α-Fe 2 O 3 /Ti 3 C 2 T x MXene Heterostructures as Photo-Fenton Catalysts Driving RAFT Polymerization for Ultrasensitive Electrochemical microRNA Sensing

α-Fe 2 O 3 /Ti 3 C 2 T x MXene Heterostructures as Photo-Fenton Catalysts Driving RAFT Polymerization for Ultrasensitive Electrochemical microRNA Sensing | 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 α-Fe 2 O 3 /Ti 3 C 2 T x MXene Heterostructures as Photo-Fenton Catalysts Driving RAFT Polymerization for Ultrasensitive Electrochemical microRNA Sensing Thao Thi Nguyen, Zhidan Tian, Weibo Huang, Qinyuan Xu, Shuaibing Yu, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7482836/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 22 Jan, 2026 Read the published version in Microchimica Acta → Version 1 posted 15 You are reading this latest preprint version Abstract α-Fe 2 O 3 nanoparticles possess a narrow band gap (~2.1 eV) and exhibit strong absorption in the visible light range, making them promising candidates for photocatalytic applications. However, their poor electrical conductivity, high electron–hole recombination rate, and short charge diffusion length limit their practical performance. To address these limitations, α-Fe 2 O 3 was integrated with Ti 3 C 2 T x MXene to construct a composite photo-Fenton catalyst that drives RAFT polymerization. In this system, the MXene substrate not only disperses α-Fe 2 O 3 nanoparticles efficiently but also facilitates the generation of abundant photoinduced electrons under visible-light excitation. This enhances the Fe 3+ /Fe 2+ redox cycling and accelerates H 2 O 2 decomposition, yielding a high concentration of hydroxyl radicals (•OH). To further explore the functional applicability of this catalyst, it was employed in a biosensing platform for the ultrasensitive detection of microRNA-144. In this design, the •OH radicals initiate RAFT polymerization, allowing for a significant amplification of the electrochemical signal. The resulting sensor exhibits a wide detection range (0.01 fM to 10 pM) and an ultralow detection limit of 4.44 aM. These findings highlight the potential of α-Fe 2 O 3 /Ti 3 C 2 T x MXene composites in photocatalysis, polymer chemistry, and biomedical sensing, offering new insights for future technological innovations. α-Fe2O3/Ti3C2TxMXene heterojunction photocatalysis biosensor Photo-Fenton Catalysts RNA detection signal amplification Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 1. Introduction Catalysts play a critical role in reversible addition-fragmentation chain-transfer (RAFT) polymerization by facilitating precise control over polymer chain growth and molecular architecture, essential for constructing functional polymeric materials in biosensing applications. However, conventional RAFT polymerization is often hindered by oxygen sensitivity and limited spatiotemporal control, which pose significant challenges for its implementation in biological and in vivo environments. To address these challenges, various strategies have been proposed, including the development of semiconducting nanoparticle photopolymerization systems [ 1 , 2 ] , enhancing oxygen tolerance [ 3 ] , and conducting polymerization under visible and even near-infrared light [ 4 , 5 ] . For instance, Erika et al. demonstrated the use of urea-based graphitic nitride (U-g-C 3 N 4 ) for heterogeneous PET-RAFT polymerization of methyl methacrylate (MMA) without prior deoxygenation of the reaction medium [ 6 ] . Similarly, Kaya et al. employed mesoporous graphitic carbon nitride (mpg-C 3 N 4 ) with FeCl 3 ·6H 2 O to establish an oxygen-tolerant visible light photo initiating system. This system works through in-situ generation of Fenton reagents using water and oxygen for free radical polymerization [ 7 ] . In comparison to these photocatalytic systems, α-Fe 2 O 3 and Ti 3 C 2 T x MXene is considered a competitive candidate due to the environmentally benign nature of hematite (α-Fe 2 O 3 ), an n-type semiconductor with a bandgap of 2.1 eV. This moderate band gap enables efficient absorption of visible light, making α-Fe 2 O 3 suitable for solar-driven photocatalytic applications [ 8 , 9 ] . Moreover, Ti 3 C 2 T x MXene, with its oxidized surface groups, has proven effective as a cocatalyst for photocatalytic reactions under visible light irradiation [ 10 , 11 ] . MXene, obtained from the MAX phase, is a two-dimensional (2D) layered material consisting of transition metal carbides, nitrides, and carbonitrides. It possesses numerous hydrophilic surface functionalities (-OH and -O), excellent electrochemical properties, a large specific surface area, and outstanding structural/chemical stability [ 12 – 15 ] . Introducing Ti 3 C 2 T x MXene into the α-Fe 2 O 3 matrix significantly increases its particular surface area [ 16 , 17 ] , broadens the light absorption spectrum [ 18 ] , and enhances the photochemical cleavage efficiency as well as the transport capacity of photogenerated carriers (electrons and holes) [ 19 – 21 ] . Therefore, in this study, α-Fe 2 O 3 and Ti 3 C 2 T x MXene heterostructures were synthesized and employed as photo-Fenton catalysts to promote RAFT polymerization. To exploit the potential of this catalytic system, it was integrated into a biosensing platform based on a RAFT-mediated signal amplification strategy, enabling ultrasensitive detection of the cancer biomarker microRNA-144. The biosensor was constructed on a gold electrode by immobilizing a polyA-DNA probe through the strong affinity between polyadenine sequences and the Au surface [ 22 , 23 ] . This interaction formed stable adenine-Au anchor points, enabling the DNA probe to adopt a longitudinal orientation perpendicular to the electrode surface. Such a spatial arrangement was crucial for reducing steric hindrance and significantly improving the hybridization efficiency with the target miRNA. Upon hybridization of polyA-DNA with miRNA, a non-hybridized nucleotide overhang was exposed. This region enabled site-specific binding of an amino-modified DNA sequence (NH 2 -DNA). The amino terminus of the NH 2 -DNA was covalently conjugated with 4-cyano-4-(thiobenzoyl) valeric acid (CPAD), a thiocarbonylthio-based RAFT agent, thereby generating a surface-immobilized RAFT initiator on the gold electrode. This initiator enabled the visible-light-driven polymerization of poly(ferrocene), catalyzed by the α-Fe 2 O 3 /Ti 3 C 2 T x MXene photo-Fenton heterostructure. The electroactive poly(ferrocene) chains produced were quantitatively analyzed via square-wave voltammetry (SWV), yielding a strongly amplified signal that displayed a linear relationship with the target miRNA concentration ( Fig. 1 ) . 2. Materials and Methods 2.1. Materials The relevant reagents and instruments can be found in the Supplementary Material. 2.2. Fabrication of Urchin-like α-Fe 2 O 3 The α-Fe 2 O 3 material was synthesized through hydrothermal method and thermal annealing. Initially, 4 mmol/L of iron(III) chloride hexahydrate (FeCl 3 ·6H 2 O) and 3 mmol/L of sodium sulfate (Na 2 SO 4 ) were dissolved in 80 mL of deionized water under vigorous stirring to ensure complete homogenization. The resulting solution was then transferred to a 100 mL Teflon-lined stainless steel autoclave and subjected to hydrothermal treatment at 120°C for 14 h. Upon completion, the autoclave was allowed to cool naturally to room temperature. The resulting yellow FeOOH precursor was collected, washed three times with deionized water to eliminate residual ions, and dried in an oven at 80°C for 4 h. The dried powder was subsequently placed in a nitrogen atmosphere, heated to 450°C at a ramp rate of 5°C/min, and annealed for 2 h to obtain the final α-Fe 2 O 3 product (Fig. 1 A). 2.3. Fabrication of Ti 3 C 2 T x MXene Nanosheets Ti 3 C 2 T x MXene nanosheets were synthesized via an in situ hydrofluoric acid (HF) generation method combined with an ultrasound-assisted etching process. Initially, 2.5 g of lithium fluoride (LiF) powder was dispersed in 50 mL of 9.0 M hydrochloric acid (HCl) and stirred for 30 min to generate HF in situ. Subsequently, Ti 3 AlC 2 powder was gradually added to the solution and stirred vigorously at 35°C for 24 h, facilitating the selective etching of the aluminum (Al) layer. Upon completion of the reaction, the resulting suspension was washed repeatedly with deionized water and subjected to successive centrifugation cycles until the pH of the supernatant approached neutrality. The resulting black precipitate was then ultrasonicated for 1.5 h to delaminate the multilayered structure, yielding monolayer Ti 3 C 2 T x MXene nanosheets. Finally, product was subjected to high-speed centrifugation at 4500 rpm for 1 h, followed by redispersion in deionized water to obtain a monolayer Ti 3 C 2 T x MXene dispersion with a final concentration of 10 mg/mL (Fig. 1 A). 2.4. Fabrication of α-Fe 2 O 3 /Ti 3 C 2 T x MXene The α-Fe 2 O 3 /Ti 3 C 2 T x MXene composites were synthesized via an electrostatic self-assembly strategy, wherein the assembly was driven by electrostatic interactions between α-Fe 2 O 3 and Ti 3 C 2 T x MXene nanosheets. Specifically, 100 mg of α-Fe 2 O 3 was uniformly dispersed in 100 mL of deionized water and subjected to combined ultrasonic treatment and magnetic stirring for 20 min to ensure thorough dispersion of the nanoparticles. Subsequently, 10µL Ti 3 C 2 T x MXene 10 mg/mL was added dropwise to the α-Fe 2 O 3 suspension under continuous magnetic stirring. This self-assembly process was maintained for 4 h to facilitate uniform interaction between the two components. The α-Fe 2 O 3 /Ti 3 C 2 T x MXene composites were isolated via centrifugal separation and subsequently dried in a vacuum oven (Fig. 1 A). 