GRAPHENE-FUNCTIONALIZED SENSOR SURFACES AND RELATED METHODS

Systems and methods of functionalizing a graphene sensor surface are provided. A representative method of functionalizing the graphene sensor surface includes providing a graphene layer including a sensor surface. The method may include binding a plurality of molecular complexes to the sensor surface of the graphene layer using a buffer solution. Each molecular complex may include a linker molecule configured to couple to the sensor surface at a first linker position, and a binding molecule coupled to the linker molecule at a second linker position different from the first linker position. The representative method further includes coupling one or more detector molecules to a first subset of the molecular complexes and coupling one or more passivation agents to a second subset of the molecular complexes. At least some of the molecular complexes of the first subset are different from those of the second subset.

TECHNICAL FIELD

The present disclosure relates generally to functionalizing a graphene-based sensor for analyzing biological test samples.

BACKGROUND

Current methods of functionalizing the surface of a graphene-based sensor pose challenges for scalable manufacturing. In particular, a standard approach to functionalizing the sensor surface includes a stepwise process in which each molecule is added to the sensor surface independently. This approach can result in binding molecules, such as antibodies, having random orientations on the sensor's surface. Random orientations affecting the molecules responsible for the basic sensing function—i.e., binding to chemical species to be detected—impairs overall sensing performance. Further, existing methods of sensor assembly and functionalization can degrade the sensor surface, further compromising overall sensor accuracy.

Accordingly, there is a need for improved approaches to functionalizing a sensor surface.

SUMMARY

The present disclosure is directed to methods of functionalizing the surface of a graphene-based sensor, as well as to the finished sensors themselves. In particular, the present disclosure describes techniques for conveniently binding molecular complexes to a graphene sensor surface. The molecular complexes control the orientation of one or more detector molecules, thereby improving the performance of the graphene-based sensor (e.g., improving the ability of a detector molecule to recognize and bind to a target biomarker). Additionally, approaches to functionalizing a sensor surface described herein reduce degradation of the graphene sensor surface by using a water-based solution (e.g., a phosphate-buffered solution) to bind the molecular complexes to a graphene sensor surface. By reducing the degradation of the graphene sensor surface, embodiments of the present invention maintain the electrical properties of the graphene-based sensor, thereby improving overall performance.

In one aspect, a method of functionalizing a graphene sensor surface is provided. The method includes, in various embodiments, providing a graphene layer including a surface and binding a plurality of molecular complexes to the surface of the graphene layer. In some embodiments, the plurality of molecular complexes are bonded to the surface using a buffer solution. Each molecular complex includes a linker molecule configured to couple to the sensor surface at a first linker position, and a binding molecule coupled to the linker molecule at a second linker position different from the first linker position. Embodiments of the method further include coupling one or more detector molecules to a first subset of the molecular complexes and coupling one or more passivation agents to a second subset of the molecular complexes. At least some of the molecular complexes of the first subset are, in various embodiments, different from molecular complexes of the second subset.

In some embodiments, the detector molecule(s) coupled to the first subset of the molecular complexes are each coupled to a binding molecule of the associated molecular complex. In some embodiments, the one or more passivation agents coupled to the second subset of the molecular complexes are each coupled to a binding molecule of the associated molecular complex.

In some embodiments, the method includes coupling the molecular complex to the sensor surface before coupling one or more detector molecules to the first subset of the molecular complexes. Alternatively, in some embodiments, the method includes coupling the molecular complex to the sensor surface after coupling one or more detector molecules to the first subset of molecular complexes. In some embodiments, the method further includes coupling the molecular complex to the sensor surface before coupling one or more passivation agents to the second subset of molecular complexes. Alternatively, in some embodiments, the method includes coupling the molecular complex to the sensor surface after coupling one or more passivation agents to the second subset of molecular complexes.

In some embodiments, binding the plurality of molecular complexes to the sensor surface of the graphene layer includes generating a molecular complex mixture by dissolving, at least in part, the molecular complexes in a buffer solution, disposing the molecular complex mixture on the sensor surface of the graphene layer, and incubating the mixture and the graphene layer for a first predetermined amount of time at a predetermined temperature. In some embodiments, the first predetermined amount of time is at least 14 hours and the predetermined temperature is 4° C. In some embodiments, the molecular complex mixture has a molar concentration of approximately 5 millimolar.

