{"paper_id":"3b6d0595-1a9a-47bd-ad99-fbad2bfb900a","body_text":"Over the past two decades, the discovery\nof two-dimensional (2D)\nnanomaterials with atomic-scale thickness has transformed fundamental\nnanoscience and nanotechnology. The inception of this discipline pertains\nto the pioneering synthesis of graphene, which paved the foundation\nfor further exploration of various 2D nanomaterials. \n , \n  Since then, many analogous materials such as hexagonal boron nitride\n(h-BN) and graphitic carbon nitride (g-C 3 N 4 )\nhave garnered significant attention due to their exceptional mechanical\nstrength, superior electrical mobility, and distinctive thermal properties. \n , \n  These unique properties have facilitated a wide array of applications,\nspanning electronics, energy storage, and biosensing.  In the area of sensing, graphene-based sensors have remarkable\nversatility in detecting analytes, encompassing environmental pollutants\nsuch as heavy metals and pesticides, as well as biological markers\nlike enzymes, proteins, and nucleic acids. \n , \n  Their\ninherent sensitivity and versatility facilitate their application\nin wearable sensors for mechanical strain and real-time physiological\nparameter monitoring. On the other hand, challenges such as graphene’s\nintrinsic lack of an electronic bandgap and certain performance limitations\nhave prompted researchers to investigate alternative 2D materials\nbeyond graphene, including silicene, germanene, borophene, transition-metal\ndichalcogenides (TMDs), MXenes, and metal–organic frameworks\n(MOFs). \n ,\nAmong these, borophene, a recently\nsynthesized monoelemental member\nof the Xene family and a two-dimensional (2D) allotrope of boron,\nhas garnered significant attention due to its unique structural, electronic,\noptical, and physicochemical properties, as well as its complex allotropic\nforms, which distinguish it from other 2D materials. \n , \n  Notably, both theoretical and experimental studies have demonstrated\nborophene’s outstanding thermal conductivity, mechanical strength,\nand tunable electronic characteristics, positioning it as a versatile\nplatform for a wide range of applications \n , , , \n  Its cohesive\natomic structure provides remarkable mechanical durability, while\nits high carrier mobility, tunable band gap, exceptional electrical\nconductivity, and intrinsic superconducting behavior collectively\nfacilitate efficient charge transport and broaden its potential for\nnext-generation nanoscale devices. \n − \n \n \n  Recent studies have highlighted\nborophene’s outstanding performance in high-performance energy\nstorage, gas sensing, and, importantly, advanced electronic and optoelectronic\napplications. \n , , \n \n  Moreover, experimental studies have\nvalidated borophene’s promise in these fields, demonstrating\nits superior electronic conductivity, optical response, and catalytic\nactivity compared to conventional materials. Furthermore, borophene\nhas emerged as a biocompatible and biodegradable material, demonstrating\nlow cytotoxicity in vitro and minimal adverse effects in vivo, making\nit highly suitable for biomedical applications. \n , , \n  Environmentally, its piezocatalytic activity\nfacilitates the efficient degradation of organic pollutants, highlighting\nits potential in sustainable environmental technologies. \n ,\nComprehensive reviews further highlight borophene’s\nbroad\napplicability and its rapidly evolving role in addressing current\nchallenges in materials science, energy conversion, nanoelectronics,\nbiomedicine, and environmental remediation. Collectively, these advances\nestablish borophene as an exceptionally promising 2D nanomaterial,\ncapable of overcoming existing limitations in multifunctional device\ndevelopment and fostering transformative innovations across both academic\nand industrial domains. \n , − \n ,\nBefore the practical implementation\nof any application, the successful\nand efficient synthesis of high-quality materials constitutes a critical\nexperimental step. Although numerous theoretical conformations of\nborophene synthesis on various metal substrates have been proposed,\nonly a limited number of experimental procedures have been reported\nthus far. \n , \n  A few studies have demonstrated the synthesis\nof atomically thin crystalline boron films on metal surfaces through\nvacuum deposition methods, such as molecular beam epitaxy (MBE) and\nchemical vapor deposition (CVD).  However,\nthese approaches present several drawbacks, including the necessity\nfor ultrahigh vacuum and low-pressure growth, an additional transfer\nstep that inevitably degrades sheet quality, and inadequate yields,\nwhich collectively constrain their practical applications.  As an alternative, liquid-phase exfoliation\nmethods have gained considerable attention for synthesizing and upscaling\n2D nanomaterials (specifically 2D nanosheets), owing to their ability\nto produce materials of high quality in large quantities. Furthermore,\nsome studies have reported the synthesis of few-layer borophene nanosheets\nvia sonication-assisted liquid-phase exfoliation in high-boiling solvents\nsuch as dimethylformamide and isopropyl alcohol. \n , \n  However, these solvents complicate purification and removal because\ntheir slow evaporation rates promote aggregation of the 2D nanomaterials,\nthereby rendering them unsuitable for biological applications. To\naddress these challenges, an alternative strategy involving low-boiling\nsolvents has been introduced. By employing low-boiling solvents such\nas water, stable dispersions of 2D nanomaterials can be achieved and\nconveniently utilized for various biological applications. \n , \n  Accordingly, in this study, we utilized Milli-Q water as a low-boiling\nsolvent to obtain stable dispersions of borophene nanosheets from\nbulk boron powder via liquid-phase exfoliation for biological application.\nThe biological interactions of borophene could be of particular interest\nnot only for developing sensing strategies but also for enhancing\nsensor performance.\nBoron atoms in borophene are electron-deficient,\nfacilitating effective\nsurface functionalization with electron-donating agents.  Moreover, its polymorphic nature enables further\ntuning of properties via various boron–boron bonding configurations.\nThis functionalization enhances biosensing capabilities by facilitating\nselective interactions with certain bioanalytes, promoting physical\nadsorption on the surface. Borophene’s anisotropic character,\nhigh electroactive surface area (stemming from its chemical and metallic\nproperties), and high electron mobility may further endow it with\nenhanced selectivity and sensitivity in biosensor development. \n , \n  Recently, several studies have employed borophene nanosheets in\ncombination with MOFs, metals, and other active organic ligands (e.g.,\npolyaniline and phthalocyanine) to synthesize borophene-based nanocomposites\nfor electrochemical biosensors.  However,\nnone of these approaches utilize pristine borophene nanosheets for\ndirect surface functionalization. Therefore, there remains a critical\ngap in the literature regarding the development of functionalization\nstrategies for pristine borophene nanosheets, either with or without\nthe need for additional functional groups.\nOur group recently\ndemonstrated that pristine borophene nanosheets\ncan be directly functionalized using a site-selective boron–sulfur\nconjugation strategy with thiol-containing amino acids like cysteine.  This work provided a deeper understanding of\ndeveloping functionalization strategies for borophene nanosheets without\nadditional surface modifications that can be applied to sensing and\ncatalysis.  Building on this foundational\napproach, we aimed to study the possibility of conjugating biomolecules,\nspecifically antibodies, onto borophene nanosheets. Although antibodies\nserve as exceptional receptors due to their specificity, flexibility,\nand reliability, their moderate long-term stability and the necessity\nfor proper orientation and high surface density pose problems for\nfunctionalization. Conventional noncovalent or covalent immobilization\ntechniques may reduce antibody immunoaffinity due to steric hindrance.  Site-specific conjugation methods, such as oxidation\nof carbohydrate moieties in the Fc region to form Schiff bases, improve\nattachment efficiency; however, they often lead to undesirable side\nreactions and reduced antibody activity.  There is a need for an effective immobilization strategy that ensures\nantibodies are attached site-specifically and in an orientation favorable\nfor antigen binding efficiency, without the use of additional chemical\nreagents and while maintaining their biological recognition activity. \n , −\nThe photochemical immobilization technique\n(PIT) is an established\nand efficient technique for antibody immobilization onto solid surfaces\nwhile ensuring optimal exposure of fragment antigen-binding (Fab)\nfragments. \n , − \n \n \n  In this study, we investigated\nthe potential of utilizing the PIT method to immobilize directly and\nspatially orient antibodies onto the borophene surface, a promising\nyet relatively unexplored approach. The PIT technique utilizes UV\nirradiation of IgG antibodies to generate four free thiol groups in\nthe Fab region, two of which are available for covalent binding to\nmetal/metallic surfaces. The thiol groups, being electron-rich, can\ninteract with the electron-deficient boron atoms of borophene, resulting\nin the formation of a boron–sulfur bond. This approach can\nuse pristine 2D borophene nanosheets to spatially orient antibodies,\ncreating a bioactive surface for sensitive biomolecule recognition.