2.5. Preparation of Electrochemical Biosensor The miRNA detection biosensor was fabricated using gold electrodes, with each modification step carefully optimized to ensure high sensitivity and specificity. Initially, the Au electrode surface was mechanically polished using 0.05 µm alumina slurry to achieve a smooth and contaminant-free surface. The electrodes were then sequentially rinsed three times each with ultrapure water and ethanol, followed by drying under a stream of high-purity nitrogen gas. Subsequently, 10 µL of polyA-DNA probe solution was drop-cast onto the electrode surface and incubated at room temperature for 2 h, promoting adsorption through the strong affinity between Au and the polyA moiety. Following this, 10 µL of tRNA was applied to hybridize with the polyA-DNA probe and incubated for an additional 2 h. To further extend the hybridization, 10 µL of NH 2 -DNA was introduced to hybridize with the remaining unpaired region of the tRNA. Meanwhile, the carboxyl group of the RAFT initiator, 2 mM CPAD, was activated using a 1:1:1 molar ratio solution of 2 mM carbodiimide hydrochloride (EDC) and 2 mM N-hydroxysuccinimide (NHS). The modified Au electrode was immersed in 300 µL of the activated CPAD solution and incubated for 2 h to facilitate covalent coupling. Subsequently, the electrode was transferred into 300 µL of an RAFT polymerization mixture containing 2 mM fluoromethyl methacrylate (FMMA), 0.05 mM hydrogen peroxide (H 2 O 2 ), and 0.05 mg/mL α-Fe 2 O 3 /Ti 3 C 2 T x MXene. The polymerization was initiated under continuous visible light irradiation (400–600 nm) for 3 h, enabling the growth of functional polymer chains on the electrode surface. All modification steps were conducted at room temperature. After each step, the electrodes were thoroughly rinsed with 0.1 M phosphate-buffered saline (PBS, pH 7.4) to eliminate unbound or unreacted species. Finally, the electrochemical performance of the modified Au electrodes was evaluated by SWV within the potential range of 0-0.7 V (Fig. 1 B). 3. Results and Discussion 3.1. Structural and Morphological Characterization of α-Fe 2 O 3 /Ti 3 C 2 T x MXene Catalysts As shown in Fig. 2 , scanning electron microscopy (SEM) was employed to examine the morphologies of Ti 3 C 2 T x MXene, α-Fe 2 O 3 , and their composite α-Fe 2 O 3 /Ti 3 C 2 T x MXene. In Fig. 2 A, Ti 3 C 2 T x MXene displays a characteristic accordion-like morphology with a multilayered architecture, confirming the successful etching of the Ti 3 AlC 2 MAX phase. This morphology significantly increases the specific surface area, enhancing interfacial interactions and providing a favorable structural basis for composite integration and performance enhancement. Figure 2 B reveals that α-Fe 2 O 3 exhibits a distinct urchin-like microstructure, comprising radially aligned needle-like features emanating from a central core. This hierarchical structure creates abundant void space, which facilitates rapid electrolyte ion transport. Such a feature is beneficial for both catalytic and electrochemical applications. In Fig. 2 C, the α-Fe 2 O 3 /Ti 3 C 2 T x MXene composite exhibits a well-integrated structure wherein the urchin-like α-Fe 2 O 3 particles are uniformly distributed and anchored within the Ti 3 C 2 T x MXene nanosheet matrix. To further confirm the compositional uniformity, elemental mapping was performed (Fig. 2 D-I), revealing the presence and homogeneous distribution of C, Ti, O, Fe, and F throughout the composite. This uniform elemental dispersion validates the successful synthesis of the α-Fe 2 O 3 /Ti 3 C 2 T x MXene composite and suggests robust interfacial contact between the two components, which is critical for maintaining structural integrity and optimizing multifunctional performance. The phase structures of α-Fe 2 O 3 , Ti 3 C 2 T x MXene, and the α-Fe 2 O 3 /Ti 3 C 2 T x MXene composites were analyzed by X-ray diffraction (XRD), as presented in Fig. S1 (A) . The diffraction peaks of Ti 3 C 2 T x MXene appeared at 2θ = 8.4°, 18.1°, 27.2°, and 61.0°, consistent with previously reported data [ 21 , 24 ] , thereby confirming the successful synthesis of Ti 3 C 2 T x MXene. Similarly, the XRD pattern of pure α-Fe 2 O 3 nanosheets exhibited distinct peaks at 24.1°, 33.1°, 35.6°, 40.9°, 49.5°, 54.1°, 57.5°, 62.5°, and 63.9°, corresponding to the characteristic reflections of the α-Fe 2 O 3 phase (JCPDS No. 00-024-0072) [ 25 ] . In the XRD patterns of the α-Fe 2 O 3 /Ti 3 C 2 T x MXene composites, the diffraction peaks were predominantly attributed to α-Fe 2 O 3 , closely matching the hematite phase (JCPDS No. 00-024-0072), which is primarily due to the high crystallinity of α-Fe 2 O 3 resulting from thermal treatment and the presence of intense diffraction peaks. The characteristic peak of Ti 3 C 2 T x MXene at 6.58° exhibited significantly reduced intensity and was nearly undetectable. However, the typical diffraction peak corresponding to the (110) plane of Ti 3 C 2 T x MXene remained prominent in the composite, indicating that the Ti 3 C 2 T x MXene nanosheets were successfully coated on the surface of the urchin-shaped α-Fe 2 O 3 structures. This coating effectively prevented the restacking of Ti 3 C 2 T x MXene layers and preserved the original layered structure of the MXene. The retention of distinct crystalline phases of both α-Fe 2 O 3 and Ti 3 C 2 T x MXene in the composite confirms the feasibility and efficiency of the adopted synthesis method. 3.2. α-Fe 2 O 3 /Ti 3 C 2 T x MXene Heterostructures as Photo-Fenton Catalysts Driving RAFT Polymerization The photo-Fenton mechanism driving the RAFT polymerization is intrinsically associated with charge-transfer dynamics within the α-Fe 2 O 3 /Ti 3 C 2 Tₓ MXene heterostructure under visible-light irradiation. The charge-transfer pathway is governed by the alignment of the band structure and Fermi level, which determines the separation and migration efficiency of photogenerated electrons, sustains the Fe 3+ /Fe 2+ redox cycling, and thereby promotes the generation of highly oxidative hydroxyl radicals (•OH). As shown in Fig. 3 A, the UV-vis diffuse reflectance spectrum (DRS) of α-Fe 2 O 3 was used to estimate its optical band gap, which was calculated to be 2.09 eV using the Kubelka-Munk function. Additionally, the valence band (VB) energy level of α-Fe 2 O 3 was determined through XPS, revealing a VB maximum at + 2.21 eV relative to the normal hydrogen electrode (NHE), in Fig. 3 B. Consequently, the conduction band (CB) potential of α-Fe 2 O 3 was calculated to be 0.12 eV using Eq. (1): $$\:{E}_{CB}(vs.\:NHE)\:=\:{E}_{VB}\:(vs.\:NHE)\:-\:{E}_{g}$$ where E CB is the conduction band potential, E VB is the valence band potential, and E g is the optical band gap [ 26 , 27 ] . Based on the above findings, the photoexcitation process within the α-Fe 2 O 3 /Ti 3 C 2 Tₓ MXene heterostructure can be described as follows (Fig. 3 C ) . Ti 3 C 2 Tₓ MXene, functioning as a semimetallic plasmonic material [ 28 , 29 ] , generates localized surface plasmon resonance under light irradiation, thereby producing hot electrons and corresponding holes. A fraction of these hot electrons can be injected into the conduction band (CB) of α-Fe 2 O 3 , while visible-light photons simultaneously excite electrons from the VB (~ 2.21 eV) to the CB (~ 0.12 eV). This process leaves positively charged holes in the VB and enables efficient charge carrier separation ( Fig. 3 C ) . The synergistic effect of hot electrons from Ti 3 C 2 Tₓand the intrinsic photoexcitation of α-Fe 2 O 3 significantly accelerates the internal Fe 3+ /Fe 2+ redox cycle of the Fenton process, thereby promoting the decomposition of H 2 O 2 and generating a high concentration of strongly oxidative hydroxyl radicals (•OH). The •OH radicals generated in this process initiate the RAFT polymerization reaction. As depicted in Fig. 4 , the RAFT mechanism proceeds through five distinct stages: 3.3. Electrochemical Characterization In this study, SWV was employed to precisely evaluate the electrochemical response of gold electrodes modified with various components, aiming to determine the specific contribution of each element within the biosensor assembly. As shown in Fig. 5 A, control experiments involving incomplete sensor configurations such as the absence of polyA-DNA probe, tRNA, NH 2 -DNA, CPAD, FMMA, α-Fe 2 O 3 /Ti 3 C 2 T x MXene, H 2 O 2 , or light irradiation failed to produce any detectable electrochemical signal. In contrast, the complete biosensor, constructed with polyA-DNA/tRNA/NH 2 -DNA/CPAD/FMMA and activated through a RAFT reaction catalyzed by α-Fe 2 O 3 /Ti 3 C 2 T x MXene under visible light in the presence of H 2 O 2 , yielded a strong electrochemical signal at 0.288 V (curve k). This result confirms the successful design and functional feasibility of the biosensor system. To further examine the surface modification process, electrochemical impedance spectroscopy (EIS) was conducted following each step of electrode modification. As depicted in Fig. 5 B, the bare gold electrode exhibited the lowest charge transfer resistance (R ct ) at approximately 207 Ω (burgundy curve), attributed to the smooth, conductive metal surface that facilitates efficient electron transfer. Upon immobilization of the polyA-DNA probe, the R ct increased markedly to ~ 502 Ω (bright red curve), due to the introduction of negatively charged phosphate groups that hinder the diffusion of [Fe(CN) 6 ] 3− /[Fe(CN) 6 ] 4− redox species. Subsequent hybridization with the target miRNA and complementary NH 2 -DNA led to further increases in R ct to ~ 726 Ω (orange curve) and ~ 1170 Ω, respectively (lime green), which can be attributed to the formation of double-stranded structures that increase both steric and electrostatic hindrance at the electrode interface. Following covalent attachment of the chain transfer initiator CPAD, the R ct rose sharply to ~ 1590 Ω (lawn green curve), indicating substantial coverage of the electrode surface, which further restricted charge transfer. Finally, polymerization of FMMA via RAFT reaction resulted in a significant increase in R ct to ~ 2310 Ω (purple curve), reflecting the deposition of a dense, hydrophobic polymer layer. This layer acts as a physical barrier, impeding the diffusion of redox species to the electrode surface and further increasing resistance. These progressive changes in impedance demonstrate the successful and sequential construction of the biosensor through RAFT-mediated surface polymerization. Complementary cyclic voltammetry (CV) measurements across a broad scan rate range (50–450 mV/s) further verified the excellent electrochemical performance of the modified electrode. The redox peaks retained a consistent shape, with a peak potential separation (ΔEp) maintained at 0.285 ± 0.02 V ( n = 10) (Fig. 5 C), confirming high redox reversibility. A kinetic analysis of the CV data further revealed that the oxidation peak current ( I pa ) and reduction peak current ( I pc ) displayed strong linear correlations with the square root of the scan rate ( v 1/2 ) over the tested range, as shown in Fig. 5 D. This linearity is characteristic of a diffusion-controlled electrochemical process involving surface-confined redox-active species and provides strong evidence that the ferrocene moieties are robustly immobilized on the electrode surface. 