A 2D/3D Nanoplatform for Electrochemical Diagnosis of miRNA-122 in Breast Cancer Cell Lines

A 2D/3D Nanoplatform for Electrochemical Diagnosis of miRNA-122 in Breast Cancer Cell Lines | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article A 2D/3D Nanoplatform for Electrochemical Diagnosis of miRNA-122 in Breast Cancer Cell Lines Ali Esmaeilian, Nastaran Arab, Fatemeh Yazdian, Morteza Hosseini, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7449624/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract A novel, label-free electrochemical biosensor was precise engineered for the ultrasensitive and selective detection of miRNA-122, a pivotal biomarker in breast cancer diagnostics. This innovative sensing platform harnesses the exceptional conductivity of Ti 3 C 2 MXene nanosheets, synergistically integrated with Au-decorated NH 2 -UiO-66 metal-organic frameworks, yielding a structurally stable and electrochemically active hybrid interface. A thiolated DNA capture probe was immobilized onto the gold-modified MOF, enabling precise hybridization with the target miRNA-122. Methylene blue functioned as a robust redox indicator, exhibiting distinct electrochemical shifts upon duplex formation through π–π stacking and electrostatic interactions. The biosensor demonstrated outstanding analytical performance with dual linear response ranges (1 pM–1 nM and 1 nM–750.0 nM) and an impressively low limit of detection of 503 fM. High sequence specificity was achieved, successfully differentiating miRNA-122 from non-complementary and closely related sequences. Validation with lysates from three distinct breast cancer cell lines (MCF-7, MDA-MB-231, and SKBR3) confirmed its efficacy in complex biological matrices. Altogether, this biosensor platform offers a powerful and reliable tool for early breast cancer detection and holds substantial potential for clinical applications in miRNA-based diagnostics. 2D nanomaterials Breast cancer miRNA-122 NH2-UiO-66 MOF Ti3C2Tx MXene Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Cancer is one of the leading causes of death worldwide, claiming millions of lives annually, as reported by the World Health Organization [1]. Among the various types of cancer, breast cancer stands out as a prevalent malignancy affecting women globally. It is the second leading cause of cancer-related illness and death, with its incidence rising annually [2]. To minimize recurrence and lower mortality, early detection, precise diagnosis, and prompt treatment are essential for the efficient control of breast cancer [3,4]. Current diagnostic techniques for breast cancer, such as MRI, mammography, X-ray imaging, ultrasound, biopsy, and CT scans, are widely recognized but come with limitations like high costs, the need for specialized personnel, complex data analysis, and the potential for false results [5]. Traditional methods like mammography require extensive hospital facilities and specialized staff, involve time-consuming processes, and often lead to overdiagnosis, which hampers early detection and effective treatment. Consequently, there is a critical need to develop more convenient, rapid, and cost-effective detection methods [6,7]. The analysis and diagnosis of cancer biomarkers is one of the most important methods for breast cancer detection. One of the biomarkers used to diagnose diseases, including breast cancer, is microRNAs (miRNAs), which are small non-coding RNAs about 19–25 nucleotides in length, characterized by their small size, relative stability in biological fluids, and their role in the post-transcriptional regulation of protein levels and gene expression [8,9]. Though the role of miRNA-122 in breast cancer is not yet fully understood, it is emerging as a crucial regulator of tumor development and carcinogenesis. miRNA-122 facilitates metastasis by increasing nutrient availability in the pre-metastatic niche [10,11]. miRNA-122 is aberrantly expressed in various tumors, including breast, lung, bladder, leukemia, liver, colorectal, ovarian, and esophageal cancers, where it can function as both a tumor promoter and suppressor [12,13]. Its deregulation in tumor development underscores its potential as a valuable biomarker for the diagnosis and prognosis of breast cancer. Additionally, miRNA-122 may enhance tumor cells' sensitivity to chemotherapy, making it a target for therapeutic intervention. Given these findings, miRNA-122 stands out as a powerful molecular biomarker and a novel therapeutic target for breast cancer, offering significant potential for advancing targeted treatments [12,14]. The quantification of miRNA within the cell nucleus is crucial for elucidating gene regulatory mechanisms and cellular processes [15,16]. These small non-coding RNAs play a pivotal role in modulating gene expression, thereby influencing tumor progression and metastasis [17,18]. MCF-7, SKBR3, and MDA-MB-231 are well-established breast cancer cell lines extensively used in research to investigate various breast cancer subtypes. MCF-7 cells, which are estrogen receptor-positive, serve as a model for hormone-dependent breast cancer. SKBR3 cells, characterized by their overexpression of HER2, are ideal for studying HER2-targeted therapies. MDA-MB-231, a triple-negative and highly aggressive cell line, is commonly employed to study metastasis and drug resistance [19]. Collectively, these cell lines provide invaluable insights into the molecular pathways underlying breast cancer and the efficacy of different therapeutic strategies [20-22]. Notably, electrochemical biosensors can provide quick and susceptible miRNA detection compared to traditional methods. To increase the sensitivity of miRNA detection for the diagnosis of many illnesses, electrochemical nanobiosensors for miRNA quantification have integrated several nanostructures [23,24]. One of the materials in this category is MXene, which was first introduced by Gogotsi and his team with the discovery of two-dimensional titanium carbide (Ti 3 C 2 ) powder, marking the beginning of the MXene family [25]. MXene nanomaterials, a class of two-dimensional transition metal carbides, nitrides, or carbonitrides, are recognized for their unique structure and exceptional electronic conductivity. These materials consist of atomically thin layers of transition metals sandwiched between ceramic-like layers [26]. In the context of electrochemical biosensors, MXenes offer several advantages, including high conductivity, a large specific surface area, and excellent electrochemical stability. These properties facilitate efficient electron transfer kinetics and provide abundant sites for biomolecule immobilization, making MXenes ideal building blocks for the development of high-performance electrochemical biosensors with enhanced sensitivity and reliability [27]. The integration of MXenes with other nanostructures is a crucial strategy for tailoring their electrical, mechanical, and chemical properties. This approach enables the design of composite materials with enhanced performance and expanded functionalities, which are highly desirable for advanced applications such as electrochemical biosensing [28,29]. Integrating MXenes with metal-organic frameworks (MOFs) creates hybrid materials that leverage the distinct advantages of both components [30,31]. MOFs are known for their high porosity, tunable chemical functionalities, and substantial surface area, which facilitate interactions with target molecules [32]. This combination results in enhanced properties for the hybrid materials, making them particularly effective for applications in biosensing, catalysis, and environmental remediation. The synergistic effects of MXenes and MOFs thus form a robust strategy for developing advanced materials tailored for specific functionalities and applications [33-35]. For example, Wang et al. using MXene@MOF for the electrochemical biosensor to detect Glycoprotein Nonmetastatic Melanoma Protein B (GPNMB). MXene@MOF was chosen for its stability, high conductivity, and enhanced electroactive surface area. The integration of 2D MOFs prevents MXene oxidation and increases sensitivity. These features make it ideal for developing a label-free, ultra-sensitive electrochemical biosensor for detecting GPNMB in Parkinson’s Disease patients' serum [36]. In another study, Kaur et al. used MOF/MXene composite as a surface with accessible pores, mechanical strength, and suitable conductivity properties to set up highly sensitive, stable, and practical electrochemical sensors [37]. In addition, Chen et al. utilized MOF/MXene. Combining MOFs with MXenes enhanced electrochemical properties, reduced stacking effects, and increased binding sites for the aptamer [38]. In this study, an advanced, label-free electrochemical biosensor was designed for the sensitive detection of miRNA-122, a crucial biomarker in breast cancer cells, utilizing a heterostructured composite of Au/NH 2 -UiO-66 and NH 2 -Ti 3 C 2 nanosheets. The synergistic combination of NH 2 -Ti 3 C 2 ’s exceptional conductivity and functionalization potential, along with the increased surface area and active sites provided by Au-decorated NH 2 -UiO-66, significantly enhances the sensor's performance. The incorporation of gold nanoparticles ensures strong immobilization of the DNA probes, leading to superior detection sensitivity. This platform dramatically amplifies electrochemical signals, establishing itself as a highly promising and reliable tool for the early and precise diagnosis of breast cancer. Scheme 1 illustrates the design of the proposed biosensor for the detection of miRNA-122. Material and methods Materials All chemicals contain titanium powder (99.98% purity, < 45 μm), graphite powder (≥ 99.99% trace metals basis,< 45 μm), aluminum powder (99.9% purity, 60 μm), Conductive carbon ink, sodium chloride (NaCl), potassium ferrocyanide (K 4 Fe(CN) 6 ), potassium chloride (KCl), potassium ferricyanide (K 3 Fe(CN) 6 ), HF (Hydrofluoric acid), Dimethyl sulfoxide (DMSO), (3-Aminopropyl) triethoxysilane (APTES), Glutaraldehyde (GA), Ethanol (C 2 H 5 OH), Phosphate buffered saline (PBS), N, N-dimethylformamide (DMF), acetic acid (CH 3 COOH), chloroauric acid tetrahydrate (HAuCl 4 ·4H 2 O), sodium tetrahydridoborate (NaBH 4 ), and bovine serum albumin (BSA) were purchased from Sigma-Aldrich. TE buffer was gained from Sinaclon. In addition, Methylene blue (MB), Zirconium tetrachloride (ZrCl 4 ), and 2-amino terephthalic acid (NH 2 -BDC) were provided from Merck. All materials utilized were of analytical reagent grade, and deionized water was sourced from a Millipore Milli-Q system. All synthetic oligonucleotide sequences listed in Table S1 were obtained from Metabion International AG (Planegg, Germany). Apparatus For the Brunauer-Emmett-Teller (BET) specific surface area study, Belsorp, Japan-made BET Mini II equipment was used. X-ray diffraction (XRD) patterns were recorded on a Philips PW1730 with Cu-Ka radiation at 40 kV and 40 mA. The morphology and the chemical composition (Energy-Dispersive X-ray Spectroscopy (EDS)) were investigated by TESCAN MIRA3 Field Emission Scanning Electron Microscope (FESEM). Transmission electron microscopy (TEM) images were recorded on a Philips EM208S. Fourier transform infrared spectra (FTIR) spectra were recorded with a Perkin-Elmer. Electrochemical measurements including the electrochemical impedance spectroscopy (EIS), cyclic voltammetry (CV), and Differential pulse voltammetry (DPV) were carried out using an AUTOLAB PGSTAT204 read out by NOVA 2.1 software. Preparation of MAX Phase (Ti 3 AlC 2 ), MXene (Ti 3 C 2 T X ) and NH 2 -Ti 3 C 2 T X The synthesis of the MAX phase process adhered to all the steps outlined in the earlier report by Xu and other comparable studies [39,40]. The selective removal of aluminum from Ti 3 AlC 2 was accomplished through a direct etching procedure. Initially, 5 g of Ti 3 AlC 2 MAX phase powder was gradually introduced into 100 mL of a 50 wt% HF solution under continuous stirring. The slow addition of the MAX phase was essential to control excessive bubbling caused by the exothermic reaction. The etching process was maintained at room temperature (~23°C) for 5 hours while stirring at 300 rpm. The resulting mixture was then subjected to centrifugation (at 3500 rpm for 5 minutes per cycle) and repeatedly washed with deionized water until the pH of the supernatant reached a nearly neutral value. Finally, the purified sample was dried in a vacuum oven at 80°C for 24 hours, yielding the desired MXene product [41]. In order to synthesize NH 2 -Ti 3 C 2 T X , initially, 0.2g of Ti 3 C 2 T x powder was dispersed in 5 mL of DMSO, which was then constantly stirred for 24 hours. The resultant Ti 3 C 2 T x precipitate was centrifuged and then redispersed in 60 mL of ultrapure water. The Ti 3 C 2 T x nanosheets were exfoliated by sonicating the dispersion for 16 hours after the solution had been purified with argon gas for 20 minutes. The nanosheets were then recovered by centrifugation at 3000 rpm for 5 minutes. After that, 1 mL of the Ti 3 C 2 T x nanosheet suspension was mixed with 20 μL of APTES, and the mixture was left to stir for a whole day at room temperature. For future usage, the resultant NH 2 -Ti 3 C 2 T x nanosheets were redispersed in 1 mL of ultrapure water after excess APTES was removed by centrifugation. Synthesis of UiO-66-NH 2 and Au@NH 2 -UiO-66 The synthesis procedure for UiO-66-NH₂ has been documented in the literature [42]. In this method, 93.2 mg of ZrCl 4 and 72.4 mg of NH 2 -BDC were dissolved in 40 mL of DMF, followed by the addition of 5.5 mL of acetic acid to facilitate the formation of UiO-66-NH 2 particles.After being moved to a 50 mL stainless steel autoclave, the mixture was heated for 24 hours to 120˚C. Following three centrifugation washes with DMF and ethanol, the UiO-66-NH 2 particles were dried at 60 °C to produce a mustard powder. Au@UiO-66-NH 2 was synthesizedthrough the reduction ofAu(III) to Au(0) nanoparticles on the pre-synthesized UiO-66-NH 2 framework[43]. For this purpose, a uniform dispersion of NH 2 -UiO-66 was first prepared by ultrasonically treating 20 mg of NH 2 -UiO-66 in 4 mL of an ethanol/water mixture (1:1 v/v). Subsequently, 0.5 mL of a 25 mM HAuCl 4 solution was introduced, and the mixture was continuously stirred for four hours. Following this, 2.4 mL of freshly prepared ice-cold NaBH 4 solution (0.05 M) was quickly added, and stirring continued for an additional hour. Finally, the resulting precipitate was collected via centrifugation and thoroughly washed multiple times to ensure purity. Fabrication of electrochemical biosensor The biosensor was fabricated using a screen-printing process on flexible PVC substrate, employing carbon paste as the printing ink. The designed screen-printed electrode (SPE) was constructed based on the common electrochemical system, with dried silver paste serving as the reference electrode (Fig. S1). To fabricate the biosensor, a precise aliquot of 40µL NH 2 -Ti 3 C 2 suspension was carefully deposited onto the working electrode via drop-casting. The electrode was then subjected to a desiccation process under an inert argon atmosphere for one hour. Subsequently, 40µL GA was dropped on the surface of the electrode and after 2 hours was rinsed with deionized water. After that 40µL Au@UiO-66-NH 2 was cast on the surface. For stabilizing the ssDNA probe, 35 μl of Thiol-ssDNA (200 nM, as probe) prepared in TE buffer was dropped on the surface of Au@NH 2 -UiO-66/NH 2 -Ti 3 C 2 at 4˚C for 16 h. Next, the electrode was rinsed with PBS (pH 7.4), followed by the application of a 1% BSA solution for 15 minutes. Afterward, it was washed again with PBS. At this stage, the nanobiosensor is prepared for hybridization with the target miRNA122. For the hybridization process, different concentrations of target miRNA122 were prepared in TE buffer. A 40.0 μL droplet of the miRNA122 solution was placed onto the modified electrode and left for 120 minutes, after which the electrode was rinsed with PBS. Finally, the electrode modified with miRNA/ssDNA hybrids was incubated in MB solution (4.0 μM MB, 0.2 M NaCl, 0.1 M phosphate-buffered saline, PBS) for 2 hours to facilitate MB binding. After incubation, any unbound MB was thoroughly removed using a washing PBS, ensuring the measured current signal derived from MB bound to the hybridized sequences. The Scheme 1illustration sequentially presents the proposed biosensor for miRNA-122 detection. miRNA-122 Detection and electrochemical measurements The biosensor fabrication and miRNA detection processes were analyzed using cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS), and differential pulse voltammetry (DPV). CV and EIS measurements were conducted in a solution of 5.0 mM [Fe(CN)₆]³⁻/⁴⁻ with 0.1 M KCl. CV scans were carried out over a scan range of -1.5 to +1.5 V, while EIS experiments were performed across a frequency range of 0.1 Hz to 10 kHz. Also, the DPV signal for the redox activity of methylene blue (MB) was recorded within the scan range of -0.6 to 0.3 V in a PBS solution at pH 7.4 with a scan rate10mV/s. Cell lysis Cells were lysed by adding 300 µL of ice-cold Triton X-100 lysis buffer (0.25% Triton X-100 in PBS) to one to three 10 cm cell culture dishes. To inhibit proteolytic and dephosphorylating activities, protease and phosphatase inhibitors were added to the lysis buffer prior to use. Cells were incubated with the lysis buffer for 30 minutes on ice with intermittent vertexing to facilitate membrane disruption and release of cellular contents. Following incubation, the lysates were centrifuged at maximum speed for 15 minutes at 4°C to pellet cellular debris. The supernatant, containing solubilized membrane and cytosolic proteins, was collected and stored at -80°C for subsequent analysis [44]. Results and Discussion Sensing mechanisms of biosensor The sensing mechanism of the developed electrochemical biosensor relies on the sequential interactions between, the immobilized thiol-modified capture probe, the target miRNA-122, and MB as the redox indicator. Initially, the capture probe is immobilized onto the Au-decorated MOF surface, facilitating the electrostatic binding of MB to the