\nTo demonstrate the practical application, the developed borophene–antibody\nconjugates were then used as capture probes to develop a lateral flow\nimmunoassay (LFIA) for women’s health diagnostics. The introduction\nof a 2D borophene nanosheets, together with a photoactivation strategy,\nenables the uniform immobilization of antibodies while preserving\ntheir antigen-binding efficacy, thereby enhancing the antigen detection\nperformance of the LFIA. To assess the efficiency of these techniques,\nalong with their sensitivity, limit of detection (LOD), and dynamic\nclinical detection range, we targeted HMGB-1,  a biomarker for endometriosis, using menstrual blood to advance\nwomen’s health diagnostics. Despite the significant potential\nof menstrual effluent  as a diagnostic\ntool for women’s health, it often faces substantial challenges\ndue to social stigma and limited access to affordable diagnostic methods.\nThese challenges contribute to delays in diagnosing endometriosis,\nwith one study of 218 women revealing an average delay of over 8 years\nin the United Kingdom and up to 12 years in the United States.  By leveraging the novel 2D material borophene,\nthis approach offers a unique opportunity to reduce menstrual stigma\nwhile advancing women’s health.\nIn a nutshell, we prepared\na stable dispersion of χ 3  borophene nanosheets from\nbulk boron powder in an appropriate volume\nof low-boiling solvent (H 2 O) via liquid-phase exfoliation.  We then employed these nanosheets to spatially\norient antibodies on their surface using PIT. The resulting nanosheets,\nbearing spatially oriented antibodies, were subsequently utilized\nto develop an ultrasensitive LFIA device capable of detecting HMGB-1\nin menstrual effluent at low concentrations within minutes, without\nrequiring pretreatment. We believe that the novelty of this approach\nand the assay’s high sensitivity will attract considerable\ninterest in advancing borophene-based biosensing applications. Moreover,\nthis study highlights the broader research opportunities essential\nfor realizing next-generation biotechnologies, with 2D materials at\ntheir core, and positions borophene as a highly promising class of\nnanomaterial for cutting-edge biosensors and advanced healthcare applications.\n\nBorophene\nnanosheets were synthesized via probe sonication-assisted liquid-phase\nexfoliation of bulk boron powder in Milli-Q water ( Figure  \n a). Water was chosen for its\nlow boiling point and compatibility with biological applications. \n , \n  While high-boiling solvents such as dimethylformamide (DMF) or  N -methylpyrrolidone (NMP) are commonly used to stabilize\n2D nanomaterial dispersions by lowering interlayer van der Waals forces,\ntheir removal is challenging and can lead to aggregation, limiting\ntheir use in biomedical contexts. \n − \n \n  In contrast, Milli-Q\nwater (18.2 MΩ cm resistivity) minimizes the generation of reactive\noxygen species (ROS) and, under probe sonication, generates intense\ncavitation microenvironments that facilitate the intercalation of\nwater molecules between boron layers, weakening interlayer attractions\nand promoting exfoliation. \n , \n  Although water is generally\nless effective than some organic solvents for liquid-phase exfoliation\ndue to its higher polarity and weaker biaxial straining effect,  it can still yield thin borophene sheets, particularly\nwhen combined with optimized sonication and postprocessing conditions. \n , , \n  Controlled sonication time under\nan inert atmosphere helps suppress oxidation, and subsequent filtration\nvia centrifugation at 3000–4000 rpm allows for the selective\nisolation of 3–6 layers of borophene nanosheets, consistent\nwith prior studies reporting layer-thickness  control via centrifugation speed. As evidenced by B–OH vibrational\nfeatures in FTIR in the literature, surface hydroxylation further\nstabilizes the exfoliated nanosheets in aqueous media and enhances\ntheir dispersibility for downstream applications \n , , , \n  This approach\naligns with recent advances in solvothermal and surfactant-assisted\nexfoliation, demonstrating that polar solvents and careful process\ncontrol can yield high-quality borophene nanosheets suitable for biological\nuse. The detailed procedure is described in  Materials\nand Methods .\n(a) Synthesis and experimental conditions for borophene\nnanosheets\nresulting in predominantly χ 3  phase. Schematic representation\nof the representative lattice arrangements of χ 3  borophene\nphase. Comparative characterization of χ 3  borophene\nnanosheets. (b) TEM image and (c) HAADF image with (d) boron EDX map,\n(e) overlay of HAADF and boron EDX map. (f) HR-TEM image of borophene.\nThe inset shows lattice fringes corresponding to predominantly χ 3  phase. (g) Calculated lattice fringes using ImageJ software.\n(h) FT-IR spectra of pristine boron powder and χ 3  borophene nanosheets in an anhydrous state. (i) Raman spectra of\nχ 3  borophene nanosheets in aqueous media.\nControlled\nprobe sonication of pristine bulk boron powder at room temperature\nfacilitated the production of free-standing synthesized 2D borophene\nnanosheets, with an average hydrodynamic diameter of 200 ± 15\nnm, as measured by dynamic light scattering (DLS) ( Figure S1 ).  The size of the synthesized\nnanosheets results from the higher localized temperature in the vicinity\nof the solvent matrix. The resulting aqueous suspension of borophene\nwas subsequently processed using centrifugation at 3000 rpm followed\nby filtration to isolate the final nanosheet suspension. Postfiltration,\nthe nanosheets were found to have an average hydrodynamic diameter\nof 160 ± 20 nm, as determined by DLS. Scanning electron microscopy\n(SEM) was employed to study and compare the morphological changes\nbetween pristine boron powder (bulk boron) and exfoliated borophene\nnanosheets ( Figure S2 ). SEM images revealed\na more flakelike two-dimensional (2D) structure in borophene, whereas\npristine boron appeared solid and three-dimensional (3D). The morphology\nof the nanosheets was further characterized using transmission electron\nmicroscopy (TEM), which revealed nanosheet-like structures measuring\napproximately 200 nm ( Figure  \n b). High-angle annular dark-field scanning transmission electron\nmicroscopy (HAADF-STEM) coupled with energy-dispersive X-ray spectroscopy\n(EDX) elemental mapping confirmed the presence of boron ( Figures  \n c–e,  S3, and S4 ). Furthermore, high-resolution transmission\nelectron microscopy (HR-TEM) images revealed lattice fringes of borophene\nobtained from liquid-phase exfoliation ( Figure  \n f). The parallel lattice fringes visible\nthroughout the sample indicate a highly ordered crystal structure.\nThe scale bar (10 nm) facilitates measurement of these fringes, which\nappear to have a moiré interference pattern spacing in the\nrange of 0.29 ± 0.01 and 0.17 ± 0.01 nm ( Figure  \n g). This lattice spacing is\nconsistent with interatomic distances reported for χ 3 -borophene in prior studies.  Moreover,\nthese spacings differ from those of β 12  borophene\nand β-rhombohedral boron. \n , , \n  The uniform fringe patterns and absence of significant defects or\ndistortions suggest the successful synthesis of high-quality borophene\nsheets predominantly in the χ 3  phase. Additionally,\nFT-IR was performed to confirm the borophene signature bonds in the\nsynthesized nanosheets. The FT-IR spectra ( Figure  \n h) observed between pristine boron powder\nand the synthesized borophene nanosheets arise from structural and\nchemical transformations during liquid-phase exfoliation. In the hydroxyl\nregion (∼3400 cm –1 ), borophene exhibits a\npronounced broad band, indicative of substantial surface hydroxylation\ndue to water interactions during aqueous exfoliation and a high tendency\nfor edge oxidation in the reaction medium. In contrast, bulk boron\nshows minimal hydroxyl signatures, consistent with its lower surface-to-volume\nratio and limited edge oxidation. Additionally, in the B–O\nbonding region (1600–2000 cm –1 ), borophene\ndisplays sharper, more intense peaks than bulk boron, reflecting increased\noxidation at the edges and surfaces of the nanosheets. This aligns\nwith the exfoliation process, which exposes reactive boron atoms to\ndissolved oxygen. With its intact 3D covalent network, pristine boron\nshows weaker B–O vibrations, indicating minimal oxidation.\nFinally, the B–B bonding region (800–1200 cm –1 ) further distinguishes the two: borophene exhibits sharper and more\nintense B–B vibrational modes, characteristic of its 2D planar\nstructure. Bulk boron shows broader, less-defined B–B peaks,\nconsistent with its 3D β-rhombohedral configuration. \n , , \n  We also utilized X-ray photoelectron\nspectroscopy (XPS) to differentiate between pristine boron powder\nand synthesized borophene nanosheets. The B1s spectrum of the pristine\nboron powder indicated only one peak corresponding to B–B (187.33\neV) ( Figure S5a ). However, the B1s spectrum\nof borophene reveals three distinct peaks at 187.4, 188.6, and 192.0\neV, corresponding to B–B, B–O, and B 2 O 3  bonds, respectively ( Figure S5b ). These peaks represent relative contents of 56% for B–B,\n36% for B–O, and 8% for B 2 O 3 . The presence\nof the B 2 O 3  peak is likely attributed to electrochemical\nreactions occurring during the exfoliation process. Notably, borophene\nexhibits a higher proportion of B–B bonds, which may result\nfrom the exposure of freshly cleaved surfaces following exfoliation.