3.4. Experimental optimization conditions During biosensor development, the mixing ratio between α-Fe 2 O 3 and Ti 3 C 2 T x MXene during the synthesis of the α-Fe 2 O 3 /Ti 3 C 2 T x MXene heterojunction plays a critical role in determining the final electrochemical performance of the RAFT photocatalytic system. Ti 3 C 2 T x MXene exhibits excellent electrical conductivity and light absorption properties, which significantly enhance the separation efficiency of photogenerated electron–hole pairs (e⁻/h⁺) generated by α-Fe 2 O 3 under visible light irradiation. Increasing the proportion of Ti 3 C 2 T x MXene in the composite material provides a larger anchoring surface for α-Fe 2 O 3 nanoparticles, preventing agglomeration and facilitating more efficient charge separation. This promotes the transfer of electrons from α-Fe 2 O 3 to Ti 3 C 2 T x MXene, where they participate in the reduction of H 2 O 2 to generate hydroxyl radicals (•OH), supplying active species required for the subsequent RAFT polymerization. To optimize the α-Fe 2 O 3 /Ti 3 C 2 T x MXene heterojunction composition, a series of biosensors were fabricated using polyA-DNA/tRNA/NH 2 -DNA/CPAD/FMMA-modified electrodes, each subjected to the RAFT reaction under varying α-Fe 2 O 3 /Ti 3 C 2 T x MXene photocatalyst conditions. Specifically, α-Fe 2 O 3 /Ti 3 C 2 T x MXene composites were synthesized at different mass ratios of α-Fe 2 O 3 to Ti 3 C 2 T x MXene: 100:1 (FM01), 75:1 (FM02), 50:1 (FM03), and 25:1 (FM04). The results (Fig. 6 A) indicated that the FM02 group exhibited the highest electrochemical signal, whereas FM01, FM03, and FM04 showed comparatively poor performance. In FM01, the low Ti 3 C 2 T x MXene content was insufficient to prevent α-Fe 2 O 3 nanoparticle aggregation, thereby limiting the catalytic activity. Conversely, in FM03 and FM04, the excess of Ti 3 C 2 T x MXene may have obstructed light absorption by α-Fe 2 O 3 , negatively impacting the generation of photogenerated carriers. Based on these findings, the FM02 composite with an α-Fe 2 O 3 : Ti 3 C 2 T x MXene ratio of 75:1 was selected as the optimal formulation for subsequent experiments due to its superior photocatalytic efficiency. Additionally, the ratio of N, N-dimethylformamide (DMF) to water in the RAFT polymerization medium was investigated as a key factor influencing polymerization efficiency. As shown in Fig. 6 B, the biosensor prepared in a 60% DMF solution produced a significantly higher electrochemical response than those in 30%, 40%, or 50% DMF. This enhancement is likely due to the polar nature of DMF, which improves the solubility and dispersion of both the monomer and the RAFT initiator, thus facilitating the propagation of polymer chains and leading to polymers with higher electrochemical activity. However, at 70% DMF, a slight decrease in performance was observed, likely due to increased solution viscosity, which hinders molecular mobility and diffusion, ultimately reducing polymerization efficiency. Therefore, a DMF concentration of 60% was determined to be optimal for RAFT-based biosensor fabrication. The duration of RAFT polymerization also significantly impacts the polymer chain length and overall product quality. Inadequate reaction times can result in incomplete polymerization, yielding low molecular weight polymers with inconsistent performance. On the other hand, excessive polymerization may lead to uncontrolled side reactions such as chain transfer or termination, compromising the living nature of RAFT polymerization and reducing the structural regularity of the final product. As demonstrated in Fig. 6 C, a polymerization time of 3 h produced the highest electrochemical signal (1.15 µA), indicating that this duration offers a balance between monomer conversion and molecular weight control. Thus, 3 h was identified as the optimal polymerization time under the given reaction conditions. 3.5. Electrochemical biosensor sensitivity Under optimized experimental conditions, the performance of the fabricated electrochemical biosensor was evaluated by analyzing its response to a series of tRNA solutions at varying concentrations. As shown in Fig. 7A , the peak current measured by SWV exhibited a clear upward trend with increasing tRNA concentration. Within a specific dynamic range of 0.01 fM to 10 pM, a strong linear relationship was observed between the peak current and the logarithm of the tRNA concentration ( Fig. 7B ). Linear regression analysis yielded the Eq. ( 5 ): $$\:I\:\left(\mu\:A\right)\:=\:2.485\:+\:0.126\:\times\:\:log\left({C}_{tRNA}\right)$$ 5 This has a correlation coefficient (R 2 ) 0.998, indicating excellent linear response performance. The detection limit of the biosensor was calculated to be as low as 4.44 aM, based on the standard criterion of a signal-to-noise ratio of 3 ( S/N = 3). Compared with many previously reported detection methods (Table 1 ), the electrochemical biosensor developed in this study demonstrated superior sensitivity and an ultra-low detection limit. Table 1 Comparison with other nucleic acid detection schemes Detection technique Analytes Linear detection range Detection limit Reference Photo-ATRP miRNA-21 10fM ~ 1nM 1.35fM [ 31 ] PET-RAFT miRNA-21 0.1nM ~ 10aM 4.48aM [ 32 ] PET-RAFT KRAS G12C-ssDNA 0fM ~ 10nM 8fM [ 33 ] CHA-HCR miRNA 21 10fM ~ 100pM 2.52fM [ 34 ] MNAzyme-CHA miRNA-21 1pM ~ 100nM 3.2fM [ 35 ] Photo-Fenton-RAFT miRNA-144 0.01fM ~ 10pM 4.44aM This work 3.6. Electrochemical biosensor selectivity Selectivity is a critical factor influencing the overall performance of a biosensor. To assess the specificity of the fabricated biosensor, its electrochemical responses to various RNA sequences were tested, including the target tRNA, a single-base mismatch (smRNA), a three-base mismatch (tmRNA), and a fully mismatched control (cRNA), each at a fixed concentration of 10 pM. As shown in Fig. S3 B , the signal responses of smRNA, tmRNA, and cRNA were 23%, 16% and 10.6% of the tRNA signal, respectively. These results demonstrate that the biosensor is capable of effectively distinguishing the target tRNA from non-complementary or mismatched sequences. The high selectivity can be attributed to the molecular recognition capability of the polyA-DNA probe, which enables efficient hybridization with fully complementary targets while discriminating against mismatched bases. 3.7. Anti-interference and detection of RNA targets in human serum Anti-interference capability is a critical performance indicator for assessing the applicability of biosensors in real biological matrices. To systematically evaluate the anti-interference performance of the developed sensor in complex serum environments, normal human serum (NHS) at varying dilution ratios (5%, 10%, 15%, 20%, and 30%) was used as the test medium, while phosphate-buffered saline (PBS) served as the control. The detection signals for tRNA were measured using SWV. As shown in Fig. S3 A , the recovery rates, calculated by comparing the current responses at each serum concentration to that of the PBS control, were 96.5% (5% NHS), 83.8% (10% NHS), 76.1% (15% NHS), 53.99% (20% NHS), and 51.6% (30% NHS), respectively. These results indicate that the sensor maintains excellent anti-interference performance at serum concentrations up to 15%, with signal recovery rates exceeding 70%. Even under higher serum concentrations (15–30%), the detection signal remained at approximately 50%, demonstrating good tolerance to matrix interference and supporting its potential use in clinical sample analysis. To further assess the sensor’s accuracy in complex biological matrices, the standard addition method was employed for the quantitative detection of target tRNA in serum samples. NHS was diluted five-fold (5%, v/v) before analysis to mitigate matrix effects. The endogenous tRNA background level was first measured, followed by spike-recovery experiments in which known concentrations of tRNA standards (1 fM, 0.1 pM, and 10 pM) were added to the serum samples. As summarized in Table S2 , the recovery rates ranged from 93.1–98.2%, meeting the acceptable criteria for bioanalytical methods (typically 80–120%). These results confirm that the biosensor maintains high detection accuracy and reliability even in complex serum matrices, highlighting its strong potential for practical application in clinical tRNA diagnostics. 4. Conclusion This study reports the development of a tRNA electrochemical biosensor based on α-Fe 2 O 3 /Ti 3 C 2 T x MXene photo-Fenton catalysts for RAFT polymerization. From a material design perspective, the construction of α-Fe 2 O 3 /Ti 3 C 2 T x MXene heterostructures significantly enhanced photocatalytic activity under visible light, thereby generating robust electrochemical signals for sensing. The biosensor demonstrated outstanding analytical performance, including an exceptionally broad linear range spanning five orders of magnitude (0.01 fM-10 pM), an ultralow detection limit of 4.44 aM for tRNA, and a selectivity coefficient above 90% in complex biological matrices. These results underscore the potential of this α-Fe 2 O 3 /Ti 3 C 2 T x MXene -based platform for translation into clinical diagnostics, offering a promising strategy for ultrasensitive and reliable detection of disease-associated biomarkers. Declarations Credit author statement Thao Thi Nguyen: Original Draft, Investigation, Methodology, Formal analysis. Zhidan Tian: Review & Editing. Weibo Huang: Review & Editing. Qinyuan Xu: Data curation. Shuaibing Yu: Data curation. Gengzhi Sun: Review & Editing. Jinming Kong: Conceptualization, Review & Editing, Funding acquisition, Resources. Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Data availability Data will be made available on request. Funding This work was financially by the National Natural Science Foundation of China (Nos. 21974068). References Qiang F, Qiushi R, Thomas G, Amin R, Junwang T, Greg G (2017)Development of a robust PET-RAFT polymerization using graphitic carbon nitride (g-C 3 N 4 ). Macromolecules 50: 7509-7516. https://doi.org/10.1021/acs.macromol.7b01651 Yiming H, Yifan Z, Eilaf E (2018) Semiconductor quantum dots as photocatalysts for controlled light-mediated radical polymerization. 