In some embodiments, coupling the detector molecule(s) to the first subset of molecular complexes includes generating a detector molecule mixture by dissolving, at least in part, the one or more detector molecules in a buffer solution, disposing the detector molecule mixture on the first subset of molecular complexes, and incubating the detector molecule mixture and the first subset of molecular complexes for a second predetermined amount of time at a predetermined temperature. In some embodiments, the detector molecule mixture has a predetermined mass concentration of 10 μg/ml.

In some embodiments, coupling the one or more passivation agents to the second subset of molecular complexes includes generating a passivation agent mixture by dissolving, at least in part, the passivation agent(s) in the buffer solution, disposing the passivation agent mixture on the second subset of molecular complexes, and incubating the passivation agent mixture and the second subset of molecular complexes for a second predetermined amount of time at a predetermined temperature. In some embodiments, the passivation agent mixture has a predetermined concentration of 3% and a pH of 8.

In some embodiments, coupling the detector molecule(s) to the first subset of molecular complexes includes generating one or more modified detector molecules by adding a tag to each detector molecule and coupling the thus-modified detector molecule(s) to one or more binding molecules of the first subset of molecular complexes via the tag(s).

In some embodiments, coupling the detector molecule(s) to the first subset of molecular complexes includes, while generating an electric field, coupling the detector molecule(s) to one or more binding molecules of the first subset of the molecular complexes.

In some embodiments, the first subset molecular complexes and the second subset of molecular complexes have a predetermined ratio. In some embodiments, the plurality of molecular complexes is configured to control the orientation of the detector molecule(s). In some embodiments, the detector molecules antibodies, enzymes, or other proteins. In some embodiments, the binding molecule includes or consist of protein A, protein G, and/or protein L. In some embodiments, the buffer solution is phosphate-buffered saline. In some embodiments, the passivation agent(s) are amino-PEG5-alcohol (APA or PEG5). In some embodiments, the linker molecule is 1-pyrenebutanoic acid succinimidyl ester (PBASE).

In another aspect, a graphene-based sensor is provided. The graphene-based sensor includes a graphene sensor surface that has been functionalized by binding a plurality of pre-conjugated molecular complexes thereto. The pre-conjugated molecular complexes may be coupled to the graphene layer using a using a buffer solution. In some embodiments, a first subset of the pre-conjugated molecular complexes includes one or more linker molecules, one or more binding molecules, and one or more detector molecules configured to detect a target biomarker. The linker molecule may be configured to couple to the sensor surface at a first linker position. The binding molecule may be configured to couple to the detector molecule, and also couple to the linker molecule at a second linker position different from the first linker position. In some embodiments, a second subset of the pre-conjugated molecular complexes includes one or more linker molecules, one or more binding molecules, and one or more passivation agents configured to block or hinder non-specific molecules from the graphene sensor surface. The linker molecule may be configured to couple to the sensor surface at a first linker position. The binding molecule may be configured to couple to the passivation agent, and couple to the linker molecule at a second linker position different from the first linker position. At least some of the molecular complexes of the first subset may be different from molecular complexes of a second subset.

Note that the various embodiments described above can be combined with any other embodiments described herein. The features and advantages described in the specification are not all inclusive and, in particular, many additional features and advantages will be apparent to one of ordinary skill in the art in view of the drawings, specification, and claims.

DETAILED DESCRIPTION

Numerous details are described herein in order to provide a thorough understanding of the example embodiments illustrated in the accompanying drawings. However, some embodiments may be practiced without many of the specific details, and the scope of the claims is only limited by those features and aspects specifically recited in the claims. Furthermore, well-known processes, components, and materials have not been described in exhaustive detail so as to avoid obscuring pertinent aspects of the embodiments described herein.