\nRaman spectroscopy was then used to identify the dominant boron phase\nin the synthesized χ 3  borophene nanosheets. Based\non the synthesis approach, the χ 3  borophene nanosheets\nexhibited an isotropic hexagonally bonded phase, defined by a triangular\nlattice with periodic holes. Raman spectral analysis revealed bands\nat 296 (A u  (Y)), 452 (B g \n 1  (Y)), 783\n(A g \n 3 ), 984 (A g \n 2 ), and\n1165 (A g \n 1 ) cm –1 , confirming\nthe predominance of the χ 3  phase in the synthesized\nnanosheets ( Figure  \n i). \n , , , \n  Moreover, the atomic force microscopy (AFM) images\nof the χ 3  borophene nanosheets revealed approximately\nfive sheet layers with an overall thickness of 1.10 ± 0.60 nm\n( Figure S6 ). The interlayer distance between\ntwo adjacent sheets was estimated to be approximately 0.2–0.3\nnm. Therefore, a few-layered borophene nanosheets were synthesized\nusing low-temperature liquid-phase exfoliation in an aqueous solution,\nsonication-assisted intercalation, and bulk boron exfoliation. The\nmethod likely involves the dispersion of bulk boron particles into\na liquid medium, where the intercalation properties of solvent molecules\nfacilitate a reduction in exfoliation energy, enabling reconstruction\nand the formation of energy-favorable structures. \n ,\nFollowing the successful synthesis, characterization, and confirmation\nof the desired borophene nanosheets, we proceeded to explore their\nfunctionalization with antibodies to develop a diagnostic assay. Antibodies\n(Abs) are the preferred choice for bioreceptors in diagnostic assay\ndevelopment due to their inherent specificity, adaptability, and reliability.\nHowever, challenges persist in achieving robust and efficient antibody\nsurface functionalization, primarily due to their moderate long-term\nstability and the critical requirement to immobilize them with proper\norientation and high surface density. The direct introduction of antibodies\nonto 2D borophene nanosheets presents additional challenges due to\nthe material’s inherent hydrophobicity, lack of reactive sites,\nand steric hindrance.\nTo overcome\nthese limitations, we employed a well-established photoinduced immobilization\ntechnique, which facilitates the generation of reactive thiol groups\nin antibodies without compromising their antigen-binding activity.\nPIT offers significant advantages over traditional methods, including\nsimplicity, speed, and effectiveness enabling the efficient tethering\nof antibodies onto surfaces with high affinity for thiol groups. The\nPIT strategy involves the UV irradiation of immunoglobulin G (IgG)\nantibodies, leading to selective photoreduction that cleaves disulfide\nbridges within cysteine–cysteine/tryptophan (Cys–Cys/Trp)\ntriads ( Figure S7 ). This process generates\nfour free thiol groups in the Fab fragments, of which two are available\nfor covalent binding to metal surfaces ( Figure S7 ). \n , \n  Moreover, studies have reported\nthat the optimal conditions for PIT with IgG antibodies include an\nirradiation time of 30 s and an antibody concentration of 50 μg/mL.\nUnder these conditions, the disulfide bridges remain open for approximately\n300 s, providing sufficient time for the activated antibodies to attach\nto the metal surface. Furthermore, it has been reported that immobilization\nof antibodies via these thiol groups promotes a side-on orientation,\nin which one Fab domain is bound to the surface, while the other Fab\ndomain adopts orientations within a range of 10° to 90°.\nThis configuration ensures effective exposure of the Fab domain to\nthe analyte in the surrounding medium, thereby enhancing binding efficiency\nand preserving functionality. \n , \n  Previous research from\nour group has highlighted the critical role of thiol groups, particularly\nthose from cysteine, an amino acid, in facilitating site-selective\nboron-sulfur conjugation. This approach leads to the formation of\nstrong covalent bonds with boron atoms, enabling the stable and selective\nattachment of biomolecules, including antibodies.\nBased on these findings, we utilized PIT for the\nselective photochemical\nreduction of disulfide bonds in immunoglobulins via UV activation\nof near-aromatic amino acids using a Trylight lamp. We picked anti-Human\nHMGB-1 IgG antibodies as a model system since the PIT approach is\nespecially successful for all IgG forms. A standard 10 mm quartz cuvette\ncontaining 500 μL solution of anti-Human HMGB-1 IgG antibody\nat a concentration of 50 μg/mL was housed inside the low-pressure\nmercury U-shaped UV lamps and irradiated for 30 s at ambient temperature\n( Figure  \n a). Considering\nthe cuvette’s proximity to the lamps and the wrapping geometry,\nwe estimated that the solution received UV irradiation of 0.3 W/cm 2 . The energy of UV photons thus released by the 6 W mercury\nUV lamp was absorbed by tryptophan residues and subsequently transmitted\nto surrounding electrophilic species, including adjacent Cys–Cys\ndisulfide bridges. This process resulted in the cleavage of the disulfide\nbonds and the formation of new reduced thiol (SH) groups. The resulting\nirradiated antibody solution (100 μL) was then combined with\nborophene nanosheets (100 μL) at a concentration of 0.175 mg/mL\nat room temperature, facilitating the functionalization of borophene\nnanosheets via the formation of boron-sulfur covalent bonds ( Figure  \n b).\n(a) Schematic representation\nof biofunctionalization of borophene\nnanosheets with anti-Human HMGB-1 IgG antibodies through PIT. Low-pressure\nmercury U-shaped UV lamps from Trylight were used to carry out the\nprocess. The UV-treated antibody produces four thiol groups (with\nonly two visible in the image). Thiol groups in one Fab region facilitate\nthe immobilization of the antibody to borophene (B–S covalent\nbond), while the other Fab region remains exposed to the environment\nfor antigen binding. (b) Graphical depiction of the developed immunoassay\nutilizing the functionalized borophene nanosheets. HMGB-1 antigen\nand antibodies were used as a model to validate the PIT-based biofunctionalization\nof borophene nanosheets (c) TEM image illustrating the sandwich formation\nover borophene nanosheets. Oriented IgG antibodies anchored onto borophene\nnanosheets form a sandwich complex through interactions between the\nFab regions, HMGB-1 antigens, and gold nanoparticles labeled with\ndetection antibodies. (d) High-resolution TEM image showing the lattice\nfringes in borophene nanosheets. (e) TEM image of the control sample\n(UV-untreated antibodies over borophene nanosheets). Gold nanoparticles\nappear separately, agglomerated, and not over borophene sheets. The\narrows indicate borophene nanosheets. The inset shows an image at\nhigher magnification. (f, g) HAADF images and EDX mappings of the\nborophene nanosheet sandwich complex showing the presence of (g) boron\n(B) and gold (Au). (h) AFM image showing the formation of a sandwich\non top of the borophene nanosheet, indicated via an arrow. (i) S 2p\nXPS spectra of the borophene nanosheets conjugated with UV-treated\nanti-human HMGB-1 IgG antibodies. The B–S bond is seen at 163.3\neV.\nFollowing the successful preparation of antibody-functionalized\nborophene nanosheets via UV-induced thiol generation and subsequent\nB–S bond formation, we proceeded to characterize the conjugation\nefficiency and binding mechanism through quantitative analytical techniques.\nTo evaluate and quantify the conjugation efficiency of UV-treated\nanti-human HMGB-1 IgG antibodies on borophene nanosheets, a Bradford\nassay using Coomassie Brilliant Blue G-250 dye was conducted ( Figure S8a ). A linear standard curve with correlation\n( R \n 2  = 0.992) was established using IgG\nconcentrations ranging from 1 to 100 μg/mL ( Figure S8b ). Following incubation of UV-irradiated antibodies\n(50 μg/mL) with borophene nanosheets (0.175 mg/mL), a conjugation\nefficiency of 48% was calculated based on the residual unbound antibody\nmeasured in the supernatant. According to the calibration curve, 25.96\nμg/mL of free IgG remained, indicating that 24.04 μg/mL\nIgG (equivalent to 192.3 μg of IgG per mg of borophene) was\nsuccessfully immobilized on the nanosheet surface. Control experiments\nusing nonirradiated antibodies exhibited negligible binding (<5%),\nverifying that UV-induced thiol generation is essential for covalent\nconjugation, presumably via B–S bond formation. Furthermore,\nthe availability of free thiol groups in anti-human HMGB-1 IgG antibodies\npost-UV irradiation and subsequent borophene binding was assessed\nby the Ellman assay. A standard calibration curve generated using\nL-cysteine concentrations ranging from 0 to 200 nM yielded a linear\ncorrelation ( R \n 2  = 0.9959) ( Figure S9 ). Samples of anti-human HMGB-1 monoclonal\nantibodies (50 μg/mL) were diluted in reaction buffer for analysis.\nUV-untreated control antibodies exhibited minimal absorbance ( A \n 412  ∼ 0.009), similar to the Ellman’s\nreagent blank, confirming the absence of accessible thiol groups in\nthe native antibody structure. Following photoinduced thiol generation\nvia exposure to 254 nm UV irradiation (Trylight UV lamp, 6 W; irradiance\n∼ 0.3 W/cm 2  for 30 s), a significant increase in\nabsorbance was detected ( A \n 412  ∼\n0.315), corresponding to 20.58 nM free thiol groups generated from\ndisulfide bond cleavage. After incubation with borophene, absorbance\ndecreased to 0.125, indicating 10.73 nM free thiol, a reduction of\napproximately 48%, suggesting substantial thiol–borophene binding,\nlikely through B–S interactions. This thiol-specific interaction\nlikely results in the antibodies adopting a side-on orientation on\nthe borophene nanosheets, leaving only one Fab arm accessible for\nantigen binding ( Figures S7  and  a). Consequently, although IgG structurally possesses\nbivalent antigen-binding sites, the functional valency is effectively\nreduced to monovalent binding due to steric constraints imposed by\nthe borophene nanosheet surface.