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Journal of Alloys and Compounds 941: 168920. https://doi.org/10.1016/j.jallcom.2023.168920 Shuaibing Y, Jian Z, Yaodong H, Lianzhi L, Jinming K, Xueji Z (2023) Ultrasensitive detection of miRNA-21 by click chemistry and fluorescein-mediated photo-ATRP signal amplification: Analytica Chimica Acta 1277: 341661. https://doi.org/10.1016/j.aca.2023.341661 Haobo S, Jingliang L, Jinming K, Jian Z, and Xueji Z (2023) Ultrasensitive miRNA-21 biosensor based on Zn (TCPP) PET-RAFT polymerization signal amplification and multiple logic gate molecular recognition. ACS Applied Materials & Interfaces 15: 17716-17725. https://doi.org/10.1021/acsami.3c02428 Ma L, Kang L, Sun Y, Liu J, Yang H, Miao M (2023) Nitrogen-doped graphene quantum dots as electrochemiluminescence-emitting species for sensitive detection of KRAS G12C mutation via PET-RAFT. Chemistry-a European Journal 29: e202301602. https://doi.org/10.1002/chem.202301602 Chen X, Huang C, Zhang J, Hu Q, Wang D, You Q, Guo Y, Chen H, Xu J, and Hu M (2024) Mini crRNA-mediated CRISPR/Cas12a system (MCM-CRISPR/Cas12a) and its application in RNA detection. Talanta 268: 125350. https://doi.org/10.1016/j.talanta.2023.125350 Li J, Lei P, Ding S, Zhang Y, Yang J, Cheng Q, Yan Y (2016) A novel surface plasmon resonance biosensor for enzyme-free and highly sensitive detection of microRNA based on multi component nucleic acid enzyme (MNAzyme)-mediated catalyzed hairpin assembly. Biosensors & Bioelectronics 80: 98-104. https://doi.org/10.1016/j.bios.2015.09.069 Additional Declarations No competing interests reported. Supplementary Files SupplementaryData.docx GraphicalAbstracts.docx Cite Share Download PDF Status: Published Journal Publication published 22 Jan, 2026 Read the published version in Microchimica Acta → Version 1 posted Editorial decision: Revision requested 25 Sep, 2025 Reviews received at journal 21 Sep, 2025 Reviews received at journal 19 Sep, 2025 Reviews received at journal 16 Sep, 2025 Reviews received at journal 16 Sep, 2025 Reviews received at journal 15 Sep, 2025 Reviewers agreed at journal 11 Sep, 2025 Reviewers agreed at journal 09 Sep, 2025 Reviewers agreed at journal 07 Sep, 2025 Reviewers agreed at journal 06 Sep, 2025 Reviewers agreed at journal 06 Sep, 2025 Reviewers invited by journal 06 Sep, 2025 Editor assigned by journal 01 Sep, 2025 Submission checks completed at journal 31 Aug, 2025 First submitted to journal 28 Aug, 2025 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. 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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-7482836","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":513897517,"identity":"f77cfaad-e1ad-4907-a360-3d8d7922b79e","order_by":0,"name":"Thao Thi Nguyen","email":"","orcid":"","institution":"Nanjing University of Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"Thao","middleName":"Thi","lastName":"Nguyen","suffix":""},{"id":513897518,"identity":"a293074f-abb3-4225-92f4-debb55f0542e","order_by":1,"name":"Zhidan Tian","email":"","orcid":"","institution":"Nanjing Medical University","correspondingAuthor":false,"prefix":"","firstName":"Zhidan","middleName":"","lastName":"Tian","suffix":""},{"id":513897522,"identity":"6515908e-0a10-42ce-916a-b4048a3366c9","order_by":2,"name":"Weibo Huang","email":"","orcid":"","institution":"Nanjing University of Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"Weibo","middleName":"","lastName":"Huang","suffix":""},{"id":513897525,"identity":"f019aad9-50dd-4b3b-b159-b4187b7eaf31","order_by":3,"name":"Qinyuan Xu","email":"","orcid":"","institution":"Nanjing University of Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"Qinyuan","middleName":"","lastName":"Xu","suffix":""},{"id":513897526,"identity":"51ebedb0-c40a-4f9f-b073-1e965f1ea7cf","order_by":4,"name":"Shuaibing Yu","email":"","orcid":"","institution":"Nanjing University of Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"Shuaibing","middleName":"","lastName":"Yu","suffix":""},{"id":513897528,"identity":"42a0aff5-d030-46bb-aef9-bd7d8ec54e69","order_by":5,"name":"Gengzhi Sun","email":"","orcid":"","institution":"Nanjing Tech University","correspondingAuthor":false,"prefix":"","firstName":"Gengzhi","middleName":"","lastName":"Sun","suffix":""},{"id":513897529,"identity":"d460f5b8-457e-40f1-a73a-c3b237b4e52e","order_by":6,"name":"Jinming Kong","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAzklEQVRIiWNgGAWjYBACCRDxgYENwuMhVgvjDJAWNlK0MINVEq1Fsr338GvbHXyJ8+c3MD5428Ygb05IizTPuTTr3DNsiRuOMTAbzm1jMNzZQECLnESOmXFuG1ALGwObNG8bQ4LBAWK0WAK1zG9jYP9NlBZpiRzjx4xALQ3HGNiYidIi2XPGjLG3jc14w7HEZsk55yQMNxDSInG8x/jDz7ZjsvObDx/88KbMRp6gLUDABoybY0CasYEBmhoIAuYPDAw1RKkcBaNgFIyCEQoAfbM6CVBbk70AAAAASUVORK5CYII=","orcid":"","institution":"Nanjing University of Science and Technology","correspondingAuthor":true,"prefix":"","firstName":"Jinming","middleName":"","lastName":"Kong","suffix":""}],"badges":[],"createdAt":"2025-08-28 18:53:16","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7482836/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7482836/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s00604-025-07669-x","type":"published","date":"2026-01-22T15:58:52+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":91161273,"identity":"4e1c74bb-e20c-494e-aa92-977aa87f2ef3","added_by":"auto","created_at":"2025-09-12 09:19:02","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":198248,"visible":true,"origin":"","legend":"\u003cp\u003e(A) Schematic diagram of the synthesis process of α-Fe\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e3\u003c/sub\u003e/Ti\u003csub\u003e3\u003c/sub\u003eC\u003csub\u003e2\u003c/sub\u003eT\u003csub\u003ex\u003c/sub\u003e MXene nanocomposites. (B) tRNA electrochemical biosensor based on α-Fe\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e3\u003c/sub\u003e/Ti\u003csub\u003e3\u003c/sub\u003eC\u003csub\u003e2\u003c/sub\u003eT\u003csub\u003ex\u003c/sub\u003e MXene photo-Fenton catalysts for RAFT polymerization.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-7482836/v1/f56ec82601b6fd90ed0fd3b6.png"},{"id":91162977,"identity":"11b12d09-399f-43e1-9122-2310e5c1c14d","added_by":"auto","created_at":"2025-09-12 09:43:02","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":537837,"visible":true,"origin":"","legend":"\u003cp\u003eSEM images of (A) Ti\u003csub\u003e3\u003c/sub\u003eC\u003csub\u003e2\u003c/sub\u003eT\u003csub\u003ex\u003c/sub\u003eMXene, (B) α-Fe\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e3\u003c/sub\u003e, (C) α-Fe\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e3\u003c/sub\u003e/Ti\u003csub\u003e3\u003c/sub\u003eC\u003csub\u003e2\u003c/sub\u003eT\u003csub\u003ex\u003c/sub\u003eMXene, (D-I) element mapping spectra of α-Fe\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e3\u003c/sub\u003e/ Ti\u003csub\u003e3\u003c/sub\u003eC\u003csub\u003e2\u003c/sub\u003eT\u003csub\u003ex\u003c/sub\u003eMXene composites\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-7482836/v1/90e8205ac2a9311f1e625c22.png"},{"id":91161274,"identity":"bd478714-a184-4da7-a151-8009c75a9ef1","added_by":"auto","created_at":"2025-09-12 09:19:02","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":120656,"visible":true,"origin":"","legend":"\u003cp\u003e(A) UV–vis diffuse reflectance spectraand determination of the indirect inter-band transition energies. (B) XPS valence band spectra. (C) Schottky-scheme charge transfer mechanism.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-7482836/v1/3a71f12f1f4e46a33f8ab6b4.png"},{"id":91161275,"identity":"b3b38a6b-020c-4a72-b287-0610a23b5fe0","added_by":"auto","created_at":"2025-09-12 09:19:02","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":71183,"visible":true,"origin":"","legend":"\u003cp\u003eThe •OH radicals generated in this process initiate the RAFT polymerization reaction\u003c/p\u003e\n\u003cp\u003eInitiation (I): The hydroxyl radical (•OH) attacks the vinyl monomer (e.g., MMA or NIPAM), cleaving the C=C double bond via a radical addition mechanism and generating a carbon-centered propagating radical (P\u003csub\u003en\u003c/sub\u003e•).\u003c/p\u003e\n\u003cp\u003ePropagation and Chain Transfer (II): The propagating radical (P\u003csub\u003en\u003c/sub\u003e•) rapidly reacts with a chain transfer agent (CTA), such as the dithioester-functionalized CPAD. This reaction forms a transient radical intermediate, which subsequently undergoes fragmentation to yield a dormant RAFT agent (3) and a new active radical (4).\u003c/p\u003e\n\u003cp\u003eReinitiation and Chain Growth (III): The new radical species (4) reinitiates polymerization by adding to another monomer molecule, continuing the chain propagation and generating a new propagating radical (P\u003csub\u003em\u003c/sub\u003e•).\u003c/p\u003e\n\u003cp\u003eReversible Chain Exchange (IV): The propagating radical (P\u003csub\u003em\u003c/sub\u003e•) can reversibly react with dormant RAFT agent (3) to form another intermediate radical (5), which can subsequently fragment to regenerate a different dormant species (6) and another propagating radical (P\u003csub\u003en\u003c/sub\u003e•), thereby maintaining the dynamic equilibrium between active and dormant chains.\u003c/p\u003e\n\u003cp\u003eTermination (V): Although RAFT polymerization is designed to minimize termination, bimolecular radical recombination between propagating chains (P\u003csub\u003em\u003c/sub\u003e• and P\u003csub\u003en\u003c/sub\u003e•) can still occur, resulting in the formation of a terminated, non-propagating polymer chain (P\u003csub\u003em+n\u003c/sub\u003e). While such termination events reduce livingness, they are typically suppressed to low levels under optimized RAFT conditions, allowing most polymer chains to retain functional end groups specifically, a dithioester moiety and a reinitiating site facilitating further chain extension or post-polymerization modifications.\u003c/p\u003e\n\u003cp\u003eThis synergistic system integrating a photoresponsive heterojunction for efficient •OH generation with a surface-bound RAFT agent enables spatially controlled, radical-mediated polymer growth with high efficiency and structural fidelity, offering significant advantages in light-triggered surface-initiated polymerizations.