The present teachings are generally directed to a graphene-based sensor that can be functionalized with a plurality of antibodies for detecting and/or analyzing one or more biomarkers. The term “biomarker” as used herein refers to a molecular species with a biological function. As discussed in more detail below, a sensor surface of the graphene-based sensor can be functionalized utilizing pre-conjugated molecules that are added and coupled to the sensor surface. The pre-conjugated molecules are configured to couple to one or more detector molecules (e.g., antibodies, enzymes, other proteins, etc.) that enable the graphene-based sensor to recognize and bind to specific target biomarkers for analysis. In particular, use of the pre-conjugated molecules improves the performance of the graphene-based sensor by controlling the orientation of the detector molecules coupled to the sensor surface, improving the detector molecules' ability to recognize and bind to specific target biomarkers. Further, the pre-conjugated molecules can be coupled to the sensor surface of the graphene-based sensor without degrading that surface.

The graphene-based sensor described herein is configured to receive a test sample and detect whether a target biomarker is present therein. For example, if the target biomarker is present in the test sample under investigation, the interaction of the biomarker with the graphene-based sensor may cause a change in at least one electrical property of the underlying graphene layer, e.g., its DC electrical resistance. Such a change in the electrical property of the graphene layer can be measured and used to quantify the amount of the biomarker in the sample.

FIG.1illustrates a graphene-based sensor100functionalized using one or more molecular complexes, in accordance with some embodiments. As described above, the graphene-based sensor100is configured to analyze a test sample. In some embodiments, the test sample is a urine sample, a blood sample, a saliva sample, and/or any other biological substance (typically in liquid or gaseous form). In some embodiments, the graphene-based sensor100analyzes the test sample to detect and/or quantify one or more target biomarkers, bacteria, allergens, pathogens, proteins, glutens, toxins, etc. In some embodiments, the graphene-based sensor100includes a graphene layer115and one or more molecular complexes. Each molecular complex can be a combination of two or more linker molecules120, one or more binding molecules125, one or more detector molecules130, and/or one or more passivating agents135. In some embodiments, one or more molecular complexes are pre-conjugated as described below in reference toFIGS.3A-3D.

In some embodiments, the graphene layer115is functionalized with one or more detector molecules130via the one or more molecular complexes. In particular, in some embodiments, one or more molecular complexes are coupled to the graphene layer115and the one or more detector molecules130are coupled to the one or more molecular complexes. The functionalization process described below with reference toFIGS.3A-5Ballows for control of the orientation of the one or more detector molecules130(when coupled to the one or more molecular complexes). By controlling the orientation of the detector molecule(s)130, the functionalization process described herein improves the performance of the graphene-based sensor100by enabling the detector molecules130to recognize and bind to a target biomarker efficiently and effectively. Further, the functionalization process described herein reduces or eliminates degradation of the graphene layer115sensor surface140when the one or more molecular complexes are coupled to the sensor surface140. By reducing the degradation of the graphene layer115, the electrical properties of the graphene layer115are improved to maintain more consistent and reliable performance of the sensor100. Additionally, the functionalization process described herein enables the manufacture of graphene-based sensor100to be reduced to a one-step functionalization of the surface (as described below in reference toFIGS.5A-5B), which makes at-scale manufacturing of the graphene-based sensor100feasible and cost-effective.

In some embodiments, a linker molecule120is a covalent and/or non-covalent chemical linker, i.e., bonds covalently and/or non-covalently (e.g., ionically or via hydrogen bonding) to the sensor surface and to a detector molecule; that is, the mode of attachment may include, one or more covalent bonds, one or more non-covalent bonds, or a combination. In some embodiments, the linker molecule is a pyrene linker, such as 1-pyrenebutanoic acid succinimidyl ester (PBASE) or any other pyrene derivative that behaves as a heterobifunctional linker. In some embodiments, the linker molecule120is configured to couple to the sensor surface140at a first linker position. The linker molecule120may also couple to a binding molecule125at a second linker position different from the first linker position. In some embodiments, the binding molecule125is a fragment crystallizable (Fc) binding peptide or aptamer. For example, the binding molecule125may be or include protein A, protein G, protein L, and/or other Fc-binding peptide or aptamer.

In some embodiments, the binding molecule125is capable of coupling (covalently or non-covalently) not only to a detector molecule130(e.g., anti-troponin antibodies) but alternatively to a passivation agent135; accordingly, the second linker position may alternatively bind a detector molecule130or the passivation agent135as shown inFIG.1. In some embodiments, the detector molecule(s)130include or consist of an antibody, enzyme, other protein, or other chemical species capable of specifically recognizing and binding (covalently or non-covalently) a target biomarker. In some embodiments, the passivation agent135is APA (PEG5), Tween, BLOTTO, bovine serum albumin (BSA), and/or gelatin. The passivation agent can inhibit, and preferably prevent, the interaction of a sample of interest introduced onto the graphene layer115with areas of the graphene layer115that are not functionalized with the one or more detector molecules130. This can in turn lower the noise in electrical signals generated as a result of the interaction of the analyte of interest with the detector molecules130.

By way of example, in this embodiment, the graphene layer115is functionalized with one or more detector molecules130that exhibit specific binding to any of troponin (e.g., a particular isoform of troponin), C-reactive protein, B-type natriuretic peptide, or myeloperoxidase. In some embodiments, the detector molecule(s)130(e.g., anti-biomarker antibodies) are monoclonal antibodies that exhibit specific binding to a particular isoform of the biomarker, e.g., a specific isoform of troponin. In other embodiments, the one or more detector molecule130(e.g., anti-biomarker antibodies) can be polyclonal antibodies that exhibit binding to multiple isoforms of the biomarker. By way of example, in some embodiments, the graphene layer115can be functionalized with cardiac troponin T (cTnT) and/or cardiac troponin I (cTnI).

In some embodiments, the one or more molecular complexes cover a fraction of, or the entire, surface of the graphene layer115. In some embodiments, the fraction can be at least about 60%, at least about 70%, at least about 80%, or 100% of the surface of the graphene layer115. The remainder of the surface of the graphene layer115(i.e., the surface areas not functionalized with the detector molecule130) can be passivated via one or more passivation agents135. Additionally, or alternatively, in some embodiments, the non-functionalized graphene areas can be passivated via a passivation layer. By way of example, passivation of the non-functionalized portions of the graphene layer115can be achieved, e.g., via incubation with 0.1% Tween. As described above, passivation can lower the noise in the electrical signals generated as a result of the interaction between the analyte of interest and the detector molecules130.

In some embodiments, the graphene-based sensor100is disposed on (e.g., bonded to) an underlying substrate. The underlying substrate can be formed of a variety of different materials, such as, silicon, plastic, metal, polymeric materials (such as polyurethane or polyethylene terephthalate), or glass, among others. In some embodiments, the graphene layer115is disposed over an underlying silicon oxide (SiO2) layer, which can in turn be formed as a thin layer of or on a silicon substrate (e.g., a layer having a thickness in a range of a 200 nm to about 10 μm). In some embodiments, the graphene layer115can be deposited on an underlying silicon substrate using any of a variety of techniques known in the art. By way of example, chemical vapor deposition (CVD) can be employed to deposit the graphene layer115on an underlying copper substrate. The graphene-coated copper substrate can then be disposed on (e.g., bonded to) a silicon oxide layer of a silicon wafer, and the copper can be removed via chemical etching. In some embodiments, the graphene layer115is deposited on the underlying substrate as an atomic monolayer, while in other embodiments the graphene layer115includes multiple atomic layers.

FIGS.2A-2Eillustrate a standard process for functionalizing a graphene-based sensor. The standard process for functionalizing the graphene-based sensor includes, in a first operation200, providing a graphene layer115. The standard process further includes, in a second operation230, disposing one or more linker molecules120on the graphene layer115. The graphene layer115is incubated with the linker molecule(s)120(e.g., a 5 nM solution of PBASE in a dimethyl sulfoxide (DMSO) solution (or a dimethylformamide (DMF) solution, or methanol) for a first time period (e.g., an hour) at a predetermined temperature (e.g., room temperature), causing the linker molecule(s) to bind to the graphene surface.

In a third operation250, the linker-modified graphene layer is then incubated with the detector molecule(s)130of interest in, e.g., a sodium carbonate bicarbonate solution (e.g., at a pH of 9.5 and with a mass concentration of at least 60 μg/ml) for a second time period (e.g., two hours) at the predetermined temperature. In the third operation250, the detector molecule(s)130bind to the linker molecules120of the linker-modified graphene layer. The detector molecule(s)130are not uniformly coupled to the linker molecules120. In particular, the orientations of the detector molecules130are not the same across different linker molecules120and, in some cases, the detector molecules130are coupled to linker molecules120at inefficient orientations. The inconsistencies in the orientations of the detector molecules130lower the performance of a graphene-based sensor functionalized using the illustrated standard process.

