Biomolecule attachment to borophene nanomaterial

Abstract

Embodiments relate to borophene-based biosensing platforms for highly sensitive, target-specific biosensing. These embodiments include borophene nanosheets functionalized with biomolecules such as antibodies and oligonucleotides. Such functionalized borophene nanosheets can be integrated into diagnostic devices designed for the targeted detection of disease biomarkers.

Description

Atty. Ref. No. 0073605-001099BIOMOLECULE ATTACHMENT TO BOROPHENE NANOMATERIALCROSS REFERENCE TO RELATED APPLICATIONS

[0001] This patent application is related to and claims the benefit of priority of U.S. Provisional Application 63 / 723,246, filed on November 21, 2024. The entire contents of this application is incorporated by reference.FIELD

[0002] Embodiments relate to biosensing platforms that integrate borophene nanosheets functionalized with biomolecules, such as antibodies and oligonucleotides, and to methods of making and using thereof.BACKGROUND

[0003] The development of two-dimensional (2D) nanomaterials has significantly progressed since the groundbreaking isolation of graphene in 2004, which opened new frontiers in nanoscience and nanotechnology. These atomically thin materials exhibit unique physical, electrical, optical, and mechanical properties that differ markedly from their bulk counterparts. Among the various 2D materials explored, graphene, hexagonal boron nitride, and transitionmetal dichalcogenides such as M0S2 have been extensively studied for applications in electronics, energy storage, and sensing. Their high surface area-to-volume ratio, exceptional electrical conductivity, and tunable properties have made them especially attractive for biosensing applications, including the detection of environmental pollutants and biological markers like proteins and nucleic acids.

[0004] More recently, research has expanded to include alternative 2D nanomaterials that can address some limitations associated with graphene and other early materials. Borophene, aAtty. Ref. No. 0073605-001099 monoel emental 2D allotrope of boron, has emerged as a promising candidate due to its distinctive structural, electronic, and optical characteristics. Unlike graphene, borophene exhibits high mechanical strength, tunable electronic properties, and intrinsic superconductivity, broadening its potential for diverse applications such as high-performance energy storage, gas sensing, and biomedical devices. Its biocompatibility and biodegradability further enhance its suitability for use in medical and environmental fields.

[0005] Despite these promising attributes, synthesizing high-quality borophene nanosheets remains a significant challenge for practical use. Conventional methods, such as molecular beam epitaxy (MBE) and chemical vapor deposition (CVD), require complex conditions, such as ultrahigh vacuum environments and low-pressure growth, that can limit scalability and pose issues related to material transfer and quality degradation. Liquid-phase exfoliation, particularly employing low-boiling solvents like water, has gained attention as a scalable and environmentally friendly approach for producing stable dispersions of borophene nanosheets.

[0006] Surface functionalization of borophene with biomolecules like DNA and RNA has also been explored to improve biosensing capabilities, leveraging its high surface reactivity, anisotropic properties, and high electron mobility. However, further development of direct surface functionalization strategies for pristine borophene remains necessary to fully exploit its potential in biosensing and biomedical applications.SUMMARY

[0007] The present disclosure relates to the development and application of 2D borophene nanosheets for biosensing and diagnostic purposes. We leveraged the unique structural, electronic, and mechanical properties of borophene, and we developed a scalable liquid-phase exfoliation method that yields stable borophene nanosheets suitable for biological applications.Atty. Ref. No. 0073605-001099

[0008] The nanosheets may be functionalized with antibodies designed to serve as biorecognition elements. A key feature involves a photoinduced immobilization technique that enables the covalent attachment of antibodies onto borophene surfaces through a UV-irradiation process. This approach exploits borophene’ s electron-deficient nature to facilitate strong covalent bonds with sulfur-containing biomolecules, resulting in well-oriented antibodies that retain their functional activity. These nanosheets can serve as the core sensing platform in various biosensing formats, including lateral flow immunoassays (LFIAs) and genosensing devices, offering enhanced sensitivity, specificity, and rapid response times.

[0009] The nanosheets may also be functionalized with thiol-modified oligonucleotides designed to serve as biorecognition elements. The electron-rich thiol groups may interact with the electron-deficient boron atoms on the borophene surface, forming covalent B-S bonds and producing functionalized borophene nanosheets. These nanosheets can similarly serve as LFIAs.

[0010] In an exemplary embodiment, a method of making a functionalized borophene nanosheet comprises providing a borophene nanosheet; UV irradiating antibodies to cleave disulfide bonds within the antibodies and to generate reactive thiol groups; and combining the borophene nanosheet and the irradiated antibodies to provide the functionalized borophene nanosheet.

[0011] In some embodiments, providing the borophene nanosheet comprises providing bulk boron material; dispersing the bulk boron material in a solvent to provide a dispersion; subjecting the dispersion to sonifi cation to facilitate cavitation; and subjecting the sonified dispersion to centrifugation to provide the borophene nanosheet.

[0012] In some embodiments, at least some of the reactive thiol groups are in Fab domains of the antibodies.Atty. Ref. No. 0073605-001099

[0013] In some embodiments, the functionalized borophene nanosheet comprises antibodies oriented to expose their Fab domains.

[0014] In an exemplary embodiment, an apparatus for detecting markers related to a pathogen of interest, the apparatus comprising a testing strip including, in sequence, a sample application region configured to receive a sample collected from a subject, wherein the sample is configured flow through the testing strip; detection elements configured to directly or indirectly attach to markers related to the pathogen of interest; a test region configured to detect the presence of markers related to the pathogen of interest, wherein at least a portion of the test region comprises the functionalized borophene nanosheet formed from the method of claim 1, wherein the functionalized borophene nanosheet is configured to immobilize the detection elements; and a control region having immobilized capture probes bound to the testing strip, wherein the immobilized capture probes are configured to immobilize the detection elements.

[0015] In some embodiments, the pathogen of interest is endometriosis.

[0016] In some embodiments, the markers are HMGB-1 proteins.

[0017] In some embodiments, the antibodies are IgG antibodies.

[0018] In an exemplary embodiment, an absorbent pad for detecting markers related to endometriosis, the absorbent pad comprising: a sample application region configured to receive a sample collected from a subject, wherein the sample is configured flow through the absorbent pad via one or more microfluidic channels; detection elements functionalized with detection probes configured to capture the markers related to the pathogen of interest; a test region configured to detect the presence of markers related to endometriosis, wherein at least a portion of the test region comprises the functionalized borophene nanosheet formed from the method of claim 1, wherein the functionalized borophene nanosheet are configured to capture the markersAtty. Ref. No. 0073605-001099 related to the pathogen of interest; and a control region having immobilized capture probes bound to the absorbent pad, wherein the immobilized capture probes are configured to capture the detection probes functionalized to the detection elements.

[0019] In some embodiments, wherein the markers are HMGB-1 proteins.

[0020] In some embodiments, the antibodies are IgG antibodies.

[0021] In some embodiments, the detection probes are anti- HMGB-1 antibodies.

[0022] In some embodiments, the immobilized capture probes are anti- HMGB-1 antibodies.

[0023] In some embodiments, the detection elements are plasmonic nanoparticles.

[0024] In an exemplary embodiment, a method of making a functionalized borophene nanosheet, the method comprising providing a borophene nanosheet; functionalizing oligonucleotides with reactive thiol groups; and combining the borophene nanosheet and the functionalized oligonucleotides to provide the functionalized borophene nanosheet.

[0025] In some embodiments, providing the borophene nanosheet comprises providing bulk boron material; dispersing the bulk boron material in a solvent to provide a dispersion; subjecting the dispersion to sonification to facilitate cavitation; and subjecting the sonified dispersion to centrifugation to provide the borophene nanosheet.

[0026] Other details, objects, and advantages of our compositions, methods, and systems will become apparent as the following description of certain exemplary embodiments thereof proceeds.BRIEF DESCRIPTION OF THE DRAWINGS

[0027] The above and other objects, aspects, features, advantages, and possible applications of embodiments of the present innovation will be more apparent from the following more particularAtty. Ref. No. 0073605-001099 description thereof, presented in conjunction with the following drawings. Like reference numbers used in the drawings may identify like components.

[0028] FIG. l is a flowchart of an exemplary method of functionalizing borophene nanosheets with antibodies.

[0029] FIG. 2 is a schematic illustration of functionalization of borophene nanosheets with antibodies through a photoinduced immobilization technique (PIT).

[0030] FIG. 3 is a flowchart of an exemplary method of functionalizing borophene nanosheets with modified oligonucleotides.

[0031] FIG. 4 is a schematic illustration of functionalization of borophene nanosheets with thiol - modified oligonucleotides.

[0032] FIG. 5 is a schematic illustration of an exemplary borophene-based lateral flow immunoassay (LFIA).

[0033] FIG. 6 is a schematic illustration of a developed LFIA utilizing functionalized borophene nanosheets. HMGB-1 antigen and antibodies were used as a model to validate the PIT-based biofunctionalization of borophene nanosheets.

[0034] FIG. 7 is a schematic illustration of an exemplary synthesis process and experimental conditions for borophene nanosheets.

[0035] FIG. 8 is a graph showing dynamic light scattering (DLS) measurements of borophene nanosheets.

[0036] FIG. 9 shows scanning electron microscopy (SEM) images for (left) pristine boron powder precursor, and (right) borophene nanosheets. The SEM images provide a comparison between the morphology of pristine boron powder (3D) and borophene nanosheets (2D) obtainedAtty. Ref. No. 0073605-001099 after liquid exfoliation. Arrows indicate the 3D structure of boron and the 2D structure of the nanosheets.

[0037] FIG. 10 shows (top-left) a transmission electron microscopy (TEM) image (top-center) a high-angle annular dark field (HAADF) image with (bottom-left) boron EDX map, (bottomcenter) overlay of HAADF and boron EDX map, (top-right) HR- TEM image of borophene with inset representing lattice fringes showing lattice fringes corresponding to predominantly %3 phase, and (bottom-right) calculated lattice fringes using ImageJ software.

[0038] FIG 11 is a schematic illustration of antibody orientation on borophene through active thiol generated by UV-irradiation.

[0039] FIG. 12 shows a Fourier-transform infrared (FT-IR) spectra of pristine boron powder and / 3 borophene nanosheets in an anhydrous state.

[0040] FIG. 13 shows a Raman spectra of %3 borophene nanosheets in aqueous media.

[0041] FIG. 14 shows (left) a schematic illustration of only the antibody and UV-treated (free thiol) antibody conjugation to borophene nanosheet, and (right) a graph illustrating a standard curve for a Bradford assay with a free antibody.

[0042] FIG. 15 is a TEM image illustrating a sandwich formation over borophene nanosheets. Oriented IgG antibodies anchored onto borophene nanosheets form a sandwich complex through interactions between the Fab regions, HMGB-1 antigens, and gold nanoparticles labeled with detection antibodies.

[0043] FIG. 16 is a high-resolution TEM image showing the lattice fringes in borophene nanosheets.

[0044] FIG. 17 is a TEM image of a control sample (UV-untreated antibodies over borophene nanosheets). Gold nanoparticles appear separately, agglomerated, and not over borophene sheets.Atty. Ref. No. 0073605-001099The arrows indicate borophene nanosheets. The inset represents an image at higher magnification.

[0045] FIG. 18 is a HAADF image along with EDX mapping of the borophene nanosheetsandwich complex.

[0046] FIG. 19 is a HAADF image showing the presence of boron and gold.

[0047] FIG. 20 is an AFM image showing the formation of a sandwich on top of the borophene nanosheet, which is indicated via an arrow.

[0048] FIG. 21 is an S 2p XPS spectra of the borophene nanosheets conjugated with UV-treated anti-human HMGB-1 IgG antibodies. The B-S bond is seen at 163.3 eV.

[0049] FIG. 22 includes graphs showing (top) the signal intensity changes in the S-S region of 500-550 for the UV-treated antibody, indicating the reduction of the disulfide bond to produce the free thiol groups, and (bottom) the disappearance of S-S vibrational bands (e.g., at 503 cm'1) alongside the emergence of thiol-associated signals (e.g., around 680 cm-1), which is characteristic of disulfide bond reduction.

[0050] FIG. 23 is a graph showing Raman spectral analysis, revealing distinct changes in the phenylalanine for the UV-treated antibody.

[0051] FIG. 24 shows graphs demonstrating isothermal titration calorimetry (ITC) thermogram of (left) borophene nanosheets with UV-treated antibodies and (right) borophene nanosheets with UV-untreated antibodies.

[0052] FIG. 25 shows graphs demonstrating binding isotherm of (left) borophene nanosheets with UV-treated antibodies, and (right) borophene nanosheets with UV-untreated antibodies.