\nIt was essential to confirm the antibodies’\nconjugation and orientation on the surface following the successful\nfunctionalization of the borophene nanosheets. To achieve this, we\ndeveloped an immunoassay-based methodology utilizing a mouse anti-human\nHMGB-1 IgG capture antibody (capture Ab), HMGB-1 antigen (HMGB-1 Ag),\na biotinylated mouse anti-human detection IgG antibody (detection\nAb), and streptavidin-coated gold nanoparticles (Strp-AuNPs). In this\napproach, 100 μL of borophene nanosheets conjugated with anti-Human\nHMGB-1 IgG capture Ab were incubated with 100 μL of HMGB-1 Ag\nat ambient conditions in a microfuge tube for 5 min. Thereafter, 10\nμL of Strp-AuNPs conjugated with detection Ab were introduced\nto the solution and incubated at ambient temperature for 15 min. Following\neach incubation phase, centrifugation was used to remove unattached\nor free-floating antigens or antibodies from the solution matrix.\nWe hypothesized that the successful conjugation of borophene nanosheets\nwith capture Ab would enable the formation of a sandwich complex in\nthe presence of anti-Human HMGB-1 IgG Ag. However, if the borophene\nnanosheets were not conjugated with the captured IgG antibodies, the\nsandwich complex would not form. We also used a control sample where\nthe borophene sheets were mixed with UV-untreated anti-Human HMGB-1\nIgG capture Ab. In this case, we hypothesize that the antibodies will\nnot conjugate onto the surface of nanosheets, and a sandwich will\nnot be formed. To validate our hypothesis, we employed TEM, AFM, and\nRaman spectroscopy. The TEM image revealed a borophene nanosheet,\nidentifiable as a lighter-colored, semitransparent structure ( Figure  \n c). Superimposed\non the nanosheet are spherical, darker regions representing gold nanoparticles\n(AuNPs), as shown in  Figure  \n c. These nanoparticles are conjugated via a sandwich complex\ncomposed of capture Ab, HMGB-1 Ag, detection Ab, and Strp-AuNPs. The\nimage provides visual confirmation of the successful conjugation of\nborophene nanosheets with the capture of IgG antibodies, as evidenced\nby the formation of the sandwich complex in the presence of the HMGB-1\nantigen. The distinct contrast between the nanosheet and the spherical\nnanoparticles highlights the functionalized borophene surface’s\nstructural integrity and the nanoparticles’ specific attachment\nthrough antigen–antibody-mediated interactions.  Figure  \n d shows an HR-TEM image of\nthe borophene nanosheet, revealing distinct lattice fringes with a\nmeasured lattice spacing of 2.92 Å.  Figure  \n e shows a TEM image of the control sample,\nwherein the gold nanoparticles are not superimposed on the borophene\nnanosheet. This observation arises from the UV-untreated capture antibodies,\nwhich lack the thiol groups necessary for conjugation to the borophene\nsurface. Consequently, the gold nanoparticles aggregate independently\nrather than adhering to the borophene nanosheets, thereby confirming\nthe absence of a sandwich structure. Furthermore, low and high-magnification\nHAADF-STEM images of the same sample revealed the borophene nanosheet\nas a light-colored sheet-like structure. In contrast, the dark spherical\nstructures were identified as gold nanoparticles ( Figure  \n f). This was further validated\nthrough EDX, which confirmed the presence of boron exclusively in\nsheet-like structures. On the contrary, gold was exclusively detected\nwithin the spherical formations ( Figure  \n g). We also utilized AFM to study the formation\nof the sandwiched complex on the surface of borophene conjugated with\ncapture antibodies. The sample containing borophene nanosheets conjugated\nwith capture Ab was mixed with HMGB-1 Ag and Strp-AuNPs labeled with\ndetection Ab. The resulting mixture was drop-cast onto cleaved mica\nto analyze the structural modifications of the 2D nanosheets induced\nby the antibody molecules using AFM. From the AFM images, the change\nin height due to the formation of the sandwich complex on top of the\nantibody-conjugated borophene nanosheets was distinguishable in comparison\nwith pristine borophene. The pristine borophene nanosheets exhibited\na surface height of 2.0 ± 0.5 nm, as observed from the height\nprofile ( Figure  \n h).\nUpon the formation of the sandwich complex on top of the antibody-conjugated\nborophene nanosheets, a height increase to 3.5 ± 0.7 nm was observed,\nindicating potential successful attachment of the antibody molecules.\nThe surface analysis further revealed the accumulation of sandwich\ncomplexes on top of the antibody-conjugated borophene nanosheets.\nFurthermore, with XPS analysis, the species detected in high-resolution\nspectra on the borophene sample included: CH \n x \n  (carbon species), C–O, C–N, CO, CF 2 , sulfonates, reduced sulfur, boron, oxidized boron, and fluorides\n( Table S1 ). The expected composition of\ntryptophan and cysteine is presented in  Table S2  for comparison. The S 2p core-level spectrum provides critical\ninsights into sulfur’s bonding environment and oxidation state.\nTypically, the S 2p signal appears as a doublet (S 2p 3/2  and S 2p 1/2 ) due to spin–orbit coupling, with\na characteristic energy separation of approximately 1.18 eV. The precise\nbinding energy varies depending on the chemical state of sulfur and\ngenerally falls within the range of ∼161–170 eV. Thiols\nare generally found at 163.5–164.0 eV in the S 2p spectrum,\nwhile metal sulfides/disulfides and metal–sulfur bonds are\ngenerally found to have lower binding energy (161–162.5 eV).\nThe formation of a boron–sulfur bond is indicated by a prominent\npeak at 163.3 eV, as confirmed by the XPS data ( Figure  \n i). This binding energy aligns with literature\nvalues ( Table S3 ) for similar heteroatom-sulfur\ncovalent bonds.  The clear distinction\nbetween this peak and those representing free thiols (R–SH\nat 164.6 eV) and oxidized sulfur species (SO 3  at 167.86\nand 169.04 eV) further supports our interpretation. The significant\nintensity of the B–S peak relative to other sulfur species\nindicates substantial covalent interaction rather than mere physical\nadsorption. Selected binding energies are presented in  Table S3 . These findings demonstrate that the\ncapture antibodies were covalently attached to the borophene surface\nvia thiol moieties generated through UV irradiation, highlighting\nthe effectiveness of the functionalization strategy and its potential\napplication in immunoassays.\nTo gain deeper insight into the molecular interactions\noccurring\nat the interface upon UV irradiation of antibodies, we leveraged the\nunique capabilities of surface-enhanced Raman spectroscopy (SERS).\nIn this study, we employed a gold substrate as the plasmonic surface.\nIgG antibodies, UV-treated and untreated, were immobilized using a\ncustom micropipette system designed to deliver a controlled flow of\nsolution across the surface (see  Materials and Methods  for details). Following deposition, the samples were analyzed via\na Raman spectrometer. The resulting SERS spectra ( Figure  \n a) were compared to the conventional\nuntreated antibody. We observed significant alterations in the intensity\nof S–S stretching bands in the UV-treated IgG compared to the\nuntreated control. A distinct spectral shift near 517 cm –1 , corresponding to the Cys76–Cys94 disulfide bridge, suggests\nthe emergence of a trans Ca–S conformation, potentially associated\nwith an increase in free thiol generation.  Additional shifts at 504, 514, 522, 527, and 544 cm –1  in the UV-irradiated samples indicate conformational changes likely\nrelated to the cleavage or rearrangement of disulfide bonds. These\nchanges, attributed to the cystine residues, exhibit characteristic\nstretching vibrations within the 505–550 cm –1  spectral range. gauche–gauche-gauche (ggg) conformations\nhave an S-S stretching band at ∼505–515 cm –1 , gauchegauche–trans (ggt) have an S-S stretching band at\n∼ 520–530 cm –1 , trans-gauche–trans\n(tgt) have an S-S stretching band at ∼540–545 cm –1 .  These observations\nsupport the hypothesis that UV exposure promotes the photoreduction\nof disulfide bridges, forming free thiol groups that can interact\nmore readily with the gold surface. Raman spectroscopy is particularly\nwell-suited for distinguishing between reduced (free thiol) and oxidized\n(disulfide) states. The disappearance of S–S vibrational bands\n(e.g., at 503 cm –1 ) alongside the emergence of thiol-associated\nsignals (e.g., around 680 cm –1 ) is  characteristic of disulfide bond reduction. ( Figure  \n b). Aromatic amino acids are\nhighly responsive to plasmonic enhancement and typically produce strong\nsignals in protein Raman spectra. In the UV-treated sample, signals\nfrom phenylalanine (Phe), tyrosine (Tyr), and tryptophan (Trp) are\nsignificantly amplified. Notably, the characteristic Phe bands at\n1122, 1240, 1335, and 1450 cm –1  show marked intensity\nincreases, likely due to their closer proximity to the plasmonic surface\n( Figure  \n c). Similarly,\nenhanced Tyr signals are observed at 830, 850, and 875 cm –1  ( Figure S10 ). The overall SERS results\nindicate that the illumination parameters used for PIT effectively\nfacilitated thiol-mediated surface attachment of the Ab molecules,\nmore so than in the untreated Ab samples. Additionally, the enhanced\nintensity of numerous vibrational modes in the UV-treated samples\nsuggests that antibody anchoring may also involve broader contact\nwith the surface.