In a fourth operation270, the linker-modified graphene layer coupled to one or more detector molecules130is further incubated with a passivation agent135. Under the standard process, the passivation agent is incubated in a phosphate-buffered solution (PBS) for a third predetermined time period (e.g., 30 minutes) at the predetermined temperature. The PBS can have a pH of, e.g., 11. Further, a fifth operation290of the standard process includes rinsing the linker-modified graphene layer coupled to one or more detector molecules130and passivation agents135with deionized (DI) water and 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 11 for another predetermined time period (e.g., 30 minutes) at the predetermined temperature).

The performance of graphene-based sensors functionalized using the illustrated standard process can vary. In particular, the inability to control the orientation of the detector molecules130can result in poor sensor performance (e.g., providing inconsistent readings, generating false positives, and/or failing to detect a target biomarker). Further, the different operations of the standard process can degrade the graphene layer115and change its electrical properties, resulting in inaccurate readings or detection of spurious electrical signals.

FIGS.3A-3Dillustrate pre-conjugation of one or more molecular complexes in accordance with some embodiments. In particular, one or more molecules (e.g., a linker molecule120, a binding molecule125, a detector molecule130, and/or a passivation agent135) are conjugated into new molecular complexes before coupling to a sensor surface140of the graphene-based sensor100(FIG.1). The coupling of molecular complexes to the sensor surface140of the graphene-based sensor100is discussed in detail below in reference toFIGS.4A-5B.

FIG.3Aillustrates a first molecular complex300in accordance with some embodiments, e.g., a linker molecule120conjugated with a binding molecule125. The first molecular complex300forms a duplex conjugate in which the binding molecule125binds to the linker module120at one linker position, and the linker module binds to the sensor surface140of the graphene-based sensor100at another (e.g., opposed) linker position.

FIG.3Billustrates conjugation of the binding molecule125with a detector molecule130to form a modified molecular complex330in which binding molecule125binds to the linker module120at one linker position and further binds to the detector molecule130at a different linker position. As described above with reference toFIG.1, in some embodiments, the binding molecule125is an Fc binding peptide or aptamer. The Fc binding peptide or aptamer is configured to bind to an Fc region of the detector molecule130, which enables the detector molecule130to be oriented on the sensor surface140to improve the performance of the graphene-based sensor100. In this configuration, the linker module120couples the sensor surface140of the graphene-based sensor100via the first linker position opposite the second linker position. In some embodiments, the modified complex330is conjugated before it is coupled to the sensor surface140. Alternatively, in some embodiments, the modified complex330is formed while the molecular complex300is coupled to the sensor surface140. In other words, a detector molecule130can be coupled to the molecular complex300before or after the complex300has been coupled to the sensor surface140.

As shown inFIG.3C, an alternative molecular complex350may be formed by conjugation of the molecular complex300with a passivation agent135. The binding molecule125binds to the linker module120and further binds to the passivation agent135. In this configuration, the linker module120is configured to couple the sensor surface140of the graphene-based sensor100via a linker position different from (e.g., opposed to) where the passivation agent135is bound. In some embodiments, the molecular complex350is formed before it is coupled to the sensor surface140. Alternatively, in some embodiments, the complex350is formed after the complex300has been coupled to the sensor surface140. In other words, a passivation agent135can be coupled to the first molecular complex300before or after the molecular complex300has been coupled to the sensor surface140.

FIG.3Dillustrates an alternative molecular complex370in which the passivation agent135directly coupled to the linker molecule120(rather than via the binding molecule125). The linker module120is still configured to couple the sensor surface140of the graphene-based sensor100via the first linker position.

Although not shown, another alternative molecular complex includes a detector molecule130directly coupled to the linker molecule120(rather than via the binding molecule125). The linker module120is still configured to couple the sensor surface140of the graphene-based sensor100via the first linker position.