[0053] FIG. 26 shows a comparative analysis between traditional gold nanoparticle-based lateral flow immunoassay and borophene-based lateral flow immunoassay strip spot assay.Atty. Ref. No. 0073605-001099

[0054] FIG. 27 includes SEM images comparing plain nitrocellulose and nitrocellulose membrane incorporated with borophene nanosheets (top-left) bare nitrocellulose membrane (topright) nitrocellulose membrane coated with borophene nanosheets. Elemental mapping shows the presence of (bottom-left) carbon in the nitrocellulose membrane, and (bottom-right) boron distribution in areas where borophene nanosheets are on the nitrocellulose membrane.

[0055] FIG. 28 is a SEM image of a nitrocellulose membrane striped with a borophene-antibody conjugate. Arrow marks indicate the region containing the conjugates.

[0056] FIG. 29 is a schematic illustration of the borophene-UV-treated antibody conjugate within the nitrocellulose matrix, illustrating the oriented antibodies on the borophene nanosheet surface.

[0057] FIG. 30 is a SEM image of nitrocellulose membrane after the formation of the sandwich complex with the nitrocellulose matrix. Arrow marks indicate the presence of the sandwich complex as identified by the presence of gold nanoparticles.

[0058] FIG. 31 is a schematic illustration of showing the formation of the sandwich complex within the nitrocellulose matrix.

[0059] FIG. 32 is a deconvoluted X-ray photoelectron spectroscopy (XPS) spectra showing the peaks in Bls.

[0060] FIG. 33 is a deconvoluted XPS spectra showing the peaks in Nls.

[0061] FIG. 34 is a deconvoluted XPS spectra showing the peaks in Ci s.

[0062] FIG. 35 is a deconvoluted XPS spectra showing the peaks in Ols.

[0063] FIG. 36 is a Raman spectra of the nitrocellulose membrane, and the nitrocellulose membrane coated with borophene nanosheets. Morphological analysis of nitrocellulose membrane surfaces used in LFIA, as characterized by atomic force microscopy (AFM).Atty. Ref. No. 0073605-001099

[0064] FIG. 37 is an image showing a nitrocellulose membrane.

[0065] FIG. 38 is an image showing a test line on the nitrocellulose membrane showing the sandwich complex of antigen, detector antibody, and capture antibody.

[0066] FIG. 39 is a schematic illustration representing the working principle for the blood separator.

[0067] FIG. 40 shows (top) detection of HMGB-1 using a traditional LFIA with a limit of detection (LOD) of 250 pg / mL, and (bottom) detection of HMGB-1 using the developed borophene-antibody conjugate LFIA with a limit of detection (LOD) of 50 pg / mL, discernible to the naked eye.

[0068] FIG. 41 shows specificity of the developed borophene-antibody conjugate LFIA against commonly found proteins in menstrual effluent.

[0069] FIG. 42 is a graph showing a calibration curve illustrating the comparative performance of borophene-antibody conjugate LFIA against the conventional LFIA across a range of HMGB- 1 concentrations from 0 to 1000 pg / ml, under optimal experimental conditions.

[0070] FIG. 43 shows an AFM image of a borophene nanosheet.

[0071] FIG. 44 is a representative diagram of borophene suspension and the atomic arrangement of %3borophene phase.

[0072] FIG. 45 is a Raman spectra for borophene, thiol-modified oligonucleotide, and borophene functionalized with thiol-modified oligonucleotide (scalebar: 100 pm).

[0073] FIG. 46 shows an ITC study for thiol-modified oligonucleotide interaction with borophene.

[0074] FIG. 47 shows an ITC study for nucleobases interaction with borophene.Atty. Ref. No. 0073605-001099

[0075] FIG. 48 is a schematic diagram of an exemplary embodiment of an LFIA for endometriosis detection.DETAILED DESCRIPTION

[0076] The following description is of exemplary embodiments and methods of use that are presently contemplated for carrying out the present invention. This description is not to be taken in a limiting sense, but is made merely for the purpose of describing the general principles and features of various aspects of the present invention. The scope of the present invention is not limited by this description.

[0077] Embodiments relate to borophene-based platforms for highly sensitive, target-specific biosensing. These embodiments include borophene nanosheets functionalized with biomolecules such as antibodies and oligonucleotides. Such functionalized borophene nanosheets can be integrated into diagnostic devices designed for the targeted detection of disease biomarkers.

[0078] Borophene is a two-dimensional material composed of boron atoms arranged in a crystalline, sheet-like structure. It can exhibit a high surface area, excellent electrical conductivity, and amphiphilic surface properties. The material contains electron-deficient boron sites that can facilitate covalent bonding with electron-donating agents, such as thiol-containing compounds. This electron deficiency can be exploited to enable covalent attachment of biomolecules bearing thiol groups, such as antibodies and thiolated oligonucleotides, through suitable chemical functionalization methods.

[0079] Functionalization Strategies for Biomolecule Attachment

[0080] In one embodiment, antibodies may be functionalized onto borophene nanosheets to serve as biorecognition elements.Atty. Ref. No. 0073605-001099

[0081] Antibodies are frequently used in sensing applications because of their inherent specificity, adaptability, and reliability. However, challenges remain in achieving robust and efficient surface immobilization, primarily due to their moderate long-term stability and the need for proper orientation and high surface density of the antibodies for effective target recognition.

[0082] To address this, a photoinduced immobilization technique has been developed to functionalize borophene nanosheets with antibodies. Specifically, UV irradiation can be employed to cleave disulfide bonds within the antibody structure, generating reactive thiol groups. These thiols can then covalently bind to borophene surfaces, forming stable boron-sulfur (B-S) bonds.

[0083] Referring to FIGS. 1 and 2, an exemplary method 100 for functionalizing antibodies onto borophene nanosheets may include a step 110 of UV irradiating the antibodies. As described above, this step cleaves disulfide bonds within the antibody, particularly within cysteinecysteine (Cys-Cys) or tryptophan (Trp) triads, resulting in the formation of free thiol groups in the Fab regions. Here, the term Fab region (Fragment Antigen-Binding region) refers to the part of the antibody responsible for recognizing and binding specific antigens.

[0084] The method further includes a step 120 of combining or mixing the irradiated antibodies with borophene nanosheets, resulting in functionalized borophene. The electron-rich thiol groups interact with the electron-deficient boron atoms on the borophene surface, leading to covalent B- S bond formation.

[0085] This functionalization yields spatially oriented antibodies that maintain their antigenbinding activity. Specifically, the antibodies are oriented to expose their Fab domains, creating a bioactive surface optimized for sensitive biomolecule recognition. Covalent immobilization via thiol groups tends to promote a side-on orientation, where one Fab fragment is bound to theAtty. Ref. No. 0073605-001099 borophene surface, while the other Fab domain adopts an orientation within approximately 10° to 90°. This configuration ensures effective exposure of the Fab domain to target analytes, thereby enhancing binding efficiency and preserving antibody functionality.

[0086] Any antibody containing disulfide bonds capable of forming free thiol groups can be selected for functionalization on borophene nanosheets. Most naturally occurring antibodies contain disulfide bonds, which are crucial for maintaining their structural integrity and stability.

[0087] The antibodies may be specific to one or more pathogens of interest. As will be further detailed, these functionalized borophene nanosheets can be incorporated into biosensing devices for targeted detection of such pathogens.

[0088] In one example, embodiments may include detection of endometriosis markers, such as HMGB-1 proteins, present in menstrual effluent. For example, embodiments may utilize Immunoglobulin G (IgG) antibodies to detect the presence of HMGB-1 proteins.

[0089] However, the scope of the present disclosure is not limited to this example. It should be understood that embodiments are highly versatile and compatible with any type of antibody for the detection of any disease-specific biomarker, without restriction.

[0090] In a further embodiment, one or more oligonucleotides may be functionalized onto borophene nanosheets as biorecognition elements.

[0091] Referring to FIGS. 3 and 4, an exemplary method 200 for functionalizing oligonucleotides onto borophene nanosheets may include a step 210 of attaching a thiol group to the oligonucleotides. This modification can be made at either the 5 ' end or the 3 ' end, resulting in oligonucleotides with free thiol groups.

[0092] The method further involves a step 220 of mixing the thiol-modified oligonucleotides with borophene nanosheets. The electron-rich thiol groups interact with the electron-deficientAtty. Ref. No. 0073605-001099 boron atoms on the borophene surface, forming covalent B-S bonds and producing functionalized borophene nanosheets.

[0093] The oligonucleotides may be designed to be complementary to target gene sequences of a pathogen of interest. For example, oligonucleotides have nucleotide sequences that complement the nucleotide sequence of a target gene (e.g., adenine (A) in an oligonucleotide sequence may complement and bind to thymine (T) in a target gene sequence, cytosine (C) in an oligonucleotide sequence may complement and bind to guanine (G) in a target gene sequence, thymine (T) in an oligonucleotide sequence may complement and bind to adenine (A) in a target gene sequence, and guanine (G) in an oligonucleotide sequence may complement and bind to cytosine (C) in a target gene sequence). This enables highly specific hybridization-based detection within biosensing platforms.

[0094] The oligonucleotides may be specific to one or more pathogens of interest, conditions of interest, etc. As will be further detailed, these functionalized borophene nanosheets can be incorporated into biosensing devices for targeted detection of such pathogens, conditions, etc.

[0095] However, the scope of the present disclosure is not limited to this example. It should be understood that embodiments are highly versatile and compatible with any type of oligonucleotide for the detection of any specific biomarker, without restriction.

[0096] Lateral Flow Immunoassays

[0097] Functionalized borophene nanosheets may be incorporated into lateral flow immunoassays (LFIA), such as dipstick assays, for targeted pathogen detection. A typical LFIA is a portable, point-of-care diagnostic device that detects specific target analytes (e.g., antigens, nucleic acid sequences, proteins, etc.) by capillary flow of a liquid sample across a test strip containing immobilized biorecognition elements, such as antibodies or oligonucleotides. TheAtty. Ref. No. 0073605-001099 assay produces a visible indicator, such as a colored line, signaling the presence or absence of the target.

[0098] As illustrated in FIG. 5, an LFIA 300 may include a testing strip 302 comprising a sample application region 304, at least one test region 306, and a control region 308. A sample collected from a subject, e.g., bodily fluid, a solution containing nucleic acids, etc., may be applied at the sample application region 304. The sample then flows along the strip (e.g., via capillary action) through the test and control regions.

[0099] The sample may be collected using various methods, including but not limited to oral, nasal, cervical, or blood-based swabs, or urine collection. The collection instrument may be a standard swab, pipette, or other suitable device.

[0100] After collection, the sample may be introduced into a sensing solution that forms an aqueous mixture. This mixture is applied at or near the sample application region and flows through the strip toward the test and control regions.

[0101] The functionalized borophene nanosheets described above may form at least a part of the surface of the testing strip 302, with the immobilized antibodies or oligonucleotides serving as biorecognition elements at the at least one test region 306. For example, the functionalized borophene nanosheets may be present at least the at least one test region 306 as a borophene nanoplatelet. The biorecognition elements are configured to capture target analytes related to pathogens of interest as the sample flows through the strip.

[0102] The LFIA 300 may incorporate detection elements configured to provide a visible signal when immobilized and accumulated on the testing strip 302. In one embodiment, the detection elements may be provided in the sensing solution. In another embodiment, the detection elements may be temporarily immobilized on the testing strip 302, such as at aAtty. Ref. No. 0073605-001099 conjugate region of the strip. For example, the detection elements may be dehydrated on the testing strip 302 and may only mobilize / flow after being rehydrated by the aqueous mixture.

[0103] In either case, the detection elements are configured to attach, either directly or indirectly, to the target antigens or gene sequences related to pathogens of interest (if present in the collected sample). In some embodiments, the detection elements may be functionalized with detection probes specifically designed to bind to the target analyte related to the pathogen of interest. For example, if the target analyte is an antigen related to the pathogen of interest, the detection elements may be functionalized with antibodies specifically designed to bind to the antigen.

[0104] The detection elements are further configured to flow along the testing strip 302 with the aqueous mixture. It is understood that if target analytes are present in the collected sample, at least some of the detection elements will attach thereto and flow through the strip, and at least some of the detection elements will remain unattached and flow through the strip. It is similarly understood that if target analytes are not present in the collected sample, the detection elements will flow through the testing strip in an unattached state.

[0105] The detection elements may be any molecule, compound, etc. able to effectuate a color change when immobilized and accumulated on the testing strip 302. For example, the detection elements may be plasmonic nanoparticles, such as gold nanoparticles.

[0106] In one embodiment, the test region 306 corresponds to a zone where borophene nanosheets are functionalized with antibodies. As the sample flows through, the antibodies capture the target analyte, such as the target antigen, if present, resulting in immobilization of the antigen at this region (see FIG. 6). As previously discussed, the antibodies may be functionalizedAtty. Ref. No. 0073605-001099 to the nanosheets such that they are oriented to expose their Fab domains, creating a bioactive surface optimized for sensitive biomolecule recognition.