\n(a–c) Raman spectral analysis: (a) Signal intensity\nchanges\nin the S–S region of 500–550 for the UV-treated antibody,\nindicating the reduction of the disulfide bond to produce the free\nthiol groups. (b) Disappearance of S–S vibrational bands (e.g.,\nat 503 cm –1 ) alongside the emergence of thiol-associated\nsignals (e.g., around 680 cm –1 ),  which is characteristic of disulfide bond reduction. (c)\nRaman spectral analysis reveals distinct changes in the phenylalanine\nfor the UV-treated antibody. (d–i) Isothermal titration calorimetry\n(ITC) and fluorescence analysis of borophene nanosheets with UV-treated\nand UV-untreated antibodies: (d) ITC thermogram of borophene nanosheets\nwith UV-treated antibodies. (e) ITC thermogram of borophene nanosheets\nwith UV-untreated antibodies. (f) Binding isotherm of borophene nanosheets\nwith UV-treated antibodies. (g) Binding isotherm of borophene nanosheets\nwith UV-untreated antibodies. (h) No fluorescence was observed for\nborophene nanosheets with UV-untreated antibodies, indicating a lack\nof antibody binding and subsequent Cy5 attachment. (i) Representative\nfluorescence image at 100 nM PfLDH concentration showing red spots\ncorresponding to fluorescence emitted by single Cy5 molecules.\nMoreover, we employed isothermal titration calorimetry\n(ITC) to\ninvestigate the thermodynamic interactions of anti-Human HMGB-1 IgG\ncapture antibodies immobilized via PIT on 2D borophene nanosheets,\ncompared to UV-untreated anti-Human HMGB-1 IgG antibodies on the same\nnanomaterial. Briefly, a 50 μM solution of borophene nanosheets\nwas loaded into a syringe for subsequent addition to the reaction\ncell containing either the UV-treated or untreated anti-Human HMGB-1\nIgG capture antibodies. Further, the concentration of HMGB-1 antibodies\n(both UV-treated and untreated) in the reaction cell was maintained\nat 1 mM for all titrations. The ITC thermograms and binding isotherms\nas shown in  Figure  \n  demonstrate a distinct difference in the binding characteristics\nof UV-treated and UV-untreated IgG antibodies on the borophene nanosheet\nsurface. For UV-treated IgG ( Figure  \n d,  f), the thermogram exhibits\nsharp, well-defined peaks, indicative of a strong and favorable interaction.\nThe binding stoichiometry ( n ) is 0.318, suggesting\npartial occupancy of available binding sites, with a dissociation\nconstant ( K \n D ) of 1.00 × 10 –9  M, reflecting high binding affinity. The enthalpy change (Δ H ) is 0.287 kcal/mol, consistent with minimal heat release,\nwhile the Gibbs free energy (Δ G ) is highly\nfavorable at −42.14 kcal/mol, confirming a thermodynamically\nstrong binding interaction.\nIn contrast, for UV-untreated IgG\n( Figure  \n e,  g), the thermogram\nreveals smaller, less defined peaks, suggesting a weaker and less\nfavorable binding process. The binding stoichiometry is significantly\nlower ( n  = 0.100), and  K \n D  increases to 4.29 × 10 –5  M, indicating a\nmuch weaker interaction. The Δ H  value is higher\nat 3.871 kcal/mol, signifying a greater heat release during binding,\nbut Δ G  is reduced to −32.97 kcal/mol,\nconfirming the relatively lower binding affinity. Collectively, these\nparameters indicate two distinct binding modes: the first reaction\nis rapid, enthalpically driven, and exhibits highly favorable free\nenergy, while the second interaction is slower, presumably influenced\nby the entropic factors, and features significantly reduced affinity.\nThe stoichiometric values suggest that both systems may involve cooperative\nor heterogeneous binding events. Thus, the results reveal that UV\ntreatment significantly enhances the binding affinity of IgG antibodies\nto borophene nanosheets, likely through the formation of boron–thiol\ncovalent bonds.\nAfter confirming the effective adhesion of UV-treated\nIgG antibodies\nto the borophene nanosheet surface, the subsequent objective was to\ndetermine their orientation. Minopoli et al. previously employed fluorescence\nmicroscopy to validate the orientation of antibodies achieved through\nPIT.  In their work, UV-induced cleavage\nof disulfide bonds in anti- P LDH antibodiestargeting\nthe malaria antigen  Plasmodium  lactate\ndehydrogenase ( P LDH)produced thiol groups\nthat covalently anchored the antibodies to a gold substrate. This\napproach ensured that at least one of the two Fab regions remained\nexposed. The accessibility and functionality of these Fab regions\nwere verified via fluorescence measurements after the binding of a\nlabeled target antigen (a P LDH-Cy5 aptamer). To confirm\nantibody orientation on borophene nanosheets, we adopted a similar\nstrategy using anti- P LDH IgG antibodies,  P LDH antigen, and a Cy5-labeled aptamer specific to  P LDH. The PIT technique was employed to introduce thiol\ngroups into IgG antibodies, which were then conjugated to the borophene\nnanosheets. The resulting antibody-functionalized nanosheets were\nincubated with  P LDH antigen bound to a Cy5-labeled\naptamer. The experimental design included borophene nanosheets functionalized\nwith anti- P LDH IgG antibodies that serve as a substrate\nfor capturing the malaria biomarker  P LDH. The Cy5-labeled\naptamer (5′-Cy5-CTG GGC GGT AGA ACC ATA GTG ACC CAG CCG TCT\nAC-3′) constituted the top layer, providing both fluorescence\nlabeling and high specificity for  P LDH at a relatively\nlow cost. Fluorescence microscopy was employed to visualize the fluorescence\nsignal, and the resulting images were processed with Image J software\nto quantify the corresponding signal intensity. As shown in  Figure  \n h, borophene nanosheets\nconjugated with UV-untreated antibodies exhibited no discernible fluorescence\nsignal in the background-corrected image, indicating limited antigen-binding\ncapacity. In contrast, borophene nanosheets functionalized with UV-treated\nantibodies display distinct red fluorescence spots ( Figure  \n i), corresponding to photons\nemitted by individual fluorophores. These results confirm that the\nPIT strategy facilitates the oriented immobilization of antibodies\non borophene nanosheets and preserves the accessibility of their Fab\nregions for antigen binding.\nBuilding upon the previously obtained results, PIT was employed to\nfunctionalize 2D borophene nanosheets with anti-Human HMGB-1 IgG antibodies,\nand the feasibility of converting the immunoassay (detailed in  Materials and Methods ) into an LFIA format was examined.\nBefore utilizing the borophene nanosheet-antibody conjugates, we wanted\nto conduct specific stability tests to ensure that borophene remains\nstably bound during assay conditions. Using a micropipette, borophene\nwas drop-cast onto the nitrocellulose (NC) strip at a 0.175 mg/mL\nconcentration. The strips were assembled by laminating the NC–borophene\nlayer with a sample pad and an absorbent pad on a backing card. Critically,\na standard assay using chase buffer was performed to assess whether\nphysically entrapped borophene would be washed away during lateral\nflow. As evidenced by photographs taken before and after the assay\n( Figure S11 ), the borophene remained visibly\nintact in its original position on the membrane following fluid flow,\nconfirming the stability of this physical immobilization approach.\nWith the immobilization stability confirmed, we next evaluated the\nefficiency of borophene nanosheets–antibody conjugate deposition\nand its impact on antigen capture performance. To this end, both UV-treated\nand UV-untreated anti-Human HMGB-1 IgG capture antibodies were mixed\nwith borophene nanosheets and deposited onto nitrocellulose membranes\nof defined dimensions. Specifically, the nitrocellulose membrane was\nsectioned into strips of 3 mm size, and 0.5 μL of borophene–antibody\nconjugates (UV-treated and conventional) were applied to each membrane\nat a concentration of 0, 25, 50, and 75 μg/mL. The borophene–antibody\nconjugates were designated as the test zone (T). After the spotting\nprocedure, the membranes were allowed to dry at 37 °C. Once dry,\nthe membranes were assembled into lateral flow test strips and analyzed\nfor HMGB-1 antigen detection following the procedures detailed in  Materials and Methods . As shown in  Figure S12 , test strips containing the UV-treated antibody–borophene\nnanosheet conjugates exhibited stronger signals than conventional\nLFIA. This enhancement can be attributed to improved antibody orientation\nand increased availability of Fab fragments for antigen binding. As\npreviously discussed, one of the intrinsic limitations of lateral\nflow assays arises from the random orientation of adsorbed antibody\nmolecules, which reduces their antigen-binding capacity and thus diminishes\nassay sensitivity. By utilizing the PIT strategy in conjunction with\nborophene nanosheets, it was possible to address this orientation\nissue and enhance the overall sensitivity of the lateral flow immunoassay.