FIGS.4A-4Dillustrate a method of functionalizing the sensor surface of a graphene-based sensor using one or more molecular complexes. A graphene layer115is provided (operation400) and, in an operation430, a plurality of molecular complexes as described above is exposed to the surface140of the graphene layer115in a buffer solution (e.g., PBS). Each molecular complex includes a linker molecule120configured to couple to the sensor surface140at a first linker position and a binding molecule125coupled to the linker molecule120at a second linker position different from the first linker position. In some embodiments, the molecular complexes coupled to the sensor surface140of the graphene layer115have the form of the molecular complex300. In some embodiments, the molecular complexes are coupled to the sensor surface140before coupling one or more detector molecules130to some or all of the plurality molecular complexes. Alternatively, the binding molecule125(e.g., an antibody-binding molecule) may be bound to the sensor surface140directly or through a linker molecule120. The linker molecule120can be pre-conjugated to the binding molecule125before it is coupled to the sensor surface140.

In some embodiments, coupling (e.g., binding) the molecular complexes to the sensor surface140of the graphene layer115includes generating a molecular complex mixture by dissolving, at least in part, the molecular complexes in a buffer solution, which is applied to the sensor surface140of the graphene layer115. The mixture is incubated for a first predetermined amount of time at a predetermined temperature, causing the molecular complexes to bind to the sensor surface140. In some embodiments, the first predetermined amount of time is at least 14 hours and the predetermined temperature is 4° C. Alternatively, the first predetermined amount of time may be overnight. In some embodiments, the molecular complex mixture has a molar concentration of approximately 5 millimolar.

In some embodiments, in operation450, detector molecules130are coupled to a first subset of the graphene-bound molecular complexes via a binding molecule125of the molecular complexes. In some embodiments, this is accomplished by dissolving, at least in part, the detector molecules in a buffer solution (e.g., PBS), applying the mixture to the bound molecular complexes, and incubating for a second predetermined amount of time at a predetermined temperature. In some embodiments, the detector molecule mixture has a mass concentration of 10 μg/ml. The molecular complexes may be selected and bound to the graphene surface so as to control the orientation of the detector molecule(s)130when they bind (e.g., such that the detector molecules130couple to the binding molecule125at the Fc region). This may be accomplished, e.g., using antibody-binding molecules to control the orientation of the antibodies or other detector molecules. In this way, the detector molecules130recognize and bind to specific target biomarkers in an optimal orientation (e.g., perpendicular to the sensor surface140).

Alternatively, coupling the detector molecule(s)130to the first subset of molecular complexes may include generating one or more modified detector molecules by adding a tag (not shown) to the detector molecule(s)140, and coupling the tagged detector molecules to binding molecules125of the first subset of molecular complexes. For example, antibody binding can be indirect through the addition of a tag in the antibody sequence, with the tag binding to the binding molecule125. In some embodiments, the tag can be bound via another molecule directly to the sensor surface140. An example of such a system includes a biotin-streptavidin system. Antibodies or other detector proteins can be biotinylated at specific sites of the molecule sequence. These modified detector molecules are then added to a streptavidin-coated sensor surface.

Alternatively, in some embodiments, coupling the detector molecule(s)130to the first subset of molecular complexes includes, while generating an electric field, coupling the detector molecule(s) to one or more binding molecules of the first subset of molecular complexes. The electric field can be used to orient the antibody or other detector molecule on the sensor surface before or during binding.

In some embodiments, at operation470, one or more passivation agents135are coupled to a second subset of the plurality of molecular complexes. The first and second subsets of molecular complexes may be completely distinct or may overlap, in the latter case with some of the molecular complexes binding to both a detector molecule and a passivation agent. In some embodiments, the passivation agents135are coupled to molecular complexes via the associated binding molecule125(seeFIG.3C). In some embodiments, the molecular complexes are coupled to the sensor surface140before the passivation agents135are coupled to the second subset thereof.