[0107] In another embodiment, the test region 306 corresponds to a zone where borophene nanosheets are functionalized with oligonucleotides. As the sample flows through, the oligonucleotides capture the target analyte, such as the target gene sequence, if present, resulting in immobilization of the gene sequence at this region.

[0108] Since the detection elements are configured to bind to their respective analytes, the immobilization of target analytes at the test region 306 will also lead to the accumulation of detection particles, creating a visible signal.

[0109] If the target analyte is absent, the detection elements will flow past the test region 306 without binding, resulting in no signal at that location.

[0110] The control region 308 contains immobilized capture probes designed to directly or indirectly bind the detection elements regardless of the presence of the target. For example, if the detection elements are functionalized with detection probes, the immobilized capture probes may be configured to bind the detection probes. This ensures that the system is functioning correctly; a visible line at the control region 308 indicates proper flow and activity.

[0111] When the detection elements are immobilized at the control region 308, a visible line or mark may appear, confirming the test’s validity. In a positive test, a line at the test region 306 indicates the presence of the antigen or gene sequence; absence of this line suggests a negative result.

[0112] Overall, the LFIA system 300 may be configured to detect the presence of one or more pathogens of interest. The lateral flow system 300 may therefore serve as a one-step detection method in a POC setting.Atty. Ref. No. 0073605-001099

[0113] Lateral Flow Immunoassays for Endometriosis Detection

[0114] In an exemplary embodiment, LFIA systems may be used to determine if a sample fluid includes endometriosis markers, such as HMGB-1 proteins. For example, the sample fluid collected from the patient may be menstrual effluent. In particular, the LFIA may be an absorbent pad configured to be worn by the user and to collect menstrual effluent from the user.

[0115] As illustrated in FIG. 48, the LFIA 600 can include an absorbent pad 602 including at least one sample application or inlet region 604, a test zone 608, and one or more microfluidic channels 606 configured to direct the sample fluid from the sample application region(s) 604 to the test zone 608. For example, a sample fluid collected from a subject, such as menstrual effluent, may be applied at the sample application region(s) 604. The sample fluid then flows to the test zone 608 via the microfluidic channels 606.

[0116] In some embodiments, the sample application region(s) 604, the microfluidics channels 606, and the test zone 608 may be laser printed into the absorbent pad 602. For example, laser printing may be used to engrave or carve into the pad 602 such that the sample fluid is maintained within these specified regions.

[0117] In other embodiments, the sample application region(s) 604, the microfluidics channels 606, and the test zone 608 may be 3D-printed on the surface of the absorbent pad 602. For example, 3D-printing techniques may be used to form 3D-structures / barriers on the surface of the pad 602 such that the sample fluid is maintained within these specified regions.

[0118] The sample application region(s) 604, the microfluidic channels 606, and the test zone may form any shape and / or design so long as the channels are configured to transport the sample fluid from the sample application region(s) 604 to the test zone 608. The configurationsAtty. Ref. No. 0073605-001099 shown in FIG. 48 are only exemplary, and the scope of the present disclosure is not limited to this embodiment.

[0119] The test zone 608 may include a test region and a control region. The test region may indicate the presence of an endometriosis marker in a patient sample, while the control region may indicate that the system is working properly.

[0120] After collection, the sample fluid may optionally be introduced into a sensing solution that forms an aqueous mixture. This mixture is applied at or near the sample application region(s) and flows through the pad toward the test and control regions.

[0121] The functionalized borophene nanosheets described above may form at least a part of the surface of the pad 602, with the immobilized antibodies serving as biorecognition elements at the test region of the test zone 608. The immobilized antibodies are configured to capture targets, such as HMGB-1 proteins related to endometriosis, as the sample fluid flows through the pad.

[0122] The LFIA 600 may incorporate detection elements configured to provide a visible signal when immobilized and accumulated on the pad 602. In one embodiment, the detection elements may be provided in the sensing solution. In another embodiment, the detection elements may be temporarily immobilized on the pad 602, such as at a conjugate region of the strip. For example, the detection elements may be dehydrated on the pad 602 and may only mobilize / flow after being rehydrated by the sample fluid or aqueous mixture.

[0123] In either case, the detection elements are configured to attach, either directly or indirectly, to the HMGB-1 proteins (if present in the collected sample).

[0124] For example, the detection elements may be functionalized with detection probes specifically designed to bind to HMGB-1 proteins. For example, if HMGB-1 proteins are presentAtty. Ref. No. 0073605-001099 in the sample fluid applied to the pad 602, the detection elements functionalized with the detection probes will bind to the HMGB-1 proteins and continue flowing through the testing strip. Alternatively, if no HMGB-1 proteins are present in the sample applied to the pad 602, the detection elements will nevertheless flow through the pad with the detection probes attached thereto.

[0125] The detection probe can be any molecule, compound, antibody, etc. capable of attaching to HMGB-1 proteins. In some embodiments, the detection probe is a detection antibody such as an anti-HNGB-1 IgG antibody.

[0126] The detection elements may be any molecule, compound, etc. able to effectuate a color change when immobilized and accumulated on the pad 602. For example, the detection elements may be plasmonic nanoparticles, such as gold nanoparticles.

[0127] In one embodiment, the test region of the test zone 608 corresponds to a region where borophene nanosheets are functionalized with antibodies, such as anti-HNGB-1 IgG antibodies. As the sample flows through, the antibodies capture the HMGB-1 proteins if present, resulting in immobilization of the HMGB-1 proteins at this region. As previously discussed, the antibodies may be functionalized to the nanosheets such that they are oriented to expose their Fab domains.

[0128] Since the detection elements are configured to bind to HMGB-1 proteins, the immobilization of HMGB-1 proteins at the test region 608 will also lead to the accumulation of detection elements, creating a visible signal.

[0129] If HMGB-1 proteins are absent, no visible signal will be generated.

[0130] The control region of the test zone 608 contains immobilized capture probes designed to directly or indirectly bind the detection elements regardless of the presence ofAtty. Ref. No. 0073605-001099HMGB-1 proteins. This ensures that the system is functioning correctly; a visible line at the control region indicates proper flow and activity.

[0131] Method of Making Borophene

[0132] The methods for synthesizing the borophene nanosheets described herein are not particularly limited and can be adapted or modified based on specific application requirements.

[0133] Referring to FIG. 7, in one embodiment, a method 500 for producing borophene nanosheets includes a step 510 of providing bulk boron material, such as boron powder.

[0134] The method further involves a step 520 of dispersing the bulk boron in a suitable solvent. In some embodiments, the solvent is a low-boiling, polar solvent (e.g., Milli-Q water) that is compatible with biological applications and minimizes the generation of reactive oxygen species.

[0135] The method further includes a step 530 of subjecting the dispersion to probe sonication to facilitate cavitation. This process promotes water molecule intercalation between the layers of boron, weakening interlayer van der Waals forces and initiating the exfoliation of borophene nanosheets. In certain embodiments, sonication is performed under an inert atmosphere to further prevent oxidation. Additionally, sonication time and parameters may be controlled to optimize exfoliation efficiency while minimizing oxidative degradation of the nanosheets.

[0136] Following sonication, the method may include a step 540 of centrifuging the mixture at a specified speed (e.g., between 3000 and 4000 rpm) to selectively isolate borophene nanosheets with a controlled number of layers.

[0137] Optionally, the method may include a surface modification step 550, such as promoting hydroxylation or other functionalization, to stabilize the nanosheets and enhance theirAtty. Ref. No. 0073605-001099 dispersibility in aqueous media, thereby improving their suitability for biological and biomedical applications.EXAMPLES

[0138] Below are examples of specific embodiments for carrying out the present invention. The examples are offered for illustrative purposes only, and are not intended to limit the scope of the present invention in any way. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperatures, etc.), but some experimental error and deviation should, of course, be allowed.EXAMPLE 1: Antibody Attachment to Borophene Nanomaterials and Use in LFIA

[0139] While this example illustrates the use of borophene nanosheets functionalized with antibodies for detection of a potential endometriosis biomarker (e.g., HMGB-1) in menstrual effluent, it is understood that this is only one potential use for embodiments described herein.Materials and MethodsMaterials

[0140] All chemicals were purchased from Sigma-Aldrich (Missouri, USA) unless otherwise stated. HMGB-1 recombinant antigen, mouse capture anti-human HMGB1, and biotinylated detection anti-human antibody (Z01LS-1122-LS68) were bought from Creative Biolabs Co., Ltd (New York, USA). A sample blood filter pad, an absorption pad (CFSP173000), and a high-flow nitrocellulose membrane (NC) (HF180) were bought from the Merch Millipore (Darmstadt, Germany).Synthesis of Borophene NanosheetsAtty. Ref. No. 0073605-001099

[0141] A total of 250 mg of boron powder was dispersed in 250 mb of Milli-Q water in a beaker and thoroughly stirred. The mixture was then subjected to probe sonication for 10 hours at a 12 pm amplitude, with a cycle of 2 seconds “on” followed by 1 second “off.” The resulting suspension was centrifuged at 3000 rpm for 3 minutes to collect the supernatant, which was then passed through a 0.45 pm PTFE filter and kept at 4°C. The final concentration of the suspension was determined and subsequently used for further analysis.Synthesis of Gold Nanoparticles

[0142] Citrate-capped AuNPs were prepared by our previously published methods.Briefly, 8.5 mg of tetrachloroauric (III) acid trihydrate (HAuCU 3H2O) was dissolved in 95 mb of deionized water. This solution was transferred to a 200 mb round-bottom flask equipped with a reflux condenser, placed in an oil bath, and brought to a boil under magnetic stirring.Subsequently, 5.0 mL of a 1% (w / v) sodium citrate solution was added rapidly. The mixture was kept at a boil and stirred for 30 minutes until it developed a wine-red color. After cooling, the final product was stored in the dark at room temperature until further use.For streptavidin conjugation to a gold nanoparticle

[0143] To prepare a streptavidin conjugate with gold nanoparticles via electrostatic interaction, adjust the pH of 5 mL colloidal gold solution to 7.0 using a freshly prepared Na2COs solution. Add a streptavidin solution prepared in 10 mM potassium phosphate buffer (pH 7.2) to the adjusted colloidal gold solution. Incubate the mixture at room temperature while stirring for 30 minutes. Then, add BSA and sucrose to final concentrations of 10% and 0.2%, respectively, and incubate the mixture for 1 hour at room temperature. Centrifuge the mixture at 11,000 rpm for 30 minutes at 4°C using a 5810R centrifuge (Eppendorf, Germany). Discard the supernatant and dissolve the resulting pellet in 10 mM Tris-HCl buffer (pH 7.2).Atty. Ref. No. 0073605-001099Dynamic Light Scattering Measurements

[0144] All samples were diluted to a concentration of 0.005 mg / mL in a disposable quartz cuvette. Dynamic light scattering (DLS) measurements were carried out using a Malvern Instruments Zetasizer Nano series system equipped with a 633 nm laser. Each sample was measured three times, and the results were averaged using the Zetasizer software. For analysis, the refractive index of water was set to 1.33, and the viscosity was taken as 0.8872, with a PDI of approximately 0.1 for each measurement.Atomic Force Microscopy (AFM)

[0145] AFM was carried out using an AFM instrument model of 56OOLs manufactured by Bruker Nano, USA. The analyses were performed in tapping mode in different sizes, using phase contrast and height modes. The raw images were processed with Nanoscope Analysis 3.0 imaging and analysis software package.HAADF-S TEM Measurements

[0146] Transmission electron microscopy (TEM) images were taken using a Talos F200X microscope. High-angle annular dark-field scanning TEM (HAADF-STEM) images, energy dispersive X-ray (EDX) mapping, and EDX line-scan profile were taken using an FEI 200 kV Titan Themis scanning transmission electron microscope.X-ray Photoelectron Spectroscopy (XPS)

[0147] XPS experiments were performed using a Physical Electronics Versa Probe ITT instrument equipped with a monochromatic Al ka x-ray source (hv = 1,486.6 eV) and a concentric hemispherical analyzer. Charge neutralization was performed using both low-energy electrons (<5 eV) and argon ions. The binding energy axis was calibrated using sputter-cleanedCu (Cu 2p32 = 932.62 eV, Cu 3p3 / 2 = 75.1 eV) and Au foils (Au 4f? / 2 = 83.96 eV).f Peaks wereAtty. Ref. No. 0073605-001099 charged referenced to CHXband in the carbon Is spectra at 284.8 eV. Measurements were made at a takeoff angle of 45° concerning the sample surface plane. This resulted in a typical sampling depth of 3-6 nm (95% of the signal originated from this depth or shallower). Quantification was done using instrumental relative sensitivity factors (RSFs) that account for the x-ray crosssection and inelastic mean free path of the electrons. The analysis size was ~100pm in diameter.Raman Spectroscopy

[0148] Borophene Spectrum Acquisition — Raman spectra were collected using a Renishaw inVia Reflex Raman Spectroscope system with the following parameters: a 785 nm laser, 45 mW (50%) power, a grating of 1200, 100* magnification, and an acquisition time of 0.3 s, with the center of Raman frequency set at 1100 cm1