negatively charged phosphate backbone of the single-stranded DNA probe. Upon introduction of miRNA-122, hybridization occurs, forming a double-stranded complex. The hybridization event significantly enhances MB binding through a dual mechanism: electrostatic interactions and intercalation. Initially, MB interacts with the negatively charged phosphate backbone of single-stranded DNA via electrostatic attraction. However, upon hybridization with miRNA-122, the formation of the double-stranded complex introduces a structurally distinct environment enriched with guanine-rich regions. Guanine possesses an extended π-electron system due to its fused bicyclic purine structure, which provides a highly conjugated and planar surface [45]. This feature enables MB, with its tricyclic aromatic core, to establish strong π–π stacking interactions with guanine bases, thereby enhancing its retention within the duplex structure [46]. Furthermore, the steric stabilization conferred by the hybridized strands limits MB diffusion away from the electrode surface, amplifying its redox signal. As a result, the electrochemical response is significantly increased, enabling highly sensitive and selective miRNA-122 detection. The interplay of these binding mechanisms ensures a stable and reproducible signal, making this approach highly effective for electrochemical biosensing [47,46]. Characterization of Ti 3 C 2 MXene The successful synthesis of Ti 3 C 2 T x was validated using FESEM, XRD, and FTIR. The FESEM images demonstrate the morphological characteristics of the synthesized Ti 3 C 2 T x . Fig. 1A displays a surface view of Ti 3 C 2 T x , highlighting its distinctive accordion-like morphology. Fig. 1B illustrates a cross-sectional view, showcasing the interlayer spacing and exfoliation of the nanosheets, with the distinct separation of layers. The two images offer a comprehensive understanding of the structural organization and layer arrangement of Ti 3 C 2 T x , confirming its characteristic two-dimensional morphology and affirming the successful synthesis of Ti 3 C 2 T x . [48]. The XRD pattern of the synthesized Ti 3 C 2 T x MXene, shown in Fig. 1C, reveals the structural transformation resulting from the selective etching of Ti 3 AlC 2 . The diffraction peaks observed at 9.1°, 18.5°, 24.1°, 34.7°, 42.1°, and 61.2° for Ti 3 C 2 T x MXenes indicate the presence of a well-crystallized nanostructure in the synthesized hybrids. The (002) peak observed at 9.1° indicates the interlayer distance (D-spacing) and verifies the incorporation of –F and –OH surface functional groups. [49,50]. The presence of broad peaks at 34.7° and 42.1°, with low intensities and corresponding to the (101) and (103) planes, confirms the successful elimination of aluminum [51,52]. The peaks observed at 2θ = 18.7° and 23.95° correspond to the (006) and (008) planes. The peaks observed at 2θ = 42.15° and 61.05°, corresponding to the (105) and (110) planes, further confirm the maintained structural integrity following the etching process. [53,54]. FTIR spectroscopy confirmed (Fig.1D) the successful functionalization of MXene with amine (-NH 2 ) groups. The spectrum showed characteristic absorption bands for amine groups, with prominent peaks in the 3200-3500 cm⁻¹ region attributed to N-H stretching vibrations and a peak around 1560-1650 cm⁻¹ corresponding to N-H bending [55]. Si-O-Si stretching vibrations in the 1000-1200 cm⁻¹ region confirmed the bonding of the APTS silane coupling agent to the MXene surface, anchoring the amine groups [56]. C-O stretching in the 1600-1700 cm⁻¹ region, associated with surface functional groups on MXene. C-H stretching in the 2800-3000 cm⁻¹ region, linked to APTS propyl chains, further reinforced successful modification. Shifts in Ti-C and Ti-O peaks around 500-800 cm⁻¹ indicated the interaction between MXene and APTS [57]. These findings highlight the structural, crystallographic, and chemical evolution of MXene, validating the efficacy of the synthesis process. Characterization of NH 2 -UiO-66 and Au@NH 2 -UiO-66 The successful synthesis of NH 2 -UiO-66 and Au@NH 2 -UiO-66 were also validated using various analytical techniques, including TEM, XRD, EDX mapping and BET surface area analysis. TEM images clearly reveal that the synthesized NH 2 -UiO-66 nanocrystals possess a polyhedral morphology and a consistent size distribution (Fig.2A and 2B). [58,43]. Fig. 2C and 2D verify the successful deposition of gold nanoparticles on the surface of the MOF nanocrystals. These images demonstrate that the crystalline structure of the NH 2 -UiO-66 is maintained intact following the decoration process with gold nanoparticles, which predominantly exhibit diameters ranging from 4 to 8 nm. XRD analysis was performed to investigate the crystallographic structures of NH 2 -UiO-66 and Au@NH 2 -UiO-66 (Fig. 2E). The XRD pattern of NH 2 -UiO-66 exhibited sharp and well-defined diffraction peaks at 2θ values of approximately 7.4°, 8.5°, 12.2°, 14.6°, 17.1°, 22.1°, 25.6°, and 30.5°, corresponding to the (111), (002), (022), (113), (222), (044), (137), and (444) crystal planes, respectively. These diffraction peaks are characteristic of the highly crystalline nature of the NH 2 -UiO-66. The XRD pattern of Au@NH 2 -UiO-66 also displayed the characteristic diffraction peaks of NH 2 -UiO-66 at 7.4°, 8.5°, 12.2°, 14.6°, 17.1°, 22.1°, 25.6°, and 30.5°, indicating that the crystalline structure of NH 2 -UiO-66 remained intact after gold nanoparticle decoration. In addition, new diffraction peaks emerged at 38.1°, 44.3°, 64.5°, and 77.5°, corresponding to the (111), (200), (220), and (311) planes of gold nanoparticles [43]. The presence of these new peaks confirmed the successful integration of gold nanoparticles within the MOF structure. The BET surface area and porosity of synthesized NH 2 -UiO-66 were determined from N 2 adsorption/desorption studies and the result is shown in Fig.2F. Typical type I isotherm with no hysteresis is observed in the figure, which confirms the microporous nature of NH 2 -UiO-66 [59]. The BET surface area for NH 2 -UiO-66 is 756.4 (m 2 /g) with a pore volume of 0.6636 cm 3 g −1 . Interestingly, similar to XRD observations, the decoration of AuNPs on Zr-MOF does not significantly change the area of NH 2 -UiO-66 based on these observations, it can be concluded that Au nanoparticles were distributed well on the surface of Zr-MOF. The XRD data, which showed the preserved peaks of NH 2 -UiO-66 alongside the new peaks corresponding to gold, suggests that the gold nanoparticles are primarily located on the surface of the MOF crystals rather than within the pores, indicating that the MOF framework integrity was maintained while introducing new catalytic sites. EDX confirms the elemental composition of the studied materials. In Fig.S2, for NH 2 -UiO-66, peaks for carbon (C), nitrogen (N), oxygen (O), and zirconium (Zr) are observed, indicating their presence in the MOF structure, with atomic percentages of 23.9% C, 8.5% N, 33.7% O, and 33.9% Zr. In Fig. 2D, for Au@UiO-66-NH 2 , peaks for the MOF elements and gold (Au) are seen, with atomic percentages of 7.6% C, 4.2% N, 14.3% O, 35.5% Zr, and 38.4% Au. The presence and high percentage of gold confirm the successful decoration of the MOF with gold nanoparticles, [43]. In addition, the mapping of NH 2 -UiO-66 and Au@NH 2 -UiO-66 show the elemental distribution of the studied materials. In Fig.2E, corresponding to NH 2 -UiO-66, the elemental maps show a uniform distribution of O, Zr, N, and C throughout the material and also the EDX maps of Au@NH 2 -UiO-66 display the distribution of the same elements (O, Zr, N, and C) along with Au (Fig.S2). Electrochemical characterization The stepwise fabrication process of the miRNA biosensors was evaluated using EIS and CV in a 5.0 mM Fe[(CN)₆]³⁻/⁴⁻ solution with 0.1 M KCl (Fig.3A and 3B). CV studies on five different electrode samples demonstrated that the modification of the electrode surface with various layers has a significant impact on the electrochemical behavior of the system. The bare electrode exhibited a pair of reversible redox peaks (curve a). Following surface modification with NH 2 -Ti 3 C 2 , a notable increase in current was observed, attributed to the exceptional conductivity of NH 2 -Ti 3 C 2 (curve b) [60]. Subsequent modification of the surface with Au@NH 2 -UiO-66 led to the emergence of clear oxidation and reduction peaks, reflecting the distinctive electrocatalytic behavior of Au@NH 2 -UiO-66 (curve c) [61]. With the attachment of DNA probes to the surface, a decrease in current was observed, indicating the occupation of part of the active surface by the probes and consequently a decrease in electron transfer (curve d). Finally, the hybridization of the target (miRNA-122) with DNA probes resulted in a further decrease in current due to the creation of a steric hindrance and a decrease in electron transfer due to the increased complexity of the surface structure (curve e). The CV results were further validated by EIS measurements (Fig.3B). The inset shows the Nyquist plot for the bare electrode, which exhibits a significantly higher impedance response compared to the modified electrodes, indicating a large charge transfer resistance (R ct ). This high impedance is attributed to the absence of conductive layers or functional groups on the electrode surface, leading to poor electron transfer kinetics. Following modification with Ti 3 C 2 T x , a nearly linear diagram was observed, reflecting enhanced electron-transfer kinetics (curve b). An increase in R ct was noted after modification with Au@NH 2 -UiO66/NH 2 -Ti 3 C 2 , showing higher charge transfer resistance compared to Ti 3 C 2 alone (curve c). This rise in resistance is attributed to the deposition of the Au@NH 2 -UiO-66 composite, which creates additional interfacial barriers and contributes to an overall increase in impedance [62]. Following the immobilization of the capture probe onto the Au@NH 2 -UiO66/NH 2 -Ti 3 C 2 surface, a significant increase in R ct was observed. This rise is due to the introduction of steric hindrance and the reduction of the electrode's active sites, which together hinder charge transfer (curve d). The impedance value continued to increase as miRNA-122 hybridization took place on the capture probe/Au@NH 2 -UiO-66/NH 2 -Ti 3 C 2 surface (curve e). This stage shows the highest impedance among the modified electrodes, reflecting the successful hybridization of miRNA with the capture probe. The significant increase in R ct at this stage is due to the formation of the biomolecular complex, which further obstructs electron flow. Overall, the stepwise increase in charge transfer resistance across the modification stages confirms the sequential electrode functionalization. The systematic progression in the Nyquist plot aligns with the expected behavior of surface modification and biomolecule immobilization, highlighting the effectiveness of the proposed biosensor in detecting specific targets like miRNA-122. Optimization of conditions In this study, comprehensive investigations were conducted to optimize various parameters of the microRNA-based electrochemical biosensor (Fig.S3), aiming to enhance sensitivity and measurement accuracy. The results demonstrated that the optimal concentration of the Au@NH 2 -UiO-66 nanocomposite, equivalent to 0.2 mg/mL, yielded the highest electrochemical response. Deviation from this value in either direction resulted in a noticeable decrease in the electrochemical signal. At lower concentrations (<0.2 mg/mL), there may be an insufficient number of conductive and catalytic sites to facilitate electron transfer, which leads to the limitation of the electron transfer process. Conversely, higher concentrations (>0.2 mg/mL) can cause the aggregation of nanoparticles, which reduces the effective electrode surface and disrupts redox reactions [63]. Furthermore, examination of the effect of DNA probe concentration revealed that a concentration of 200 nM generated the highest current intensity, and increasing concentrations beyond this value led to a reduction in response. Weak responses observed at concentrations below 200 nM can be attributed to an insufficient amount of immobilized probe on the SPE surface. Furthermore, at concentrations exceeding 200 nM, the decline in response signals is likely due to excessive accumulation of ssDNA, which hinders efficient electron transfer and sensor performance [64]. In the experiment concerning the probe immobilization time on the electrode surface, 16 hours was identified as the optimal time, as the highest signal was obtained within this time frame without a significant drop. The DPV response increased progressively with longer immobilization times, reaching its peak at 16 hours. A notable rise in the biosensor signal was observed between 8 and 16 hours, indicating a higher degree of probe attachment to the modified SPE surface. Beyond this point, no significant enhancement in signal response was detected, suggesting that the surface had reached saturation. Consequently, 16 hours was selected as the optimal immobilization time to ensure efficient and stable probe attachment on the SPE surface [65]. In the investigation of the hybridization time between miRNA-122 and the probe, it was observed that 120 minutes produced the highest current response, and increasing the time beyond this value did not have a significant impact on the signal intensity. At shorter hybridization times, the number of formed duplexes between miRNA-122 and the capture probe was limited, leading to a lower current response. As the incubation time increased, more hybridization events occurred, enhancing the signal until reaching 120 minutes, where saturation was achieved. Beyond this point, no further increase in signal intensity was observed, indicating that all available binding sites were occupied and the hybridization process had reached equilibrium [66]. Finally, regarding the MB incubation time, 15 minutes was determined as the optimal time, as the signal intensity reached a steady state after this time frame and no significant change in response was observed. At shorter incubation times, an insufficient amount of MB molecules interacted with the hybridized miRNA-122, leading to a lower signal response. Extending the incubation period beyond 15 minutes did not further enhance the electrochemical signal, suggesting that equilibrium in MB binding had been achieved, and excess MB molecules were removed during the washing step [47]. These results confirm the successful optimization of the experimental conditions to enhance the accuracy and efficiency of the proposed biosensor. MB as the Redox Indicator for miRNA Detection To evaluate the feasibility of the proposed miRNA detection method, MB was employed as an electrochemical indicator to track the hybridization interaction with the target miRNA. MB, a phenothiazine dye, is known to exhibit varying binding affinities for single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA) [67,68]. This is due to MB binding to ssDNA primarily through electrostatic interactions, while its interaction with dsDNA involves both intercalation and electrostatic forces. As a result, these differences produce distinct electrochemical responses when MB interacts with ssDNA compared to dsDNA [69,70]. To study the interaction of MB with ssDNA and dsDNA, CV was conducted on the electrode functionalized with the capture probe (Fig. 4A) and the capture probe after hybridization with the target miRNA (Fig. 4B) at different scan rates. In both cases, an increase in scan rate resulted in a proportional increase in peak currents. However, a significant difference was observed in the magnitude of the redox peaks. The target system consistently exhibited higher peak currents compared to the capture system across all scan rates. This enhancement in current suggests successful hybridization between the capture probe and the target miRNA, leading to improved electron transfer kinetics at the electrode surface. The higher peak currents observed in the target system can be attributed to several factors. Hybridization may enhance the local electron density near the electrode surface, facilitate faster electron transfer pathways, or reduce the distance for electron hopping within the immobilized biomolecular complex. Additionally, the increase in current for the target system is largely due to the interaction of MB with the miRNA-DNA hybrid [71,47]. In the presence of the target, MB binds not only electrostatically to single-stranded DNA but also intercalates into the double-stranded miRNA-DNA complex. This dual binding mechanism results in a higher accumulation of MB, thereby increasing the overall current response. In contrast, for the capture system, MB binds primarily through electrostatic interactions with single-stranded DNA, leading to a relatively lower current. This differential binding explains the significant enhancement in the electrochemical signal observed after target hybridization, reinforcing the sensitivity and specificity of the biosensor for miRNA detection. The observed increase in electrochemical response indicated greater absorption of MB molecules on the probe surface, providing strong evidence for the formation of ssDNA/miRNA hybrids on the electrode. This finding aligns with previous studies [72,73]. Additionally, for further analysis, the anodic and cathodic peak currents of the two prepared electrodes were plotted as a function of scan rate. As shown in Fig. 4C, the anodic and cathodic peak currents exhibit a linear relationship with the scan rate over the range of 0.025 to 0.8 V/s, suggesting that the process is surface-confined. The surface coverage of electroactive MB (Γ MB ) on the electrode was estimated using the equation [74]: Ip = F²n²AΓv / 4RT where F is Faraday’s constant (96,485 C mol⁻¹), n represents the number of electrons transferred (n = 2 for MB), A is the electrode area (cm²), Γ is the surface coverage, v is the scan rate (V/s), R is the gas constant (8.314 J K⁻¹ mol⁻¹), and T is the temperature. Based on the slope of the I pc versus v plot in Figure 4B, the calculated surface coverage (Γ MB ) for the target was 553.23 nM/cm², which is notably higher than the 250.76 nM/cm² measured for the capture probe. This suggests that more MB binds to the miRNA/ssDNA duplexes, enhancing electron transfer from MB to the electrode. These results confirm the effectiveness of the biosensor for miRNA detection by monitoring the reduction response of MB. Performance of electrochemical biosensor The quantification performance of the biosensor for detecting different concentrations of miRNA-122 was assessed using DPV in PBS buffer, measuring the oxidation peak current of methylene blue intercalated within double-stranded complexes of capture probes and miRNA-122 (Fig.5A). DPV curves are shown for miRNA-122 concentrations ranging from 1 pM to 0.75 µM, demonstrating an increase in peak current with increasing miRNA concentration. This increase is attributed to the hybridization of target miRNA with the capture probe, resulting in greater accumulation of MB molecules at the electrode surface, thereby enhancing the electrochemical signal. The direct correlation between miRNA concentration and current highlights the biosensor’s high sensitivity for detecting low levels of target miRNA. Fig. 5B presents the calibration plot of peak current against the miRNA concentration, revealing two distinct linear ranges. In the low concentration range (1 pM to 1 nM), the biosensor exhibits a steeper slope, reflecting higher sensitivity at lower miRNA concentrations, which is critical for early biomarker detection. In the high concentration range (1 nM to 0.75 µM), the slope is less steep, with slight deviation from linearity at higher concentrations, likely due to binding site saturation or diffusion limitations affecting analyte transport to the electrode surface. The limit of detection (LOD) was calculated based on the IUPAC definition [75] (LOD = y_blank + 3 δ_blank), where y_blank represents the mean blank signal and δ_blank is the standard deviation of the blank response. With experimentally determined values of y_blank = 0.26 and δ_blank = 0.08167, the LOD was found to be 0.503 pM, underscoring the biosensor’s exceptional sensitivity. These results confirm the efficiency of the fabricated biosensor for miRNA-122 detection. The linear relationship between current and miRNA concentration, combined with a wide dynamic range and low detection limit, underscores the biosensor’s reliability and precision. This makes it a promising tool for early disease diagnosis and point-of-care applications. The proposed biosensor exhibited a broad linear range and a LOD 503 fM, demonstrating competitive sensitivity compared to other reported methods (Tabel S2). While certain approaches achieve lower LODs in the attomolar range, the current platform stands out due to its stable hybrid nanostructure, excellent signal amplification, and reliable performance in biological environments. So, positioning it as a strong candidate for practical miRNA-122 detection in breast cancer diagnostics. Selectivity To evaluate the selectivity of the proposed biosensor, DPV responses were investigated after incubating the biosensor with miRNA-122, as well as sequences with one and three base mismatches, and four non-complementary sequences (miRNA-141, miRNA-109, miRNA-21, miRNA-103) at a concentration of 100 pM (Fig.6). The results demonstrated that the biosensor incubated with the complementary miRNA-122 produced the highest signal. In contrast, hybridization with the sequence containing one base mismatch showed a significant reduction in the response compared to the fully complementary target. With an increase in the number of base mismatches in the target sequence, the electrochemical response of the biosensor further decreased, indicating weaker hybridization events. Notably, the DPV signal for the non-complementary sequence was almost equal to the control sample without ssDNA, which demonstrates the high selectivity of the biosensor for the specific detection of miRNA-122. As the number of mismatched nucleotide base pairs in the target sequence increases and also non complementary sequence, the electrochemical response of the DNA biosensor declines further due to the reduced hybridization efficiency of mismatched sequences during the detection process [51,66]. Repeatability, reproducibility, and stability for miRNA-122 detection The reproducibility of the biosensor was inspected by intra and inter-assay precision by detecting 100 pM of miRNA-122 (Fig. S4A and Fig. S4B, respectively). The RSD% for intra- and inter-assay precision were separately determined as 2.3 and 4.1%, respectively, demonstrating the good reproducibility of the proposed biosensor. The long-term stability (Fig. S4C) of the fabricated biosensor was investigated by storing it at 4°C and evaluating its performance at various time intervals (days 1, 5, 9, and 14). The biosensor was used to measure the concentration of miRNA-122 under identical conditions. As shown in the bar graph, the current responses remained remarkably stable, with only a slight decrease in signal intensity observed over 14 days. Specifically, the current on the 14 days retained more than 97% of the initial value recorded on the first day. This minimal decrease in signal indicates the reliable performance of the biosensor over an extended period. The high stability observed can be attributed to the robust design and efficient immobilization of recognition elements on the electrode surface. These results demonstrate that the developed biosensor possesses excellent long-term stability . Real sample analysis To assess the diagnostic feasibility of our sensing platform for miRNA-122 detection in biologically relevant systems, we analyzed its performance across three established breast cancer cell lines (MCF-7, SKBR3, and MDA-MB-231) and human foreskin fibroblasts (HFF) as a non-malignant cellular baseline. Following cell lysis and target extraction, DPV analysis revealed significantly elevated peak current intensity in the MDA-MB-231 lysate (Fig.7), indicative of pronounced miRNA-122 overexpression in this aggressive triple-negative breast cancer (TNBC) model. This heightened expression aligns with the unique biological profile of MDA-MB-231 cells, characterized by their metastatic propensity and metabolic reprogramming. MicroRNA-122 is a known regulator of critical pathways driving these phenotypes, including PI3K/AKT signaling and the epithelial-to-mesenchymal transition (EMT) [76]. Furthermore, the distinct membrane composition and enhanced electron transfer characteristics inherent to these mesenchymal cells facilitate a more robust interaction with the methylene blue (MB) redox reporter, amplifying the electrochemical signal compared to the receptor-driven MCF-7 (ER+) and SKBR3 (HER2+) lines [77,78]. Crucially, analysis of HFF lysate generated only a minimal detectable signal, primarily attributed to residual electrostatic adsorption of MB to the capture probe. This stark contrast underscores the biosensor's specificity for miRNA-122 within the complex cellular milieu. These results demonstrate the exceptional capability of our biosensor to precisely detect and quantify miRNA-122 expression across diverse cellular models. The platform successfully delineates significant differences linked to breast cancer subtype and aggressiveness, highlighting its robust analytical performance and significant potential for advancing breast cancer diagnostics and biomarker research. Conclusions This study presents the development of a highly sensitive and selective electrochemical biosensor for the detection of miRNA-122, an important biomarker for breast cancer. By integrating functionalized MXene (NH 2 -Ti 3 C 2 ) and Au-decorated UiO-66 MOF, the proposed sensing platform offers an exceptionally efficient electron transfer pathway and a highly conductive environment, which significantly enhance detection performance. The incorporation of gold nanoparticles provides a stable surface for the immobilization of thiolated DNA probes, ensuring strong hybridization efficiency with the target microRNA. The biosensor utilizes MB as a redox indicator, where its dual electrostatic and intercalative interactions with the hybridized duplex enable precise electrochemical readout. The biosensor demonstrated an impressive linear detection range spanning from 1 pM to 1 nM and 50 nM to 1 µM, with a low detection limit of 503 fM, highlighting its capability for trace-level miRNA detection. Selectivity studies confirmed high discrimination ability against mismatched and non-complementary sequences, proving the robustness of the sensing strategy. Furthermore, validation using MCF-7, MDA-MB-231, and SKBR3 breast cancer cell lysates established its strong applicability for real biological samples, demonstrating its feasibility for clinical diagnostics. Beyond its outstanding sensitivity and specificity, this biosensor offers additional advantages such as label-free detection, ease of fabrication, and excellent reproducibility, making it a highly practical candidate for early-stage breast cancer diagnosis. The synergistic effect of MXene’s large surface area and high conductivity with the hierarchical porosity and stability of MOFs has unlocked new potential for designing next-generation biosensors. The promising results achieved in this work highlight the biosensor’s strong translational potential for point-of-care cancer diagnostics and microRNA-based disease monitoring, paving the way for further advancements in electrochemical biosensing technologies. Declarations Funding Declaration This work has been financially supported by the Research Council of the University of Tehran. Author Contributions Ali Esmaeilian: Conceptualization, Methodology, Designing the analysis, Formal analysis, Visualization, Data curation, Writing–original draft. Nastaran Arab: Methodology, Co-supervisor, Writing – review & editing. Fatemeh Yazdian: Supervision, Project administration, Funding acquisition, Writing – review & editing. Morteza Hosseini: Methodology, Project administration, Funding acquisition, Writing – review & editing. Seyed Mohammad Reza Mortazavi: Methodology, Designing the analysis, Mohammad Reza Ganjali : review & editing. Declarations Conflict of interest The authors declare that they have no competing interests. All the experiments have been carried out in compliance with ethical standards in a prescribed format. Clinical Trial Number Clinical trial number: not applicable. Reference Siegel RL, Miller KD, Jemal A (2019) Cancer statistics. CA: a cancer journal for clinicians 69 (1):7–34 Ahirwar R (2021) Recent advances in nanomaterials-based electrochemical immunosensors and aptasensors for HER2 assessment in breast cancer. Microchimica Acta 188 (10):317 Senel M, Dervisevic M, Kokkokoğlu F (2019) Electrochemical DNA biosensors for label-free breast cancer gene marker detection. Analytical and bioanalytical chemistry 411:2925–2935 Lahoura V, Singh H, Aggarwal A, Sharma B, Mohammed MA, Damaševičius R, Kadry S, Cengiz K (2021) Cloud computing-based framework for breast cancer diagnosis using extreme learning machine. Diagnostics 11 (2):241 Irfan M, Habiba U, Maryam A (2025) Next-generation cancer therapeutics: unveiling the potential of liposome-based nanoparticles through bioinformatics. Microchimica Acta 192 (7):428 Ranjan P, Parihar A, Jain S, Kumar N, Dhand C, Murali S, Mishra D, Sanghi SK, Chaurasia J, Srivastava AK (2020) Biosensor-based diagnostic approaches for various cellular biomarkers of breast cancer: A comprehensive review. Analytical biochemistry 610:113996 Ryser MD, Lange J, Inoue LY, O’Meara ES, Gard C, Miglioretti DL, Bulliard J-L, Brouwer AF, Hwang ES, Etzioni RB (2022) Estimation of breast cancer overdiagnosis in a US breast screening cohort. Annals of internal medicine 175 (4):471–478 Pan A, Gu X, Ming W, Zhang J, Jiang W, Zhang Y, Wang S, Qin Y, Wu L (2025) miRNA biomarkers: advancing early detection of acute myocardial infarction. Microchimica Acta 192 (8):1–24 Tran HV, Piro B (2021) Recent trends in application of nanomaterials for the development of electrochemical microRNA biosensors. Microchimica Acta 188 (4):128 Fong MY, Zhou W, Liu L, Alontaga AY, Chandra M, Ashby J, Chow A, O’Connor STF, Li S, Chin AR (2015) Breast-cancer-secreted miR-122 reprograms glucose metabolism in premetastatic niche to promote metastasis. Nature cell biology 17 (2):183–194 Mortazavi SMR, Hosseini M, Xu G, Naderi-Manesh H, Ganjali MR (2024) Highly specific detection of ROR1 cancer biomarker with bipolar electrochemiluminescence. Microchimica Acta 191 (12):734 Faramin Lashkarian M, Hashemipour N, Niaraki N, Soghala S, Moradi A, Sarhangi S, Hatami M, Aghaei-Zarch F, Khosravifar M, Mohammadzadeh A (2023) MicroRNA-122 in human cancers: from mechanistic to clinical perspectives. Cancer Cell International 23 (1):29 Su Z, Zhang L, Yu Y, Lin B, Wang Y, Guo M, Cao Y (2023) An electrochemical determination strategy for miRNA based on bimetallic nanozyme and toehold-mediated DNA replacement procedure. Microchimica Acta 190 (4):149 Wang B, Wang H, Yang Z (2012) MiR-122 inhibits cell proliferation and tumorigenesis of breast cancer by targeting IGF1R. Nemati F, Hosseini M (2022) Fluorescence turn-on detection of miRNA-155 based on hybrid Ce-MOF/PtNPs/graphene oxide serving as fluorescence quencher. Journal of Photochemistry and Photobiology A: Chemistry 429:113943 Lee J, Na H-K, Lee S, Kim W-K (2022) Advanced graphene oxide-based paper sensor for colorimetric detection of miRNA. Microchimica Acta 189 (1):35 Baranwal S, Alahari SK (2010) miRNA control of tumor cell invasion and metastasis. International journal of cancer 126 (6):1283–1290 Dehnoei M, Hosseini M, Ahmadi-Sangachin E, Mousavizadegan M (2024) A “turn-on” nano-biosensor based on the Ti3C2 (OH) 2 MXene nanosheets and DNA-silver nanoclusters for miRNA detection. Microchemical Journal 204:110964 Green TM, Alpaugh ML, Barsky SH, Rappa G, Lorico A (2015) Breast cancer‐derived extracellular vesicles: characterization and contribution to the metastatic phenotype. BioMed research international 2015 (1):634865 Adriá-Cebrián J, Guaita-Esteruelas S, Lam EW-F, Rodríguez-Balada M, Capellades J, Girona J, Jimenez-Santamaria AM, Yanes O, Masana L, Gumà J (2021) MCF-7 drug resistant cell lines switch their lipid metabolism to triple negative breast Cancer signature. Cancers 13 (23):5871 Yu X, Ghamande S, Liu H, Xue L, Zhao S, Tan W, Zhao L, Tang S-C, Wu D, Korkaya H (2018) Targeting EGFR/HER2/HER3 with a three-in-one aptamer-siRNA chimera confers superior activity against HER2+ breast cancer. Molecular Therapy-Nucleic Acids 10:317–330 Osborne CK, Hobbs K, Trent JM (1987) Biological differences among MCF-7 human breast cancer cell lines from different laboratories. Breast cancer research and treatment 9:111–121 Azimzadeh M, Rahaie M, Nasirizadeh N, Daneshpour M, Naderi-Manesh H (2017) Electrochemical miRNA biosensors: the benefits of nanotechnology. Nanomedicine Research Journal 2 (1):36–48 Esmaeilian A, Yazdian F, Saberi A, Hosseini A, Rahdar A, Fathi-karkan S, Pandey S (2025) Innovations in Self-Powered glucose Biosensors: From energy harvesting to Continuous, Non-Invasive monitoring. Inorganic Chemistry Communications:114076 Lorencova L, Kasak P, Kosutova N, Jerigova M, Noskovicova E, Vikartovska A, Barath M, Farkas P, Tkac J (2024) MXene-based electrochemical devices applied for healthcare applications. Microchimica Acta 191 (2):88 Qu L, Wu M, Zhao L, Li J, Pan H (2023) A sandwich electrochemical immunosensor based on MXene@dual MOFs for detection of tumor marker CA125. Microchimica Acta 190 (4):147 Amara U, Hussain I, Ahmad M, Mahmood K, Zhang K (2023) 2D MXene‐based biosensing: a review. Small 19 (2):2205249 Adel S, Firoozbakhtian A, Rabbani H, Hosseini M, Pebdeni AB, Sadeghi N, Gilnezhad J, Ganjali MR (2024) COVID-19 electrochemical immunosensor with Ag-MOF: Rapid and high-selectivity nasal swab testing for effective detection. Analytical Biochemistry 689:115500 Gan T, Hu S (2011) Electrochemical sensors based on graphene materials. Microchimica Acta 175 (1):1–19 Sharma K, Hasija V, Patial S, Singh P, Nguyen V-H, Nadda AK, Thakur S, Nguyen-Tri P, Nguyen CC, Kim SY (2023) Recent progress on MXenes and MOFs hybrids: structure, synthetic strategies and catalytic water splitting. International Journal of Hydrogen Energy 48 (17):6560–6574 Chen Z, Li X, Yang C, Cheng K, Tan T, Lv Y, Liu Y (2021) Hybrid porous crystalline materials from metal organic frameworks and covalent organic frameworks. Advanced Science 8 (20):2101883 Anik Ü, Timur S, Dursun Z (2019) Metal organic frameworks in electrochemical and optical sensing platforms: a review. Microchimica Acta 186 (3):196 Xu M-M, Xie L-H, Li J-R (2021) Metal–Organic Framework/Polymer Hybrid Materials. Yusuf VF, Malek NI, Kailasa SK (2022) Review on metal–organic framework classification, synthetic approaches, and influencing factors: applications in energy, drug delivery, and wastewater treatment. ACS omega 7 (49):44507–44531 Saini H, Srinivasan N, Sedajova V, Majumder M, Dubal DP, Otyepka M, Zboril R, Kurra N, Fischer RA, Jayaramulu K (2021) Emerging MXene@ Metal–organic framework hybrids: design strategies toward versatile applications. ACS nano 15 (12):18742–18776 Wang Y, Yang W, Cao J, Liu Y, Shao M, Xu X, Chen H (2024) Enhanced electron transfer and stability via hetero-interface engineering of MXene@MOF: Electrochemical sensors for direct detection of GPNMB in serum of PD. Sensors and Actuators B: Chemical:136066 Kaur G, Sharma S, Bhardwaj N, Nayak MK, Deep A (2024) Highly sensitive and selective electrochemical detection of Aflatoxin B1 in water and Pistachio samples with MOF/MXene composite based sensor. Food Control 165:110694 Chen J, Li S, Chen Y, Yang J, Dong J (2022) Highly selective detection of adenine and guanine by NH 2 -MIL-53 (Fe)/CS/MXene nanocomposites with excellent electrochemical performance. Microchimica Acta 189 (9):328 Xu W, Li S, Hu S, Yu W, Zhou Y (2021) Effect of Ti 3 AlC 2 precursor and processing conditions on microwave absorption performance of resultant Ti 3 C 2 T x MXene. Journal of Materials Science 56:9287–9301 Gilnezhad J, Firoozbakhtian A, Hosseini M, Adel S, Xu G, Ganjali MR (2023) An enzyme-free Ti 3 C 2 /Ni/Sm-LDH-based screen-printed-electrode for real-time sweat detection of glucose. Analytica Chimica Acta 1250:340981 Adibah NA, Zaine SA, Shukur MFA Synthesis of Ti3C2 mxene through in situ hf and direct hf etching procedures as electrolyte fillers in dye-sensitized solar cell. In: Materials Science Forum, 2021. Trans Tech Publ, pp 15–20 Cheng G, Ding Q, Sun Y, Zhang Y, Zhang W, Li G (2024) Electrochemiluminescence resonance energy transfer detection of HBsAg based on Co doped 3D porous luminol-based conjugates and quencher UiO-66-NH 2 @Au. Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy:124574 Zhai Y, Li Y, Huang X, Hou J, Li H, Ai S (2022) Colorimetric and ratiometric fluorescent dual-mode sensitive detection of Hg 2+ based on UiO-66-NH 2 @Au composite. Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 275:121187 Gilbert C, Svejstrup JQ (2006) RNA immunoprecipitation for determining RNA‐protein associations in vivo. Current protocols in molecular biology 75 (1):27.24. 21–27.24. 11 Rohs R, Sklenar H (2001) Methylene blue binding to DNA with alternating GC base sequence: continuum treatment of salt effects. Kara P, Kerman K, Ozkan D, Meric B, Erdem A, Ozkan Z, Ozsoz M (2002) Electrochemical genosensor for the detection of interaction between methylene blue and DNA. Electrochemistry Communications 4 (9):705–709 Han S, Liu W, Yang S, Wang R (2019) Facile and label-free electrochemical biosensors for microRNA detection based on DNA origami nanostructures. ACS omega 4 (6):11025–11031 Arab N, Hosseini M, Xu G (2024) Emerging trends and recent advances in MXene/MXene-based nanocomposites toward electrochemiluminescence sensing and biosensing. Biosensors and Bioelectronics:116623 Naguib M, Kurtoglu M, Presser V, Lu J, Niu J, Heon M, Hultman L, Gogotsi Y, Barsoum MW (2023) Two-dimensional nanocrystals produced by exfoliation of Ti 3 AlC 2 . In: MXenes. Jenny Stanford Publishing, pp 15–29 Dixit P, Maiti T (2022) A facile pot synthesis of (Ti 3 AlC 2 ) MAX phase and its derived MXene (Ti 3 C 2 T x ). Ceramics International 48 (24):36156–36165 Ranjbari S, Rezayi M, Arefinia R, Aghaee-Bakhtiari SH, Hatamluyi B, Pasdar A (2023) A novel electrochemical biosensor based on signal amplification of Au HFGNs/PnBA-MXene nanocomposite for the detection of miRNA-122 as a biomarker of breast cancer. Talanta 255:124247 Nayak P, Jiang Q, Mohanraman R, Anjum D, Hedhili MN, Alshareef HN (2018) Inherent electrochemistry and charge transfer properties of few-layered two-dimensional Ti 3 C 2 T x MXene. Nanoscale 10 (36):17030–17037 Alhabeb M, Maleski K, Anasori B, Lelyukh P, Clark L, Sin S, Gogotsi Y (2017) Guidelines for synthesis and processing of two-dimensional titanium carbide (Ti3C2T x MXene). Chemistry of Materials 29 (18):7633–7644 Damirchi Z, Firoozbakhtian A, Hosseini M, Ganjali MR (2024) Ti 3 C 2 /Ni/Sm-based electrochemical glucose sensor for sweat analysis using bipolar electrochemistry. Microchimica Acta 191 (3):137 Kumar AN, Pal K (2022) Amine-functionalized stable Nb 2 CT x MXene toward room temperature ultrasensitive NO 2 gas sensor. Materials Advances 3 (12):5151–5162 Ji J, Zhao L, Shen Y, Liu S, Zhang Y (2019) Covalent stabilization and functionalization of MXene via silylation reactions with improved surface properties. FlatChem 17:100128 Parker T, Zhang D, Bugallo D, Shevchuk K, Downes M, Valurouthu G, Inman A, Chacon B, Zhang T, Shuck CE (2024) Fourier-transform infrared spectral library of MXenes. Chemistry of Materials 36 (17):8437–8446 Wu L, Pu H, Huang L, Sun D-W (2020) Plasmonic nanoparticles on metal-organic framework: A versatile SERS platform for adsorptive detection of new coccine and orange II dyes in food. Food chemistry 328:127105 Chen Q, He Q, Lv M, Xu Y, Yang H, Liu X, Wei F (2015) Selective adsorption of cationic dyes by UiO-66-NH 2 . Applied Surface Science 327:77–85 Kumar S, Lei Y, Alshareef NH, Quevedo-Lopez M, Salama KN (2018) Biofunctionalized two-dimensional Ti 3 C 2 MXenes for ultrasensitive detection of cancer biomarker. Biosensors and Bioelectronics 121:243–249 Hatamluyi B, Sadeghian R, Sany SBT, Alipourfard I, Rezayi M (2021) Dual-signaling electrochemical ratiometric strategy for simultaneous quantification of anticancer drugs. Talanta 234:122662 Chai X, Ye S, Wang F, Yuan H, Liu M, Fan F, Zhang L, Zhang X, Wang T, Fu Y (2023) Au nanoparticles@ UiO-66 composite film-coated carbon cloth substrate for high-performance H 2 O 2 electrochemical sensing. Inorganic Chemistry 62 (27):10694–10703 Bajpai VK, Haldorai Y, Khan I, Sonwal S, Singh MP, Yadav S, Paray BA, Jan BL, Kang S-M, Huh YS (2021) Au@ Zr-based metal–organic framework composite as an immunosensing platform for determination of hepatitis B virus surface antigen. Microchimica Acta 188:1–13 Mahmoodi P, Rezayi M, Rasouli E, Avan A, Gholami M, Ghayour Mobarhan M, Karimi E, Alias Y (2020) Early-stage cervical cancer diagnosis based on an ultra-sensitive electrochemical DNA nanobiosensor for HPV-18 detection in real samples. Journal of nanobiotechnology 18:1–12 Rahman M, Heng LY, Futra D, Chiang CP, Rashid ZA, Ling TL (2017) A highly sensitive electrochemical DNA biosensor from acrylic-gold nano-composite for the determination of arowana fish gender. Nanoscale research letters 12:1–10 Li S, Zhu J, Fu H, Wan J, Hu Z, Liu S, Li J, Tie Y, Xing R, Zhu J (2012) Hepato-specific microRNA-122 facilitates accumulation of newly synthesized miRNA through regulating PRKRA. Nucleic acids research 40 (2):884–891 Zhu L, Zhao R, Wang K, Xiang H, Shang Z, Sun W (2008) Electrochemical behaviors of methylene blue on DNA modified electrode and its application to the detection of PCR product from NOS sequence. Sensors 8 (9):5649–5660 Jin Y, Yao X, Liu Q, Li J (2007) Hairpin DNA probe based electrochemical biosensor using methylene blue as hybridization indicator. Biosensors and Bioelectronics 22 (6):1126–1130 Silvestrini M, Fruk L, Moretto LM, Ugo P (2015) Detection of DNA hybridization by methylene blue electrochemistry at activated nanoelectrode ensembles. Journal of Nanoscience and Nanotechnology 15 (5):3437–3442 Li Y, Chang Y, Yuan R, Chai Y (2018) Highly efficient target recycling-based netlike Y-DNA for regulation of electrocatalysis toward methylene blue for sensitive DNA detection. ACS applied materials & interfaces 10 (30):25213–25218 Talemi RP, Mashhadizade MH (2017) A novel optical DNA biosensor for detection of trace concentration of Methylene blue using Gold nano-particles and Guanine rich single strand DNA. International Journal of Nano Dimension 8 (3):224–233 Yan F, Erdem A, Meric B, Kerman K, Ozsoz M, Sadik OA (2001) Electrochemical DNA biosensor for the detection of specific gene related to Microcystis species. Electrochemistry communications 3 (5):224–228 Gu J, Lu X, Ju H (2002) DNA sensor for recognition of native yeast DNA sequence with methylene blue as an electrochemical hybridization indicator. Electroanalysis: An International Journal Devoted to Fundamental and Practical Aspects of Electroanalysis 14 (13):949–954 Bard AJ, Faulkner LR, White HS (2022) Electrochemical methods: fundamentals and applications. John Wiley & Sons, McNaught AD, Wilkinson A (1997) Compendium of chemical terminology, vol 1669. Blackwell Science Oxford, Zhang J, Cui Y, Lin X, Zhang G, Li Z (2017) MiR-122-3p sensitizes breast cancer cells to ionizing radiation via controlling of cell apoptosis, migration and invasion. Int J Clin Exp Pathol 10 (1):215–223 Song B, Hou G, Xu M, Chen M (2024) Exosomal miR-122-3p represses the growth and metastasis of MCF-7/ADR cells by targeting GRK4-mediated activation of the Wnt/β-catenin pathway. Cellular Signalling 117:111101 Zhang W, Jiang H, Chen Y, Ren F (2019) Resveratrol chemosensitizes adriamycin‐resistant breast cancer cells by modulating miR‐122‐5p. Journal of Cellular Biochemistry 120 (9):16283–16292 Scheme Scheme 1 is available in the Supplementary Files section. Additional Declarations No competing interests reported. Supplementary Files SupportingInformation.docx Scheme1.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7449624","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":511583438,"identity":"4696370b-42b9-4144-be48-a7bd841510a1","order_by":0,"name":"Ali Esmaeilian","email":"","orcid":"","institution":"University of Tehran","correspondingAuthor":false,"prefix":"","firstName":"Ali","middleName":"","lastName":"Esmaeilian","suffix":""},{"id":511583439,"identity":"43e862fe-a993-4b39-b7bd-21ab1b1e3c83","order_by":1,"name":"Nastaran Arab","email":"","orcid":"","institution":"University of Tehran","correspondingAuthor":false,"prefix":"","firstName":"Nastaran","middleName":"","lastName":"Arab","suffix":""},{"id":511583440,"identity":"349ae616-a476-4f78-8c5a-2de49cf3a4a8","order_by":2,"name":"Fatemeh Yazdian","email":"","orcid":"","institution":"University of Tehran","correspondingAuthor":false,"prefix":"","firstName":"Fatemeh","middleName":"","lastName":"Yazdian","suffix":""},{"id":511583443,"identity":"7e9c40ab-a13f-4f29-bf36-f45337fb70df","order_by":3,"name":"Morteza Hosseini","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAxklEQVRIiWNgGAWjYBACAyjJw8DAfAAixEy8FrYEhgPEawEDHgOGA8Q4zJy9/fGHDwV3ZBjEznyT/sBgJ8/AzvsArxbLnjNmkjMMnvEwSOdukzjAkGzYwMxugFeLwY0cNmYeg8MwLcwJDMxsBPxyI/3x5z9gLTnPgFrqidGSYCDNANHCBtRymAgtZ4B+6QFrSTO2OGNw3LCNoJbjwBD78eewPYN08sMbFRXV8vz8x/BrgQP7A2ATgDFKpIZRMApGwSgYBXgAALxBOgJcD9/lAAAAAElFTkSuQmCC","orcid":"","institution":"University of Tehran","correspondingAuthor":true,"prefix":"","firstName":"Morteza","middleName":"","lastName":"Hosseini","suffix":""},{"id":511583445,"identity":"dcc2976c-d9e9-486b-b3e5-2310e63f2f09","order_by":4,"name":"Seyed Mohammad Reza Mortazavi","email":"","orcid":"","institution":"University of Tehran","correspondingAuthor":false,"prefix":"","firstName":"Seyed","middleName":"Mohammad Reza","lastName":"Mortazavi","suffix":""},{"id":511583447,"identity":"7a3d359c-e860-4dc4-82f0-ee4017505ebe","order_by":5,"name":"Mohammad Reza Ganjali","email":"","orcid":"","institution":"University of Tehran","correspondingAuthor":false,"prefix":"","firstName":"Mohammad","middleName":"Reza","lastName":"Ganjali","suffix":""}],"badges":[],"createdAt":"2025-08-25 04:38:21","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7449624/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7449624/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":90991843,"identity":"8bf2bd7c-7eec-461f-93c7-e3c6c467ea07","added_by":"auto","created_at":"2025-09-10 11:28:30","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":196449,"visible":true,"origin":"","legend":"\u003cp\u003e(A, B) FESEM images with different magnification (C) XRD pattern and (D) FTIR of NH\u003csub\u003e2\u003c/sub\u003e-Ti\u003csub\u003e3\u003c/sub\u003eC\u003csub\u003e2\u003c/sub\u003eT\u003csub\u003ex\u003c/sub\u003e\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-7449624/v1/9ff45872d131581f2235425e.png"},{"id":90991848,"identity":"96f1fdd9-4de0-406d-8e71-ba384db3ae2c","added_by":"auto","created_at":"2025-09-10 11:28:30","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":367206,"visible":true,"origin":"","legend":"\u003cp\u003e(A, B) TEM images of NH\u003csub\u003e2\u003c/sub\u003e-UiO-66 (C, D) TEM images of Au@NH\u003csub\u003e2\u003c/sub\u003e-UiO-66 (E) XRD pattern of NH\u003csub\u003e2\u003c/sub\u003e-UiO-66 and Au@NH\u003csub\u003e2\u003c/sub\u003e-UiO-66 (F) N\u003csub\u003e2\u003c/sub\u003e adsorption - desorption isotherms of NH\u003csub\u003e2\u003c/sub\u003e-UiO-66 and Au@NH\u003csub\u003e2\u003c/sub\u003e-UiO-66\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-7449624/v1/632cd044e099ced3729cc42a.png"},{"id":90991968,"identity":"1dcb1a05-88e6-4365-9ea5-affb157798f2","added_by":"auto","created_at":"2025-09-10 11:36:30","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":101472,"visible":true,"origin":"","legend":"\u003cp\u003e(A) CVs and (B) EISs of various modified electrodes in 5 mM [Fe(CN)\u003csub\u003e6\u003c/sub\u003e]\u003csup\u003e3-/4-\u003c/sup\u003e comprising 0.1 M KCl.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-7449624/v1/56fff186b382a6a5962789af.png"},{"id":90991967,"identity":"a7dd6f14-9d8e-465a-988d-1e072fc4eb04","added_by":"auto","created_at":"2025-09-10 11:36:30","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":109877,"visible":true,"origin":"","legend":"\u003cp\u003e(A) Cyclic voltammograms of capture probe/Au@NH\u003csub\u003e2\u003c/sub\u003e-UiO-66/NH\u003csub\u003e2\u003c/sub\u003e-Ti\u003csub\u003e3\u003c/sub\u003eC\u003csub\u003e2\u003c/sub\u003e/SPE (B) Cyclic voltammograms of 1 nM of miRNA-122/capture probe/Au@NH\u003csub\u003e2\u003c/sub\u003e-UiO-66/NH\u003csub\u003e2\u003c/sub\u003e-Ti\u003csub\u003e3\u003c/sub\u003eC\u003csub\u003e2\u003c/sub\u003e/SPE at different scan rate (from 25 to 800 mV s\u003csup\u003e-1\u003c/sup\u003e). (C) Dependence of the oxidation and reduction peak currents of MB with scan rate recorded on capture probe/Au@NH\u003csub\u003e2\u003c/sub\u003e-UiO-66/NH\u003csub\u003e2\u003c/sub\u003e-Ti\u003csub\u003e3\u003c/sub\u003eC\u003csub\u003e2\u003c/sub\u003e/SPE (red) and miRNA-122/capture probe/Au@NH\u003csub\u003e2\u003c/sub\u003e-UiO-66/NH\u003csub\u003e2\u003c/sub\u003e-Ti\u003csub\u003e3\u003c/sub\u003eC\u003csub\u003e2\u003c/sub\u003e/SPE (purple).\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-7449624/v1/7b386e6f53ab0861ffcef586.png"},{"id":90992623,"identity":"1c9f83a6-9231-4dd3-a330-87aa44d9cf1e","added_by":"auto","created_at":"2025-09-10 11:44:30","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":130529,"visible":true,"origin":"","legend":"\u003cp\u003e(A) DPV response of various concentration of miRNA-122 in 0.1 M PBS (pH 7.4), (B) Corresponding calibration plot in the linear range 1pM – 1 nM (inset) and 1 nM- 750.0 nM concentration of miRNA122.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-7449624/v1/40a2f75c4ad235fd4d2cece1.png"},{"id":90991850,"identity":"6f3b0bfe-c786-427a-bfbb-cbc1496831f0","added_by":"auto","created_at":"2025-09-10 11:28:30","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":72416,"visible":true,"origin":"","legend":"\u003cp\u003eSpecificity of the assay for microRNA-122 (50 nM) against 1-mismatch target, 3- mismatch target, miRNA-141, miRNA-109, miRNA-21, miRNA-103 (750 nM)\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-7449624/v1/b93faf9cffa2f57a4cd397e2.png"},{"id":91290012,"identity":"23b7c466-a6e9-47d9-9022-b6362b85ba95","added_by":"auto","created_at":"2025-09-14 23:46:25","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1821601,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7449624/v1/e888ce28-d5a2-4423-a907-0e223d3aed5b.pdf"},{"id":90991970,"identity":"d0bf2346-fd0a-4378-9663-a0a001667f6f","added_by":"auto","created_at":"2025-09-10 11:36:30","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":1443459,"visible":true,"origin":"","legend":"","description":"","filename":"SupportingInformation.docx","url":"https://assets-eu.researchsquare.com/files/rs-7449624/v1/4c5e4155cb84ac6faa794024.docx"},{"id":90991844,"identity":"8df2a858-6fcb-4284-bbea-929ff7fe3ddf","added_by":"auto","created_at":"2025-09-10 11:28:30","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":504935,"visible":true,"origin":"","legend":"","description":"","filename":"Scheme1.docx","url":"https://assets-eu.researchsquare.com/files/rs-7449624/v1/8b5df0b30067d95f83ad2e4e.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"A 2D/3D Nanoplatform for Electrochemical Diagnosis of miRNA-122 in Breast Cancer Cell Lines","fulltext":[{"header":"Introduction","content":"\u003cp\u003eCancer is one of the leading causes of death worldwide, claiming millions of lives annually, as reported by the World Health Organization [1]. Among the various types of cancer, breast cancer stands out as a prevalent malignancy affecting women globally. It is the second leading cause of cancer-related illness and death, with its incidence rising annually [2]. To minimize recurrence and lower mortality, early detection, precise diagnosis, and prompt treatment are essential for the efficient control of breast cancer [3,4]. Current diagnostic techniques for breast cancer, such as MRI, mammography, X-ray imaging, ultrasound, biopsy, and CT scans, are widely recognized but come with limitations like high costs, the need for specialized personnel, complex data analysis, and the potential for false results [5]. Traditional methods like mammography require extensive hospital facilities and specialized staff, involve time-consuming processes, and often lead to overdiagnosis, which hampers early detection and effective treatment. Consequently, there is a critical need to develop more convenient, rapid, and cost-effective detection methods [6,7].\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe analysis and diagnosis of cancer biomarkers is one of the most important methods for breast cancer detection. One of the biomarkers used to diagnose diseases, including breast cancer, is microRNAs (miRNAs), which are small non-coding RNAs about 19–25 nucleotides in length, characterized by their small size, relative stability in biological fluids, and their role in the post-transcriptional regulation of protein levels and gene expression [8,9]. Though the role of miRNA-122 in breast cancer is not yet fully understood, it is emerging as a crucial regulator of tumor development and carcinogenesis. miRNA-122 facilitates metastasis by increasing nutrient availability in the pre-metastatic niche [10,11].\u0026nbsp;miRNA-122 is aberrantly expressed in various tumors, including breast, lung, bladder, leukemia, liver, colorectal, ovarian, and esophageal cancers, where it can function as both a tumor promoter and suppressor\u0026nbsp;[12,13]. Its deregulation in tumor development underscores its potential as a valuable biomarker for the diagnosis and prognosis of breast cancer. Additionally, miRNA-122 may enhance tumor cells' sensitivity to chemotherapy, making it a target for therapeutic intervention. Given these findings, miRNA-122 stands out as a powerful molecular biomarker and a novel therapeutic target for breast cancer, offering significant potential for advancing targeted treatments\u0026nbsp;[12,14].