In some embodiments, coupling the passivation agents135to the second subset of molecular complexes includes generating a passivation agent mixture by dissolving, at least in part, the passivation agents in a buffer solution (e.g., PBS), applying the mixture to the molecular complexes, and incubating for a second predetermined amount of time at a predetermined temperature. In some embodiments, the passivation agent mixture has a concentration of 3% and a pH of 8. In some embodiments, the first subset of the plurality of molecular complexes and the second subset of the plurality of molecular complexes have a predetermined ratio. For example, the first subset of molecular complexes (coupled to the detector molecule(s)130) can cover 30%, 60%, 80%, or 100% of the graphene surface and the second subset (coupled to the one or more passivation agents135) can cover 70%, 40%, 20%, or 0% of the graphene surface, respectively. The molecular complexes binding passivation agents may be bound to the graphene surface in regions distinct from the regions where molecular complexes binding detector molecules are bound, or they may be interspersed.

FIGS.5A and5Billustrate a single-step method of functionalizing a sensor surface of a graphene-based sensor using molecular complexes that have been pre-conjugated. In this way, the process for functionalizing the sensor surface140of a graphene-based sensor100is streamlined.

In some embodiments, molecular complexes are coupled to the sensor surface140after detector molecules130have been coupled to a first subset of the molecular complexes (FIGS.4A-4D). For example, one or more molecular complexes510(e.g., having the form of the molecular complex330) can be prepared prior to being coupled to the sensor surface140. Similarly, in some embodiments, the molecular complexes are coupled to the sensor surface140after one or more passivation agents135(FIGS.4A-4D) have been coupled to a second subset thereof. For example, one or more molecular complexes530(e.g., having the form of the molecular complex350) can be prepared prior to being coupled to the sensor surface140.

In operation500, a mixture of molecular complexes510,530is dissolved in a water-based solution, such as PBS, which is then applied to the sensor surface140. The mixture is incubated over the surface for a predefined period of time at a suitable temperature (e.g., at least 15 minutes at a temperature of 4° C.). This allows the sensor surface140of the graphene-based sensor100to be functionalized in a single step.

FIG.6illustrates a method600of functionalizing a sensor surface. Operations (e.g., steps) of the method600may be performed to functionalize a graphene sensor surface100as described above with reference toFIGS.3and4. The foregoing method descriptions and the process flow diagrams are provided merely as illustrative examples and are not intended to require or imply that the operations of the various embodiments must be performed in the order presented. As will be appreciated by one of skill in the art the steps in the foregoing embodiments may be performed in any order. Words such as “then,” “next,” etc. are not intended to limit the order of the steps; these words are simply used to guide the reader through the description of the methods. Although process flow diagrams may describe the operations as a sequential process, many of the operations can be performed in parallel or concurrently. In addition, the order of the operations may be re-arranged. A process may correspond to a method, a function, a procedure, a subroutine, a subprogram, etc. When a process corresponds to a function, its termination may correspond to a return of the function to the calling function or the main function.

The method600includes providing (step610) a graphene layer including a sensor surface. The method600further includes binding (step620) molecular complexes to the sensor surface of the graphene layer using a buffer solution. Each molecular complex includes a linker molecule configured to couple (step630) to the sensor surface at a first linker position, and a binding molecule coupled (step640) to the linker molecule at a second linker position different from the first linker position. For example, as shown and described above in reference toFIG.3A, in some embodiments, a molecular complex includes at least a linker molecule120and a binding molecule125. In some embodiments, the linker molecule is a pyrene linker, such as PBASE or any other pyrene derivative that operates as a heterobifunctional linker. In some embodiments, the binding molecule125is an Fc binding peptide or aptamer. In some embodiments, the binding molecule125is one of protein A, protein G, or protein L.

Binding the plurality of molecular complexes to the sensor surface of the graphene layer may include generating a molecular complex mixture by dissolving, at least in part, the molecular complexes in a buffer solution, applying the mixture to the sensor surface of the graphene layer, and incubating for a first predetermined amount of time at a predetermined temperature. In some embodiments, the first predetermined amount of time is overnight, and the predetermined temperature is 4° C. In some embodiments, overnight means at between 12-20 hours. In some embodiments, the molecular complex mixture has a molar concentration of approximately 5 millimolar. In some embodiments, the buffer solution is phosphate-buffered saline.