[0149] SERS Experiment - Raman measurements were performed using a Horiba LabRAM HR Evolution spectrometer equipped with a 633 nm excitation laser focused through a 100* objective lens (NA 0.9), delivering an incident laser power of 400 pW on the sample.Samples were deposited onto a gold (Au) substrate, and fast mapping was conducted with an integration time of 0.9 seconds, a confocal hole size of 100 pm, and a 300 gr / mm grating coupled to a BIDD Si-array detector (Horiba - Synapse). The spectrometer was calibrated using the Raman response of a single-crystal silicon standard at 520 cm Acquired spectra were processed by subtracting background signals using an 8th-order polynomial fit and subsequently averaged across all spectra obtained within the mapped region.FTIR Spectroscopy

[0150] FTIR in the attenuated total reflectance (ATR) mode was performed in an Agilent Cary 630 FTIR spectrometer from 4000 to 650 cm1at room temperature on a diamond detector.Scanning Electron MicroscopyAtty. Ref. No. 0073605-001099

[0151] The surface characterization of the test strip to ensure the establishment of conjugated probes on the NC membrane was performed using SEM. Verios G4 SEM was used to characterize the test strip NC membrane before and after loading of the HMGB-1 sample. Lateral Flow Test Strip Preparation

[0152] The lateral flow assay test strips were prepared by a Claremont antibody stripping machine and a Guillotine Paper Cutter. Photographs of strips and liquid samples in containers were taken using an ESI quant and the Image was analyzed using Image Studio 4.0 The complete LFA strip consists of a blood filter sample pad, an absorbent pad, and a nitrocellulose (NC) membrane featuring one test (T) line and one control (C) line. These components were assembled on a backing pad with overlapping ends to ensure a continuous flow of the developing solutions. Biotinylated anti-HMGB-1 antibodies conjugated with streptavidin-AuNp (1 pg / strip) were immobilized on the conjugation pad by incubation at 37°C for 1 hour. For the test and control lines, UV-treated anti-HMGB-1 IgG antibodies conjugated with borophene nanosheets and anti-mouse IgG antibodies were immobilized on the NC membrane, respectively. The anti- HMGB-l-borophene conjugate was prepared by mixing 50 pg / mL UV-treated anti-HMGB-1 IgG with 0.15 mg / mL borophene nanosheets, followed by incubation at room temperature for 5 minutes. This conjugate was then sprayed onto the NC membrane using an antibody dispenser and dried at 37°C for 1 hour. The test and control lines were spaced 5 mm apart. The fully assembled LFA strip measured 3 60 mm and was stored in a sealed bag at room temperature until use. In conventional LFIA strip preparation, borophene is excluded from the test line, while the same nanoparticles are present on the conjugation pad.Detection of HMGB-1 using borophene-based LFIAAtty. Ref. No. 0073605-001099

[0153] A 10 pl sample of menstrual blood spiked with HMGB-1 standards was applied to the blood filter sample pad. After 30 seconds, 120 pl of PBST (1% BSA in PBS containing 0.05% Tween 20) was sequentially dispensed onto the pad. The sample then interacted with biotinylated anti -HMGB-1 antibodies conjugated to streptavidin-AuNp complexes on the conjugation pad. Subsequently, the complex bound to borophene and the capture anti-HMGB-1 antibody on the NC membrane, forming the test line, while unbound complexes migrated to form the control line. After approximately 15 minutes of incubation at room temperature, images of the strips were captured using an ESE Quant Flex. For quantitative analysis, the images were analyzed using Image Studio software, and the pixel intensity of the test line regions was measured to determine the color signal intensity.Bradford Assay

[0154] Borophene-antibody conjugation was performed by mixing 1 mL of borophene nanosheet suspension (0.125 mg / mL in ultrapure water) with anti-human HMGB-1 IgG antibody at a final concentration of 50pg / mL. Prior to conjugation, the antibody solution was irradiated at 254 nm using a Trylight® UV lamp (6 W, irradiance ~0.3 W / cm2) for 30 seconds to generate reactive thiol groups. Following UV irradiation, antibodies were incubated with borophene for 3 minutes at 25°C. The conjugation mixture was subsequently centrifuged at 3,000 x g for 10 minutes at 4°C to pellet the antibody-borophene complexes. The pellet was resuspended in 1 mL of ultrapure water for further analysis. A control group using non-irradiated antibodies was processed identically. To quantify antibody conjugation, a Bradford assay was performed using Coomassie Brilliant Blue G-250 dye. Briefly, IgG standards (1-100 pg / mL in phosphate- buffered saline (PBS), pH 7.4) and sample supernatants (150 pL) were mixed with 50 pL of Bradford dye reagent in a 96-well plate and incubated for 5 minutes at room temperature.Atty. Ref. No. 0073605-001099Absorbance was measured at 595 nm using a BioTek Synergy Hl microplate reader. The linear standard calibration curve determined free IgG concentration in the supernatants. Conjugation efficiency (%) was calculated using the formula: Conjugation (%) = [1 - (Free IgG in supernatant / Total IgG initially added)] x 100. The resulting conjugation efficiency was approximately 48%, corresponding to 24.04 pg / mL of IgG immobilized on the borophene surface.Ellman ’s Assay

[0155] Ellman’ s assay was used to quantify free thiol groups. The reaction buffer consisted of 0.1 M sodium phosphate buffer (pH 8.0) containing 1 mM EDTA. Ellman’s reagent (DTNB) was freshly prepared by dissolving 4 mg of DTNB in 1 mb of reaction buffer. A calibration curve was established using L-cysteine standards ranging from 0 to 200 nM. Antihuman HMGB-1 IgG antibodies were diluted to 50 pg / mL in the reaction buffer and exposed to UV irradiation (254 nm, Tiylight® UV lamp, 6 W, irradiance ~0.3 W / cm2) for 30 s. A nonirradiated control group was maintained under identical conditions without UV exposure. Postirradiation, aliquots of 150 pL from each sample or standard were transferred into a 96-well plate, and 50 pL of Ellman’s reagent was added. The plate was incubated at room temperature for 15 min. After UV activation, antibodies were incubated with borophene nanosheets as described above. Post-conjugation samples underwent the same Ellman’s assay procedure to assess residual free thiol concentration. Background absorbance from borophene alone was subtracted from all sample measurements. Absorbance values were recorded at 412 nm using a UV-Vis spectrophotometer, and the concentration of free thiols was determined using the established standard curve.Results and DiscussionAtty. Ref. No. 0073605-0010993D Bulk Boron to 2D Borophene Nanosheets

[0156] Borophene nanosheets were synthesized via probe sonication-assisted liquidphase exfoliation of bulk boron powder in Milli-Q water (FIG. 7). Water was chosen for its low boiling point and compatibility with biological applications. While high-boiling solvents such as Dimethylformamide (DMF) or N-Methyl pyrrolidone (NMP) are commonly used to stabilize 2D nanomaterial dispersions by lowering interlayer van der Waals forces, their removal is challenging and can lead to aggregation, limiting their use in biomedical contexts. In contrast, Milli-Q water (18.2 MQ-cm resistivity) minimizes the generation of reactive oxygen species (ROS) and, under probe sonication, generates intense cavitation microenvironments that facilitate the intercalation of water molecules between boron layers, weakening interlayer attractions and promoting exfoliation. Although water is generally less effective than some organic solvents for liquid-phase exfoliation due to its higher polarity and weaker biaxial straining effect, it can still yield thin borophene sheets, particularly when combined with optimized sonication and post-processing conditions. Controlled sonication time under an inert atmosphere helps suppress oxidation, and subsequent filtration via centrifugation at 3000 - 4000 rpm allows for the selective isolation of 3-6 layers of borophene nanosheets, consistent with prior studies reporting layer-thickness control via centrifugation speed.Borophene Nanosheets Charcterization

[0157] Controlled probe sonication of pristine bulk boron powder at room temperature facilitated the production of free-standing synthesized 2D borophene nanosheets, with an average hydrodynamic diameter of 200 ± 15 nm, as measured by dynamic light scattering (DLS) (FIG. 8). The size of the synthesized nanosheets results from the higher localized temperature in the vicinity of the solvent matrix. The resulting aqueous suspension of borophene was subsequentlyAtty. Ref. No. 0073605-001099 processed using centrifugation at 3000 rpm followed by filtration to isolate the final nanosheet suspension. Post-filtration, the nanosheets were found to have an average hydrodynamic diameter of 160 ± 20 nm, as determined by DLS. Scanning electron microscopy (SEM) was employed to study and compare the morphological changes between pristine boron powder (bulk boron) and exfoliated borophene nanosheets (FIG. 9). SEM images revealed a more flakelike two-dimensional (2D) structure in borophene, whereas pristine boron appeared solid and three- dimensional (3D). The morphology of the nanosheets was further characterized using transmission electron microscopy (TEM), which revealed nanosheet-like structures measuring approximately 200 nm (FIG. 10). High-angle annular dark-field scanning transmission electron microscopy (HAADF-STEM) coupled with elemental mapping confirmed the presence of boron (FIG. 10). Furthermore, high-resolution transmission electron microscopy (HR-TEM) images revealed lattice fringes of borophene obtained from liquid-phase exfoliation (FIG. 10). The parallel lattice fringes visible throughout the sample indicate a highly ordered crystal structure. The scale bar (10 nm) facilitates measurement of these fringes, which appear to have a Moire interference pattern spacing in the range of 0.29 ± 0.01 and 0.17 ± 0.01 nm (FIG. 10). This lattice spacing is consistent with interatomic distances reported for jA-borophene in prior studies. Moreover, these spacings differ from those of P12 borophene and P-rhombohedral boron. The uniform fringe patterns and absence of significant defects or distortions suggest the successful synthesis of high-quality borophene sheets predominantly in the %3 phase. Additionally, FT-TR was performed to confirm the borophene signature bonds in the synthesized nanosheets. The FT- IR spectra (FIG. 10) observed between pristine boron powder and the synthesized borophene nanosheets arise from structural and chemical transformations during liquid-phase exfoliation. In the hydroxyl region (-3400 cm'1), borophene exhibits a pronounced broad band, indicative ofAtty. Ref. No. 0073605-001099 substantial surface hydroxylation due to water interactions during aqueous exfoliation and a high tendency for edge oxidation in the reaction medium. In contrast, bulk boron shows minimal hydroxyl signatures, consistent with its lower surface-to-volume ratio and limited edge oxidation. Additionally, in the B-0 bonding region (1600-2000 cm '), borophene displays sharper, more intense peaks than bulk boron, reflecting increased oxidation at the edges and surfaces of the nanosheets. This aligns with the exfoliation process, which exposes reactive boron atoms to dissolved oxygen. With its intact 3D covalent network, pristine boron shows weaker B-0 vibrations, indicating minimal oxidation. Finally, the B-B bonding region (800- 1200 cm’1) further distinguishes the two: borophene exhibits sharper and more intense B-B vibrational modes, characteristic of its 2D planar structure. Bulk boron shows broader, less- defined B-B peaks, consistent with its 3D P-rhombohedral configuration. We also utilized X-ray photoelectron spectroscopy (XPS) to differentiate between pristine boron powder and synthesized borophene nanosheets. The Bls spectrum of the pristine boron powder indicated only one peak corresponding to B-B (187.33 eV). However, the Bls spectrum of borophene reveals three distinct peaks at 187.4, 188.6, and 192.0 eV, corresponding to B-B, B-O, and B2O3 bonds, respectively. These peaks represent relative contents of 56% for B-B, 36% for B-O, and 8% for B2O3. The presence of the B2O3 peak is likely attributed to electrochemical reactions occurring during the exfoliation process. Notably, borophene exhibits a higher proportion of B-B bonds, which may result from the exposure of freshly cleaved surfaces following exfoliation. Raman spectroscopy was then used to identify the dominant boron phase in the synthesized %3 borophene nanosheets. Based on the synthesis approach, the borophene nanosheets exhibited an isotropic hexagonally bonded phase, defined by a triangular lattice with periodic holes.Raman spectral analysis revealed bands at 296 (Au(Y)), 452 (Bg1(Y)), 783 (Ag3), 984 (Ag2), andAtty. Ref. No. 0073605-0010991165 (Ag1) cm'1, confirming the predominance of the phase in the synthesized nanosheets(FIG. 10) Moreover, the atomic force microscopy (AFM) images of the borophene nanosheets revealed approximately five sheet layers with an overall thickness of 1.10 ± 0.60 nm. The interlayer distance between two adjacent sheets was estimated to be approximately 0.2-0.3 nm. Therefore, a few-layered borophene nanosheets were synthesized using low-temperature liquidphase exfoliation in an aqueous solution, sonication-assisted intercalation, and bulk boron exfoliation. The method likely involves the dispersion of bulk boron particles into a liquid medium, where the intercalation properties of solvent molecules facilitate a reduction in exfoliation energy, enabling reconstruction and the formation of energy-favorable structures.Functionalization of Borophene Nanosheets with IgG Antibodies

[0158] Following the successful synthesis, characterization, and confirmation of the desired borophene nanosheets, we proceeded to explore their functionalization with antibodies to develop a diagnostic assay. We employed a well-established photoinduced immobilization technique (PIT), which facilitates the generation of reactive thiol groups in antibodies without compromising their antigen-binding activity. PIT offers significant advantages over traditional methods, including simplicity, speed, and effectiveness enabling the efficient tethering of antibodies onto surfaces with high affinity for thiol groups. The PIT strategy involves the UV irradiation of IgG antibodies, leading to selective photoreduction that cleaves disulfide bridges within cysteine-cysteine / tryptophan (Cys-Cys / Trp) triads (FIG. 11). This process generates four free thiol groups in the Fab fragments, of which two are available for covalent binding to metal surfaces (FIG. 11). Moreover, the optimal conditions for PIT with IgG antibodies include an irradiation time of 30 seconds and an antibody concentration of 50 pg / ml. Under these conditions, the disulfide bridges remain open for approximately 300 seconds, providingAtty. Ref. No. 0073605-001099 sufficient time for the activated antibodies to attach to the metal surface. Furthermore, immobilization of antibodies via these thiol groups promotes a side-on orientation, in which one fragment antigen-binding (Fab) domain is bound to the surface, while the other Fab domain adopts orientations within a range of 10° to 90°. This configuration ensures effective exposure of the Fab domain to the analyte in the surrounding medium, thereby enhancing binding efficiency and preserving functionality. Previous research from our group has highlighted the critical role of thiol groups, particularly those from cysteine, an amino acid, in facilitating site-selective boronsulfur conjugation. This approach leads to the formation of strong covalent bonds with boron atoms, enabling the stable and selective attachment of biomolecules, including antibodies.

