphate buffered solution (PBS). In order to quench the unreacted succinimidyl ester groups, the modified graphene layer can be incubated with ethanolamine (e.g., 0.1 M solution at a pH of 9 for 1 hour). 
     Subsequently, the non-functionalized areas of the graphene layer can be passivated via a passivation layer  20 , as shown in  FIG. 4B . By way of example, the passivation of the non-functionalized portions of the graphene layer can be achieved, e.g., via incubation with 0.1% Tween 20 or BLOTTO, gelatin and/or amino-PEGS-alcohol (pH 7.4). Further details regarding the use of linkers for attaching antibody molecules to a graphene layer and passivating the non-functionalized portions of the graphene layer can be found, e.g., in U.S. Pat. No. 9,664,674 B2, which is herein incorporated by reference in its entirety. 
     Referring again to  FIGS. 1A and 1B , two metallic pads  22 / 24  in electrical contact with the graphene layer  14  allow measuring the electrical resistance of the graphene layer, and particularly, a change in the electrical resistance of the graphene layer in response to exposure thereof to a sample containing a pathogen, e.g.,  Listeria, Chlamydia,  gonorrhea bacteria and/or HPV. 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. 
     The device  100  further includes a microfluidic structure  1008  having two reservoirs  1008   a/   1008   b  and a fluid channel  1008   c  that fluidly connects the two reservoirs. As shown more clearly in  FIG. 1B , the fluid channel can be arranged such that a portion thereof is in fluid contact with a portion of the graphene layer  14 . 
     In some embodiments, in use, a sample suspected of containing a pathogen of interest, e.g.,  Listeria  bacteria, can be introduced into one of the reservoirs  1008   a/   1008   b  and can be made to flow, e.g., via application of hydrodynamic pressure thereto, to the other reservoir through the microfluidic channel  1008   c.  In this embodiment, a pump (such as pump  3010  depicted in the embodiment of  FIG. 3 ) can be coupled to reservoir  1008   b  to facilitate the flow of the sample to the other reservoir. In other embodiments, the pump may be coupled to the other reservoir and/or to a fluid channel connecting those reservoirs. The passage of the sample through the channel  1008   c  brings the pathogen, if any, present in the sample into contact with the antibody-functionalized graphene layer  14 . Without being limited to any particular theory, the interaction of the pathogen ,e.g.,  Listeria  bacteria with the antibodies to which they can bind can mediate a change in the electrical conductivity (and hence resistance) of the underlying graphene layer  14 , e.g., via charge transfer or other mechanisms. This change in the electrical conductivity of the graphene layer  14  can in turn be measured to detect the presence of the pathogen in the sample under study. 
     In some embodiments, a four-point measurement technique can be used to measure the resistance of the antibody-functionalized graphene layer in response to exposure thereof to a sample under investigation. 
     By way of example,  FIG. 5  schematically depicts a voltage measuring circuitry  701  that can be employed in some embodiments of the present teachings. This figures shows a sensor  702  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 VR 1  ground via a resistor R 1 . The output of the operational amplifier  704  (Voutt) is coupled to one end of the sensor  702  and the end of the resistor R 1  that is not connected to VR 1  ground 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. 6A , 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 a pathogen of interest. 
     By way of example, as shown schematically in  FIG. 6B , 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 . 
     Referring to  FIG. 3 , in some embodiments, the sensor  100  according to an embodiment can include a reference electrode  3001  disposed in proximity of the antibody-functionalized graphene layer  14 , e.g., at a distance in a range of about 50 micrometers to about a few millimeters (e.g., 1-2 millimeters) above the functionalized graphene layer. In some embodiments, the distance of the reference electrode relative to the functionalized graphene layer  14  can be in a range of about 100 microns to about 1 millimeter, or in a range of about 200 microns to about 0.5 millimeter. Further, in some embodiments, rather than being positioned above the graphene layer, the reference electrode can be positioned in the same plane as the graphene layer. 
     The reference electrode can be utilized to generate a time-varying electric field at the interface of the functionalized graphene layer and a liquid sample, e.g., a liquid sample suspected of containing one or more pathogens, that is brought into 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. 
     The AC reference electrode  3001  can be formed of any suitable electrical conductor. Some examples of suitable conductors include, without limitation, silver, copper, and gold. In some embodiments, the thickness of the reference electrode  3001  can be, for example, in a range of about 100 nm to about 400 micrometers (microns), e.g., in a range of about 1 microns to about 100 microns, though other thicknesses can also be employed. 
     The application of such a time-varying electric field via the reference electrode to the interface between the graphene layer  14  and a liquid sample 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 pathogen present in the sample that exhibits specific binding to the antibody of the functionalized graphene layer. 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 1 MHz, e.g., in a range of about 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. 
     Without being limited to any particular theory, in some embodiments, it is expected that the application of such a 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 a respective pathogen. In some cases, the effective capacitance of the sample can be due to ions present in the sample. 