\nHaving established\nthe feasibility of integrating the HMGB-1 immunoassay into a lateral\nflow assay format, the next step involved the development of a fully\noperational lateral flow immunoassay. To ensure optimal performance,\nit was necessary to investigate and characterize the molecular interactions\noccurring on the nitrocellulose membrane surface, particularly those\ninvolving the immobilized borophene–antibody conjugates. This\nwas accomplished by designing a sandwich immunoassay in a lateral\nflow format incorporating gold nanoparticles. In brief, anti-Human\nHMGB-1 IgG antibodies (50 μg/mL) subjected to UV treatment were\nmixed with borophene nanosheets at (0.175 mg/mL) and incubated at\nroom temperature for 5 min. As a control, borophene nanosheets were\ncombined with UV-untreated anti-Human HMGB-1 IgG antibodies, coated\nonto a nitrocellulose membrane using an antibody striping machine,\nand then dried in an incubator at 37 °C for 2 h. The lateral\nflow assay was subsequently assembled following the protocols detailed\nin  Materials and Methods . As shown in  Figure S13 , the assay incorporating UV-treated\nantibodies generated a significantly stronger signal than strips lacking\nUV-irradiated antibodies, indicating successful covalent linkage of\nUV-treated antibodies to borophene nanosheets. These results suggest\nthat UV-untreated antibodies do not effectively adhere to the borophene\nsurface during the assay, likely due to the absence of thiol groups\nnecessary for stable binding.  We utilized\nscanning electron microscopy (SEM) to investigate the role of 2D borophene\nnanosheets in increasing the surface area of nitrocellulose membranes\nand facilitating the formation of a sandwich structure in the lateral\nflow assay’s test zone. From our previous work, depositing\nan aqueous borophene suspension onto a glass slide and allowing it\nto evaporate yielded a heterogeneous array of rough-edged microparticles\nof varying sizes ( Figure S14 ). At higher\nmagnification, the SEM images revealed that these larger borophene\nsheets consisted of smaller aggregates, which in turn were formed\nby the aggregation and stacking of nanometer-scale particles, indicating\na hierarchical structure. Building on these findings, we coated the\ntest line zone of the nitrocellulose membrane with 2D borophene nanosheets\nand compared it with a plain NC membrane using SEM analysis ( Figure  \n a–d). The\nplain NC membrane exhibited a three-dimensional open-pore configuration\ncomposed of interconnected fibrous threads, along with spherical features\nmeasuring 2–4 μm in diameter fused onto these fibrous\nstructures ( Figure  \n a). In contrast, the region coated with 2D borophene nanosheets displayed\nevidence of borophene sheets ( Figure  \n b), suggesting that these nanosheets predominantly\naccumulate on the top layer of the nitrocellulose pore network. Furthermore,\ncross-sectional SEM images of both borophene-coated and plain NC membranes\nwere acquired at various magnifications ( Figure S15 ). Although individual nanosheets could not be discerned,\nthe data indicate that borophene particles primarily permeate the\ntop 60–70 μm of the 130 μm-thick NC membrane ( Figure S15 ). Collectively, these SEM observations\nprovide qualitative evidence that the borophene nanosheets enhance\nthe surface area of the nitrocellulose strips’ top layer. Elemental\nanalysis via EDX conducted on different regions of the NC membrane\nshowed an average composition of 54.35% carbon ( Figure  \n c), 29.72% boron ( Figure  \n d), 6.90% nitrogen, and 4.80% oxygen ( Table S4 and Figure S16  ).\n(a, b) SEM images comparing\n(a) a bare nitrocellulose (NC) membrane\nand (b) an NC membrane incorporated with borophene nanosheets. (c,\nd) Elemental mappings showing (c) the presence of carbon in the NC\nmembrane and (d) the boron distribution in areas where borophene nanosheets\nare on the NC membrane. (e) SEM image of an NC membrane striped with\na borophene–antibody conjugate. Arrows indicate the region\ncontaining the conjugates. (f) Schematic representation of the borophene–UV-treated\nantibody conjugate within the NC matrix, illustrating the oriented\nantibodies on the borophene nanosheet surface. (g) SEM image of the\nNC membrane after the formation of the sandwich complex with the NC\nmatrix. Arrows indicate the presence of the sandwich complex as identified\nby the presence of gold nanoparticles. (h) Schematic illustration\nshowing the formation of the sandwich complex within the NC matrix.\nAs discussed previously, the sensitivity of LFIAs\nis constrained\nby the orientation of the capture antibodies within the nitrocellulose\nmembrane. Beyond orientation challenges, the capture molecules in\nthe NC matrix tend to distribute uniformly throughout the membrane’s\nthickness (approximately 120–180 μm) without forming\nany noticeable gradient. Consequently, analyte recognition occurs\nat the membrane surface and within the three-dimensional open-pore\nstructure (∼3–20 μm) of the NC matrix. Therefore,\na substantial fraction of the resulting detection signal may be concealed\nfrom automated readers or operators due to the membrane’s opacity,\nleading to diminished signal intensities and higher detection limits\ncompared with scenarios where all binding events occurred at the surface.\nTo address this limitation, the introduction of a 2D nanomaterial–antibody\nconjugate with a high surface-to-volume ratio as the detection element\nin the NC strip’s test region could potentially enhance both\nthe immobilization density and the antigen–antibody interaction\nefficiency nearer to the membrane surface, thereby improving the assay’s\noverall sensitivity.\nFurther, the SEM images showed that the\nimmobilizing UV-treated\nanti-Human HMGB-1 IgG antibodies over the 2D borophene nanosheets\non the test line region of the NC membrane showed a densely packed\narrangement of antibodies ( Figure  \n e).  Figure  \n f shows the graphical representation of the borophene–antibody\nconjugate in an NC matrix highlighting that the photoinduced immobilization\nstrategy enables strong covalent anchoring of the antibody to the\nmetal surface, positioning one Fab region side-on orientation while\nexposing the other Fab to the environment for effective antigen binding.\nFurther, upon the addition of the HMGB-1 antigen, the sandwich is\nformed ( Figure  \n g,h).\nThe sandwich structure consists of a borophene-capture antibody in\nthe bottom layer followed by HMGB-1 antigen and then streptavidin\ngold nanoparticles labeled with biotinylated detection antibody ( Figure  \n h). The SEM image\nconfirmed the presence of sandwich structure in the test zone ( Figure  \n g). Moreover, the\nsandwiched structures appeared to be formed in a concentrated area\nindicating the densely packed antibodies over the borophene surface\n( Figure  \n g).\nMoreover, we also utilized XPS to understand the chemical composition\nof the sandwich structure formed on the test line. Our group previously\nreported the conjugation of cysteine to borophene  for chiral induction to the 2D material. The interaction\nof B atoms with the thiol group of cysteine results in B–S\nbond formation, which was established with XPS as provided ( Figure S17 ). This knowledge was leveraged for\nthe conjugation of antibodies to the borophene surface for lateral\nflow sensing technology. XPS analysis was carried out with the lateral\nflow nitrocellulose strip containing the borophene antibody sandwich\nand compared with the control strip without the sandwiched complex.\nThe XPS spectra of the pristine nitrocellulose membrane ( Figure S18 ) and borophene–antibody sandwich\ncontaining nitrocellulose membrane gave an understanding of the B,\nC, N, O, and S presence. The B 1s spectrum, while not visible in the\ncontrol nitrocellulose surface ( Figure S18 ), is present in the borophene antibody sandwich spotted nitrocellulose\nstrip. Three prominent peaks were observed at 187.5 eV for B–B\nbonds, whereas the B–O bond at 200.5 eV \n , \n  ( Figure  \n a). The\nO 1s peaks for N–O are observed at 532.9 eV, C–O–N\nis observed in 534.2 eV with a higher intensity and C–O–C\nat 532.9 eV. A peak at 531.5 eV is observed for B–O from the\nborophene-attached moiety ( Figure  \n b). The high-resolution XPS data of the NC membrane\nshows the C 1s region with peaks at 284.6, 287.0, and 288.6 eV ( Figure  \n c). These peaks can\nbe assigned to the C–C, C–O–C, and C–O–N\nbonds.  The peak at 289.6 eV arises from\nthe carboxyl group in the antibodies. The S 2p spectrum shows two\npeaks at 168.2 and 169.5 eV. The peak of N 1s for nitrocellulose is\nconsistent with the NO 2  peak at 407.9 eV ( Figure  \n d). For the borophene antibody\nsandwich on NC, two new peaks arise at 404.8 eV for NH 2  and another peak at 400.3 eV for amide groups from the antibody.  Raman spectra conformed the peaks appearing\nin nitrocellulose strip containing antibodies immobilized onto borophene\nnanosheets ( Figure  \n e). AFM analysis of the borophene-coated nitrocellulose (NC) membrane\nwith the sandwich complex revealed an average roughness height ( S \n a ) of 1.23 nm and a root-mean-square (RMS) roughness\n( S \n q ) of 1.72 nm ( Figure  \n f). In comparison, the NC membrane without\nthe sandwich complex exhibited  S \n a  and  S \n q  values of 0.94 and 1.19 nm, respectively ( Figure  \n g). These changes\nin surface roughness are in agreement with the presence of immobilized\nbiomolecules. However, we acknowledge that AFM alone cannot definitively\nresolve antibody orientation, particularly on topographically irregular\nsurfaces like borophene. Therefore, while the increased roughness\nmay suggest surface functionalization, the effective sandwich formation\nin the presence of HMGB-1 antigen could be due to the accessibility\nand binding functionality of the antibody Fab regions.