[0159] Based on these findings, we utilized the PIT method for the selective photochemical reduction of disulfide bonds in immunoglobulins (Ig) via UV activation of neararomatic amino acids using a Trylight® lamp. We picked HMGB-1 IgG antibodies as a model system since the PIT approach is especially successful for all Immunoglobulin G (IgG) forms. A standard 10 mm quartz cuvette containing 500 pL solution of HMGB-1 IgG antibody at a concentration of 50 pg / mL was housed inside the low-pressure mercury U-shaped UV lamps and irradiated for 30 seconds at ambient temperature (FIG. 2). Considering the cuvette's proximity to the lamps and the wrapping geometry, we estimated that the solution received UV irradiation of 0.3 W / cm2The energy of UV photons thus released by the 6 W mercury UV lamp was absorbed by tryptophan residues and subsequently transmitted to surrounding electrophilic species, including adjacent cysteine-cysteine (Cys-Cys) disulfide bridges. This process resulted in the cleavage of the disulfide bonds and the formation of new reduced thiol (SH) groups. The resulting irradiated antibody solution (lOOpL) was then combined with borophene nanosheets (100 pL) at a concentration of 0.175 mg / mL at room temperature, facilitating theAtty. Ref. No. 0073605-001099 functionalization of borophene nanosheets via the formation of boron-sulfur covalent bonds (FIG. 6).Quantifying Antibody Functionalization on Borophene Nanosheet Surface

[0160] Following the successful preparation of antibody-functionalized borophene nanosheets via UV-induced thiol generation and subsequent B-S bond formation, we proceeded to characterize the conjugation efficiency and binding mechanism through quantitative analytical techniques. To evaluate and quantify the conjugation efficiency of UV-treated anti-human HMGB-1 IgG antibodies on borophene nanosheets, a Bradford assay using Coomassie Brilliant Blue G-250 dye was conducted (FIG. 14). A linear standard curve with correlation (R2= 0.998) was established using IgG concentrations ranging from 1 to 100 pg / mL (FIG. 14). Following incubation of UV-irradiated antibodies (50 pg / mL) with borophene nanosheets (0.125 mg / mL), a conjugation efficiency of 48% was calculated based on the residual unbound antibody measured in the supernatant. According to the calibration curve, 25.96 pg / mL of free IgG remained, indicating that 24.04 pg / mL IgG (equivalent to 192.3 pg IgG per mg of borophene) was successfully immobilized on the nanosheet surface. Control experiments using non-irradiated antibodies exhibited negligible binding (<5%), verifying that UV-induced thiol generation is essential for covalent conjugation, presumably via boron-sulfur (B-S) bond formation.Furthermore, the availability of free thiol groups in anti-human HMGB-1 IgG antibodies post- UV irradiation and subsequent borophene binding was assessed by Ellman’s assay. A standard calibration curve generated using L-cysteine concentrations ranging from 0 to 200 nM yielded a linear correlation (R2= 0.9959). Samples of anti-human HMGB-1 monoclonal antibodies (50 pg / mL) were diluted in reaction buffer for analysis. UV-untreated control antibodies exhibited minimal absorbance (A412 -0.009), similar to the Ellman’s reagent blank, confirming the absenceAtty. Ref. No. 0073605-001099 of accessible thiol groups in the native antibody structure. Following photoinduced thiol generation via exposure to 254 nm UV irradiation (Trylight® UV lamp, 6 W; irradiance ~0.3 W / cm2for 30 s), a significant increase in absorbance was detected (A412 -0.315), corresponding to 20.58 nM free thiol groups generated from disulfide bond cleavage. After incubation with borophene, absorbance decreased to 0.125, indicating 10.73 nM free thiol, a reduction of approximately 48%, suggesting substantial thiol-borophene binding, likely through B-S interactions. This thiol-specific interaction likely results in the antibodies adopting a side-on orientation on the borophene nanosheets, leaving only one Fab arm accessible for antigen binding (FIGS. 2 and 11). Consequently, although IgG structurally possesses bivalent antigenbinding sites, the functional valency is effectively reduced to monovalent binding due to steric constraints imposed by the borophene nanosheet surface.Antibody-Functionalized Borophene Nanosheets for Biosensing Application

[0161] It was essential to confirm the antibodies' conjugation and orientation on the surface following the successful functionalization of the borophene nanosheets. To achieve this, we developed an immunoassay -based methodology utilizing a mouse anti -human HMGB-1 IgG capture antibody (capture Ab), HMGB-1 antigen (HMGB-1 Ag), a biotinylated mouse antihuman detection IgG antibody (detection Ab), and streptavidin-coated gold nanoparticles (Strp- AuNPs). In this approach, 100 pL of borophene nanosheets conjugated with HMGB-1 capture Ab were incubated with 100 pL of HMGB-1 Ag at ambient conditions in a microfuge tube for 5 minutes. Thereafter, 10 pL of Strp-AuNPs conjugated with detection Ab were introduced to the solution and incubated at ambient temperature for 15 minutes. Following each incubation phase, centrifugation was used to remove unattached or free-floating antigens or antibodies from the solution matrix. We hypothesized that the successful conjugation of borophene nanosheets withAtty. Ref. No. 0073605-001099 capture Ab would enable the formation of a sandwich complex in the presence of HMGB-1 Ag. However, if the borophene nanosheets were not conjugated with the captured IgG antibodies, the sandwich complex would not form. We also used a control sample where the borophene sheets were mixed with UV-untreated HMGB-1 capture Ab. In this case, we hypothesize that the antibodies won't conjugate onto the surface of nanosheets, and a sandwich will not be formed. To validate our hypothesis, we employed TEM, AFM, and Raman Spectroscopy. The TEM image revealed a borophene nanosheet, identifiable as a lighter-colored, semi-transparent structure (FIG. 15). Superimposed on the nanosheet are spherical, darker regions representing gold nanoparticles (AuNPs), as shown in FIG. 15. These nanoparticles are conjugated via a sandwich complex composed of capture Ab, HMGB-1 Ag, detection Ab, and Strp-AuNPs. The image provides visual confirmation of the successful conjugation of borophene nanosheets with the capture of IgG antibodies, as evidenced by the formation of the sandwich complex in the presence of the HMGB-1 antigen. The distinct contrast between the nanosheet and the spherical nanoparticles highlights the functionalized borophene surface's structural integrity and the nanoparticles' specific attachment through antigen-antibody-mediated interactions. FIG. 16 represents a high-resolution TEM (HR- TEM) image of the borophene nanosheet, revealing distinct lattice fringes with a measured lattice spacing of 2.92 A. FIG. 17 illustrates a TEM image of the control sample, wherein the gold nanoparticles are not superimposed on the borophene nanosheet. This observation arises from the UV-untreated capture antibodies, which lack the thiol groups necessary for conjugation to the borophene surface. Consequently, the gold nanoparticles aggregate independently rather than adhering to the borophene nanosheets, thereby confirming the absence of a sandwich structure. Furthermore, low- and high-magnification high- angle annular dark-field scanning TEM (HAADF-STEM) images of the same sample revealedAtty. Ref. No. 0073605-001099 the borophene nanosheet as a light-colored sheet-like structure. In contrast, the dark spherical structures were identified as gold nanoparticles (FIG. 18). This was further validated through energy-dispersive X-ray spectroscopy (EDX), which confirmed the presence of boron exclusively in sheet-like structures. On the contrary, gold was exclusively detected within the spherical formations (FIG. 19). We also utilized AFM to study the formation of the sandwiched complex on the surface of borophene conjugated with capture antibodies. The sample containing borophene nanosheets conjugated with capture Ab was mixed with HMGB-1 Ag and Strp- AuNPs labeled with detection Ab. The resulting mixture was drop-cast onto cleaved mica to analyze the structural modifications of the 2D nanosheets induced by the antibody molecules using AFM. From the AFM images, the change in height due to the formation of the sandwich complex on top of the antibody-conjugated borophene nanosheets was distinguishable in comparison with pristine borophene. The pristine borophene nanosheets exhibited a surface height of 2.0 ± 0.5 nm, as observed from the height profile (FIG. 20). Upon the formation of the sandwich complex on top of the antibody-conjugated borophene nanosheets, a height increase to 3.5 ± 0.7 nm was observed, indicating potential successful attachment of the antibody molecules. The surface analysis further revealed the accumulation of sandwich complexes on top of the antibody-conjugated borophene nanosheets. Furthermore, with XPS analysis, the species detected in high-resolution spectra on the borophene sample included: CHx (carbon species), C- O, C-N, C=O, CF2, sulfonates, reduced sulfur, boron, oxidized boron, and fluorides. The S 2p core-level spectrum provides critical insights into sulfur's bonding environment and oxidation state. Typically, the S 2p signal appears as a doublet-S 2pv2 and S 2pv -due to spin-orbit coupling, with a characteristic energy separation of approximately 1.18 eV. The precise binding energy varies depending on the chemical state of sulfur and generally falls within the range ofAtty. Ref. No. 0073605-001099-161-170 eV. Thiols are generally found at 163.5-164.0 eV in the S 2p spectra, while metal sulfides / disulfides and metal-sulfur bonds are generally found to have lower binding energy (161-162.5 eV). The formation of a boron-sulfur (B-S) bond is indicated by a prominent peak at 163.3 eV, as confirmed by the XPS data (FIG. 21). This binding energy aligns with literature values for similar heteroatom-sulfur covalent bonds. The clear distinction between this peak and those representing free thiols (R-SH at 164.6 eV) and oxidized sulfur species (SOs at 167.86 and 169.04 eV) further supports our interpretation. The significant intensity of the B-S peak relative to other sulfur species indicates substantial covalent interaction rather than mere physical adsorption. These findings demonstrate that the capture antibodies were covalently attached to the borophene surface via thiol moieties generated through UV irradiation, highlighting the effectiveness of the functionalization strategy and its potential application in immunoassays.Analysis of Molecular Interaction and Antibody Orientation

[0162] To gain deeper insight into the molecular interactions occurring at the interface upon UV irradiation of antibodies, we leveraged the unique capabilities of Surface-Enhanced Raman Spectroscopy (SERS). In this example, we employed a gold substrate as the plasmonic surface. IgG antibodies, UV-treated and untreated, were immobilized using a custom micropipette system designed to deliver a controlled flow of solution across the surface (see Methods section for details). Following deposition, the samples were analyzed via a Raman spectrometer. The resulting SERS spectra (FIG. 22) were compared to the conventional untreated antibody. We observed significant alterations in the intensity of S-S stretching bands in the UV- treated IgG compared to the untreated control. A distinct spectral shift near 517 cm’1, corresponding to the Cys76-Cys94 disulfide bridge, suggests the emergence of a trans Ca-S conformation, potentially associated with an increase in free thiol generation. Additional shifts atAtty. Ref. No. 0073605-001099504, 522, 527, and 544 cm'1in the UV-irradiated samples indicate conformational changes likely related to the cleavage or rearrangement of disulfide bonds. These changes, attributed to the cystine residues, exhibit characteristic stretching vibrations within the 505-550 cm'1spectral range, gauche-gauche-gauche (ggg) conformations have an S-S stretching band at ~ 505-515 cm gauchegauche-trans (ggt) have an S-S stretching band at ~ 520-530 cm trans-gauche- trans (tgt) have an S-S stretching band at ~ 540-545 cm'1. These observations support the hypothesis that UV exposure promotes the photoreduction of disulfide bridges, forming free thiol groups that can interact more readily with the gold surface. Raman spectroscopy is particularly well-suited for distinguishing between reduced (free thiol) and oxidized (disulfide) states. The disappearance of S-S vibrational bands (e.g., at 503 cm'1) alongside the emergence of thiol- associated signals (e.g., around 680 cm'1) is characteristic of disulfide bond reduction. (FIG. 22). Aromatic amino acids are highly responsive to plasmonic enhancement and typically produce strong signals in protein Raman spectra. In the UV-treated sample, signals from phenylalanine (Phe), tyrosine (Tyr), and tryptophan (Trp) are significantly amplified. Notably, the characteristic Phe bands at 1122, 1240, 1335, and 1450 cm'1show marked intensity increases, likely due to their closer proximity to the plasmonic surface (FIG. 23). Similarly, enhanced Tyr signals are observed at 830, 850, and 875 cm1. The overall SERS results indicate that the illumination parameters used for PIT effectively facilitated thiol-mediated surface attachment of the antibody (Ab) molecules, more so than in the untreated Ab samples. Additionally, the enhanced intensity of numerous vibrational modes in the UV-treated samples suggests that antibody anchoring may also involve broader contact with the surface.