     The present teachings can be applied to detect a variety of pathogens, such as those discussed above, in a variety of different samples. Some examples of samples that can be interrogated include, without limitation, food samples and bodily fluids, such as blood, urine, saliva, etc. 
     In some embodiments, a sensor according to the present teachings is capable of detecting pathogens in a variety of different sample types including, without limitation, urine, mucous and/or blood. In some cases, e.g., when the detection of  Chlamydia  is desired, the sample can be obtained by a swab, e.g., an endocervical swab. Other methods known in the art for obtaining samples can also be utilized. 
     With reference to  FIG. 7 , in some embodiments, a graphene-based sensor  800  according to the present teachings can include a plurality of sensing units  802   a,    802   b,    802   c,  and  802   d,  (herein collectively referred to as sensing units  802 ), each of which is configured for the detection of a different pathogen. In this embodiment, the sensing units  802  are formed on a single underlying substrate  804 , e.g., a semiconductor (such as silicon), glass or plastic. Each sensing unit includes an antibody-functionalized graphene layer, fabricated in a manner discussed above. In this embodiment, at least two sensing units are functionalized with two different antibodies suitable for the detection of two different pathogens. For example, one sensing unit can be functionalized with an antibody that specifically binds to  Listeria  bacterium and another sensing unit can be functionalized with an antibody that specifically binds to  Chlamydia  bacterium. In some embodiments, each of the sensing units is functionalized with an antibody suitable for the detection of a different pathogen. By way of example, the number of sensing units can range from 2 to 20, though other numbers can also be employed. 
     The sensing units  802  are electrically insulated from one another via an electrically insulating network  803 . By way of example, the electrically-insulating network  803  can be in the form of a plurality of electrically-insulating strips formed, e.g., of silicon oxide (such as SiO 2 ), which can electrically isolate different sensing units from one another. Further, in this embodiment, each of the sensing units  800  includes a reference electrode  806   a,    806   b,    806   c,  and  806   d  (herein referred to collectively as reference electrodes  806 ) to which AC signals can be applied, e.g., in a manner discussed above, to facilitate the detection of a pathogen of interest. While in some embodiments, the frequency and amplitude of the AC signal applied to the different sensing units can be the same, in other embodiments, the AC signals applied to at least two different sensing units can exhibit different frequencies and/or amplitudes. 
     In some embodiments, rather than employing multiple reference electrodes, a single reference electrode can be employed for applying a reference AC voltage to multiple, or all, of the sensing units  802 . The frequency and the amplitude of the applied reference AC voltage can be, for example, in the ranges discussed above. 
     With continued reference to  FIG. 7 , a voltage measuring device  808  can be employed to measure a voltage across each of the sensing units in response to application of a predefined constant current thereto, e.g., in a manner discussed above in connection with the voltage measuring device  701 . In this embodiment, a multiplexer  810  can be employed to selectively measure the voltage induced across the sensing units in response to application of a predefined current thereto, e.g., in a time-wise sequential manner. An analyzer  812 , such as the above analyzer  600 , can receive the voltage data from the voltage measuring device  808  and can calculate the change in the electrical resistance of the underlying graphene layer to determine whether one or more of the pathogens are present in the sample under study. 
     With reference to  FIG. 8 , in some embodiments, a microfluidic device  900  can be fluidly coupled to the sensor  800  for delivering a liquid sample suspected of containing one or more pathogens of interest to the sensor  800 . In this embodiment, the microfluidic device  900  includes a body  902  having an input port  903  for receiving a sample. Each of a plurality of internal channels  904   a,    904   b,    904   c,    904   d  and  904   e  (herein collectively referred to as channels  904 ) in fluid communication with the input port  903  can receive a portion of the sample via the input port  903  and guide those portions to output ports  906   a,    906   b,    906   c,  and  906   d  (herein collectively referred to as output ports  906 ), which in turn deliver those sample portions to the sensing units for analysis. 
       FIG. 9  schematically depicts another embodiment of a sensor  1000  according to the present teachings, which includes sensing units  1002 ,  1004 , and  1006  are disposed. Each of the sensing units  1002 ,  1004 , and  1006  includes a graphene layer that is functionalized with an antibody that exhibits specific binding for a different pathogen, in a manner discussed above. In this embodiment, the graphene layers of the sensing units  1002 ,  1004 , and  1006  are deposited on the top surface of a single underlying substrate  1007 , e.g., a silicon substrate. In this embodiment, the sensing units are separated from one another by a plurality of electrically insulating strips  1009 , which can be formed, e.g., from silicon oxide. 