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-7482836/v1/7ae79789be5159f3b728010d.png"},{"id":91161282,"identity":"eb2003d2-9b44-4396-8f58-0b7597d20ab8","added_by":"auto","created_at":"2025-09-12 09:19:02","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":427773,"visible":true,"origin":"","legend":"\u003cp\u003e(A) SWV of gold electrode surface with different modifications. (B) Nyquist diagram corresponding to gold electrodes with different modifications. (C) The CV of modified gold electrode scan rate from 50–450 mV/s. (D) The linear correlation between cathodic and anodic peak currents with scan rate.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-7482836/v1/41968e483bca33b559d48bf5.png"},{"id":91162474,"identity":"95e94d49-b201-425c-adc9-a71fb367a06f","added_by":"auto","created_at":"2025-09-12 09:35:02","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":334986,"visible":true,"origin":"","legend":"\u003cp\u003e\u0026nbsp;(A) Influence of the ratio of α-Fe\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e3\u003c/sub\u003e to Ti\u003csub\u003e3\u003c/sub\u003eC\u003csub\u003e2\u003c/sub\u003eT\u003csub\u003ex\u003c/sub\u003eMXene. (B) Effect of DMF to water ratio. (C) Effect of polymerization time on RAFT reaction\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-7482836/v1/5a33f46c5899e92e7ff45555.png"},{"id":91161280,"identity":"270a1411-1fb3-47e8-a6e1-24c75a42a0d7","added_by":"auto","created_at":"2025-09-12 09:19:02","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":380305,"visible":true,"origin":"","legend":"\u003cp\u003e(A) SWV curves of different target RNA concentrations to 0.01fM for10 pM) in the biosensor. (B) Linear relationship between tRNA concentration and current intensity\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-7482836/v1/8892c1b8cf49ae6746b6c60d.png"},{"id":101152054,"identity":"3f2de9ec-a963-4097-b5a3-ce331dc14ccf","added_by":"auto","created_at":"2026-01-26 16:09:31","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2853699,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7482836/v1/661505e8-d849-43c5-a764-eee90d026951.pdf"},{"id":91162049,"identity":"29657e77-acce-47d8-805a-7d3cedd95c1c","added_by":"auto","created_at":"2025-09-12 09:27:02","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":1409637,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryData.docx","url":"https://assets-eu.researchsquare.com/files/rs-7482836/v1/9afd72ea176ac7c7aa265afb.docx"},{"id":91162472,"identity":"2428a2cc-ef3e-4604-83a9-f153488b5ec8","added_by":"auto","created_at":"2025-09-12 09:35:02","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":206721,"visible":true,"origin":"","legend":"","description":"","filename":"GraphicalAbstracts.docx","url":"https://assets-eu.researchsquare.com/files/rs-7482836/v1/955747d7553708e044a2b5bc.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"α-Fe 2 O 3 /Ti 3 C 2 T x MXene Heterostructures as Photo-Fenton Catalysts Driving RAFT Polymerization for Ultrasensitive Electrochemical microRNA Sensing","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eCatalysts play a critical role in reversible addition-fragmentation chain-transfer (RAFT) polymerization by facilitating precise control over polymer chain growth and molecular architecture, essential for constructing functional polymeric materials in biosensing applications. However, conventional RAFT polymerization is often hindered by oxygen sensitivity and limited spatiotemporal control, which pose significant challenges for its implementation in biological and in vivo environments. To address these challenges, various strategies have been proposed, including the development of semiconducting nanoparticle photopolymerization systems \u003csup\u003e[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]\u003c/sup\u003e, enhancing oxygen tolerance \u003csup\u003e[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]\u003c/sup\u003e, and conducting polymerization under visible and even near-infrared light \u003csup\u003e[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]\u003c/sup\u003e. For instance, Erika et al. demonstrated the use of urea-based graphitic nitride (U-g-C\u003csub\u003e3\u003c/sub\u003eN\u003csub\u003e4\u003c/sub\u003e) for heterogeneous PET-RAFT polymerization of methyl methacrylate (MMA) without prior deoxygenation of the reaction medium \u003csup\u003e[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]\u003c/sup\u003e. Similarly, Kaya et al. employed mesoporous graphitic carbon nitride (mpg-C\u003csub\u003e3\u003c/sub\u003eN\u003csub\u003e4\u003c/sub\u003e) with FeCl\u003csub\u003e3\u003c/sub\u003e\u0026middot;6H\u003csub\u003e2\u003c/sub\u003eO to establish an oxygen-tolerant visible light photo initiating system. This system works through in-situ generation of Fenton reagents using water and oxygen for free radical polymerization \u003csup\u003e[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]\u003c/sup\u003e. In comparison to these photocatalytic systems, α-Fe\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e3\u003c/sub\u003e and Ti\u003csub\u003e3\u003c/sub\u003eC\u003csub\u003e2\u003c/sub\u003eT\u003csub\u003ex\u003c/sub\u003e MXene is considered a competitive candidate due to the environmentally benign nature of hematite (α-Fe\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e3\u003c/sub\u003e), an n-type semiconductor with a bandgap of 2.1 eV. This moderate band gap enables efficient absorption of visible light, making α-Fe\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e3\u003c/sub\u003e suitable for solar-driven photocatalytic applications \u003csup\u003e[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]\u003c/sup\u003e. Moreover, Ti\u003csub\u003e3\u003c/sub\u003eC\u003csub\u003e2\u003c/sub\u003eT\u003csub\u003ex\u003c/sub\u003e MXene, with its oxidized surface groups, has proven effective as a cocatalyst for photocatalytic reactions under visible light irradiation \u003csup\u003e[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]\u003c/sup\u003e. MXene, obtained from the MAX phase, is a two-dimensional (2D) layered material consisting of transition metal carbides, nitrides, and carbonitrides. It possesses numerous hydrophilic surface functionalities (-OH and -O), excellent electrochemical properties, a large specific surface area, and outstanding structural/chemical stability \u003csup\u003e[\u003cspan additionalcitationids=\"CR13 CR14\" citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]\u003c/sup\u003e. Introducing Ti\u003csub\u003e3\u003c/sub\u003eC\u003csub\u003e2\u003c/sub\u003eT\u003csub\u003ex\u003c/sub\u003e MXene into the α-Fe\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e3\u003c/sub\u003e matrix significantly increases its particular surface area \u003csup\u003e[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]\u003c/sup\u003e, broadens the light absorption spectrum \u003csup\u003e[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]\u003c/sup\u003e, and enhances the photochemical cleavage efficiency as well as the transport capacity of photogenerated carriers (electrons and holes) \u003csup\u003e[\u003cspan additionalcitationids=\"CR20\" citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eTherefore, in this study, α-Fe\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e3\u003c/sub\u003e and Ti\u003csub\u003e3\u003c/sub\u003eC\u003csub\u003e2\u003c/sub\u003eT\u003csub\u003ex\u003c/sub\u003e MXene heterostructures were synthesized and employed as photo-Fenton catalysts to promote RAFT polymerization. To exploit the potential of this catalytic system, it was integrated into a biosensing platform based on a RAFT-mediated signal amplification strategy, enabling ultrasensitive detection of the cancer biomarker microRNA-144. The biosensor was constructed on a gold electrode by immobilizing a polyA-DNA probe through the strong affinity between polyadenine sequences and the Au surface \u003csup\u003e[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]\u003c/sup\u003e. This interaction formed stable adenine-Au anchor points, enabling the DNA probe to adopt a longitudinal orientation perpendicular to the electrode surface. Such a spatial arrangement was crucial for reducing steric hindrance and significantly improving the hybridization efficiency with the target miRNA. Upon hybridization of polyA-DNA with miRNA, a non-hybridized nucleotide overhang was exposed. This region enabled site-specific binding of an amino-modified DNA sequence (NH\u003csub\u003e2\u003c/sub\u003e-DNA). The amino terminus of the NH\u003csub\u003e2\u003c/sub\u003e-DNA was covalently conjugated with 4-cyano-4-(thiobenzoyl) valeric acid (CPAD), a thiocarbonylthio-based RAFT agent, thereby generating a surface-immobilized RAFT initiator on the gold electrode. This initiator enabled the visible-light-driven polymerization of poly(ferrocene), catalyzed by the α-Fe\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e3\u003c/sub\u003e/Ti\u003csub\u003e3\u003c/sub\u003eC\u003csub\u003e2\u003c/sub\u003eT\u003csub\u003ex\u003c/sub\u003e MXene photo-Fenton heterostructure. The electroactive poly(ferrocene) chains produced were quantitatively analyzed via square-wave voltammetry (SWV), yielding a strongly amplified signal that displayed a linear relationship with the target miRNA concentration \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e\u003cb\u003e)\u003c/b\u003e.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1. Materials\u003c/h2\u003e\u003cp\u003eThe relevant reagents and instruments can be found in the Supplementary Material.