[0163] Moreover, we employed isothermal titration calorimetry (ITC) to investigate the thermodynamic interactions of HMGB-1 capture antibodies immobilized via photoinducedAtty. Ref. No. 0073605-001099 immobilization (PIT) on 2D borophene nanosheets, compared to UV-untreated HMGB-1 antibodies on the same nanomaterial. Briefly, a 50 pM solution of borophene nanosheets was loaded into a syringe for subsequent addition to the reaction cell containing either the UV-treated or untreated HMGB-1 capture IgG antibodies. Further, the concentration of HMGB-1 antibodies (both UV-treated and untreated) in the reaction cell was maintained at 1 mM for all titrations. The ITC thermograms and binding isotherms as shown in FIGS. 22-25 demonstrate a distinct difference in the binding characteristics of UV-treated and UV-untreated IgG antibodies on the borophene nanosheet surface. For UV-treated IgG (FIGS. 24 and 25), the thermogram exhibits sharp, well-defined peaks, indicative of a strong and favorable interaction. The binding stoichiometry (n) is 0.318, suggesting partial occupancy of available binding sites, with a dissociation constant (Kp) of 1.00 x 109M, reflecting high binding affinity. The enthalpy change (AH) is 0.287 kcal / mol, consistent with minimal heat release, while the Gibbs free energy (AG) is highly favorable at - 42.14 kcal / mol, confirming a thermodynamically strong binding interaction.

[0164] In contrast, for UV-untreated IgG (FIGS. 24 and 25), the thermogram reveals smaller, less defined peaks, suggesting a weaker and less favorable binding process. The binding stoichiometry (n) is significantly lower at 0.100, and the dissociation constant (KD) increases to 4.29 x 105M, indicating a much weaker interaction. The enthalpy change (AH) is higher at 3.871 kcal / mol, signifying a greater heat release during binding, but the Gibbs free energy (AG) is reduced to -32.97 kcal / mol, confirming the relatively lower binding affinity. Collectively, these parameters indicate two distinct binding modes: the first reaction is rapid, enthalpically driven, and exhibits highly favorable free energy, while the second interaction is slower, presumably influenced by the entropic factors, and features significantly reduced affinity. TheAtty. Ref. No. 0073605-001099 stoichiometric values suggest that both systems may involve cooperative or heterogeneous binding events. Thus, the results reveal that UV treatment significantly enhances the binding affinity of IgG antibodies to borophene nanosheets, likely through the formation of boron-thiol covalent bonds.

[0165] After confirming the effective adhesion of UV-treated IgG antibodies to the borophene nanosheet surface, the subsequent objective was to determine their orientation. The accessibility and functionality of these Fab regions were verified via fluorescence measurements after the binding of a labeled target antigen (aPLDH-Cy5 aptamer). To confirm antibody orientation on borophene nanosheets, we adopted a similar strategy using anti-PLDH IgG antibodies, F’LDH antigen, and a Cy5-labeled aptamer specific to PLDH. The PIT technique was employed to introduce thiol groups into IgG antibodies, which were then conjugated to the borophene nanosheets. The resulting antibody-functionalized nanosheets were incubated with LDH antigen bound to a Cy5-labeled aptamer. The experimental design included borophene nanosheets functionalized with anti- LDH IgG antibodies that serve as a substrate for capturing the malaria biomarker LDH. The Cy5-labeled aptamer (5'-Cy5-CTG GGC GGT AGA ACC ATA GTG ACC CAG CCG TCT AC-3') constituted the top layer, providing both fluorescence labeling and high specificity for PLDH at a relatively low cost. Fluorescence microscopy was employed to visualize the fluorescence signal, and the resulting images were processed with Image J software to quantify the corresponding signal intensity. Borophene nanosheets conjugated with UV-untreated antibodies exhibited no discernible fluorescence signal in the background-corrected image, indicating limited antigen-binding capacity. In contrast, borophene nanosheets functionalized with UV-treated antibodies display distinct red fluorescence spots, corresponding to photons emitted by individual fluorophores. These results confirm that the PITAtty. Ref. No. 0073605-001099 strategy facilitates the oriented immobilization of antibodies on borophene nanosheets and preserves the accessibility of their Fab regions for antigen binding.Assessment of Borophene Nanosheet-Antibody Conjugate Immobilization on Nitrocellulose Membrane and Antigen Capture

[0166] Building upon the previously obtained results, the PIT method was employed to functionalize two-dimensional (2D) borophene nanosheets with HMGB-1 IgG antibodies, and the feasibility of converting the immunoassay (detailed in the Materials and Methods section) into a lateral flow immunoassay (LFIA) format was examined. Before utilizing the borophene nanosheet-antibody conjugates, we wanted to conduct specific stability tests to ensure that borophene remains stably bound during assay conditions. Using a micropipette, Borophene was drop-cast onto the nitrocellulose strip at a 0.125 mg / ml concentration. The strips were assembled by laminating the NC-borophene layer with a sample pad and an absorbent pad on a backing card. Critically, a standard assay using chase buffer was performed to assess whether physically entrapped borophene would be washed away during lateral flow. As evidenced by photographs taken before and after the assay, the borophene remained visibly intact in its original position on the membrane following fluid flow, confirming the stability of this physical immobilization approach. With the immobilization stability confirmed, we next evaluated the efficiency of borophene nanosheets-antibody conjugate deposition and its impact on antigen capture performance. To this end, both UV-treated and UV-untreated HMGB-1 TgG capture antibodies were mixed with borophene nanosheets and deposited onto nitrocellulose membranes of defined dimensions. Specifically, the nitrocellulose membrane was sectioned into strips of 3 mm size, and 0.5 pL of borophene-antibody conjugates (UV-treated and conventional) were applied to each membrane at a concentration of 0, 25, 50, and 75 pg / ml. The borophene-antibodyAtty. Ref. No. 0073605-001099 conjugates were designated as the test zone (T). After the spotting procedure, the membranes were allowed to dry at 37°C. Once dry, the membranes were assembled into lateral flow test strips and analyzed for HMGB-1 antigen detection following the procedures detailed in the Methods section. As shown in FIG. 26, test strips containing the UV-treated antibody-borophene nanosheet conjugates exhibited stronger signals than conventional LFIA. This enhancement can be attributed to improved antibody orientation and increased availability of Fab fragments for antigen binding. As previously discussed, one of the intrinsic limitations of lateral flow assays arises from the random orientation of adsorbed antibody molecules, which reduces their antigenbinding capacity and thus diminishes assay sensitivity. By utilizing the PIT strategy in conjunction with borophene nanosheets, it was possible to address this orientation issue and enhance the overall sensitivity of the lateral flow immunoassay.Characterization of the Immobilized Borophene-Antibody Conjugates on Nitrocellulose Membranes

[0167] Having established the feasibility of integrating the HMGB-1 immunoassay into a lateral flow assay format, the next step involved the development of a fully operational lateral flow immunoassay. To ensure optimal performance, it was necessary to investigate and characterize the molecular interactions occurring on the nitrocellulose membrane surface, particularly those involving the immobilized borophene-antibody conjugates. This was accomplished by designing a sandwich immunoassay in a lateral flow format incorporating gold nanoparticles. In brief, HMGB-1 IgG antibodies (50 pg / mL) subjected to UV treatment were mixed with borophene nanosheets at (0.15 mg / ml) and incubated at room temperature for 5 minutes. As a control, borophene nanosheets were combined with UV-untreated HMGB-1 IgG antibodies, coated onto a nitrocellulose membrane using an antibody striping machine, and thenAtty. Ref. No. 0073605-001099 dried in an incubator at 37°C for 2 hours. The lateral flow assay was subsequently assembled following the protocols detailed in the Methods section. The assay incorporating UV-treated antibodies generated a significantly stronger signal than strips lacking UV-irradiated antibodies, indicating successful covalent linkage of UV-treated antibodies to borophene nanosheets. These results suggest that UV-untreated antibodies do not effectively adhere to the borophene surface during the assay, likely due to the absence of thiol groups necessary for stable binding. We utilized scanning electron microscopy (SEM) to investigate the role of 2D borophene nanosheets in increasing the surface area of nitrocellulose membranes and facilitating the formation of a sandwich structure in the lateral flow assay’s test zone. Depositing an aqueous borophene suspension onto a glass slide and allowing it to evaporate yielded a heterogeneous array of rough-edged microparticles of varying sizes. At higher magnification, the SEM images revealed that these larger borophene sheets consisted of smaller aggregates, which in turn were formed by the aggregation and stacking of nanometer-scale particles, indicating a hierarchical structure. Building on these findings, we coated the test line zone of the nitrocellulose (NC) membrane with 2D borophene nanosheets and compared it with a plain NC membrane using SEM analysis (FIG. 27). The plain NC membrane exhibited a three-dimensional open-pore configuration composed of interconnected fibrous threads, along with spherical features measuring 2-4 pm in diameter fused onto these fibrous structures (FIG. 27). In contrast, the region coated with 2D borophene nanosheets displayed evidence of borophene sheets (FIG. 27), suggesting that these nanosheets predominantly accumulate on the top layer of the nitrocellulose pore network. Furthermore, cross-sectional SEM images of both borophene-coated and plain NC membranes were acquired at various magnifications. Although individual nanosheets could not be discerned, the data indicate that borophene particles primarily permeate the top 60-70 pm of the 130 pm-Atty. Ref. No. 0073605-001099 thick NC membrane. Collectively, these SEM observations provide qualitative evidence that the borophene nanosheets enhance the surface area of the nitrocellulose strips’ top layer. Elemental analysis via energy-dispersive X-ray spectroscopy (EDS) conducted on different regions of the NC membrane showed an average composition of 54.35% carbon (FIG. 27), 29.72% boron (FIG. 27), 6.90% nitrogen, and 4.80% oxygen.

[0168] Previously, as discussed, the sensitivity of LFIAs is constrained by the orientation of the capture antibodies within the nitrocellulose (NC) membrane. Beyond orientation challenges, the capture molecules in the NC matrix tend to distribute uniformly throughout the membrane’s thickness (approximately 120-180 pm) without forming any noticeable gradient. Consequently, analyte recognition occurs at the membrane surface and within the three- dimensional open-pore structure (~3-20 pm) of the NC matrix. Therefore, a substantial fraction of the resulting detection signal may be concealed from automated readers or operators due to the membrane’s opacity, leading to diminished signal intensities and higher detection limits compared with scenarios where all binding events occurred at the surface. To address this limitation, the introduction of a 2D nanomaterial-antibody conjugate with a high surface-to- volume ratio as the detection element in the NC strip’s test region could potentially enhance both the immobilization density and the antigen-antibody interaction efficiency nearer to the membrane surface, thereby improving the assay’s overall sensitivity.

[0169] Further, the SEM images showed that the immobilizing UV-treated HMGB-1 antibodies over the 2D borophene nanosheets on the test line region of the NC membrane showed a densely packed arrangement of antibodies (FIG. 28). FIG. 29 shows the graphical representation of the borophene-antibody conjugate in an NC matrix highlighting that the photoinduced immobilization strategy enables strong covalent anchoring of the antibody to theAtty. Ref. No. 0073605-001099 metal surface, positioning one Fab region side-on orientation while exposing the other Fab to the environment for effective antigen binding. Further, upon the addition of the HMGB-1 antigen, the sandwich is formed (FIGS. 30 and 31). The sandwich structure consists of a borophene- capture antibody in the bottom layer followed by HMGB-1 antigen and then streptavidin gold nanoparticles labeled with biotinylated detection antibody (FIG. 31). The SEM image confirmed the presence of sandwich structure in the test zone (FIG. 30). Moreover, the sandwiched structures appeared to be formed in a concentrated area indicating the densely packed antibodies over the borophene surface (FIG. 30).