\nDeconvoluted XPS spectra\nshowing the (a) boron peaks in B 1s (b)\nN 1s (c) C 1s, and (d) O 1s from the borophene content in the nitrocellulose\n(NC) membrane. (e) Raman spectra of the NC membrane and the NC membrane\ncoated with borophene nanosheets. (f, g) Morphological analysis of\nthe NC membrane surfaces used in LFIA, as characterized by AFM: (f)\nNC membrane; (g) test line on the NC membrane showing the sandwich\ncomplex of antigen, detector antibody, and capture antibody.\nHaving characterized the\nmolecular interactions at the test line of our developed lateral flow\nimmunoassay, we proceeded to evaluate its capacity for detecting HMGB-1\nin whole menstrual effluent. In this study, we employed photoinduced\nimmobilization to covalently attach anti-Human HMGB-1 IgG capture\nantibodies onto 2D borophene nanosheets and compared the assay performance\nto that of a conventional LFIA in a sandwich-based format ( Figure  \n ). In the borophene-based\nLFIA, the test line of the nitrocellulose membrane was coated with\nUV-treated anti-Human HMGB-1 IgG capture antibodies on 2D borophene\nnanosheets. In the conventional LFIA, only the HMGB-1 capture antibody\nwas immobilized on the nitrocellulose membrane ( Figure  \n a). Both assays utilized the same Anti-mouse\nIgG at the control line and a conjugation pad containing gold-labeled\nanti-Human HMGB-1 IgG detection antibodies. Various concentrations\nof HMGB-1 (0–1000 pg/mL) were spiked into whole menstrual blood\nand applied to the sample pad, which incorporated a blood separator.\nThe blood separator (a glass fiber filter pad) effectively separates\nblood components, trapping red blood cells and larger cellular debris\non the surface while allowing only plasma containing the target HMGB-1\nanalyte and proteins to flow through to the nitrocellulose membrane\n( Figure  \n b). This physical\nseparation step significantly reduces matrix interference before the\nsample reaches the detection zone. As the sample flows, HMGB-1 binds\nto the gold-labeled detection antibodies, and this complex then migrates\nto the test line, where it interacts with the immobilized capture\nantibodies to form a visible sandwich complex. Unbound detection antibodies\nproceeded to the control line, yielding a separate signal. Visual\nanalysis of the borophene-based LFIA indicated that test lines could\nbe observed at HMGB-1 concentrations as low as 50 pg/mL ( Figure  \n d), whereas the conventional\nLFIA ( Figure  \n c) required\nat least 250 pg/mL to produce a visible band. These findings suggest\nthat the borophene-based LFIA achieves a naked-eye detection limit\nthat is approximately 80% lower (i.e., more sensitive) than the conventional\nassay. Subsequently, to evaluate potential cross-reactivity, the specificity\nof the lateral flow assay was examined using albumin, fibrinogen,\nand gamma globulinproteins commonly present in whole menstrual\neffluent. Each protein (20 μg/mL) was individually mixed with\n120 μL of chase buffer and applied to the lateral flow assay\nstrip. None of these proteins elicited a signal at the test line as\nshown in  Figure  \n e.\nThese observations confirmed the specificity of the lateral flow assay,\nwhich exclusively recognized its target recombinant protein HMGB-1\nand did not interact with other recombinant proteins found in menstrual\neffluent. Furthermore, quantitative assessments were performed using\nan ESE Quant Flex reader, and the color intensities at the test line\nwere analyzed with Studio 4.0 software. As expected, the signal intensities\nincreased for both LFIA formats in proportion to increasing HMGB-1\nconcentrations.  Figure  \n f shows the calibration curves for the borophene-based and conventional\nLFIAs, which yielded high correlation coefficients ( R \n 2  = 0.9772 and 0.9531, respectively). To calculate the\nanalytical limit of detection of the borophene LFIA, we use LOD =\n3.3 s \n \n y \n / y . The LOD was approximately 40 pg/mL for the borophene-based LFIA\nand 240 pg/mL for the conventional LFIA. The rationale for targeting\nlow concentrations, such as 40 pg/mL, is supported by clinical evidence\nindicating that HMGB-1 levels in menstrual blood are significantly\nelevated in individuals with endometriosis compared to healthy controls.\nHowever, early stage or asymptomatic cases may present with only modest\nincreases. The incorporation of two-dimensional borophene nanosheets,\ncombined with photoinduced antibody immobilization, enhances the assay’s\nanalytical performance, resulting in an approximately 500% increase\nin sensitivity relative to conventional LFIA platforms. While ELISAs\ncan detect lower HMGB-1 levels, our borophene-based LFIA achieves\nclinically relevant sensitivity (40 pg/mL) in a rapid point-of-care\nformat, enabling noninvasive monitoring of localized inflammatory\nactivity. This threshold aligns with reported menstrual fluid HMGB-1\nlevels in endometriosis progression, where early detection is critical\nfor timely intervention.  Unlike lab-based\nELISA, our approach balances sensitivity with practicality for decentralized\nsettings, addressing unmet needs in endometriosis screening.\n(a) Schematic\nrepresentation of the developed borophene–antibody\nconjugate LFIA. (b) Schematic illustration representing the working\nprinciple for the blood separator. (c) Detection of HMGB-1 using a\ntraditional LFIA with a limit of detection (LOD) of 250 pg/mL (d)\nDetection of HMGB-1 using the developed borophene–antibody\nconjugate LFIA with an LOD of 50 pg/mL, discernible to the naked eye.\n(e) Specificity of the developed borophene–antibody conjugate\nLFIA against commonly found proteins in menstrual effluent. (f) Calibration\ncurves illustrating the comparative performance of the borophene–antibody\nconjugate LFIA against the conventional LFIA across a range of HMGB-1\nconcentrations from 0 to 1000 pg/mL under optimal experimental conditions.\n\nIn this work, we have\nsuccessfully developed a simple and efficient\nmethod for the immobilization of antibodies utilizing borophene combined\nwith a unique functionalization procedure (PIT) to spatially orient\nantibodies on their surface to enhance their interaction with target\nanalytes. This approach enabled the construction of a functional lateral\nflow assay to detect HMGB-1, a key biomarker for endometriosis in\nmenstrual effluent at low concentrations. This success was achieved\nby exploiting the high surface-area-to-volume ratio of borophene and\nensuring robust antibody orientation via photoactivation. The surface\nfunctionalization approach used in this platform yields spatially\noriented antibodies, resulting in a densely packed distribution. This\nensures optimal utilization of the biosensor’s interacting\narea, which may contribute to its exceptional sensitivity and an impressive\ndetection limit of 40 pg/mL, surpassing conventional colorimetric\nassays. These findings tackle a key challenge in endometriosis diagnostics,\nwhere current methods often suffer from limited sensitivity. We expect\nthat the proposed method will serve as a simple and universal platform\nfor analyzing low-concentration analytes with high specificity and\nno cross-reactivity. Its clinical reliability makes it well-suited\nfor real-time diagnostic and biosensing applications. A key innovation\nof this approach is the integration of LFIA strips into menstrual\npads, enabling women to discreetly and conveniently monitor HMGB-1\nlevels at home. This advancement enhances accessibility to cutting-edge\ndiagnostics, decentralizing the platform to empower patients while\nfacilitating widespread use in resource-limited settings. Future research\nwill focus on scaling up larger clinical studies, enhancing the device’s\nsensitivity, and expanding the assay to detect additional disease\nbiomarkers. By integrating advanced 2D nanotechnology with user-friendly\ndiagnostic formats, this borophene-based LFIA paves the way for more\naccessible, noninvasive, and highly precise detection strategies,\nwith the potential to revolutionize reproductive health and beyond.\nAdditionally, it highlights key research opportunities that must be\nexplored to drive the development of next-generation biotechnologies\ncentered on 2D materials.\n\nAll\nchemicals were purchased from Sigma-Aldrich\n(St. Louis, MO, USA) unless otherwise stated. HMGB-1 recombinant antigen,\nmouse capture anti-human HMGB-1 IgG, and biotinylated detection anti-human\nHMGB-1 IgG antibody (Z01LS-1122-LS68) were bought from Creative Biolabs\nCo., Ltd. (New York, NY, USA). Whole blood filter sample pad (Fusion-5,\nCytiva, USA), an absorption pad (CFSP173000), and a high-flow nitrocellulose\nmembrane (HF180) were bought from the Merck Millipore (Darmstadt,\nGermany).\nA total of 250 mg\nof boron powder was dispersed in 250 mL of Milli-Q water in a beaker\nand thoroughly stirred. The mixture was then subjected to probe sonication\nfor 10 h at a 12 μm amplitude, with a cycle of 2 s “on”\nfollowed by 1 s “off.” The resulting suspension was\ncentrifuged at 3000 rpm for 3 min to collect the supernatant, which\nwas then passed through a 0.45 μm PTFE filter and kept at 4\n°C. The final concentration of the suspension was determined\nand subsequently used for further analysis.\nCitrate-capped AuNPs\nwere prepared by our previously published methods. \n − \n \n  Briefly, 8.5\nmg of tetrachloroauric­(III) acid trihydrate (HAuCl 4 ·3H 2 O) was dissolved in 95 mL of deionized water. This solution\nwas transferred to a 200 mL round-bottom flask equipped with a reflux\ncondenser, placed in an oil bath, and brought to a boil under magnetic\nstirring. Subsequently, 5.0 mL of a 1% (w/v) sodium citrate solution\nwas added rapidly. The mixture was kept at a boil and stirred for\n30 min until it developed a wine-red color. After cooling, the final\nproduct was stored in the dark at room temperature until further use.