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e2.2. Fabrication of Urchin-like α-Fe\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e3\u003c/sub\u003e\u003c/h2\u003e\u003cp\u003eThe α-Fe\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e3\u003c/sub\u003e material was synthesized through hydrothermal method and thermal annealing. Initially, 4 mmol/L of iron(III) chloride hexahydrate (FeCl\u003csub\u003e3\u003c/sub\u003e\u0026middot;6H\u003csub\u003e2\u003c/sub\u003eO) and 3 mmol/L of sodium sulfate (Na\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e) were dissolved in 80 mL of deionized water under vigorous stirring to ensure complete homogenization. The resulting solution was then transferred to a 100 mL Teflon-lined stainless steel autoclave and subjected to hydrothermal treatment at 120\u0026deg;C for 14 h. Upon completion, the autoclave was allowed to cool naturally to room temperature. The resulting yellow FeOOH precursor was collected, washed three times with deionized water to eliminate residual ions, and dried in an oven at 80\u0026deg;C for 4 h. The dried powder was subsequently placed in a nitrogen atmosphere, heated to 450\u0026deg;C at a ramp rate of 5\u0026deg;C/min, and annealed for 2 h to obtain the final α-Fe\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e3\u003c/sub\u003e product (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003e2.3. Fabrication of Ti\u003csub\u003e3\u003c/sub\u003eC\u003csub\u003e2\u003c/sub\u003eT\u003csub\u003ex\u003c/sub\u003e MXene Nanosheets\u003c/h2\u003e\u003cp\u003eTi\u003csub\u003e3\u003c/sub\u003eC\u003csub\u003e2\u003c/sub\u003eT\u003csub\u003ex\u003c/sub\u003e MXene nanosheets were synthesized via an in situ hydrofluoric acid (HF) generation method combined with an ultrasound-assisted etching process. Initially, 2.5 g of lithium fluoride (LiF) powder was dispersed in 50 mL of 9.0 M hydrochloric acid (HCl) and stirred for 30 min to generate HF in situ. Subsequently, Ti\u003csub\u003e3\u003c/sub\u003eAlC\u003csub\u003e2\u003c/sub\u003e powder was gradually added to the solution and stirred vigorously at 35\u0026deg;C for 24 h, facilitating the selective etching of the aluminum (Al) layer. Upon completion of the reaction, the resulting suspension was washed repeatedly with deionized water and subjected to successive centrifugation cycles until the pH of the supernatant approached neutrality. The resulting black precipitate was then ultrasonicated for 1.5 h to delaminate the multilayered structure, yielding monolayer Ti\u003csub\u003e3\u003c/sub\u003eC\u003csub\u003e2\u003c/sub\u003eT\u003csub\u003ex\u003c/sub\u003e MXene nanosheets. Finally, product was subjected to high-speed centrifugation at 4500 rpm for 1 h, followed by redispersion in deionized water to obtain a monolayer Ti\u003csub\u003e3\u003c/sub\u003eC\u003csub\u003e2\u003c/sub\u003eT\u003csub\u003ex\u003c/sub\u003e MXene dispersion with a final concentration of 10 mg/mL (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003e2.4. Fabrication of α-Fe\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e3\u003c/sub\u003e/Ti\u003csub\u003e3\u003c/sub\u003eC\u003csub\u003e2\u003c/sub\u003eT\u003csub\u003ex\u003c/sub\u003e MXene\u003c/h2\u003e\u003cp\u003eThe α-Fe\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e3\u003c/sub\u003e/Ti\u003csub\u003e3\u003c/sub\u003eC\u003csub\u003e2\u003c/sub\u003eT\u003csub\u003ex\u003c/sub\u003e MXene composites were synthesized via an electrostatic self-assembly strategy, wherein the assembly was driven by electrostatic interactions between α-Fe\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e3\u003c/sub\u003e and Ti\u003csub\u003e3\u003c/sub\u003eC\u003csub\u003e2\u003c/sub\u003eT\u003csub\u003ex\u003c/sub\u003e MXene nanosheets. Specifically, 100 mg of α-Fe\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e3\u003c/sub\u003e was uniformly dispersed in 100 mL of deionized water and subjected to combined ultrasonic treatment and magnetic stirring for 20 min to ensure thorough dispersion of the nanoparticles. Subsequently, 10\u0026micro;L Ti\u003csub\u003e3\u003c/sub\u003eC\u003csub\u003e2\u003c/sub\u003eT\u003csub\u003ex\u003c/sub\u003e MXene 10 mg/mL was added dropwise to the α-Fe\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e3\u003c/sub\u003e suspension under continuous magnetic stirring. This self-assembly process was maintained for 4 h to facilitate uniform interaction between the two components. The α-Fe\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e3\u003c/sub\u003e/Ti\u003csub\u003e3\u003c/sub\u003eC\u003csub\u003e2\u003c/sub\u003eT\u003csub\u003ex\u003c/sub\u003e MXene composites were isolated via centrifugal separation and subsequently dried in a vacuum oven (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\u003ch2\u003e2.5. Preparation of Electrochemical Biosensor\u003c/h2\u003e\u003cp\u003eThe miRNA detection biosensor was fabricated using gold electrodes, with each modification step carefully optimized to ensure high sensitivity and specificity. Initially, the Au electrode surface was mechanically polished using 0.05 \u0026micro;m alumina slurry to achieve a smooth and contaminant-free surface. The electrodes were then sequentially rinsed three times each with ultrapure water and ethanol, followed by drying under a stream of high-purity nitrogen gas. Subsequently, 10 \u0026micro;L of polyA-DNA probe solution was drop-cast onto the electrode surface and incubated at room temperature for 2 h, promoting adsorption through the strong affinity between Au and the polyA moiety. Following this, 10 \u0026micro;L of tRNA was applied to hybridize with the polyA-DNA probe and incubated for an additional 2 h. To further extend the hybridization, 10 \u0026micro;L of NH\u003csub\u003e2\u003c/sub\u003e-DNA was introduced to hybridize with the remaining unpaired region of the tRNA. Meanwhile, the carboxyl group of the RAFT initiator, 2 mM CPAD, was activated using a 1:1:1 molar ratio solution of 2 mM carbodiimide hydrochloride (EDC) and 2 mM N-hydroxysuccinimide (NHS). The modified Au electrode was immersed in 300 \u0026micro;L of the activated CPAD solution and incubated for 2 h to facilitate covalent coupling. Subsequently, the electrode was transferred into 300 \u0026micro;L of an RAFT polymerization mixture containing 2 mM fluoromethyl methacrylate (FMMA), 0.05 mM hydrogen peroxide (H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e), and 0.05 mg/mL α-Fe\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e3\u003c/sub\u003e/Ti\u003csub\u003e3\u003c/sub\u003eC\u003csub\u003e2\u003c/sub\u003eT\u003csub\u003ex\u003c/sub\u003e MXene. The polymerization was initiated under continuous visible light irradiation (400\u0026ndash;600 nm) for 3 h, enabling the growth of functional polymer chains on the electrode surface. All modification steps were conducted at room temperature. After each step, the electrodes were thoroughly rinsed with 0.1 M phosphate-buffered saline (PBS, pH 7.4) to eliminate unbound or unreacted species. Finally, the electrochemical performance of the modified Au electrodes was evaluated by SWV within the potential range of 0-0.7 V (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB).\u003c/p\u003e\u003c/div\u003e"},{"header":"3. Results and Discussion","content":"\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\n \u003ch2\u003e3.1. Structural and Morphological Characterization of \u0026alpha;-Fe\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e3\u003c/sub\u003e/Ti\u003csub\u003e3\u003c/sub\u003eC\u003csub\u003e2\u003c/sub\u003eT\u003csub\u003ex\u003c/sub\u003e MXene Catalysts\u003c/h2\u003e\n \u003cp\u003eAs shown in Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e, scanning electron microscopy (SEM) was employed to examine the morphologies of Ti\u003csub\u003e3\u003c/sub\u003eC\u003csub\u003e2\u003c/sub\u003eT\u003csub\u003ex\u003c/sub\u003e MXene, \u0026alpha;-Fe\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e3\u003c/sub\u003e, and their composite \u0026alpha;-Fe\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e3\u003c/sub\u003e/Ti\u003csub\u003e3\u003c/sub\u003eC\u003csub\u003e2\u003c/sub\u003eT\u003csub\u003ex\u003c/sub\u003e MXene. In Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eA, Ti\u003csub\u003e3\u003c/sub\u003eC\u003csub\u003e2\u003c/sub\u003eT\u003csub\u003ex\u003c/sub\u003e MXene displays a characteristic accordion-like morphology with a multilayered architecture, confirming the successful etching of the Ti\u003csub\u003e3\u003c/sub\u003eAlC\u003csub\u003e2\u003c/sub\u003e MAX phase. This morphology significantly increases the specific surface area, enhancing interfacial interactions and providing a favorable structural basis for composite integration and performance enhancement. Figure \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eB reveals that \u0026alpha;-Fe\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e3\u003c/sub\u003e exhibits a distinct urchin-like microstructure, comprising radially aligned needle-like features emanating from a central core. This hierarchical structure creates abundant void space, which facilitates rapid electrolyte ion transport. Such a feature is beneficial for both catalytic and electrochemical applications. In Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eC, the \u0026alpha;-Fe\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e3\u003c/sub\u003e/Ti\u003csub\u003e3\u003c/sub\u003eC\u003csub\u003e2\u003c/sub\u003eT\u003csub\u003ex\u003c/sub\u003e MXene composite exhibits a well-integrated structure wherein the urchin-like \u0026alpha;-Fe\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e3\u003c/sub\u003e particles are uniformly distributed and anchored within the Ti\u003csub\u003e3\u003c/sub\u003eC\u003csub\u003e2\u003c/sub\u003eT\u003csub\u003ex\u003c/sub\u003e MXene nanosheet matrix. To further confirm the compositional uniformity, elemental mapping was performed (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eD-I), revealing the presence and homogeneous distribution of C, Ti, O, Fe, and F throughout the composite. This uniform elemental dispersion validates the successful synthesis of the \u0026alpha;-Fe\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e3\u003c/sub\u003e/Ti\u003csub\u003e3\u003c/sub\u003eC\u003csub\u003e2\u003c/sub\u003eT\u003csub\u003ex\u003c/sub\u003e MXene composite and suggests robust interfacial contact between the two components, which is critical for maintaining structural integrity and optimizing multifunctional performance.