[0170] Moreover, we also utilized X-ray photoelectron spectroscopy (XPS) to understand the chemical composition of the sandwich structure formed on the test line. Previously, from our lab, we have reported the conjugation of cysteine conjugated to borophene for chiral induction to the 2D material. The interaction of B atoms with the thiol group of cysteine results in B-S bond formation, which was established with XPS as provided. This knowledge was leveraged for the conjugation of antibodies to the borophene surface for lateral flow sensing technology. XPS analysis was carried out with the lateral flow nitrocellulose strip containing the borophene antibody sandwich and compared with the control strip without the sandwiched complex. The XPS spectra of the pristine nitrocellulose membrane and borophene-antibody sandwich containing nitrocellulose membrane gave an understanding of the B, C, N, O, and S presence. The Bl s while not visible in the control nitrocellulose surface, are present in the borophene antibody sandwich spotted nitrocellulose strip. Three prominent peaks were observed at 187.5 eV for B-B bonds whereas the B-S bond is visible at 191.6 eV and the B-0 bond at 200.5 eV (FIG. 32). The Ols peaks for NO are observed at 532.9 eV, CON is observed in 534.2eV with a higher intensity and COC at 532.9 eV. A peak at 531.5 eV is observed for BO from theAtty. Ref. No. 0073605-001099 borophene-attached moiety (FIG. 33). The high-resolution XPS data of the NC membrane shows the Cis with peaks at 284.6, 287.0, and 288.6 eV (FIG. 34). These peaks can be assigned to the C-C, C-O-C, and C-O-N bonds. The peak at 289.6 eV arises from the carboxyl group in the antibodies. The S2p shows two peaks at 168.2 and 169.5 eV. The peak of Nls for nitrocellulose is consistent with the NO2 peak at 407.9 eV (FIG. 35). For the borophene antibody sandwich on NC, two new peaks arise at 404.8 eV for NH2 and another peak at 400.3 eV for amide groups from the antibody Raman spectra conformed the peaks appearing in nitrocellulose strip containing antibodies immobilized onto borophene nanosheets. (FIG. 36). AFM analysis of the borophene-coated nitrocellulose (NC) membrane with the sandwich complex revealed an average roughness height (Sa) of 0.94 nm and a root mean square (RMS) roughness (Sq) of 1.72 nm (FIG. 37). In comparison, the NC membrane without the sandwich complex exhibited Sa and Sq values of 0.94 nm and 1.19 nm, respectively (FIG. 38). These changes in surface roughness are in agreement with the presence of immobilized biomolecules. However, we acknowledge that AFM alone cannot definitively resolve antibody orientation, particularly on topographically irregular surfaces like borophene. Therefore, while the increased roughness may suggest surface functionalization, the effective sandwich formation in the presence of HMGB-1 antigen could be due to the accessibility and binding functionality of the antibody Fab regions.Borophene-Antibody Conjugate Lateral Flow Assay for HMGB-1 Detection in Menstrual Effluent

[0171] Having characterized the molecular interactions at the test line of our developed lateral flow immunoassay (LFIA), we proceeded to evaluate its capacity for detecting HMGB-1 in whole menstrual effluent. In this example, we employed photoinduced immobilization to covalently attach HMGB-1 IgG capture antibodies onto two-dimensional (2D) boropheneAtty. Ref. No. 0073605-001099 nanosheets and compared the assay performance to that of a conventional LFIA in a sandwichbased format. In the borophene-based LFIA, the test line of the nitrocellulose membrane was coated with UV-treated HMGB-1 capture antibodies on 2D borophene nanosheets. In the conventional LFIA, only the HMGB-1 capture antibody was immobilized on the nitrocellulose membrane (FIG. 5). Both assays utilized the same rabbit IgG at the control line and a conjugation pad containing gold-labeled HMGB-1 detection antibodies. Various concentrations of HMGB-1 (0-1000 pg / mL) were spiked into whole menstrual blood and applied to the sample pad, which incorporated a blood separator. The blood separator (a glass fiber filter pad) effectively separates blood components, trapping red blood cells and larger cellular debris on the surface while allowing only plasma containing the target HMGB-1 analyte and proteins to flow through to the nitrocellulose membrane (FIG. 39). This physical separation step significantly reduces matrix interference before the sample reaches the detection zone. As the sample flows, HMGB-1 binds to the gold-labeled detection antibodies, and this complex then migrates to the test line, where it interacts with the immobilized capture antibodies to form a visible sandwich complex. Unbound detection antibodies proceeded to the control line, yielding a separate signal. Visual analysis of the borophene-based LFIA indicated that test lines could be observed at HMGB-1 concentrations as low as 50 pg / mL (FIG. 40), whereas the conventional LFIA required at least 250 pg / mL to produce a visible band. These findings suggest that the borophene-based LFIA achieves a naked-eye detection limit that is approximately 80% lower (i.e., more sensitive) than the conventional assay. Subsequently, to evaluate potential cross-reactivity, the specificity of the lateral flow assay was examined using albumin, fibrinogen, and gamma globulin — proteins commonly present in whole menstrual effluent. Each protein (20 pg / mL) was individually mixed with 120 pL of chase buffer and applied to the lateral flow assay strip. NoneAtty. Ref. No. 0073605-001099 of these proteins elicited a signal at the test line as shown in FIG. 40. These observations confirmed the specificity of the lateral flow assay, which exclusively recognized its target recombinant protein HMGB-1 and did not interact with other recombinant proteins found in menstrual effluent. Furthermore, quantitative assessments were performed using an ESE Quant Flex reader, and the color intensities at the test line were analyzed with Studio 4.0 software. As expected, the signal intensities increased for both LFIA formats in proportion to increasing HMGB-1 concentrations. FIG. 41 shows the calibration curves for the borophene-based and conventional LFIAs, which yielded high correlation coefficients (R2= 0.9960 and R2= 0.9816, respectively). To calculate the analytical limit of detection of the borophene LFIA, we use LOD= 3.3*(— ), the (LOD) was approximately 40 pg / mL for the borophene-based LFIA and 240 pg / mL for the conventional LFIA. The rationale for targeting low concentrations, such as 40 pg / mL, is supported by clinical evidence indicating that HMGB1 levels in menstrual blood are significantly elevated in individuals with endometriosis compared to healthy controls. However, early-stage or asymptomatic cases may present with only modest increases. The incorporation of two- dimensional borophene nanosheets, combined with photoinduced antibody immobilization, enhances the assay’s analytical performance, resulting in an approximately 500% increase in sensitivity relative to conventional LFIA platforms. While ELISAs can detect lower HMGB1 levels, our borophene-based LFIA achieves clinically relevant sensitivity (40 pg / mL) in a rapid, POC format, enabling non-invasive monitoring of localized inflammatory activity. This threshold aligns with reported menstrual fluid HMGB1 levels in endometriosis progression, where early detection is critical for timely intervention. Unlike lab-based ELISA, our approachAtty. Ref. No. 0073605-001099 balances sensitivity with practicality for decentralized settings, addressing unmet needs in endometriosis screening.EXAMPLE 2: Oligonucleotide Attachment to Borophene NanomaterialsSyntheses of / 3 Borophene (Bph) nanosheet

[0172] 250 mg of boron powder was added to 250 mL of Milli-Q water in a beaker and mixed well. This solution was then subjected to probe sonication for 10 h with an amplitude of 12 pm with 2 s “on” time period and 1 s “off’ time period. The suspension was thereafter centrifuged at 3000 rpm for 3 min, and the supernatant was collected. This supernatant was then filtered using a 0.45 pm PTFE filter and stored in a 4 °C refrigerator. The resulting concentration of the suspension was calculated and used for further analysis.Synthetic Oligonucleotides (ODN and ODN-SH)

[0173] HIV viral DNA sequence used was purchased from Sigma Oligos and purified using HPLC. To impart thiol functionality on the oligonucleotide, sequence the 3’ end was protected with (CH2)6S-S or ThiC6-SS. Such a disulfide form was used to prevent spontaneous, uncontrolled oxidation, which in turn would lead to dimer formation. The disulfide bond of these oligos were broken by incubating the aqueous solution of the oligonucleotides with Cleland’s reagent, dithiothreitol (DTT), for 1 h at room temperature and passing the reaction mixture through Nap-10 columns, all the while maintaining the pH at 6.0.Syntheses of Oligo-Bph Conjugates

[0174] 5 pM of ODN-SH and FAM-ODN-SH was mixed with 50-500 nM of as- synthesized Bphs suspensions. The reaction mixtures were then stirred for 30 min at room temperature. The resulting suspension was then centrifuged at 6000 rpm for 5 min to precipitate the oligonucleotide attached Bph. The supernatant with free oligonucleotide was then replacedAtty. Ref. No. 0073605-001099 with fresh milli-Q water. This centrifugation process was continued for two consecutive cycles to get Bph- ODN-SH and Bph-FAM-ODN-SH products. All the sample suspensions were sonicated for 2 min in a bath sonicator before use.Characterization of the Oligonucleotide Functionalized Borophene for Target Specific Biosensing Platform

[0175] To obtain a comprehensive understanding of the modes of interaction of the oligonucleotides with the Bph surface, we took a dilute suspension of Bph and incubated it with the thiolated oligonucleotides under constant shaking at 37°C for 30 min. The well-reacted mixture was thereafter washed with Milli-Q water three times and studied thoroughly. To understand the change in the hydrodynamic diameter, comparative DLS with pristine Bph, Bph-ODN, and Bph- ODN-SH suspension was conducted. The data exhibited changes in the size after the interactions of Bph with ODN-SH containing a thiol group in comparison to that without a thiol functional group. Pristine Bph in its native form shows a hydrodynamic diameter of around 295 ± 10 d.nm with a low poly dispersity index (PDI < 0.05), which embodies a few layers of Bph nanosheets. However, when ODN-SH is added to the same suspension of Bph and allowed to incubate for 30 min, the size increases significantly to 342 ± 5 d.nm (PDI < 0.05), proving attachment of the strongly bonded thiol group with the Bph nanosheets.

[0176] Based on the fundamental working principle of the antisense oligonucleotide, as the target gene comes in the vicinity, the attachment of nucleobases adenosine (A), Thymine (T), G and C with their complementary strand causes the hairpin oligonucleotide to open. This conclusion was evidenced from the DLS measurement of the Bph-ODN-SH solution after the target was added, giving an increase in the value to 396 ± 20 d.nm with a broad spectrum (PDI < 0.05). Since the ODN-SH is attached to the surface of the Bph, the target added to the suspensionAtty. Ref. No. 0073605-001099 gets attached to the Bph-ODN-SH moiety and opens the hairpin loop to give a greater hydrodynamic diameter. This conjectures to the fact that the incubation process causes the ODN- SH to interact with the surface due to the thiol affinity of the Bph moiety and plausibly also shows intercalation of thiolated oligonucleotides between the layers of Bph. This allows credibility to our hypotheses that Bph, containing electron-deficient boron molecules, when comes in close proximity to the thiolated group of ODN-SH, a favorable interaction between the molecules is established, further confirmed by the attached target gene. This necessitated more research into the mechanism by which nearby Bph nanosheets may affect the photophysical characteristics of modified oligonucleotides and how this phenomenon might be advantageously applied for possible sensing applications. For sensing purposes, when oligonucleotides are attached to the surface of the Bph, electrokinetic potential changes from -9.83 ± 6.2 mV to -45 ± 2.0 mV, exhibiting a more stable colloidal suspension for nano sensors. The Raman spectra of pristine Bph and pure ODN- SH were compared with the Bph-ODN-SH molecule (FIG. 45). From the Raman spectra peaks at 181 (Bg2), 297 (Au(Y)), 450 (Bg1(Y)), 783 (Ag3) and 1230 (Ag1) 1164 cm'1was obtained from the vibrational modes of interaction present in %3 Bph phase. Similar bands were observed in the Bph- ODN-SH molecule. The oligonucleotides in the presence of thiol functional group show Raman peaks under a broad spectrum for 675, 723, 739, 776 cm1attributed G, A, T and C ring breathing, respectively. The peaks at 786 and 1097 cm'1can be assigned to PO2' and that at 1327 cm'1to A. Smaller peaks at 1570 cm'1can be assigned to A and G and those at 1650 cm'1can be assigned to T and C. When compared with the Raman spectra of the Bph-ODN-SH, there appear the signals of both Bph and ODN-SH present.