\nTo prepare\na streptavidin conjugate with gold nanoparticles via electrostatic\ninteraction, adjust the pH of 5 mL colloidal gold solution to 7.0\nusing a freshly prepared Na 2 CO 3  solution. Add\na streptavidin solution prepared in 10 mM potassium phosphate buffer\n(pH 7.2) to the adjusted colloidal gold solution. Incubate the mixture\nat room temperature while stirring for 30 min. Then, add BSA and sucrose\nto final concentrations of 10% and 0.2%, respectively, and incubate\nthe mixture for 1 h at room temperature. Centrifuge the mixture at\n11,000 rpm for 30 min at 4 °C using a 5810R centrifuge (Eppendorf,\nGermany). Discard the supernatant and dissolve the resulting pellet\nin 10 mM Tris-HCl buffer (pH 7.2).\nAll samples were diluted to\na concentration of 0.005 mg/mL in a disposable quartz cuvette. DLS\nmeasurements were carried out using a Malvern Instruments Zetasizer\nNano series system equipped with a 633 nm laser. Each sample was measured\nthree times, and the results were averaged using the Zetasizer software.\nFor analysis, the refractive index of water was set to 1.33, and the\nviscosity was taken as 0.8872, with a PDI of approximately 0.1 for\neach measurement.\nAFM was\ncarried out using a\nmodel 5600Ls atomic force microscope manufactured by Bruker Nano,\nUSA. The analyses were performed in tapping mode in different sizes,\nusing phase contrast and height modes. The raw images were processed\nwith Nanoscope Analysis 3.0 imaging and analysis software package.\nTEM images were taken using\na Talos F200X microscope. HAADF-STEM images, EDX mapping, and EDX\nline-scan profiles were taken using an FEI 200 kV Titan Themis scanning\ntransmission electron microscope.\nXPS experiments were\nperformed using a Physical Electronics Versa Probe III instrument\nequipped with a monochromatic Al Kα X-ray source ( h ν = 1486.6 eV) and a concentric hemispherical analyzer. Charge\nneutralization was performed using both low-energy electrons (<5\neV) and argon ions. The binding energy axis was calibrated using sputter-cleaned\nCu (Cu 2p 3/2  = 932.62 eV, Cu 3p 3/2  = 75.1 eV)\nand Au (Au 4f 7/2  = 83.96 eV) foils. Peaks were referenced\nto the CH \n x \n  band in the C 1s spectrum at\n284.8 eV. Measurements were made at a takeoff angle of 45° concerning\nthe sample surface plane. This resulted in a typical sampling depth\nof 3–6 nm (95% of the signal originated from this depth or\nshallower). Quantification was done using instrumental relative sensitivity\nfactors (RSFs) that account for the X-ray cross-section and inelastic\nmean free path of the electrons. The analysis size was ∼100\nμm in diameter.\nRaman spectra were collected\nusing a Renishaw inVia Reflex Raman Spectroscope system with the following\nparameters: a 785 nm laser, 45 mW (50%) power, a grating of 1200,\n100× magnification, and an acquisition time of 0.3 s, with the\ncenter of Raman frequency set at 1100 cm –1 .\nRaman measurements were performed using\na Horiba LabRAM HR Evolution spectrometer equipped with a 633 nm excitation\nlaser focused through a 100× objective lens (NA 0.9), delivering\nan incident laser power of 400 μW on the sample. Samples were\ndeposited onto a gold (Au) substrate, and fast mapping was conducted\nwith an integration time of 0.9 s, a confocal hole size of 100 μm,\nand a 300 gr/mm grating coupled to a BIDD Si-array detector (Horiba\nSynapse). The spectrometer was calibrated using the Raman response\nof a single-crystal silicon standard at 520 cm –1 . Acquired spectra were processed by subtracting background signals\nusing an eighth-order polynomial fit and subsequently averaged across\nall spectra obtained within the mapped region.\nFTIR in the attenuated total reflectance\n(ATR) mode was performed in an Agilent Cary 630 FTIR spectrometer\nfrom 4000 to 650 cm –1  at room temperature on a diamond\ndetector.\nThe\nsurface characterization\nof the test strip to ensure the establishment of conjugated probes\non the NC membrane was performed using SEM. A Verios G4 scanning electron\nmicroscope was used to characterize the test strip NC membrane before\nand after loading of the HMGB-1 sample.\nThe lateral flow\nassay test strips were prepared by a Claremont antibody stripping\nmachine and a Guillotine paper cutter. Photographs of strips and liquid\nsamples in containers were taken using an ESI quant, and the Image\nwas analyzed using Image Studio 4.0 The complete LFA strip consists\nof a blood filter sample pad, an absorbent pad, and a nitrocellulose\nmembrane featuring one test (T) line and one control (C) line. These\ncomponents were assembled on a backing pad with overlapping ends to\nensure a continuous flow of the developing solutions. Biotinylated\nanti-Human HMGB-1 IgG antibodies conjugated with streptavidin-AuNP\n(1 μg/strip) were immobilized on the conjugation pad by incubation\nat 37 °C for 1 h. For the test and control lines, UV-treated\nanti-Human HMGB-1 IgG antibodies conjugated with borophene nanosheets\nand anti-mouse IgG antibodies were immobilized on the NC membrane,\nrespectively. The anti-Human HMGB-1 IgG antibody-borophene conjugate\nwas prepared by mixing 50 μg/mL UV-treated anti-Human HMGB-1\nIgG with 0.175 mg/mL borophene nanosheets, followed by incubation\nat room temperature for 5 min. This conjugate was then sprayed onto\nthe NC membrane using an antibody dispenser and dried at 37 °C\nfor 1 h. The test and control lines were spaced 5 mm apart. The fully\nassembled LFA strip measured 3 × 60 mm and was stored in a sealed\nbag at room temperature until use. In conventional LFIA strip preparation,\nborophene is excluded from the test line, while the same nanoparticles\nare present on the conjugation pad.\nA 10\nμL sample of menstrual blood spiked with HMGB-1 standards was\napplied to the blood filter sample pad. After 30 s, 120 μL of\nPBST (1% BSA in PBS containing 0.05% Tween 20) was sequentially dispensed\nonto the pad. The sample then interacted with biotinylated anti-Human\nHMGB-1 IgG antibodies conjugated to streptavidin-AuNp complexes on\nthe conjugation pad. Subsequently, the complex bound to borophene\nand the capture anti-Human HMGB-1 IgG antibody on the NC membrane,\nforming the test line, while unbound complexes migrated to form the\ncontrol line. After approximately 15 min of incubation at room temperature,\nimages of the strips were captured using an ESE Quant Flex. For quantitative\nanalysis, the images were analyzed using Image Studio software, and\nthe pixel intensity of the test line regions was measured to determine\nthe color signal intensity.\nBorophene–antibody\nconjugation\nwas performed by mixing 1 mL of borophene nanosheet suspension (0.175\nmg/mL in ultrapure water) with anti-human HMGB-1 IgG antibody at a\nfinal concentration of 50 μg/mL. Prior to conjugation, the antibody\nsolution was irradiated at 254 nm using a Trylight UV lamp (6 W, irradiance\n∼ 0.3 W/cm 2 ) for 30 s to generate reactive thiol\ngroups. Following UV irradiation, antibodies were incubated with borophene\nfor 3 min at 25 °C. The conjugation mixture was subsequently\ncentrifuged at 3000 g  for 10 min at 4 °C to pellet\nthe antibody-borophene complexes. The pellet was resuspended in 1\nmL of ultrapure water for further analysis. A control group using\nnonirradiated antibodies was processed identically. To quantify antibody\nconjugation, a Bradford assay was performed using Coomassie Brilliant\nBlue G-250 dye. Briefly, IgG standards (1–100 μg/mL in\nphosphate-buffered saline (PBS), pH 7.4) and sample supernatants (150\nμL) were mixed with 50 μL of Bradford dye reagent in a\n96-well plate and incubated for 5 min at room temperature. Absorbance\nwas measured at 595 nm using a BioTek Synergy H1 microplate reader.\nThe linear standard calibration curve determined free IgG concentration\nin the supernatants. The conjugation efficiency (CE, %) was calculated\nusing the following formula: CE = [1 – (Free IgG in supernatant/Total\nIgG initially added)] × 100%. The resulting conjugation efficiency\nwas approximately 48%, corresponding to 24.04 μg/mL IgG immobilized\non the borophene surface.\nThe Ellman assay was\nused to quantify\nfree thiol groups. The reaction buffer consisted of 0.1 M sodium phosphate\nbuffer (pH 8.0) containing 1 mM EDTA. Ellman’s reagent (DTNB)\nwas freshly prepared by dissolving 4 mg of DTNB in 1 mL of reaction\nbuffer. A calibration curve was established using  l -cysteine\nstandards ranging from 0 to 200 nM. Anti-human HMGB-1 IgG antibodies\nwere diluted to 50 μg/mL in the reaction buffer and exposed\nto UV irradiation (254 nm, Trylight UV lamp, 6 W, irradiance ∼\n0.3 W/cm 2 ) for 30 s. A nonirradiated control group was\nmaintained under identical conditions without UV exposure. Postirradiation,\naliquots of 150 μL from each sample or standard were transferred\ninto a 96-well plate, and 50 μL of Ellman’s reagent was\nadded. The plate was incubated at room temperature for 15 min. After\nUV activation, antibodies were incubated with borophene nanosheets\nas described above. Postconjugation samples underwent the same Ellman\nassay procedure to assess residual free thiol concentration. Background\nabsorbance from borophene alone was subtracted from all sample measurements.\nAbsorbance values were recorded at 412 nm using a UV–vis spectrophotometer,\nand the concentration of free thiols was determined using the established\nstandard curve.","source_license":"CC-BY-4.0","license_restricted":false}