\u003c/p\u003e\n \u003cp\u003eThe phase structures of \u0026alpha;-Fe\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e3\u003c/sub\u003e, Ti\u003csub\u003e3\u003c/sub\u003eC\u003csub\u003e2\u003c/sub\u003eT\u003csub\u003ex\u003c/sub\u003e MXene, and the \u0026alpha;-Fe\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e3\u003c/sub\u003e/Ti\u003csub\u003e3\u003c/sub\u003eC\u003csub\u003e2\u003c/sub\u003eT\u003csub\u003ex\u003c/sub\u003e MXene composites were analyzed by X-ray diffraction (XRD), as presented in \u003cstrong\u003eFig. \u003cspan class=\"InternalRef\"\u003eS1\u003c/span\u003e (A)\u003c/strong\u003e. The diffraction peaks of Ti\u003csub\u003e3\u003c/sub\u003eC\u003csub\u003e2\u003c/sub\u003eT\u003csub\u003ex\u003c/sub\u003e MXene appeared at 2\u0026theta;\u0026thinsp;=\u0026thinsp;8.4\u0026deg;, 18.1\u0026deg;, 27.2\u0026deg;, and 61.0\u0026deg;, consistent with previously reported data \u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e24\u003c/span\u003e]\u003c/sup\u003e, thereby confirming the successful synthesis of Ti\u003csub\u003e3\u003c/sub\u003eC\u003csub\u003e2\u003c/sub\u003eT\u003csub\u003ex\u003c/sub\u003e MXene. Similarly, the XRD pattern of pure \u0026alpha;-Fe\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e3\u003c/sub\u003e nanosheets exhibited distinct peaks at 24.1\u0026deg;, 33.1\u0026deg;, 35.6\u0026deg;, 40.9\u0026deg;, 49.5\u0026deg;, 54.1\u0026deg;, 57.5\u0026deg;, 62.5\u0026deg;, and 63.9\u0026deg;, corresponding to the characteristic reflections of the \u0026alpha;-Fe\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e3\u003c/sub\u003e phase (JCPDS No. 00-024-0072) \u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e25\u003c/span\u003e]\u003c/sup\u003e. In the XRD patterns of the \u0026alpha;-Fe\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e3\u003c/sub\u003e/Ti\u003csub\u003e3\u003c/sub\u003eC\u003csub\u003e2\u003c/sub\u003eT\u003csub\u003ex\u003c/sub\u003e MXene composites, the diffraction peaks were predominantly attributed to \u0026alpha;-Fe\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e3\u003c/sub\u003e, closely matching the hematite phase (JCPDS No. 00-024-0072), which is primarily due to the high crystallinity of \u0026alpha;-Fe\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e3\u003c/sub\u003e resulting from thermal treatment and the presence of intense diffraction peaks. The characteristic peak of Ti\u003csub\u003e3\u003c/sub\u003eC\u003csub\u003e2\u003c/sub\u003eT\u003csub\u003ex\u003c/sub\u003e MXene at 6.58\u0026deg; exhibited significantly reduced intensity and was nearly undetectable. However, the typical diffraction peak corresponding to the (110) plane of Ti\u003csub\u003e3\u003c/sub\u003eC\u003csub\u003e2\u003c/sub\u003eT\u003csub\u003ex\u003c/sub\u003e MXene remained prominent in the composite, indicating that the Ti\u003csub\u003e3\u003c/sub\u003eC\u003csub\u003e2\u003c/sub\u003eT\u003csub\u003ex\u003c/sub\u003e MXene nanosheets were successfully coated on the surface of the urchin-shaped \u0026alpha;-Fe\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e3\u003c/sub\u003e structures. This coating effectively prevented the restacking of Ti\u003csub\u003e3\u003c/sub\u003eC\u003csub\u003e2\u003c/sub\u003eT\u003csub\u003ex\u003c/sub\u003e MXene layers and preserved the original layered structure of the MXene. The retention of distinct crystalline phases of both \u0026alpha;-Fe\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e3\u003c/sub\u003e and Ti\u003csub\u003e3\u003c/sub\u003eC\u003csub\u003e2\u003c/sub\u003eT\u003csub\u003ex\u003c/sub\u003e MXene in the composite confirms the feasibility and efficiency of the adopted synthesis method.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\n \u003ch2\u003e3.2. \u0026alpha;-Fe\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e3\u003c/sub\u003e/Ti\u003csub\u003e3\u003c/sub\u003eC\u003csub\u003e2\u003c/sub\u003eT\u003csub\u003ex\u003c/sub\u003e MXene Heterostructures as Photo-Fenton Catalysts Driving RAFT Polymerization\u003c/h2\u003e\n \u003cp\u003eThe photo-Fenton mechanism driving the RAFT polymerization is intrinsically associated with charge-transfer dynamics within the \u0026alpha;-Fe\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e3\u003c/sub\u003e/Ti\u003csub\u003e3\u003c/sub\u003eC\u003csub\u003e2\u003c/sub\u003eTₓ MXene heterostructure under visible-light irradiation. The charge-transfer pathway is governed by the alignment of the band structure and Fermi level, which determines the separation and migration efficiency of photogenerated electrons, sustains the Fe\u003csup\u003e3+\u003c/sup\u003e/Fe\u003csup\u003e2+\u003c/sup\u003e redox cycling, and thereby promotes the generation of highly oxidative hydroxyl radicals (\u0026bull;OH).\u003c/p\u003e\n \u003cp\u003eAs shown in Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eA, the UV-vis diffuse reflectance spectrum (DRS) of \u0026alpha;-Fe\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e3\u003c/sub\u003e was used to estimate its optical band gap, which was calculated to be 2.09 eV using the Kubelka-Munk function. Additionally, the valence band (VB) energy level of \u0026alpha;-Fe\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e3\u003c/sub\u003e was determined through XPS, revealing a VB maximum at +\u0026thinsp;2.21 eV relative to the normal hydrogen electrode (NHE), in Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eB. Consequently, the conduction band (CB) potential of \u0026alpha;-Fe\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e3\u003c/sub\u003e was calculated to be 0.12 eV using Eq. (1):\u003c/p\u003e\n \u003cdiv id=\"Equa\" class=\"Equation\"\u003e\n \u003cdiv class=\"mathdisplay\" id=\"FileID_Equa\" name=\"EquationSource\"\u003e$$\\:{E}_{CB}(vs.\\:NHE)\\:=\\:{E}_{VB}\\:(vs.\\:NHE)\\:-\\:{E}_{g}$$\u003c/div\u003e\n \u003c/div\u003e\n \u003cp\u003ewhere \u003cem\u003eE\u003c/em\u003e\u003csub\u003e\u003cem\u003eCB\u003c/em\u003e\u003c/sub\u003e is the conduction band potential, \u003cem\u003eE\u003c/em\u003e\u003csub\u003e\u003cem\u003eVB\u003c/em\u003e\u003c/sub\u003e is the valence band potential, and \u003cem\u003eE\u003c/em\u003e\u003csub\u003e\u003cem\u003eg\u003c/em\u003e\u003c/sub\u003e is the optical band gap \u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e27\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e\n \u003cp\u003eBased on the above findings, the photoexcitation process within the \u0026alpha;-Fe\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e3\u003c/sub\u003e/Ti\u003csub\u003e3\u003c/sub\u003eC\u003csub\u003e2\u003c/sub\u003eTₓ MXene heterostructure can be described as follows (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eC\u003cstrong\u003e)\u003c/strong\u003e. Ti\u003csub\u003e3\u003c/sub\u003eC\u003csub\u003e2\u003c/sub\u003eTₓ MXene, functioning as a semimetallic plasmonic material \u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e29\u003c/span\u003e]\u003c/sup\u003e, generates localized surface plasmon resonance under light irradiation, thereby producing hot electrons and corresponding holes. A fraction of these hot electrons can be injected into the conduction band (CB) of \u0026alpha;-Fe\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e3\u003c/sub\u003e, while visible-light photons simultaneously excite electrons from the VB (~\u0026thinsp;2.21 eV) to the CB (~\u0026thinsp;0.12 eV). This process leaves positively charged holes in the VB and enables efficient charge carrier separation \u003cstrong\u003e(\u003c/strong\u003eFig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eC\u003cstrong\u003e)\u003c/strong\u003e. The synergistic effect of hot electrons from Ti\u003csub\u003e3\u003c/sub\u003eC\u003csub\u003e2\u003c/sub\u003eTₓand the intrinsic photoexcitation of \u0026alpha;-Fe\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e3\u003c/sub\u003esignificantly accelerates the internal Fe\u003csup\u003e3+\u003c/sup\u003e/Fe\u003csup\u003e2+\u003c/sup\u003e redox cycle of the Fenton process, thereby promoting the decomposition of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e and generating a high concentration of strongly oxidative hydroxyl radicals (\u0026bull;OH).\u003c/p\u003e\n \u003cp\u003eThe \u0026bull;OH radicals generated in this process initiate the RAFT polymerization reaction. As depicted in Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e, the RAFT mechanism proceeds through five distinct stages:\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\n \u003ch2\u003e3.3. Electrochemical Characterization\u003c/h2\u003e\n \u003cp\u003eIn this study, SWV was employed to precisely evaluate the electrochemical response of gold electrodes modified with various components, aiming to determine the specific contribution of each element within the biosensor assembly. As shown in Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eA, control experiments involving incomplete sensor configurations such as the absence of polyA-DNA probe, tRNA, NH\u003csub\u003e2\u003c/sub\u003e-DNA, CPAD, FMMA, \u0026alpha;-Fe\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e3\u003c/sub\u003e/Ti\u003csub\u003e3\u003c/sub\u003eC\u003csub\u003e2\u003c/sub\u003eT\u003csub\u003ex\u003c/sub\u003eMXene, H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, or light irradiation failed to produce any detectable electrochemical signal. In contrast, the complete biosensor, constructed with polyA-DNA/tRNA/NH\u003csub\u003e2\u003c/sub\u003e-DNA/CPAD/FMMA and activated through a RAFT reaction catalyzed by \u0026alpha;-Fe\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e3\u003c/sub\u003e/Ti\u003csub\u003e3\u003c/sub\u003eC\u003csub\u003e2\u003c/sub\u003eT\u003csub\u003ex\u003c/sub\u003eMXene under visible light in the presence of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, yielded a strong electrochemical signal at 0.288 V (curve k). This result confirms the successful design and functional feasibility of the biosensor system.