\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe quantification of miRNA within the cell nucleus is crucial for elucidating gene regulatory mechanisms and cellular processes\u0026nbsp;[15,16]. These small non-coding RNAs play a pivotal role in modulating gene expression, thereby influencing tumor progression and metastasis [17,18]. MCF-7, SKBR3, and MDA-MB-231 are well-established breast cancer cell lines extensively used in research to investigate various breast cancer subtypes. MCF-7 cells, which are estrogen receptor-positive, serve as a model for hormone-dependent breast cancer. SKBR3 cells, characterized by their overexpression of HER2, are ideal for studying HER2-targeted therapies. MDA-MB-231, a triple-negative and highly aggressive cell line, is commonly employed to study metastasis and drug resistance\u0026nbsp;[19]. Collectively, these cell lines provide invaluable insights into the molecular pathways underlying breast cancer and the efficacy of different therapeutic strategies\u0026nbsp;[20-22].\u003c/p\u003e\n\u003cp\u003eNotably, electrochemical biosensors can provide quick and susceptible miRNA detection compared to traditional methods. To increase the sensitivity of miRNA detection for the diagnosis of many illnesses, electrochemical nanobiosensors for miRNA quantification have integrated several nanostructures [23,24]. One of the materials in this category is MXene, which was first introduced by Gogotsi and his team with the discovery of two-dimensional titanium carbide (Ti\u003csub\u003e3\u003c/sub\u003eC\u003csub\u003e2\u003c/sub\u003e) powder, marking the beginning of the MXene family [25]. MXene nanomaterials, a class of two-dimensional transition metal carbides, nitrides, or carbonitrides, are recognized for their unique structure and exceptional electronic conductivity. These materials consist of atomically thin layers of transition metals sandwiched between ceramic-like layers [26]. In the context of electrochemical biosensors, MXenes offer several advantages, including high conductivity, a large specific surface area, and excellent electrochemical stability. These properties facilitate efficient electron transfer kinetics and provide abundant sites for biomolecule immobilization, making MXenes ideal building blocks for the development of high-performance electrochemical biosensors with enhanced sensitivity and reliability [27]. The integration of MXenes with other nanostructures is a crucial strategy for tailoring their electrical, mechanical, and chemical properties. This approach enables the design of composite materials with enhanced performance and expanded functionalities, which are highly desirable for advanced applications such as electrochemical biosensing [28,29]. Integrating MXenes with metal-organic frameworks (MOFs) creates hybrid materials that leverage the distinct advantages of both components [30,31]. MOFs are known for their high porosity, tunable chemical functionalities, and substantial surface area, which facilitate interactions with target molecules [32]. This combination results in enhanced properties for the hybrid materials, making them particularly effective for applications in biosensing, catalysis, and environmental remediation. The synergistic effects of MXenes and MOFs thus form a robust strategy for developing advanced materials tailored for specific functionalities and applications [33-35]. For example, Wang et al. using MXene@MOF for the electrochemical biosensor to detect Glycoprotein Nonmetastatic Melanoma Protein B (GPNMB). MXene@MOF was chosen for its stability, high conductivity, and enhanced electroactive surface area. The integration of 2D MOFs prevents MXene oxidation and increases sensitivity. These features make it ideal for developing a label-free, ultra-sensitive electrochemical biosensor for detecting GPNMB in Parkinson’s Disease patients' serum [36]. In another study, Kaur et al. used MOF/MXene composite as a surface with accessible pores, mechanical strength, and suitable conductivity properties to set up highly sensitive, stable, and practical electrochemical sensors [37]. In addition, Chen et al. utilized MOF/MXene. Combining MOFs with MXenes enhanced electrochemical properties, reduced stacking effects, and increased binding sites for the aptamer [38].\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn this study, an advanced, label-free electrochemical biosensor was designed for the sensitive detection of miRNA-122, a crucial biomarker in breast cancer cells, utilizing a heterostructured composite of Au/NH\u003csub\u003e2\u003c/sub\u003e-UiO-66 and NH\u003csub\u003e2\u003c/sub\u003e-Ti\u003csub\u003e3\u003c/sub\u003eC\u003csub\u003e2\u0026nbsp;\u003c/sub\u003enanosheets. The synergistic combination of NH\u003csub\u003e2\u003c/sub\u003e-Ti\u003csub\u003e3\u003c/sub\u003eC\u003csub\u003e2\u003c/sub\u003e’s exceptional conductivity and functionalization potential, along with the increased surface area and active sites provided by Au-decorated NH\u003csub\u003e2\u003c/sub\u003e-UiO-66, significantly enhances the sensor's performance. The incorporation of gold nanoparticles ensures strong immobilization of the DNA probes, leading to superior detection sensitivity. This platform dramatically amplifies electrochemical signals, establishing itself as a highly promising and reliable tool for the early and precise diagnosis of breast cancer. Scheme 1 illustrates the design of the proposed biosensor for the detection of miRNA-122.\u003c/p\u003e"},{"header":"Material and methods ","content":"\u003ch2\u003eMaterials\u003c/h2\u003e\n\u003cp\u003eAll chemicals contain titanium powder (99.98% purity, \u0026lt; 45 \u0026mu;m), graphite powder (\u0026ge; 99.99% trace metals basis,\u0026lt; 45 \u0026mu;m), aluminum powder (99.9% purity, 60 \u0026mu;m), Conductive carbon ink, sodium chloride (NaCl), \u0026nbsp;potassium ferrocyanide (K\u003csub\u003e4\u003c/sub\u003eFe(CN)\u003csub\u003e6\u003c/sub\u003e), potassium chloride (KCl), potassium ferricyanide (K\u003csub\u003e3\u003c/sub\u003eFe(CN)\u003csub\u003e6\u003c/sub\u003e), \u0026nbsp;HF (Hydrofluoric acid), Dimethyl sulfoxide (DMSO), (3-Aminopropyl) triethoxysilane (APTES), Glutaraldehyde (GA), Ethanol (C\u003csub\u003e2\u003c/sub\u003eH\u003csub\u003e5\u003c/sub\u003eOH), Phosphate buffered saline (PBS), N, N-dimethylformamide (DMF), acetic acid (CH\u003csub\u003e3\u003c/sub\u003eCOOH), chloroauric acid tetrahydrate (HAuCl\u003csub\u003e4\u003c/sub\u003e\u0026middot;4H\u003csub\u003e2\u003c/sub\u003eO), sodium tetrahydridoborate (NaBH\u003csub\u003e4\u003c/sub\u003e), and bovine serum albumin (BSA) were purchased from Sigma-Aldrich.\u0026nbsp;TE buffer was gained from Sinaclon. In addition, Methylene blue (MB), Zirconium tetrachloride (ZrCl\u003csub\u003e4\u003c/sub\u003e), and 2-amino terephthalic acid (NH\u003csub\u003e2\u003c/sub\u003e-BDC) were provided from Merck. All materials utilized were of analytical reagent grade, and deionized water was sourced from a Millipore Milli-Q system. All synthetic oligonucleotide sequences listed in Table S1 were obtained from Metabion International AG (Planegg, Germany).\u003c/p\u003e\n\u003ch2\u003eApparatus\u003c/h2\u003e\n\u003cp\u003eFor the Brunauer-Emmett-Teller (BET) specific surface area study, Belsorp, Japan-made BET Mini II equipment was used. X-ray diffraction (XRD) patterns were recorded on a Philips PW1730 with Cu-Ka radiation at 40 kV and 40 mA. The morphology and the chemical composition (Energy-Dispersive X-ray Spectroscopy (EDS)) were investigated by TESCAN MIRA3 Field Emission Scanning Electron Microscope (FESEM). Transmission electron microscopy (TEM) images were recorded on a Philips EM208S.\u0026nbsp;Fourier transform infrared spectra (FTIR) spectra were recorded with a Perkin-Elmer.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;Electrochemical measurements including the electrochemical impedance spectroscopy (EIS), cyclic voltammetry (CV), and Differential pulse voltammetry (DPV) were carried out using an AUTOLAB PGSTAT204 read out by NOVA 2.1 software.\u003c/p\u003e\n\u003ch2\u003ePreparation\u0026nbsp;of MAX Phase (Ti\u003csub\u003e3\u003c/sub\u003eAlC\u003csub\u003e2\u003c/sub\u003e), MXene (Ti\u003csub\u003e3\u003c/sub\u003eC\u003csub\u003e2\u003c/sub\u003eT\u003csub\u003eX\u003c/sub\u003e) and NH\u003csub\u003e2\u003c/sub\u003e-Ti\u003csub\u003e3\u003c/sub\u003eC\u003csub\u003e2\u003c/sub\u003eT\u003csub\u003eX\u003c/sub\u003e\u003c/h2\u003e\n\u003cp\u003eThe synthesis of the MAX phase process adhered to all the steps outlined in the earlier report by Xu and other comparable studies [39,40]. The selective removal of aluminum from Ti\u003csub\u003e3\u003c/sub\u003eAlC\u003csub\u003e2\u003c/sub\u003e was accomplished through a direct etching procedure. Initially, 5 g of Ti\u003csub\u003e3\u003c/sub\u003eAlC\u003csub\u003e2\u003c/sub\u003e MAX phase powder was gradually introduced into 100 mL of a 50 wt% HF solution under continuous stirring. The slow addition of the MAX phase was essential to control excessive bubbling caused by the exothermic reaction. The etching process was maintained at room temperature (~23\u0026deg;C) for 5 hours while stirring at 300 rpm. The resulting mixture was then subjected to centrifugation (at 3500 rpm for 5 minutes per cycle) and repeatedly washed with deionized water until the pH of the supernatant reached a nearly neutral value. Finally, the purified sample was dried in a vacuum oven at 80\u0026deg;C for 24 hours, yielding the desired MXene product [41].\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn order to synthesize NH\u003csub\u003e2\u003c/sub\u003e-Ti\u003csub\u003e3\u003c/sub\u003eC\u003csub\u003e2\u003c/sub\u003eT\u003csub\u003eX\u003c/sub\u003e, initially, 0.2g of Ti\u003csub\u003e3\u003c/sub\u003eC\u003csub\u003e2\u003c/sub\u003eT\u003csub\u003ex\u003c/sub\u003e powder was dispersed in 5 mL of DMSO, which was then constantly stirred for 24 hours. The resultant Ti\u003csub\u003e3\u003c/sub\u003eC\u003csub\u003e2\u003c/sub\u003eT\u003csub\u003ex\u003c/sub\u003e precipitate was centrifuged and then redispersed in 60 mL of ultrapure water. The Ti\u003csub\u003e3\u003c/sub\u003eC\u003csub\u003e2\u003c/sub\u003eT\u003csub\u003ex\u003c/sub\u003e nanosheets were exfoliated by sonicating the dispersion for 16 hours after the solution had been purified with argon gas for 20 minutes. The nanosheets were then recovered by centrifugation at 3000 rpm for 5 minutes. After that, 1 mL of the Ti\u003csub\u003e3\u003c/sub\u003eC\u003csub\u003e2\u003c/sub\u003eT\u003csub\u003ex\u003c/sub\u003e nanosheet suspension was mixed with 20 \u0026mu;L of APTES, and the mixture was left to stir for a whole day at room temperature. For future usage, the resultant NH\u003csub\u003e2\u003c/sub\u003e-Ti\u003csub\u003e3\u003c/sub\u003eC\u003csub\u003e2\u003c/sub\u003eT\u003csub\u003ex\u003c/sub\u003e nanosheets were redispersed in 1 mL of ultrapure water after excess APTES was removed by centrifugation.\u003c/p\u003e\n\u003ch2\u003eSynthesis of UiO-66-NH\u003csub\u003e2\u003c/sub\u003e and Au@NH\u003csub\u003e2\u003c/sub\u003e-UiO-66\u003c/h2\u003e\n\u003cp\u003eThe synthesis procedure for UiO-66-NH₂ has been documented in the literature [42]. In this method, 93.2 mg of ZrCl\u003csub\u003e4\u003c/sub\u003e and 72.4 mg of NH\u003csub\u003e2\u003c/sub\u003e-BDC were dissolved in 40 mL of DMF, followed by the addition of 5.5 mL of acetic acid to facilitate the formation of UiO-66-NH\u003csub\u003e2\u003c/sub\u003e particles.After being moved to a 50 mL stainless steel autoclave, the mixture was heated for 24 hours to 120˚C. Following three centrifugation washes with DMF and ethanol, the UiO-66-NH\u003csub\u003e2\u003c/sub\u003e particles were dried at 60 \u0026deg;C to produce a mustard powder.\u003c/p\u003e\n\u003cp\u003eAu@UiO-66-NH\u003csub\u003e2\u003c/sub\u003ewas synthesizedthrough the reduction ofAu(III) to Au(0) nanoparticles on the pre-synthesized UiO-66-NH\u003csub\u003e2\u003c/sub\u003e framework[43]. For this purpose, a uniform dispersion of NH\u003csub\u003e2\u003c/sub\u003e-UiO-66 was first prepared by ultrasonically treating 20 mg of NH\u003csub\u003e2\u003c/sub\u003e-UiO-66 in 4 mL of an ethanol/water mixture (1:1 v/v). Subsequently, 0.5 mL of a 25 mM HAuCl\u003csub\u003e4\u003c/sub\u003e solution was introduced, and the mixture was continuously stirred for four hours. Following this, 2.4 mL of freshly prepared ice-cold NaBH\u003csub\u003e4\u003c/sub\u003e solution (0.05 M) was quickly added, and stirring continued for an additional hour. Finally, the resulting precipitate was collected via centrifugation and thoroughly washed multiple times to ensure purity.\u003c/p\u003e\n\u003ch2\u003eFabrication of electrochemical biosensor\u003c/h2\u003e\n\u003cp\u003eThe biosensor was fabricated using a screen-printing process on flexible PVC substrate, employing carbon paste as the printing ink. The designed screen-printed electrode (SPE) was constructed based on the common electrochemical system, with dried silver paste serving as the reference electrode (Fig. S1). To fabricate the biosensor, a precise aliquot of 40\u0026micro;L NH\u003csub\u003e2\u003c/sub\u003e-Ti\u003csub\u003e3\u003c/sub\u003eC\u003csub\u003e2\u003c/sub\u003e suspension was carefully deposited onto the working electrode via drop-casting. The electrode was then subjected to a desiccation process under an inert argon atmosphere for one hour. Subsequently, 40\u0026micro;L GA was dropped on the surface of the electrode and after 2 hours was rinsed with deionized water. After that 40\u0026micro;L Au@UiO-66-NH\u003csub\u003e2\u003c/sub\u003e was cast on the surface. For stabilizing the ssDNA probe, 35 \u0026mu;l of Thiol-ssDNA\u0026nbsp;(200 nM, as probe) prepared in TE buffer was dropped on the surface of Au@NH\u003csub\u003e2\u003c/sub\u003e-UiO-66/NH\u003csub\u003e2\u003c/sub\u003e-Ti\u003csub\u003e3\u003c/sub\u003eC\u003csub\u003e2\u003c/sub\u003e at 4˚C for 16 h.\u0026nbsp;Next, the electrode was rinsed with PBS (pH 7.4), followed by the application of a 1% BSA solution for 15 minutes. Afterward, it was washed again with PBS. At this stage, the nanobiosensor is prepared for hybridization with the target miRNA122. For the hybridization process, different concentrations of target miRNA122 were prepared in TE buffer. A 40.0 \u0026mu;L droplet of the miRNA122\u0026nbsp;solution was placed onto the modified electrode and left for 120 minutes, after which the electrode was rinsed with PBS.\u0026nbsp;Finally, the electrode modified with miRNA/ssDNA hybrids was incubated in MB solution (4.0 \u0026mu;M MB, 0.2 M NaCl, 0.1 M phosphate-buffered saline, PBS) for 2 hours to facilitate MB binding. After incubation, any unbound MB was thoroughly removed using a washing PBS, ensuring the measured current signal derived from MB bound to the hybridized sequences. The Scheme 1illustration sequentially presents the proposed biosensor for miRNA-122 detection.\u003c/p\u003e\n\u003ch2\u003emiRNA-122 Detection and electrochemical measurements\u003c/h2\u003e\n\u003cp\u003eThe biosensor fabrication and miRNA detection processes were analyzed using cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS), and differential pulse voltammetry (DPV). CV and EIS measurements were conducted in a solution of 5.0 mM [Fe(CN)₆]\u0026sup3;⁻/⁴⁻ with 0.1 M KCl. CV scans were carried out over a scan range of -1.5 to +1.5 V, while EIS experiments were performed across a frequency range of 0.1 Hz to 10 kHz. Also, the DPV signal for the redox activity of methylene blue (MB) was recorded within the scan range of -0.6 to 0.3 V in a PBS solution at pH 7.4 with a scan rate10mV/s.\u003c/p\u003e\n\u003ch2\u003eCell lysis\u0026nbsp;\u003c/h2\u003e\n\u003cp\u003eCells were lysed by adding 300 \u0026micro;L of ice-cold Triton X-100 lysis buffer (0.25% Triton X-100 in PBS) to one to three 10 cm cell culture dishes. To inhibit proteolytic and dephosphorylating activities, protease and phosphatase inhibitors were added to the lysis buffer prior to use. Cells were incubated with the lysis buffer for 30 minutes on ice with intermittent vertexing to facilitate membrane disruption and release of cellular contents. Following incubation, the lysates were centrifuged at maximum speed for 15 minutes at 4\u0026deg;C to pellet cellular debris. The supernatant, containing solubilized membrane and cytosolic proteins, was collected and stored at -80\u0026deg;C for subsequent analysis [44].\u003c/p\u003e"},{"header":"Results and Discussion","content":"\u003ch2\u003eSensing mechanisms of biosensor\u0026nbsp;\u003c/h2\u003e\n\u003cp\u003eThe sensing mechanism of the developed electrochemical biosensor relies on the sequential interactions between, the immobilized thiol-modified capture probe, the target miRNA-122, and MB as the redox indicator. Initially, the capture probe is immobilized onto the Au-decorated MOF surface, facilitating the electrostatic binding of MB to the negatively charged phosphate backbone of the single-stranded DNA probe. Upon introduction of miRNA-122, hybridization occurs, forming a double-stranded complex. The hybridization event significantly enhances MB binding through a dual mechanism: electrostatic interactions and intercalation. Initially, MB interacts with the negatively charged phosphate backbone of single-stranded DNA via electrostatic attraction. However, upon hybridization with miRNA-122, the formation of the double-stranded complex introduces a structurally distinct environment enriched with guanine-rich regions. Guanine possesses an extended \u0026pi;-electron system due to its fused bicyclic purine structure, which provides a highly conjugated and planar surface [45]. This feature enables MB, with its tricyclic aromatic core, to establish strong \u0026pi;\u0026ndash;\u0026pi; stacking interactions with guanine bases, thereby enhancing its retention within the duplex structure [46]. Furthermore, the steric stabilization conferred by the hybridized strands limits MB diffusion away from the electrode surface, amplifying its redox signal. As a result, the electrochemical response is significantly increased, enabling highly sensitive and selective miRNA-122 detection. The interplay of these binding mechanisms ensures a stable and reproducible signal, making this approach highly effective for electrochemical biosensing\u0026nbsp;[47,46].