     With continued reference to  FIG. 9 , in this embodiment, a microfluidic device  1010  is coupled to the sensing units  1002 ,  1004 , and  1006  for delivering a sample under investigation to the sensing units. In this embodiment, the microfluidic device  1010  includes three fluidic channels  1011 ,  1013 , and  1015 , e.g., each of which has a diameter in a range of about 10 microns to about 100 microns and is in fluid communication with the antibody-functionalized graphene layer of one of the sensing units. The channels  1011 ,  1013 , and  1015  are fluidically coupled via an input manifold  1017  to an input port  1019  through which a sample can be introduced into the microfluidic device  1010 . The input manifold  1017  can distribute a portion of the received sample to each of the channels  1011 ,  1013  and  1015 . The channels  1011 ,  1013  and  1015  terminate in an return manifold  1020  that is in turn fluidly coupled to a return channel  1021 , which can direct the fluid back to the input manifold, thereby providing closed-loop fluid circulation. A input pump  1022 , e.g., a gear pump, can facilitate the flow of the sample into the input manifold, and a return pump  1024  can facilitate the return of the fluid exiting the channels  1011 ,  1013 , and  1015  back to the input manifold  1017 . 
     In some embodiments, rather than employing a single return microfluidic channel, a plurality of return microfluidic channels, each associated with one of the sensing units, can be employed. 
     Similar to the previous embodiment, a change of the electrical resistance of the antibody-functionalized graphene layer of each sensing unit can be measured and correlated with the presence of a particular pathogen for which that sensing unit is configured. 
     Although in the above embodiments, the resistance of the antibody-functionalized graphene layer is measured via application of a predefined current to the graphene layer following by measuring a voltage induced across the graphene layer, in other embodiments, a predefined voltage can be applied across the graphene layer and a current flow through the graphene layer caused by the applied voltage can be measured. The values of the voltage and current can then be employed, for example, in a manner discussed above, to arrive at the resistance of the graphene layer (e.g., using Ohm&#39;s law). 
     A sensor according to the present teachings can be employed in a variety of settings. By way of example, a sensor according to the present teachings can be employed in a medical setting. Further, a sensor according to the present teachings can be employed for home use. In such cases, the analyzer can be implemented on a mobile device. In addition or alternatively, the analyzer can be implemented on a remote server that can be in communication with the sensor via a network, e.g., the Internet, to receive sensing data, such as a voltage measured across the antibody-functionalized graphene layer. The analyzer can employ the sensing data to determine whether a pathogen of interest is present in a sample under study in a manner discussed above. 
     The following example is provided for further elucidation of various aspects of the invention and is not intended to provide necessarily the optimal way of practicing the present teachings or optical results that can be obtained. 
     EXAMPLE 
     A microfluidic chip based on the design depicted in  FIGS. 1A and 1B  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 heat-killed  Listeria monocytogenes.    
     The graphene layer was functionalized with  Listeria monocytogenes Antibody LZF 7 (BGN/0884/67), which is an IgG 2   a  Mouse anti  Listeria monocytogenes  Monoclonal Antibody and was purchased from Bio-Rad. The functionalization process included covalently attaching 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 B2, which is herein incorporated by reference in its entirety, were followed. 
     A freeze-dried heat-killed preparation of  Listeria monocytogenes  (HKLM) was purchased from InvivoGen of San Diego, Calif. After mixing the  Listeria  preparation with endotoxin-free water, 1 mL of the resultant solution contained  10   7  to  10   8  bacteria. The bacteria were then stained by 5% violet crystal solution purchased from Sigma-Aldrich and washed 4 times by deionized (DI) water to remove the excessive dies in the solution. Then, in two steps, the solution was diluted to  10   4  to  10   3  bacteria per mL. 
     Subsequently, 1 mL of the sample was mixed with 9 mL of the Phosphate-buffered saline (PBS) buffer to achieve a concentration of  10   3  to  10   2  bacteria per mL. The sample was then injected into the chip by using a syringe pump at the rate of 1 mL/hour for two hours (phase 1). Then the syringe was disconnected and blank PBS buffer solution was pumped in to the chip for another two hours (Phase 2). Then the vial was disconnected and  Listeria  sample again was injected into the chip for another 2 hours. 
     The electrical resistance of the graphene was monitored using a four-probe based circuit such that the one described in the aforementioned U.S. Pat. No. 9.664,674 and data was recorded on the connected computer. The results are shown in the graph presented in  FIG. 10 . The data shows that the resistance of the graphene layer decreases during phase 1 as the bacterial cells interact with the antibody-functionalized graphene layer. During phase 2, no change in the resistance of the graphene layer is observed. This shows that the change in the resistance of the graphene layer observed during phase 1 can be attributed to the interaction of the  Listeria  bacteria with the antibody-functionalized graphene. As the sample containing bacterial cells is reintroduced into the chip during phase 3, the resistance of the graphene layer begins to decrease again, albeit at a smaller slope relative to the change of resistance observed during phase 1. Without being limited to any particular theory, the smaller slope observed during phase 2 may be due to a lower number of available sites on the antibody-functionalized graphene with which the  Listeria  bacteria can interact. 
     The above data clearly shows that the detection of  Listeria  bacteria, even at small concentrations, is feasible by using the present teachings. 
     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.