The method600includes coupling (step650) detector molecules to a first subset of the plurality of molecular complexes. In some embodiments, the detector molecule includes or consists of an antibody, an enzyme, another type of protein, or other molecule capable of specifically recognizing and binding a target biomarker. In some embodiments, the molecular complexes control the orientation of the detector molecule when coupled thereto. For example, as shown and described above with reference toFIGS.1and3A-5B, the molecular complexes control the orientation of the detector molecules130such that the Fc region of the detector molecules130couples to the plurality of molecular complexes. By controlling the orientation of the detector molecules130, the method600improves the overall performance of the sensor.

In some embodiments, coupling the detector molecules to the first subset of molecular complexes includes generating a by dissolving, at least in part, the detector molecules in a buffer solution, applying the mixture to the first subset of molecular complexes, and incubating for a second predetermined amount of time at a predetermined temperature. In some embodiments, the second predetermined amount of time is at least 15 minutes, and the predetermined temperature is 4° C. In some embodiments, the detector molecule mixture has a predetermined mass concentration of 10 μg/ml. In some embodiments, the buffer solution is phosphate-buffered saline.

In some embodiments, the detector molecules coupled to the first subset of molecular complexes are each coupled to a binding molecule of the associated molecular complex. For example, in some embodiments, coupling the detector molecules to the first subset of molecular complexes includes coupling the detector molecules to one or more binding molecules thereof. For example, as shown and described above with reference toFIGS.1and3A-5B, the detector molecules130each couple to one or more binding molecules125.

In some embodiments, the method600includes coupling the molecular complex to the sensor surface before coupling one or more detector molecules to the first subset of molecular complexes. Alternatively, the method600may include coupling the molecular complex to the sensor surface after coupling one or more detector molecules to the first subset thereof.

In some embodiments, coupling the detector molecules to the first subset of molecular complexes includes generating one or more modified detector molecules by adding a tag thereto and coupling the tagged detector molecules to a binding molecule (e.g., via the tag), of the molecular complex. Alternatively, the tagged detector molecules can be bound directly to the sensor surface.

In some embodiments, coupling the detector molecules to the first subset of molecular complexes occurs in the presence of an electric field. Alternatively, the electric field can be used to bind the detector molecules directly to the sensor surface.

The method600further includes coupling (step660) one or more passivation agents to a second subset of the molecular complexes, which may be distinct from or overlap the first subset of the molecular complexes. In some embodiments, the passivation agent(s) include APA (PEG5), amino-PEG5-alcohol, or other species that keeps molecules other than the target away from the sensor surface.

In some embodiments, coupling the one or more passivation agents to the second subset of the molecular complexes includes generating a passivation agent mixture by dissolving, at least in part, the one or more passivation agents in a buffer solution, applying the mixture to the second subset of molecular complexes, and incubating for a second predetermined amount of time at a predetermined temperature. In some embodiments, the second predetermined amount of time is at least 15 minutes, and the predetermined temperature is 4° C. In some embodiments, the passivation agent mixture has a concentration of 3% and a pH balance 8.

In some embodiments, the passivation agents are coupled to a binding molecule of the associated molecular complex. For example, coupling the passivation agent(s) to the second subset of molecular complexes may include coupling to one or more binding molecules of the second subset of molecular complexes. As shown and described above with reference toFIGS.1and3A-5B, the passivation agents135may couple to one or more binding molecules125. Alternatively, the passivation agents135may couple to one or more linker molecules120.

In some embodiments, the method600includes coupling the molecular complexes to the sensor surface before coupling one or more passivation agents to the second subset thereof. In some embodiments, the method600includes coupling the molecular complexes to the sensor surface after coupling one or more passivation agents to the second subset thereof.

The first and second subsets of molecular complexes may be present in a predetermined ratio. For example, in some embodiments, the population of the first subset of molecular complexes can be less than, equal to, or greater than the population of the second subset of molecular complexes. In some embodiments, the predetermined ratio is 60% of the first subset of molecular complexes to 40% of the second subset of molecular complexes, 80% of the first subset of molecular complexes to 20% of the second subset of molecular complexes, 100% of the first subset of molecular complexes to 0% of the second subset of molecular complexes, 40% of the first subset of molecular complexes to 60% of the second subset of molecular complexes, etc.