\u003c/p\u003e\n\u003ch2\u003eCharacterization of Ti\u003csub\u003e3\u003c/sub\u003eC\u003csub\u003e2\u003c/sub\u003e MXene\u003c/h2\u003e\n\u003cp\u003eThe successful synthesis of Ti\u003csub\u003e3\u003c/sub\u003eC\u003csub\u003e2\u003c/sub\u003eT\u003csub\u003ex\u003c/sub\u003e was validated using FESEM, XRD, and FTIR. The FESEM images demonstrate the morphological characteristics of the synthesized Ti\u003csub\u003e3\u003c/sub\u003eC\u003csub\u003e2\u003c/sub\u003eT\u003csub\u003ex\u003c/sub\u003e. Fig. 1A displays a surface view of Ti\u003csub\u003e3\u003c/sub\u003eC\u003csub\u003e2\u003c/sub\u003eT\u003csub\u003ex\u003c/sub\u003e, highlighting its distinctive accordion-like morphology. Fig. 1B illustrates a cross-sectional view, showcasing the interlayer spacing and exfoliation of the nanosheets, with the distinct separation of layers. The two images offer a comprehensive understanding of the structural organization and layer arrangement of Ti\u003csub\u003e3\u003c/sub\u003eC\u003csub\u003e2\u003c/sub\u003eT\u003csub\u003ex\u003c/sub\u003e, confirming its characteristic two-dimensional morphology and affirming the successful synthesis of Ti\u003csub\u003e3\u003c/sub\u003eC\u003csub\u003e2\u003c/sub\u003eT\u003csub\u003ex\u003c/sub\u003e. [48].\u003c/p\u003e\n\u003cp\u003eThe XRD pattern of the synthesized Ti\u003csub\u003e3\u003c/sub\u003eC\u003csub\u003e2\u003c/sub\u003eT\u003csub\u003ex\u003c/sub\u003e MXene, shown in Fig. 1C, reveals the structural transformation resulting from the selective etching of Ti\u003csub\u003e3\u003c/sub\u003eAlC\u003csub\u003e2\u003c/sub\u003e. The diffraction peaks observed at 9.1\u0026deg;, 18.5\u0026deg;, 24.1\u0026deg;, 34.7\u0026deg;, 42.1\u0026deg;, and 61.2\u0026deg; for Ti\u003csub\u003e3\u003c/sub\u003eC\u003csub\u003e2\u003c/sub\u003eT\u003csub\u003ex\u003c/sub\u003e MXenes indicate the presence of a well-crystallized nanostructure in the synthesized hybrids. The (002) peak observed at 9.1\u0026deg; indicates the interlayer distance (D-spacing) and verifies the incorporation of \u0026ndash;F and \u0026ndash;OH surface functional groups. [49,50].\u0026nbsp;The presence of broad peaks at 34.7\u0026deg; and 42.1\u0026deg;, with low intensities and corresponding to the (101) and (103) planes, confirms the successful elimination of aluminum\u0026nbsp;[51,52].\u0026nbsp;The peaks observed at 2\u0026theta; = 18.7\u0026deg; and 23.95\u0026deg; correspond to the (006) and (008) planes.\u0026nbsp;The peaks observed at 2\u0026theta; = 42.15\u0026deg; and 61.05\u0026deg;, corresponding to the (105) and (110) planes, further confirm the maintained structural integrity following the etching process.\u0026nbsp;[53,54].\u003c/p\u003e\n\u003cp\u003eFTIR spectroscopy confirmed (Fig.1D) the successful functionalization of MXene with amine (-NH\u003csub\u003e2\u003c/sub\u003e) groups. The spectrum showed characteristic absorption bands for amine groups, with prominent peaks in the 3200-3500 cm⁻\u0026sup1; region attributed to N-H stretching vibrations and a peak around 1560-1650 cm⁻\u0026sup1; corresponding to N-H bending [55]. Si-O-Si stretching vibrations in the 1000-1200 cm⁻\u0026sup1; region confirmed the bonding of the APTS silane coupling agent to the MXene surface, anchoring the amine groups [56]. C-O stretching in the 1600-1700 cm⁻\u0026sup1; region, associated with surface functional groups on MXene. C-H stretching in the 2800-3000 cm⁻\u0026sup1; region, linked to APTS propyl chains, further reinforced successful modification. Shifts in Ti-C and Ti-O peaks around 500-800 cm⁻\u0026sup1; indicated the interaction between MXene and APTS [57].\u003c/p\u003e\n\u003cp\u003eThese findings highlight the structural, crystallographic, and chemical evolution of MXene, validating the efficacy of the synthesis process.\u003c/p\u003e\n\u003ch2\u003eCharacterization of NH\u003csub\u003e2\u003c/sub\u003e-UiO-66 and Au@NH\u003csub\u003e2\u003c/sub\u003e-UiO-66\u003c/h2\u003e\n\u003cp\u003eThe successful synthesis of NH\u003csub\u003e2\u003c/sub\u003e-UiO-66 and Au@NH\u003csub\u003e2\u003c/sub\u003e-UiO-66 were also validated using various analytical techniques, including TEM, XRD, EDX mapping and BET surface area analysis. TEM images clearly reveal that the synthesized NH\u003csub\u003e2\u003c/sub\u003e-UiO-66 nanocrystals possess a polyhedral morphology and a consistent size distribution (Fig.2A and 2B).\u0026nbsp;[58,43]. Fig. 2C and 2D verify the successful deposition of gold nanoparticles on the surface of the MOF nanocrystals. These images demonstrate that the crystalline structure of the NH\u003csub\u003e2\u003c/sub\u003e-UiO-66 is maintained intact following the decoration process with gold nanoparticles, which predominantly exhibit diameters ranging from 4 to 8 nm.\u003c/p\u003e\n\u003cp\u003eXRD analysis was performed to investigate the crystallographic structures of NH\u003csub\u003e2\u003c/sub\u003e-UiO-66 and Au@NH\u003csub\u003e2\u003c/sub\u003e-UiO-66 (Fig. 2E). The XRD pattern of NH\u003csub\u003e2\u003c/sub\u003e-UiO-66 exhibited sharp and well-defined diffraction peaks at 2\u0026theta; values of approximately 7.4\u0026deg;, 8.5\u0026deg;, 12.2\u0026deg;, 14.6\u0026deg;, 17.1\u0026deg;, 22.1\u0026deg;, 25.6\u0026deg;, and 30.5\u0026deg;, corresponding to the (111), (002), (022), (113), (222), (044), (137), and (444) crystal planes, respectively. These diffraction peaks are characteristic of the highly crystalline nature of the NH\u003csub\u003e2\u003c/sub\u003e-UiO-66. The XRD pattern of Au@NH\u003csub\u003e2\u003c/sub\u003e-UiO-66 also displayed the characteristic diffraction peaks of NH\u003csub\u003e2\u003c/sub\u003e-UiO-66 at 7.4\u0026deg;, 8.5\u0026deg;, 12.2\u0026deg;, 14.6\u0026deg;, 17.1\u0026deg;, 22.1\u0026deg;, 25.6\u0026deg;, and 30.5\u0026deg;, indicating that the crystalline structure of NH\u003csub\u003e2\u003c/sub\u003e-UiO-66 remained intact after gold nanoparticle decoration. In addition, new diffraction peaks emerged at 38.1\u0026deg;, 44.3\u0026deg;, 64.5\u0026deg;, and 77.5\u0026deg;, corresponding to the (111), (200), (220), and (311) planes of gold nanoparticles [43]. The presence of these new peaks confirmed the successful integration of gold nanoparticles within the MOF structure.\u003c/p\u003e\n\u003cp\u003eThe BET surface area and porosity of synthesized NH\u003csub\u003e2\u003c/sub\u003e-UiO-66 were determined from N\u003csub\u003e2\u003c/sub\u003e adsorption/desorption studies and the result is shown in Fig.2F. Typical type I isotherm with no hysteresis is observed in the figure, which confirms the microporous nature of NH\u003csub\u003e2\u003c/sub\u003e-UiO-66 [59]. The BET surface area for NH\u003csub\u003e2\u003c/sub\u003e-UiO-66 is 756.4 (m\u003csup\u003e2\u003c/sup\u003e/g) with a pore volume of 0.6636 cm\u003csup\u003e3\u003c/sup\u003eg\u003csup\u003e\u0026minus;1\u003c/sup\u003e. Interestingly, similar to XRD observations, the decoration of AuNPs on Zr-MOF does not significantly change the area of NH\u003csub\u003e2\u003c/sub\u003e-UiO-66 based on these observations, it can be concluded that Au nanoparticles were distributed well on the surface of Zr-MOF. The XRD data, which showed the preserved peaks of NH\u003csub\u003e2\u003c/sub\u003e-UiO-66 alongside the new peaks corresponding to gold, suggests that the gold nanoparticles are primarily located on the surface of the MOF crystals rather than within the pores, indicating that the MOF framework integrity was maintained while introducing new catalytic sites.\u003c/p\u003e\n\u003cp\u003eEDX confirms the elemental composition of the studied materials. In Fig.S2, for NH\u003csub\u003e2\u003c/sub\u003e-UiO-66, peaks for carbon (C), nitrogen (N), oxygen (O), and zirconium (Zr) are observed, indicating their presence in the MOF structure, with atomic percentages of 23.9% C, 8.5% N, 33.7% O, and 33.9% Zr. In Fig. 2D, for Au@UiO-66-NH\u003csub\u003e2\u003c/sub\u003e, peaks for the MOF elements and gold (Au) are seen, with atomic percentages of 7.6% C, 4.2% N, 14.3% O, 35.5% Zr, and 38.4% Au. The presence and high percentage of gold confirm the successful decoration of the MOF with gold nanoparticles, [43]. In addition, the mapping of NH\u003csub\u003e2\u003c/sub\u003e-UiO-66 and Au@NH\u003csub\u003e2\u003c/sub\u003e-UiO-66 show the elemental distribution of the studied materials. In Fig.2E, corresponding to NH\u003csub\u003e2\u003c/sub\u003e-UiO-66, the elemental maps show a uniform distribution of O, Zr, N, and C throughout the material and also the EDX maps of Au@NH\u003csub\u003e2\u003c/sub\u003e-UiO-66 display the distribution of the same elements (O, Zr, N, and C) along with Au (Fig.S2). \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eElectrochemical characterization\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe stepwise fabrication process of the miRNA\u0026nbsp;biosensors\u0026nbsp;was evaluated using EIS and CV in a 5.0 mM Fe[(CN)₆]\u0026sup3;⁻/⁴⁻ solution with 0.1 M KCl (Fig.3A and 3B). CV studies on five different electrode samples demonstrated that the modification of the electrode surface with various layers has a significant impact on the electrochemical behavior of the system. The bare electrode exhibited a pair of reversible redox peaks (curve a). Following surface modification with NH\u003csub\u003e2\u003c/sub\u003e-Ti\u003csub\u003e3\u003c/sub\u003eC\u003csub\u003e2\u003c/sub\u003e, a notable increase in current was observed, attributed to the exceptional conductivity of NH\u003csub\u003e2\u003c/sub\u003e-Ti\u003csub\u003e3\u003c/sub\u003eC\u003csub\u003e2\u0026nbsp;\u003c/sub\u003e(curve b) [60]. Subsequent modification of the surface with Au@NH\u003csub\u003e2\u003c/sub\u003e-UiO-66 led to the emergence of clear oxidation and reduction peaks, reflecting the distinctive electrocatalytic behavior of Au@NH\u003csub\u003e2\u003c/sub\u003e-UiO-66 (curve c) [61]. With the attachment of DNA probes to the surface, a decrease in current was observed, indicating the occupation of part of the active surface by the probes and consequently a decrease in electron transfer (curve d). Finally, the hybridization of the target (miRNA-122) with DNA probes resulted in a further decrease in current due to the creation of a steric hindrance and a decrease in electron transfer due to the increased complexity of the surface structure (curve e). The CV results were further validated by EIS measurements (Fig.3B). The inset shows the Nyquist plot for the bare electrode, which exhibits a significantly higher impedance response compared to the modified electrodes, indicating a large charge transfer resistance (R\u003csub\u003ect\u003c/sub\u003e). This high impedance is attributed to the absence of conductive layers or functional groups on the electrode surface, leading to poor electron transfer kinetics. Following modification with Ti\u003csub\u003e3\u003c/sub\u003eC\u003csub\u003e2\u003c/sub\u003eT\u003csub\u003ex\u003c/sub\u003e, a nearly linear diagram was observed, reflecting enhanced electron-transfer kinetics (curve b). An increase in R\u003csub\u003ect\u003c/sub\u003e was noted after modification with Au@NH\u003csub\u003e2\u003c/sub\u003e-UiO66/NH\u003csub\u003e2\u003c/sub\u003e-Ti\u003csub\u003e3\u003c/sub\u003eC\u003csub\u003e2\u003c/sub\u003e, showing higher charge transfer resistance compared to Ti\u003csub\u003e3\u003c/sub\u003eC\u003csub\u003e2\u003c/sub\u003e alone (curve c). This rise in resistance is attributed to the deposition of the Au@NH\u003csub\u003e2\u003c/sub\u003e-UiO-66 composite, which creates additional interfacial barriers and contributes to an overall increase in impedance [62]. Following the immobilization of the capture probe onto the Au@NH\u003csub\u003e2\u003c/sub\u003e-UiO66/NH\u003csub\u003e2\u003c/sub\u003e-Ti\u003csub\u003e3\u003c/sub\u003eC\u003csub\u003e2\u003c/sub\u003e surface, a significant increase in R\u003csub\u003ect\u003c/sub\u003e was observed. This rise is due to the introduction of steric hindrance and the reduction of the electrode\u0026apos;s active sites, which together hinder charge transfer (curve d). The impedance value continued to increase as miRNA-122 hybridization took place on the capture probe/Au@NH\u003csub\u003e2\u003c/sub\u003e-UiO-66/NH\u003csub\u003e2\u003c/sub\u003e-Ti\u003csub\u003e3\u003c/sub\u003eC\u003csub\u003e2\u003c/sub\u003e surface (curve e). This stage shows the highest impedance among the modified electrodes, reflecting the successful hybridization of miRNA with the capture probe. The significant increase in R\u003csub\u003ect\u003c/sub\u003e at this stage is due to the formation of the biomolecular complex, which further obstructs electron flow. Overall, the stepwise increase in charge transfer resistance across the modification stages confirms the sequential electrode functionalization. The systematic progression in the Nyquist plot aligns with the expected behavior of surface modification and biomolecule immobilization, highlighting the effectiveness of the proposed biosensor in detecting specific targets like miRNA-122.\u003c/p\u003e\n\u003ch2\u003eOptimization of\u003cspan dir=\"RTL\"\u003e\u0026nbsp;\u003c/span\u003econditions\u003c/h2\u003e\n\u003cp\u003eIn this study, comprehensive investigations were conducted to optimize various parameters of the microRNA-based electrochemical biosensor (Fig.S3), aiming to enhance sensitivity and measurement accuracy. The results demonstrated that the optimal concentration of the Au@NH\u003csub\u003e2\u003c/sub\u003e-UiO-66 nanocomposite, equivalent to 0.2 mg/mL, yielded the highest electrochemical response. Deviation from this value in either direction resulted in a noticeable decrease in the electrochemical signal. At lower concentrations (\u0026lt;0.2 mg/mL), there may be an insufficient number of conductive and catalytic sites to facilitate electron transfer, which leads to the limitation of the electron transfer process. Conversely, higher concentrations (\u0026gt;0.2 mg/mL) can cause the aggregation of nanoparticles, which reduces the effective electrode surface and disrupts redox reactions [63].\u003c/p\u003e\n\u003cp\u003eFurthermore, examination of the effect of DNA probe concentration revealed that a concentration of 200 nM generated the highest current intensity, and increasing concentrations beyond this value led to a reduction in response. Weak responses observed at concentrations below 200 nM can be attributed to an insufficient amount of immobilized probe on the SPE surface. Furthermore, at concentrations exceeding 200 nM, the decline in response signals is likely due to excessive accumulation of ssDNA, which hinders efficient electron transfer and sensor performance [64]. In the experiment concerning the probe immobilization time on the electrode surface, 16 hours was identified as the optimal time, as the highest signal was obtained within this time frame without a significant drop. The DPV response increased progressively with longer immobilization times, reaching its peak at 16 hours. A notable rise in the biosensor signal was observed between 8 and 16 hours, indicating a higher degree of probe attachment to the modified SPE surface. Beyond this point, no significant enhancement in signal response was detected, suggesting that the surface had reached saturation. Consequently, 16 hours was selected as the optimal immobilization time to ensure efficient and stable probe attachment on the SPE surface [65]. In the investigation of the hybridization time between miRNA-122 and the probe, it was observed that 120 minutes produced the highest current response, and increasing the time beyond this value did not have a significant impact on the signal intensity. At shorter hybridization times, the number of formed duplexes between miRNA-122 and the capture probe was limited, leading to a lower current response. As the incubation time increased, more hybridization events occurred, enhancing the signal until reaching 120 minutes, where saturation was achieved. Beyond this point, no further increase in signal intensity was observed, indicating that all available binding sites were occupied and the hybridization process had reached equilibrium [66]. Finally, regarding the MB incubation time, 15 minutes was determined as the optimal time, as the signal intensity reached a steady state after this time frame and no significant change in response was observed. At shorter incubation times, an insufficient amount of MB molecules interacted with the hybridized miRNA-122, leading to a lower signal response. Extending the incubation period beyond 15 minutes did not further enhance the electrochemical signal, suggesting that equilibrium in MB binding had been achieved, and excess MB molecules were removed during the washing step [47]. These results confirm the successful optimization of the experimental conditions to enhance the accuracy and efficiency of the proposed biosensor.\u003c/p\u003e\n\u003ch2\u003eMB as the Redox Indicator for miRNA Detection\u003c/h2\u003e\n\u003cp\u003eTo evaluate the feasibility of the proposed miRNA detection method, MB was employed as an electrochemical indicator to track the hybridization interaction with the target miRNA. MB, a phenothiazine dye, is known to exhibit varying binding affinities for single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA) [67,68]. This is due to MB binding to ssDNA primarily through electrostatic interactions, while its interaction with dsDNA involves both intercalation and electrostatic forces. As a result, these differences produce distinct electrochemical responses when MB interacts with ssDNA compared to dsDNA [69,70].