[0177] The Bph nanosheets were studied under TEM at a lOx dilution of the stock Bph solution, giving the final concentration of 2 mM, which was consistently used throughout theAtty. Ref. No. 0073605-001099 study. From images obtained it was observed that the pristine Bph nanosheets were present, with sheet-like structures visible having definite boundaries achieved from the liquid phase exfoliation process. Next, 5 uM of the ODN-SH solution was added to the 2 mM Bph suspension. From the TEM studies of the oligonucleotide decorated Bph, the size increased to an average dimension of 342 ± 5 d.nm as observed from the pristine structure of 295 ± 10 d.nm. There were subtle changes observed under TEM, one of which was the formation of a layer of oligonucleotides, or stringlike attachments, with the nanosheet structure. Under higher magnification, the attachment of oligonucleotides with Bph was confirmed with fringe patterns of 0.27 nm, obtained in HRTEM images. From the EDX mapping, a clear signal of sulfur on the Bph surface was visible, confirming the attachments of thiolated ODN-SH. In the presence of ODN-SH, agglomerations of Bph nanosheets in their pristine state become scarce. Hence, the presence of non-agglomerated Bph nanosheet entities can be attributed to the oligonucleotide’s binding interaction with the surface, providing the structures with greater stability. The resistance of the nanoparticles against the aggregation as explained in the context of Derjaguin-Landau-Verwey-Overbeek (DLVO) theory relates to DNA or oligonucleotide decorated surface of the nanoparticle causing the electrostatic repulsion between the negative phosphate backbones. Chemical compositions of the ODN-SH, Bph, Bph-ODN-SH were analyzed with X-ray photoelectron spectroscopy (XPS). The peak at 187.3 eV can be assigned to the B-B bond, and the peak at 188.5 eV for B-0 (FIG. 47). In case of Bph-ODN-SH the presence of the P 2s peak of ODN-SH overshadows the B peak. However, there is a peak shift to 186.7, 187.6, and 188.2 eV interaction of an electron-rich SH group with boron. On the other hand, the S 2p3 / 2 peak appears at 163.2 eV, and S 2pi / 2 appears at 164.4 eV, which has an area ratio of 2: 1 (FIG. 46). These peaks undergo a blue shift to 163.5 and 164.8 eV, respectively, due to the interaction with boron atoms (FIG. 47). The peak at 168.3 eV arises dueAtty. Ref. No. 0073605-001099 to oxidized sulfur from aerial oxidation, which diminishes when boron from Bph attaches to S, thereby blocking the possibility of oxidation at the air-water interface.Reaction Kinetics and Thermodynamic Spontaneity of Interactions

[0178] ITC is used to confirm binding interactions and investigate further biomolecular interactions in quantitative ITC is a unique method that provides a comprehensive thermodynamic profile and kinetic aspects of the interaction in buffer solutions. ITC can, in principle, provide enthalpy (AH) which is the total heat associated with the interaction of the two molecules, K which is the binding constant or association constant, Kd also known as the dissociation constant and the stoichiometry n of the interaction by assessing the degree of complex formation while altering the ligand concentration, ODN-SH in this case, throughout the titration in the system. The binding or Gibbs free energy (AG) can be obtained from the system’s change in AH and entropy (AS) to provide a comprehensive prediction of the spontaneity of the reaction, considering both the heat content and the disorder of the system. For a reaction process the thermodynamic parameters,AG = AH -TAS . equation 1AG = -RT In K . equation 2Kd - I K . equation 3

[0179] Where T is the absolute temperature in Kelvin, and R is the universal gas constant, and K is the equilibrium constant for the binding process. The heat released or absorbed during binding at constant pressure in each injection Qinj, is proportional to the molar enthalpy change associated with that process, AH, and the amount of macromolecule-ligand complex formed. The Brandt's c parameter is defined to be equal to the total macromolecule concentration multiplied by the equilibrium constant K. equation 4Atty. Ref. No. 0073605-001099 c = n K [M] ceil equation 5

[0180] Where Vmj is the volume of the injection, [M]inj is the concentration in molarity of the ligand, ODN-SH in this case, tried against the concentration inside the cell [M]ceii of the macromolecule, Bph in this case.

[0181] To explore the possible interactions between Bph and thiolated oligonucleotides, calorimetric measurements, ODN-SH, and the nucleobases were considered in different sets. The buffer medium matched for this study was water. The ODN-SH having a concentration of 0.1 mM, was tried against 200 pL of 0.001 mM Bph suspension present in the well. The thermodynamic parameters, upon binding, were derived from the known concentrations of Bph and the titrant ODN-SH (FIG. 46). From this study, the number of sites was calculated to be n = 6.47 which gave an understanding of the binding sites achieved. From the graph, the binding constant was found to be Kd = 6.38 pM. The low value of Kd justifies the appropriate binding of the Bph and thiolated oligonucleotide. It was also observed that endothermic binding with AH of 25 kcal mol'1was taking place between the two molecules, which implied an intake of heat on interaction of oligonucleotides with the Bph surface and an increase of entropy AS by 107.6 cal mol'1K'1.

[0182] The overall binding reaction being endothermic indicates that conformational changes in the DNA and ligand binding require the absorption of heat. The primary binding site, Bph, starts to become partially saturated with the DNA; additional binding may occur cooperatively or follow a multi-site binding model. From the ODN-SH solution, the endothermic DNA binding would lead to a AH > 0, from which we can assume that AS must be high since negative Gibbs’ free energy is indispensable for the reaction to occur spontaneously. Biomolecular interactions can be affected by electrostatic force, hydrogen bonding, and hydrophobic forces, which may be present in the borophene system. The presence of water adsorbed on possibleAtty. Ref. No. 0073605-001099 hydrophobic pockets of borophene is unstable and there is an increase in entropy when the water molecules are displaced with thiolated oligonucleotides. Water is arranged in a 3D network of hydrogen bonds, forming stiff hydration shells on the surfaces of. When these layers are displaced, during ligand binding or adsorption, water molecules are released into the bulk, where they acquire rotational and translational freedom, leading to an overall increase in entropy. In the case of Bph, ODN-SH attaching on the surface of the nanosheets displaces ordered water, resulting in entropy gains offsetting hybridization enthalpy penalties as observed in ITC. The oligonucleotide backbones have charged regions, such as amine, phosphates and thiol modification in this case, which can attract counterions within the solution. Upon binding with the borophene, these counterions are released, resulting in an increase of entropy with enhanced disorder in the bulk phase. These mechanisms of increase in entropy possibly leads to thermodynamically spontaneous interaction of Bph and ODN-SH.

[0183] We also conducted ITC studies on independent nucleobases and observed that only G exhibited an exothermic binding with Bph (FIG. 47). However, the overall interaction with the antisense oligonucleotide provides evidence of an endothermic reaction that surpasses the exothermic interaction of G. This endothermic reaction results from ODN-SH primarily interacting with Bph through sulfur interaction with the boron atom of Bph, which pushes out water molecules and creates a more disordered system overall. Hence the G-Bph interaction was precluded from being considered the primary mode of interaction, given the overall binding was driven by the B- S interaction. The pKa of the nucleobases are known to be sensitive to environmental change in the experimental medium. According to Henderson-Hasselbalch Equation pH = pKa+ log([HA] / [A ]) equation 6Atty. Ref. No. 0073605-001099

[0184] When pH of the environment is equal to the pKavalue, equimolar concentrations of acid and conjugate base exist. When pH is less than the value of pKa, or an acidic condition exists, protonated form of the molecule dominates, whereas in basic medium or when the pH is greater than the pKa, deprotonated form of the molecule dominates. From the pKavalues of purines A’s N1 is 4.1 and G’s N1 is 3.2-3.3 and N7 is 9.2-9.6. In case of pyrimidines, C’s N3 is 4.4 and N4 is 12.2, whereas T’s N3 is 9.9. In the case of Bph suspension the solution has a pH of 7.1 which is mildly acidic. In this case A, G and C have the N1 or N3 in the unprotonated state. It is important to mention here that while the pH of the A, C, T solution was around 6.4, 6.3 and 5.4, respectively, in case of G the solution was made acidic at pH 1.1 to improve solubility. This caused both the N 1 and N7 to be in a protonated state. However, we observed a strong binding in this case due to the presence of the exocyclic primary amine, which has a lone pair of electrons to cause favorable interaction with electron-deficient boron in Bph.

[0185] It should be understood that modifications to the embodiments disclosed herein can be made to meet a particular set of design criteria. For instance, the number of or configuration of components or parameters may be used to meet a particular objective.

[0186] It will be apparent to those skilled in the art that numerous modifications and variations of the described examples and embodiments are possible in light of the above teachings of the disclosure. The disclosed examples and embodiments are presented for purposes of illustration only. Other alternative embodiments may include some or all of the features of the various embodiments disclosed herein. For instance, it is contemplated that a particular feature described, either individually or as part of an embodiment, can be combined with other individually described features, or parts of other embodiments. The elements and acts of theAtty. Ref. No. 0073605-001099 various embodiments described herein can therefore be combined to provide further embodiments.

[0187] It is the intent to cover all such modifications and alternative embodiments as may come within the true scope of this invention, which is to be given the full breadth thereof.Additionally, the disclosure of a range of values is a disclosure of every numerical value within that range, including the end points. Thus, while certain exemplary embodiments of the apparatus and process and / or utilization and methods of making and using the same have been discussed and illustrated herein, it is to be distinctly understood that the invention is not limited thereto but may be otherwise variously embodied and practiced within the scope of the following claims.

Claims

Atty. Ref. No. 0073605-001099What is claimed is:

1. A method of making a functionalized borophene nanosheet, the method comprising: providing a borophene nanosheet;UV irradiating antibodies to cleave disulfide bonds within the antibodies and to generate reactive thiol groups; and combining the borophene nanosheet and the irradiated antibodies to provide the functionalized borophene nanosheet.

2. The method of claim 1, wherein providing the borophene nanosheet comprises: providing bulk boron material; dispersing the bulk boron material in a solvent to provide a dispersion; subjecting the dispersion to sonifi cation to facilitate cavitation; and subjecting the sonified dispersion to centrifugation to provide the borophene nanosheet.

3. The method of claim 1, wherein at least some of the reactive thiol groups are in Fab domains of the antibodies.

4. The method of claim 3, wherein the functionalized borophene nanosheet comprises antibodies oriented to expose their Fab domains.

5. An apparatus for detecting markers related to a pathogen of interest, the apparatus comprising a testing strip including, in sequence:Atty. Ref. No. 0073605-001099 a sample application region configured to receive a sample collected from a subject, wherein the sample is configured flow through the testing strip; detection elements configured to directly or indirectly attach to markers related to the pathogen of interest; a test region configured to detect the presence of markers related to the pathogen of interest, wherein at least a portion of the test region comprises the functionalized borophene nanosheet formed from the method of claim 1, wherein the functionalized borophene nanosheet is configured to immobilize the detection elements; and a control region having immobilized capture probes bound to the testing strip, wherein the immobilized capture probes are configured to immobilize the detection elements.

6. The apparatus of claim 5, wherein the pathogen of interest is endometriosis.

7. The apparatus of claim 6, wherein the markers are HMGB-1 proteins.

8. The apparatus of claim 7, wherein the antibodies are IgG antibodies.

9. An absorbent pad for detecting markers related to endometriosis, the absorbent pad comprising: a sample application region configured to receive a sample collected from a subject, wherein the sample is configured flow through the absorbent pad via one or more microfluidic channels;Atty. Ref. No. 0073605-001099 detection elements functionalized with detection probes configured to capture the markers related to the pathogen of interest; a test region configured to detect the presence of markers related to endometriosis, wherein at least a portion of the test region comprises the functionalized borophene nanosheet formed from the method of claim 1, wherein the functionalized borophene nanosheet are configured to capture the markers related to the pathogen of interest; and a control region having immobilized capture probes bound to the absorbent pad, wherein the immobilized capture probes are configured to capture the detection probes functionalized to the detection elements.

10. The absorbent pad of claim 9, wherein the markers are HMGB-1 proteins.

11. The absorbent pad of claim 10, wherein the antibodies are IgG antibodies.

12. The absorbent pad of claim 10, wherein the detection probes are anti- HMGB-1 antibodies.

13. The absorbent pad of claim 12, wherein the immobilized capture probes are anti- HMGB- 1 antibodies.

14. The absorbent pad of claim 9, wherein the detection elements are plasmonic nanoparticles.Atty. Ref. No. 0073605-00109915. A method of making a functionalized borophene nanosheet, the method comprising: providing a borophene nanosheet; functionalizing oligonucleotides with reactive thiol groups; and combining the borophene nanosheet and the functionalized oligonucleotides to provide the functionalized borophene nanosheet.

16. The method of claim 15, wherein providing the borophene nanosheet comprises: providing bulk boron material; dispersing the bulk boron material in a solvent to provide a dispersion; subjecting the dispersion to sonifi cation to facilitate cavitation; and subjecting the sonified dispersion to centrifugation to provide the borophene nanosheet.

Images