\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTo study the interaction of MB with ssDNA and dsDNA, CV was conducted on the electrode functionalized with the capture probe (Fig. 4A) and the capture probe after hybridization with the target miRNA (Fig. 4B) at different scan rates. In both cases, an increase in scan rate resulted in a proportional increase in peak currents. However, a significant difference was observed in the magnitude of the redox peaks. The target system consistently exhibited higher peak currents compared to the capture system across all scan rates. This enhancement in current suggests successful hybridization between the capture probe and the target miRNA, leading to improved electron transfer kinetics at the electrode surface. The higher peak currents observed in the target system can be attributed to several factors. Hybridization may enhance the local electron density near the electrode surface, facilitate faster electron transfer pathways, or reduce the distance for electron hopping within the immobilized biomolecular complex. Additionally, the increase in current for the target system is largely due to the interaction of MB with the miRNA-DNA hybrid\u003cspan dir=\"RTL\"\u003e\u0026nbsp;\u003c/span\u003e[71,47]. In the presence of the target, MB binds not only electrostatically to single-stranded DNA but also intercalates into the double-stranded miRNA-DNA complex. This dual binding mechanism results in a higher accumulation of MB, thereby increasing the overall current response. In contrast, for the capture system, MB binds primarily through electrostatic interactions with single-stranded DNA, leading to a relatively lower current. This differential binding explains the significant enhancement in the electrochemical signal observed after target hybridization, reinforcing the sensitivity and specificity of the biosensor for miRNA detection.\u003c/p\u003e\n\u003cp\u003eThe observed increase in electrochemical response indicated greater absorption of MB molecules on the probe surface, providing strong evidence for the formation of ssDNA/miRNA hybrids on the electrode. This finding aligns with previous studies [72,73]. Additionally, for further analysis, the anodic and cathodic peak currents of the two prepared electrodes were plotted as a function of scan rate. As shown in Fig. 4C, the anodic and cathodic peak currents exhibit a linear relationship with the scan rate over the range of 0.025 to 0.8 V/s, suggesting that the process is surface-confined. The surface coverage of electroactive MB (\u0026Gamma;\u003csub\u003eMB\u003c/sub\u003e) on the electrode was estimated using the equation [74]: \u0026nbsp;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIp = F\u0026sup2;n\u0026sup2;A\u0026Gamma;v / 4RT \u0026nbsp; \u0026nbsp; \u0026nbsp;\u003c/p\u003e\n\u003cp\u003ewhere F is Faraday\u0026rsquo;s constant (96,485 C mol⁻\u0026sup1;), n represents the number of electrons transferred (n = 2 for MB), A is the electrode area (cm\u0026sup2;), \u0026Gamma; is the surface coverage, v is the scan rate (V/s), R is the gas constant (8.314 J K⁻\u0026sup1; mol⁻\u0026sup1;), and T is the temperature. Based on the slope of the I\u003csub\u003epc\u003c/sub\u003e versus v plot in Figure 4B, the calculated surface coverage (\u0026Gamma;\u003csub\u003eMB\u003c/sub\u003e) for the target was 553.23 nM/cm\u0026sup2;, which is notably higher than the 250.76 nM/cm\u0026sup2; measured for the capture probe. This suggests that more MB binds to the miRNA/ssDNA duplexes, enhancing electron transfer from MB to the electrode. These results confirm the effectiveness of the biosensor for miRNA detection by monitoring the reduction response of MB.\u003c/p\u003e\n\u003ch2\u003ePerformance of electrochemical biosensor\u003c/h2\u003e\n\u003cp\u003eThe quantification performance of the biosensor for detecting different concentrations of miRNA-122 was assessed using DPV in PBS buffer, measuring the oxidation peak current of methylene blue intercalated within double-stranded complexes of capture probes and miRNA-122 (Fig.5A). DPV curves are shown for miRNA-122 concentrations ranging from 1 pM to 0.75 \u0026micro;M, demonstrating an increase in peak current with increasing miRNA concentration. This increase is attributed to the hybridization of target miRNA with the capture probe, resulting in greater accumulation of MB molecules at the electrode surface, thereby enhancing the electrochemical signal. The direct correlation between miRNA concentration and current highlights the biosensor\u0026rsquo;s high sensitivity for detecting low levels of target miRNA. Fig. 5B presents the calibration plot of peak current against the miRNA concentration, revealing two distinct linear ranges. In the low concentration range (1 pM to 1 nM), the biosensor exhibits a steeper slope, reflecting higher sensitivity at lower miRNA concentrations, which is critical for early biomarker detection. In the high concentration range (1 nM to 0.75 \u0026micro;M), the slope is less steep, with slight deviation from linearity at higher concentrations, likely due to binding site saturation or diffusion limitations affecting analyte transport to the electrode surface.\u003cspan dir=\"RTL\"\u003e\u0026nbsp;\u003c/span\u003eThe limit of detection (LOD) was calculated based on the IUPAC definition [75] (LOD = y_blank + 3 \u0026delta;_blank), where y_blank represents the mean blank signal and \u0026delta;_blank is the standard deviation of the blank response. With experimentally determined values of y_blank = 0.26 and \u0026delta;_blank = 0.08167, the LOD was found to be 0.503 pM, underscoring the biosensor\u0026rsquo;s exceptional sensitivity. These results confirm the efficiency of the fabricated biosensor for miRNA-122 detection. The linear relationship between current and miRNA concentration, combined with a wide dynamic range and low detection limit, underscores the biosensor\u0026rsquo;s reliability and precision. This makes it a promising tool for early disease diagnosis and point-of-care applications.\u003c/p\u003e\n\u003cp\u003eThe proposed biosensor exhibited a broad linear range and a LOD 503 fM, demonstrating competitive sensitivity compared to other reported methods (Tabel S2). While certain approaches achieve lower LODs in the attomolar range, the current platform stands out due to its stable hybrid nanostructure, excellent signal amplification, and reliable performance in biological environments. So, positioning it as a strong candidate for practical miRNA-122 detection in breast cancer diagnostics.\u003c/p\u003e\n\u003ch2\u003eSelectivity\u003c/h2\u003e\n\u003cp\u003eTo evaluate the selectivity of the proposed biosensor, DPV responses were investigated after incubating the biosensor with miRNA-122, as well as sequences with one and three base mismatches, and four non-complementary sequences (miRNA-141, miRNA-109, miRNA-21, miRNA-103) at a concentration of 100 pM (Fig.6). The results demonstrated that the biosensor incubated with the complementary miRNA-122 produced the highest signal. In contrast, hybridization with the sequence containing one base mismatch showed a significant reduction in the response compared to the fully complementary target. With an increase in the number of base mismatches in the target sequence, the electrochemical response of the biosensor further decreased, indicating weaker hybridization events. Notably, the DPV signal for the non-complementary sequence was almost equal to the control sample without ssDNA, which demonstrates the high selectivity of the biosensor for the specific detection of miRNA-122. As the number of mismatched nucleotide base pairs in the target sequence increases and also non complementary sequence, the electrochemical response of the DNA biosensor declines further due to the reduced hybridization efficiency of mismatched sequences during the detection process [51,66].\u003c/p\u003e\n\u003ch2\u003eRepeatability, reproducibility, and stability for miRNA-122 detection\u003c/h2\u003e\n\u003cp\u003eThe reproducibility of the biosensor was inspected by intra and inter-assay precision by detecting 100 pM of miRNA-122 (Fig. S4A and Fig. S4B, respectively). The RSD% for intra- and inter-assay precision were separately determined as 2.3 and 4.1%, respectively, demonstrating the good reproducibility of the proposed biosensor.\u003c/p\u003e\n\u003cp\u003eThe long-term stability (Fig. S4C) of the fabricated biosensor was investigated by storing it at 4\u0026deg;C and evaluating its performance at various time intervals (days 1, 5, 9, and 14). The biosensor was used to measure the concentration of miRNA-122 under identical conditions. As shown in the bar graph, the current responses remained remarkably stable, with only a slight decrease in signal intensity observed over 14 days. Specifically, the current on the 14 days retained more than 97% of the initial value recorded on the first day. This minimal decrease in signal indicates the reliable performance of the biosensor over an extended period. The high stability observed can be attributed to the robust design and efficient immobilization of recognition elements on the electrode surface. These results demonstrate that the developed biosensor possesses excellent long-term stability\u003cspan dir=\"RTL\"\u003e.\u003c/span\u003e\u003c/p\u003e\n\u003ch2\u003eReal sample analysis\u003c/h2\u003e\n\u003cp\u003eTo assess the diagnostic feasibility of our sensing platform for miRNA-122 detection in biologically relevant systems, we analyzed its performance across three established breast cancer cell lines (MCF-7, SKBR3, and MDA-MB-231) and human foreskin fibroblasts (HFF) as a non-malignant cellular baseline. Following cell lysis and target extraction, DPV analysis revealed significantly elevated peak current intensity in the MDA-MB-231 lysate (Fig.7), indicative of pronounced miRNA-122 overexpression in this aggressive triple-negative breast cancer (TNBC) model. This heightened expression aligns with the unique biological profile of MDA-MB-231 cells, characterized by their metastatic propensity and metabolic reprogramming. MicroRNA-122 is a known regulator of critical pathways driving these phenotypes, including PI3K/AKT signaling and the epithelial-to-mesenchymal transition (EMT) [76]. Furthermore, the distinct membrane composition and enhanced electron transfer characteristics inherent to these mesenchymal cells facilitate a more robust interaction with the methylene blue (MB) redox reporter, amplifying the electrochemical signal compared to the receptor-driven MCF-7 (ER+) and SKBR3 (HER2+) lines [77,78]. Crucially, analysis of HFF lysate generated only a minimal detectable signal, primarily attributed to residual electrostatic adsorption of MB to the capture probe. This stark contrast underscores the biosensor\u0026apos;s specificity for miRNA-122 within the complex cellular milieu.\u003c/p\u003e\n\u003cp\u003eThese results demonstrate the exceptional capability of our biosensor to precisely detect and quantify miRNA-122 expression across diverse cellular models. The platform successfully delineates significant differences linked to breast cancer subtype and aggressiveness, highlighting its robust analytical performance and significant potential for advancing breast cancer diagnostics and biomarker research.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eThis study presents the development of a highly sensitive and selective electrochemical biosensor for the detection of miRNA-122, an important biomarker for breast cancer. By integrating functionalized MXene (NH\u003csub\u003e2\u003c/sub\u003e-Ti\u003csub\u003e3\u003c/sub\u003eC\u003csub\u003e2\u003c/sub\u003e) and Au-decorated UiO-66 MOF, the proposed sensing platform offers an exceptionally efficient electron transfer pathway and a highly conductive environment, which significantly enhance detection performance. The incorporation of gold nanoparticles provides a stable surface for the immobilization of thiolated DNA probes, ensuring strong hybridization efficiency with the target microRNA. The biosensor utilizes MB as a redox indicator, where its dual electrostatic and intercalative interactions with the hybridized duplex enable precise electrochemical readout. The biosensor demonstrated an impressive linear detection range spanning from 1 pM to 1 nM and 50 nM to 1 µM, with a low detection limit of 503 fM, highlighting its capability for trace-level miRNA detection. Selectivity studies confirmed high discrimination ability against mismatched and non-complementary sequences, proving the robustness of the sensing strategy. Furthermore, validation using MCF-7, MDA-MB-231, and SKBR3 breast cancer cell lysates established its strong applicability for real biological samples, demonstrating its feasibility for clinical diagnostics. Beyond its outstanding sensitivity and specificity, this biosensor offers additional advantages such as label-free detection, ease of fabrication, and excellent reproducibility, making it a highly practical candidate for early-stage breast cancer diagnosis. The synergistic effect of MXene’s large surface area and high conductivity with the hierarchical porosity and stability of MOFs has unlocked new potential for designing next-generation biosensors. The promising results achieved in this work highlight the biosensor’s strong translational potential for point-of-care cancer diagnostics and microRNA-based disease monitoring, paving the way for further advancements in electrochemical biosensing technologies.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding Declaration\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work has been financially supported by the Research Council of the University of Tehran.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAli Esmaeilian:\u003c/strong\u003e Conceptualization, Methodology, Designing the analysis, Formal analysis, Visualization, Data curation, Writing–original draft. \u003cstrong\u003eNastaran Arab:\u003c/strong\u003e Methodology, Co-supervisor, Writing – review \u0026amp; editing. \u003cstrong\u003eFatemeh Yazdian:\u003c/strong\u003e Supervision, Project administration, Funding acquisition, Writing – review \u0026amp; editing. \u003cstrong\u003eMorteza Hosseini:\u0026nbsp;\u003c/strong\u003eMethodology, Project administration, Funding acquisition, Writing – review \u0026amp; editing.\u003cstrong\u003e\u0026nbsp;Seyed Mohammad Reza Mortazavi:\u003c/strong\u003e Methodology, Designing the analysis,\u0026nbsp;\u003cstrong\u003eMohammad Reza Ganjali\u003c/strong\u003e: review \u0026amp; editing.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclarations Conflict of interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;The authors declare that they have no competing interests. All the experiments have been carried out in compliance with ethical standards in a prescribed format.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eClinical Trial Number\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eClinical trial number: not applicable.\u003c/p\u003e"},{"header":"Reference","content":"\u003col\u003e\n \u003cli\u003eSiegel RL, Miller KD, Jemal A (2019) Cancer statistics. CA: a cancer journal for clinicians 69 (1):7\u0026ndash;34\u003c/li\u003e\n \u003cli\u003eAhirwar R (2021) Recent advances in nanomaterials-based electrochemical immunosensors and aptasensors for HER2 assessment in breast cancer. Microchimica Acta 188 (10):317\u003c/li\u003e\n \u003cli\u003eSenel M, Dervisevic M, Kokkokoğlu F (2019) Electrochemical DNA biosensors for label-free breast cancer gene marker detection. Analytical and bioanalytical chemistry 411:2925\u0026ndash;2935\u003c/li\u003e\n \u003cli\u003eLahoura V, Singh H, Aggarwal A, Sharma B, Mohammed MA, Dama\u0026scaron;evičius R, Kadry S, Cengiz K (2021) Cloud computing-based framework for breast cancer diagnosis using extreme learning machine. Diagnostics 11 (2):241\u003c/li\u003e\n \u003cli\u003eIrfan M, Habiba U, Maryam A (2025) Next-generation cancer therapeutics: unveiling the potential of liposome-based nanoparticles through bioinformatics. Microchimica Acta 192 (7):428\u003c/li\u003e\n \u003cli\u003eRanjan P, Parihar A, Jain S, Kumar N, Dhand C, Murali S, Mishra D, Sanghi SK, Chaurasia J, Srivastava AK (2020) Biosensor-based diagnostic approaches for various cellular biomarkers of breast cancer: A comprehensive review. Analytical biochemistry 610:113996\u003c/li\u003e\n \u003cli\u003eRyser MD, Lange J, Inoue LY, O\u0026rsquo;Meara ES, Gard C, Miglioretti DL, Bulliard J-L, Brouwer AF, Hwang ES, Etzioni RB (2022) Estimation of breast cancer overdiagnosis in a US breast screening cohort. Annals of internal medicine 175 (4):471\u0026ndash;478\u003c/li\u003e\n \u003cli\u003ePan A, Gu X, Ming W, Zhang J, Jiang W, Zhang Y, Wang S, Qin Y, Wu L (2025) miRNA biomarkers: advancing early detection of acute myocardial infarction. Microchimica Acta 192 (8):1\u0026ndash;24\u003c/li\u003e\n \u003cli\u003eTran HV, Piro B (2021) Recent trends in application of nanomaterials for the development of electrochemical microRNA biosensors. Microchimica Acta 188 (4):128\u003c/li\u003e\n \u003cli\u003eFong MY, Zhou W, Liu L, Alontaga AY, Chandra M, Ashby J, Chow A, O\u0026rsquo;Connor STF, Li S, Chin AR (2015) Breast-cancer-secreted miR-122 reprograms glucose metabolism in premetastatic niche to promote metastasis. Nature cell biology 17 (2):183\u0026ndash;194\u003c/li\u003e\n \u003cli\u003eMortazavi SMR, Hosseini M, Xu G, Naderi-Manesh H, Ganjali MR (2024) Highly specific detection of ROR1 cancer biomarker with bipolar electrochemiluminescence. Microchimica Acta 191 (12):734\u003c/li\u003e\n \u003cli\u003eFaramin Lashkarian M, Hashemipour N, Niaraki N, Soghala S, Moradi A, Sarhangi S, Hatami M, Aghaei-Zarch F, Khosravifar M, Mohammadzadeh A (2023) MicroRNA-122 in human cancers: from mechanistic to clinical perspectives. Cancer Cell International 23 (1):29\u003c/li\u003e\n \u003cli\u003eSu Z, Zhang L, Yu Y, Lin B, Wang Y, Guo M, Cao Y (2023) An electrochemical determination strategy for miRNA based on bimetallic nanozyme and toehold-mediated DNA replacement procedure. Microchimica Acta 190 (4):149\u003c/li\u003e\n \u003cli\u003eWang B, Wang H, Yang Z (2012) MiR-122 inhibits cell proliferation and tumorigenesis of breast cancer by targeting IGF1R.\u003c/li\u003e\n \u003cli\u003eNemati F, Hosseini M (2022) Fluorescence turn-on detection of miRNA-155 based on hybrid Ce-MOF/PtNPs/graphene oxide serving as fluorescence quencher. Journal of Photochemistry and Photobiology A: Chemistry 429:113943\u003c/li\u003e\n \u003cli\u003eLee J, Na H-K, Lee S, Kim W-K (2022) Advanced graphene oxide-based paper sensor for colorimetric detection of miRNA. Microchimica Acta 189 (1):35\u003c/li\u003e\n \u003cli\u003eBaranwal S, Alahari SK (2010) miRNA control of tumor cell invasion and metastasis. International journal of cancer 126 (6):1283\u0026ndash;1290\u003c/li\u003e\n \u003cli\u003eDehnoei M, Hosseini M, Ahmadi-Sangachin E, Mousavizadegan M (2024) A \u0026ldquo;turn-on\u0026rdquo; nano-biosensor based on the Ti3C2 (OH) 2 MXene nanosheets and DNA-silver nanoclusters for miRNA detection. Microchemical Journal 204:110964\u003c/li\u003e\n \u003cli\u003eGreen TM, Alpaugh ML, Barsky SH, Rappa G, Lorico A (2015) Breast cancer‐derived extracellular vesicles: characterization and contribution to the metastatic phenotype. BioMed research international 2015 (1):634865\u003c/li\u003e\n \u003cli\u003eAdri\u0026aacute;-Cebri\u0026aacute;n J, Guaita-Esteruelas S, Lam EW-F, Rodr\u0026iacute;guez-Balada M, Capellades J, Girona J, Jimenez-Santamaria AM, Yanes O, Masana L, Gum\u0026agrave; J (2021) MCF-7 drug resistant cell lines switch their lipid metabolism to triple negative breast Cancer signature. Cancers 13 (23):5871\u003c/li\u003e\n \u003cli\u003eYu X, Ghamande S, Liu H, Xue L, Zhao S, Tan W, Zhao L, Tang S-C, Wu D, Korkaya H (2018) Targeting EGFR/HER2/HER3 with a three-in-one aptamer-siRNA chimera confers superior activity against HER2+ breast cancer. Molecular Therapy-Nucleic Acids 10:317\u0026ndash;330\u003c/li\u003e\n \u003cli\u003eOsborne CK, Hobbs K, Trent JM (1987) Biological differences among MCF-7 human breast cancer cell lines from different laboratories. Breast cancer research and treatment 9:111\u0026ndash;121\u003c/li\u003e\n \u003cli\u003eAzimzadeh M, Rahaie M, Nasirizadeh N, Daneshpour M, Naderi-Manesh H (2017) Electrochemical miRNA biosensors: the benefits of nanotechnology. Nanomedicine Research Journal 2 (1):36\u0026ndash;48\u003c/li\u003e\n \u003cli\u003eEsmaeilian A, Yazdian F, Saberi A, Hosseini A, Rahdar A, Fathi-karkan S, Pandey S (2025) Innovations in Self-Powered glucose Biosensors: From energy harvesting to Continuous, Non-Invasive monitoring. Inorganic Chemistry Communications:114076\u003c/li\u003e\n \u003cli\u003eLorencova L, Kasak P, Kosutova N, Jerigova M, Noskovicova E, Vikartovska A, Barath M, Farkas P, Tkac J (2024) MXene-based electrochemical devices applied for healthcare applications. Microchimica Acta 191 (2):88\u003c/li\u003e\n \u003cli\u003eQu L, Wu M, Zhao L, Li J, Pan H (2023) A sandwich electrochemical immunosensor based on MXene@dual MOFs for detection of tumor marker CA125. Microchimica Acta 190 (4):147\u003c/li\u003e\n \u003cli\u003eAmara U, Hussain I, Ahmad M, Mahmood K, Zhang K (2023) 2D MXene‐based biosensing: a review. Small 19 (2):2205249\u003c/li\u003e\n \u003cli\u003eAdel S, Firoozbakhtian A, Rabbani H, Hosseini M, Pebdeni AB, Sadeghi N, Gilnezhad J, Ganjali MR (2024) COVID-19 electrochemical immunosensor with Ag-MOF: Rapid and high-selectivity nasal swab testing for effective detection. Analytical Biochemistry 689:115500\u003c/li\u003e\n \u003cli\u003eGan T, Hu S (2011) Electrochemical sensors based on graphene materials. Microchimica Acta 175 (1):1\u0026ndash;19\u003c/li\u003e\n \u003cli\u003eSharma K, Hasija V, Patial S, Singh P, Nguyen V-H, Nadda AK, Thakur S, Nguyen-Tri P, Nguyen CC, Kim SY (2023) Recent progress on MXenes and MOFs hybrids: structure, synthetic strategies and catalytic water splitting. International Journal of Hydrogen Energy 48 (17):6560\u0026ndash;6574\u003c/li\u003e\n \u003cli\u003eChen Z, Li X, Yang C, Cheng K, Tan T, Lv Y, Liu Y (2021) Hybrid porous crystalline materials from metal organic frameworks and covalent organic frameworks. Advanced Science 8 (20):2101883\u003c/li\u003e\n \u003cli\u003eAnik \u0026Uuml;, Timur S, Dursun Z (2019) Metal organic frameworks in electrochemical and optical sensing platforms: a review. Microchimica Acta 186 (3):196\u003c/li\u003e\n \u003cli\u003eXu M-M, Xie L-H, Li J-R (2021) Metal\u0026ndash;Organic Framework/Polymer Hybrid Materials.\u003c/li\u003e\n \u003cli\u003eYusuf VF, Malek NI, Kailasa SK (2022) Review on metal\u0026ndash;organic framework classification, synthetic approaches, and influencing factors: applications in energy, drug delivery, and wastewater treatment. ACS omega 7 (49):44507\u0026ndash;44531\u003c/li\u003e\n \u003cli\u003eSaini H, Srinivasan N, Sedajova V, Majumder M, Dubal DP, Otyepka M, Zboril R, Kurra N, Fischer RA, Jayaramulu K (2021) Emerging MXene@ Metal\u0026ndash;organic framework hybrids: design strategies toward versatile applications. ACS nano 15 (12):18742\u0026ndash;18776\u003c/li\u003e\n \u003cli\u003eWang Y, Yang W, Cao J, Liu Y, Shao M, Xu X, Chen H (2024) Enhanced electron transfer and stability via hetero-interface engineering of MXene@MOF: Electrochemical sensors for direct detection of GPNMB in serum of PD. Sensors and Actuators B: Chemical:136066\u003c/li\u003e\n \u003cli\u003eKaur G, Sharma S, Bhardwaj N, Nayak MK, Deep A (2024) Highly sensitive and selective electrochemical detection of Aflatoxin B1 in water and Pistachio samples with MOF/MXene composite based sensor. Food Control 165:110694\u003c/li\u003e\n \u003cli\u003eChen J, Li S, Chen Y, Yang J, Dong J (2022) Highly selective detection of adenine and guanine by NH\u003csub\u003e2\u003c/sub\u003e-MIL-53 (Fe)/CS/MXene nanocomposites with excellent electrochemical performance. Microchimica Acta 189 (9):328\u003c/li\u003e\n \u003cli\u003eXu W, Li S, Hu S, Yu W, Zhou Y (2021) Effect of Ti\u003csub\u003e3\u003c/sub\u003eAlC\u003csub\u003e2\u003c/sub\u003e precursor and processing conditions on microwave absorption performance of resultant Ti 3 C 2 T x MXene. Journal of Materials Science 56:9287\u0026ndash;9301\u003c/li\u003e\n \u003cli\u003eGilnezhad J, Firoozbakhtian A, Hosseini M, Adel S, Xu G, Ganjali MR (2023) An enzyme-free Ti\u003csub\u003e3\u003c/sub\u003eC\u003csub\u003e2\u003c/sub\u003e/Ni/Sm-LDH-based screen-printed-electrode for real-time sweat detection of glucose. Analytica Chimica Acta 1250:340981\u003c/li\u003e\n \u003cli\u003eAdibah NA, Zaine SA, Shukur MFA Synthesis of Ti3C2 mxene through in situ hf and direct hf etching procedures as electrolyte fillers in dye-sensitized solar cell. In: Materials Science Forum, 2021. Trans Tech Publ, pp 15\u0026ndash;20\u003c/li\u003e\n \u003cli\u003eCheng G, Ding Q, Sun Y, Zhang Y, Zhang W, Li G (2024) Electrochemiluminescence resonance energy transfer detection of HBsAg based on Co doped 3D porous luminol-based conjugates and quencher UiO-66-NH\u003csub\u003e2\u003c/sub\u003e@Au. Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy:124574\u003c/li\u003e\n \u003cli\u003eZhai Y, Li Y, Huang X, Hou J, Li H, Ai S (2022) Colorimetric and ratiometric fluorescent dual-mode sensitive detection of Hg\u003csup\u003e2+\u003c/sup\u003e based on UiO-66-NH\u003csub\u003e2\u003c/sub\u003e@Au composite. Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 275:121187\u003c/li\u003e\n \u003cli\u003eGilbert C, Svejstrup JQ (2006) RNA immunoprecipitation for determining RNA‐protein associations in vivo. Current protocols in molecular biology 75 (1):27.24. 21\u0026ndash;27.24. 11\u003c/li\u003e\n \u003cli\u003eRohs R, Sklenar H (2001) Methylene blue binding to DNA with alternating GC base sequence: continuum treatment of salt effects.\u003c/li\u003e\n \u003cli\u003eKara P, Kerman K, Ozkan D, Meric B, Erdem A, Ozkan Z, Ozsoz M (2002) Electrochemical genosensor for the detection of interaction between methylene blue and DNA. Electrochemistry Communications 4 (9):705\u0026ndash;709\u003c/li\u003e\n \u003cli\u003eHan S, Liu W, Yang S, Wang R (2019) Facile and label-free electrochemical biosensors for microRNA detection based on DNA origami nanostructures. ACS omega 4 (6):11025\u0026ndash;11031\u003c/li\u003e\n \u003cli\u003eArab N, Hosseini M, Xu G (2024) Emerging trends and recent advances in MXene/MXene-based nanocomposites toward electrochemiluminescence sensing and biosensing. Biosensors and Bioelectronics:116623\u003c/li\u003e\n \u003cli\u003eNaguib M, Kurtoglu M, Presser V, Lu J, Niu J, Heon M, Hultman L, Gogotsi Y, Barsoum MW (2023) Two-dimensional nanocrystals produced by exfoliation of Ti\u003csub\u003e3\u003c/sub\u003eAlC\u003csub\u003e2\u003c/sub\u003e. In: MXenes. Jenny Stanford Publishing, pp 15\u0026ndash;29\u003c/li\u003e\n \u003cli\u003eDixit P, Maiti T (2022) A facile pot synthesis of (Ti\u003csub\u003e3\u003c/sub\u003eAlC\u003csub\u003e2\u003c/sub\u003e) MAX phase and its derived MXene (Ti\u003csub\u003e3\u003c/sub\u003eC\u003csub\u003e2\u003c/sub\u003eT\u003csub\u003ex\u003c/sub\u003e). Ceramics International 48 (24):36156\u0026ndash;36165\u003c/li\u003e\n \u003cli\u003eRanjbari S, Rezayi M, Arefinia R, Aghaee-Bakhtiari SH, Hatamluyi B, Pasdar A (2023) A novel electrochemical biosensor based on signal amplification of Au HFGNs/PnBA-MXene nanocomposite for the detection of miRNA-122 as a biomarker of breast cancer. Talanta 255:124247\u003c/li\u003e\n \u003cli\u003eNayak P, Jiang Q, Mohanraman R, Anjum D, Hedhili MN, Alshareef HN (2018) Inherent electrochemistry and charge transfer properties of few-layered two-dimensional Ti 3 C 2 T x MXene. Nanoscale 10 (36):17030\u0026ndash;17037\u003c/li\u003e\n \u003cli\u003eAlhabeb M, Maleski K, Anasori B, Lelyukh P, Clark L, Sin S, Gogotsi Y (2017) Guidelines for synthesis and processing of two-dimensional titanium carbide (Ti3C2T x MXene). Chemistry of Materials 29 (18):7633\u0026ndash;7644\u003c/li\u003e\n \u003cli\u003eDamirchi Z, Firoozbakhtian A, Hosseini M, Ganjali MR (2024) Ti\u003csub\u003e3\u003c/sub\u003eC\u003csub\u003e2\u003c/sub\u003e/Ni/Sm-based electrochemical glucose sensor for sweat analysis using bipolar electrochemistry. Microchimica Acta 191 (3):137\u003c/li\u003e\n \u003cli\u003eKumar AN, Pal K (2022) Amine-functionalized stable Nb\u003csub\u003e2\u003c/sub\u003eCT\u003csub\u003ex\u003c/sub\u003e MXene toward room temperature ultrasensitive NO 2 gas sensor. Materials Advances 3 (12):5151\u0026ndash;5162\u003c/li\u003e\n \u003cli\u003eJi J, Zhao L, Shen Y, Liu S, Zhang Y (2019) Covalent stabilization and functionalization of MXene via silylation reactions with improved surface properties. FlatChem 17:100128\u003c/li\u003e\n \u003cli\u003eParker T, Zhang D, Bugallo D, Shevchuk K, Downes M, Valurouthu G, Inman A, Chacon B, Zhang T, Shuck CE (2024) Fourier-transform infrared spectral library of MXenes. Chemistry of Materials 36 (17):8437\u0026ndash;8446\u003c/li\u003e\n \u003cli\u003eWu L, Pu H, Huang L, Sun D-W (2020) Plasmonic nanoparticles on metal-organic framework: A versatile SERS platform for adsorptive detection of new coccine and orange II dyes in food. Food chemistry 328:127105\u003c/li\u003e\n \u003cli\u003eChen Q, He Q, Lv M, Xu Y, Yang H, Liu X, Wei F (2015) Selective adsorption of cationic dyes by UiO-66-NH\u003csub\u003e2\u003c/sub\u003e. Applied Surface Science 327:77\u0026ndash;85\u003c/li\u003e\n \u003cli\u003eKumar S, Lei Y, Alshareef NH, Quevedo-Lopez M, Salama KN (2018) Biofunctionalized two-dimensional Ti\u003csub\u003e3\u003c/sub\u003eC\u003csub\u003e2\u003c/sub\u003e MXenes for ultrasensitive detection of cancer biomarker. Biosensors and Bioelectronics 121:243\u0026ndash;249\u003c/li\u003e\n \u003cli\u003eHatamluyi B, Sadeghian R, Sany SBT, Alipourfard I, Rezayi M (2021) Dual-signaling electrochemical ratiometric strategy for simultaneous quantification of anticancer drugs. Talanta 234:122662\u003c/li\u003e\n \u003cli\u003eChai X, Ye S, Wang F, Yuan H, Liu M, Fan F, Zhang L, Zhang X, Wang T, Fu Y (2023) Au nanoparticles@ UiO-66 composite film-coated carbon cloth substrate for high-performance H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e electrochemical sensing. Inorganic Chemistry 62 (27):10694\u0026ndash;10703\u003c/li\u003e\n \u003cli\u003eBajpai VK, Haldorai Y, Khan I, Sonwal S, Singh MP, Yadav S, Paray BA, Jan BL, Kang S-M, Huh YS (2021) Au@ Zr-based metal\u0026ndash;organic framework composite as an immunosensing platform for determination of hepatitis B virus surface antigen. Microchimica Acta 188:1\u0026ndash;13\u003c/li\u003e\n \u003cli\u003eMahmoodi P, Rezayi M, Rasouli E, Avan A, Gholami M, Ghayour Mobarhan M, Karimi E, Alias Y (2020) Early-stage cervical cancer diagnosis based on an ultra-sensitive electrochemical DNA nanobiosensor for HPV-18 detection in real samples. Journal of nanobiotechnology 18:1\u0026ndash;12\u003c/li\u003e\n \u003cli\u003eRahman M, Heng LY, Futra D, Chiang CP, Rashid ZA, Ling TL (2017) A highly sensitive electrochemical DNA biosensor from acrylic-gold nano-composite for the determination of arowana fish gender. Nanoscale research letters 12:1\u0026ndash;10\u003c/li\u003e\n \u003cli\u003eLi S, Zhu J, Fu H, Wan J, Hu Z, Liu S, Li J, Tie Y, Xing R, Zhu J (2012) Hepato-specific microRNA-122 facilitates accumulation of newly synthesized miRNA through regulating PRKRA. Nucleic acids research 40 (2):884\u0026ndash;891\u003c/li\u003e\n \u003cli\u003eZhu L, Zhao R, Wang K, Xiang H, Shang Z, Sun W (2008) Electrochemical behaviors of methylene blue on DNA modified electrode and its application to the detection of PCR product from NOS sequence. Sensors 8 (9):5649\u0026ndash;5660\u003c/li\u003e\n \u003cli\u003eJin Y, Yao X, Liu Q, Li J (2007) Hairpin DNA probe based electrochemical biosensor using methylene blue as hybridization indicator. Biosensors and Bioelectronics 22 (6):1126\u0026ndash;1130\u003c/li\u003e\n \u003cli\u003eSilvestrini M, Fruk L, Moretto LM, Ugo P (2015) Detection of DNA hybridization by methylene blue electrochemistry at activated nanoelectrode ensembles. Journal of Nanoscience and Nanotechnology 15 (5):3437\u0026ndash;3442\u003c/li\u003e\n \u003cli\u003eLi Y, Chang Y, Yuan R, Chai Y (2018) Highly efficient target recycling-based netlike Y-DNA for regulation of electrocatalysis toward methylene blue for sensitive DNA detection. ACS applied materials \u0026amp; interfaces 10 (30):25213\u0026ndash;25218\u003c/li\u003e\n \u003cli\u003eTalemi RP, Mashhadizade MH (2017) A novel optical DNA biosensor for detection of trace concentration of Methylene blue using Gold nano-particles and Guanine rich single strand DNA. International Journal of Nano Dimension 8 (3):224\u0026ndash;233\u003c/li\u003e\n \u003cli\u003eYan F, Erdem A, Meric B, Kerman K, Ozsoz M, Sadik OA (2001) Electrochemical DNA biosensor for the detection of specific gene related to Microcystis species. Electrochemistry communications 3 (5):224\u0026ndash;228\u003c/li\u003e\n \u003cli\u003eGu J, Lu X, Ju H (2002) DNA sensor for recognition of native yeast DNA sequence with methylene blue as an electrochemical hybridization indicator. Electroanalysis: An International Journal Devoted to Fundamental and Practical Aspects of Electroanalysis 14 (13):949\u0026ndash;954\u003c/li\u003e\n \u003cli\u003eBard AJ, Faulkner LR, White HS (2022) Electrochemical methods: fundamentals and applications. John Wiley \u0026amp; Sons,\u003c/li\u003e\n \u003cli\u003eMcNaught AD, Wilkinson A (1997) Compendium of chemical terminology, vol 1669. Blackwell Science Oxford,\u003c/li\u003e\n \u003cli\u003eZhang J, Cui Y, Lin X, Zhang G, Li Z (2017) MiR-122-3p sensitizes breast cancer cells to ionizing radiation via controlling of cell apoptosis, migration and invasion. Int J Clin Exp Pathol 10 (1):215\u0026ndash;223\u003c/li\u003e\n \u003cli\u003eSong B, Hou G, Xu M, Chen M (2024) Exosomal miR-122-3p represses the growth and metastasis of MCF-7/ADR cells by targeting GRK4-mediated activation of the Wnt/\u0026beta;-catenin pathway. Cellular Signalling 117:111101\u003c/li\u003e\n \u003cli\u003eZhang W, Jiang H, Chen Y, Ren F (2019) Resveratrol chemosensitizes adriamycin‐resistant breast cancer cells by modulating miR‐122‐5p. Journal of Cellular Biochemistry 120 (9):16283\u0026ndash;16292\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Scheme","content":"\u003cp\u003eScheme 1 is available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"2D nanomaterials, Breast cancer, miRNA-122, NH2-UiO-66 MOF, Ti3C2Tx MXene","lastPublishedDoi":"10.21203/rs.3.rs-7449624/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7449624/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eA novel, label-free electrochemical biosensor was precise engineered for the ultrasensitive and selective detection of miRNA-122, a pivotal biomarker in breast cancer diagnostics. This innovative sensing platform harnesses the exceptional conductivity of Ti\u003csub\u003e3\u003c/sub\u003eC\u003csub\u003e2\u003c/sub\u003e MXene nanosheets, synergistically integrated with Au-decorated NH\u003csub\u003e2\u003c/sub\u003e-UiO-66 metal-organic frameworks, yielding a structurally stable and electrochemically active hybrid interface. A thiolated DNA capture probe was immobilized onto the gold-modified MOF, enabling precise hybridization with the target miRNA-122. Methylene blue functioned as a robust redox indicator, exhibiting distinct electrochemical shifts upon duplex formation through π–π stacking and electrostatic interactions. The biosensor demonstrated outstanding analytical performance with dual linear response ranges (1 pM–1 nM and 1 nM–750.0 nM) and an impressively low limit of detection of 503 fM. High sequence specificity was achieved, successfully differentiating miRNA-122 from non-complementary and closely related sequences. Validation with lysates from three distinct breast cancer cell lines (MCF-7, MDA-MB-231, and SKBR3) confirmed its efficacy in complex biological matrices. Altogether, this biosensor platform offers a powerful and reliable tool for early breast cancer detection and holds substantial potential for clinical applications in miRNA-based diagnostics.\u003c/p\u003e","manuscriptTitle":"A 2D/3D Nanoplatform for Electrochemical Diagnosis of miRNA-122 in Breast Cancer Cell Lines","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-09-10 11:28:25","doi":"10.21203/rs.3.rs-7449624/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"b5bef2f7-cdf7-420d-80c5-7d0333ce0b12","owner":[],"postedDate":"September 10th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-09-14T23:38:16+00:00","versionOfRecord":[],"versionCreatedAt":"2025-09-10 11:28:25","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7449624","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7449624","identity":"rs-7449624","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}