\u003c/p\u003e\n \u003cp\u003eTo further examine the surface modification process, electrochemical impedance spectroscopy (EIS) was conducted following each step of electrode modification. As depicted in Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eB, the bare gold electrode exhibited the lowest charge transfer resistance (R\u003csub\u003ect\u003c/sub\u003e) at approximately 207 Ω (burgundy curve), attributed to the smooth, conductive metal surface that facilitates efficient electron transfer. Upon immobilization of the polyA-DNA probe, the R\u003csub\u003ect\u003c/sub\u003e increased markedly to ~\u0026thinsp;502 Ω (bright red curve), due to the introduction of negatively charged phosphate groups that hinder the diffusion of [Fe(CN)\u003csub\u003e6\u003c/sub\u003e]\u003csup\u003e3\u0026minus;\u003c/sup\u003e/[Fe(CN)\u003csub\u003e6\u003c/sub\u003e]\u003csup\u003e4\u0026minus;\u003c/sup\u003e redox species. Subsequent hybridization with the target miRNA and complementary NH\u003csub\u003e2\u003c/sub\u003e-DNA led to further increases in R\u003csub\u003ect\u003c/sub\u003e to ~\u0026thinsp;726 Ω (orange curve) and ~\u0026thinsp;1170 Ω, respectively (lime green), which can be attributed to the formation of double-stranded structures that increase both steric and electrostatic hindrance at the electrode interface. Following covalent attachment of the chain transfer initiator CPAD, the R\u003csub\u003ect\u003c/sub\u003e rose sharply to ~\u0026thinsp;1590 Ω (lawn green curve), indicating substantial coverage of the electrode surface, which further restricted charge transfer. Finally, polymerization of FMMA via RAFT reaction resulted in a significant increase in R\u003csub\u003ect\u003c/sub\u003e to ~\u0026thinsp;2310 Ω (purple curve), reflecting the deposition of a dense, hydrophobic polymer layer. This layer acts as a physical barrier, impeding the diffusion of redox species to the electrode surface and further increasing resistance. These progressive changes in impedance demonstrate the successful and sequential construction of the biosensor through RAFT-mediated surface polymerization.\u003c/p\u003e\n \u003cp\u003eComplementary cyclic voltammetry (CV) measurements across a broad scan rate range (50\u0026ndash;450 mV/s) further verified the excellent electrochemical performance of the modified electrode. The redox peaks retained a consistent shape, with a peak potential separation (\u0026Delta;Ep) maintained at 0.285\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02 V (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;10) (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eC), confirming high redox reversibility. A kinetic analysis of the CV data further revealed that the oxidation peak current (\u003cem\u003eI\u003c/em\u003e\u003csub\u003e\u003cem\u003epa\u003c/em\u003e\u003c/sub\u003e) and reduction peak current (\u003cem\u003eI\u003c/em\u003e\u003csub\u003e\u003cem\u003epc\u003c/em\u003e\u003c/sub\u003e) displayed strong linear correlations with the square root of the scan rate (\u003cem\u003ev\u003c/em\u003e\u003csup\u003e\u003cem\u003e1/2\u003c/em\u003e\u003c/sup\u003e) over the tested range, as shown in Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eD. This linearity is characteristic of a diffusion-controlled electrochemical process involving surface-confined redox-active species and provides strong evidence that the ferrocene moieties are robustly immobilized on the electrode surface.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\n \u003ch2\u003e3.4. Experimental optimization conditions\u003c/h2\u003e\n \u003cp\u003eDuring biosensor development, the mixing ratio between \u0026alpha;-Fe\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e3\u003c/sub\u003e and Ti\u003csub\u003e3\u003c/sub\u003eC\u003csub\u003e2\u003c/sub\u003eT\u003csub\u003ex\u003c/sub\u003e MXene during the synthesis of the \u0026alpha;-Fe\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e3\u003c/sub\u003e/Ti\u003csub\u003e3\u003c/sub\u003eC\u003csub\u003e2\u003c/sub\u003eT\u003csub\u003ex\u003c/sub\u003e MXene heterojunction plays a critical role in determining the final electrochemical performance of the RAFT photocatalytic system. Ti\u003csub\u003e3\u003c/sub\u003eC\u003csub\u003e2\u003c/sub\u003eT\u003csub\u003ex\u003c/sub\u003e MXene exhibits excellent electrical conductivity and light absorption properties, which significantly enhance the separation efficiency of photogenerated electron\u0026ndash;hole pairs (e⁻/h⁺) generated by \u0026alpha;-Fe\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e3\u003c/sub\u003e under visible light irradiation. Increasing the proportion of Ti\u003csub\u003e3\u003c/sub\u003eC\u003csub\u003e2\u003c/sub\u003eT\u003csub\u003ex\u003c/sub\u003e MXene in the composite material provides a larger anchoring surface for \u0026alpha;-Fe\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e3\u003c/sub\u003e nanoparticles, preventing agglomeration and facilitating more efficient charge separation. This promotes the transfer of electrons from \u0026alpha;-Fe\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e3\u003c/sub\u003e to Ti\u003csub\u003e3\u003c/sub\u003eC\u003csub\u003e2\u003c/sub\u003eT\u003csub\u003ex\u003c/sub\u003e MXene, where they participate in the reduction of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e to generate hydroxyl radicals (\u0026bull;OH), supplying active species required for the subsequent RAFT polymerization.\u003c/p\u003e\n \u003cp\u003eTo optimize the \u0026alpha;-Fe\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e3\u003c/sub\u003e/Ti\u003csub\u003e3\u003c/sub\u003eC\u003csub\u003e2\u003c/sub\u003eT\u003csub\u003ex\u003c/sub\u003e MXene heterojunction composition, a series of biosensors were fabricated using polyA-DNA/tRNA/NH\u003csub\u003e2\u003c/sub\u003e-DNA/CPAD/FMMA-modified electrodes, each subjected to the RAFT reaction under varying \u0026alpha;-Fe\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e3\u003c/sub\u003e/Ti\u003csub\u003e3\u003c/sub\u003eC\u003csub\u003e2\u003c/sub\u003eT\u003csub\u003ex\u003c/sub\u003e MXene photocatalyst conditions. Specifically, \u0026alpha;-Fe\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e3\u003c/sub\u003e/Ti\u003csub\u003e3\u003c/sub\u003eC\u003csub\u003e2\u003c/sub\u003eT\u003csub\u003ex\u003c/sub\u003e MXene composites were synthesized at different mass ratios of \u0026alpha;-Fe\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e3\u003c/sub\u003e to Ti\u003csub\u003e3\u003c/sub\u003eC\u003csub\u003e2\u003c/sub\u003eT\u003csub\u003ex\u003c/sub\u003e MXene: 100:1 (FM01), 75:1 (FM02), 50:1 (FM03), and 25:1 (FM04). The results (Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eA) indicated that the FM02 group exhibited the highest electrochemical signal, whereas FM01, FM03, and FM04 showed comparatively poor performance. In FM01, the low Ti\u003csub\u003e3\u003c/sub\u003eC\u003csub\u003e2\u003c/sub\u003eT\u003csub\u003ex\u003c/sub\u003e MXene content was insufficient to prevent \u0026alpha;-Fe\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e3\u003c/sub\u003e nanoparticle aggregation, thereby limiting the catalytic activity. Conversely, in FM03 and FM04, the excess of Ti\u003csub\u003e3\u003c/sub\u003eC\u003csub\u003e2\u003c/sub\u003eT\u003csub\u003ex\u003c/sub\u003e MXene may have obstructed light absorption by \u0026alpha;-Fe\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e3\u003c/sub\u003e, negatively impacting the generation of photogenerated carriers. Based on these findings, the FM02 composite with an \u0026alpha;-Fe\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e3\u003c/sub\u003e : Ti\u003csub\u003e3\u003c/sub\u003eC\u003csub\u003e2\u003c/sub\u003eT\u003csub\u003ex\u003c/sub\u003e MXene ratio of 75:1 was selected as the optimal formulation for subsequent experiments due to its superior photocatalytic efficiency.\u003c/p\u003e\n \u003cp\u003eAdditionally, the ratio of N, N-dimethylformamide (DMF) to water in the RAFT polymerization medium was investigated as a key factor influencing polymerization efficiency. As shown in Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eB, the biosensor prepared in a 60% DMF solution produced a significantly higher electrochemical response than those in 30%, 40%, or 50% DMF. This enhancement is likely due to the polar nature of DMF, which improves the solubility and dispersion of both the monomer and the RAFT initiator, thus facilitating the propagation of polymer chains and leading to polymers with higher electrochemical activity. However, at 70% DMF, a slight decrease in performance was observed, likely due to increased solution viscosity, which hinders molecular mobility and diffusion, ultimately reducing polymerization efficiency. Therefore, a DMF concentration of 60% was determined to be optimal for RAFT-based biosensor fabrication.\u003c/p\u003e\n \u003cp\u003eThe duration of RAFT polymerization also significantly impacts the polymer chain length and overall product quality. Inadequate reaction times can result in incomplete polymerization, yielding low molecular weight polymers with inconsistent performance. On the other hand, excessive polymerization may lead to uncontrolled side reactions such as chain transfer or termination, compromising the living nature of RAFT polymerization and reducing the structural regularity of the final product. As demonstrated in Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eC, a polymerization time of 3 h produced the highest electrochemical signal (1.15 \u0026micro;A), indicating that this duration offers a balance between monomer conversion and molecular weight control. Thus, 3 h was identified as the optimal polymerization time under the given reaction conditions.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\n \u003ch2\u003e3.5. Electrochemical biosensor sensitivity\u003c/h2\u003e\n \u003cp\u003eUnder optimized experimental conditions, the performance of the fabricated electrochemical biosensor was evaluated by analyzing its response to a series of tRNA solutions at varying concentrations. As shown in \u003cstrong\u003eFig.\u0026nbsp;7A\u003c/strong\u003e, the peak current measured by SWV exhibited a clear upward trend with increasing tRNA concentration. Within a specific dynamic range of 0.01 fM to 10 pM, a strong linear relationship was observed between the peak current and the logarithm of the tRNA concentration (\u003cstrong\u003eFig.\u0026nbsp;7B\u003c/strong\u003e). Linear regression analysis yielded the Eq.\u0026nbsp;(\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e):\u003c/p\u003e\n \u003cdiv id=\"Equ1\" class=\"Equation\"\u003e\n \u003cdiv class=\"mathdisplay\" id=\"FileID_Equ1\" name=\"EquationSource\"\u003e$$\\:I\\:\\left(\\mu\\:A\\right)\\:=\\:2.485\\:+\\:0.126\\:\\times\\:\\:log\\left({C}_{tRNA}\\right)$$\u003c/div\u003e\n \u003cdiv class=\"EquationNumber\"\u003e5\u003c/div\u003e\n \u003c/div\u003e\n \u003cp\u003eThis has a correlation coefficient (R\u003csup\u003e2\u003c/sup\u003e) 0.998, indicating excellent linear response performance. The detection limit of the biosensor was calculated to be as low as 4.44 aM, based on the standard criterion of a signal-to-noise ratio of 3 (\u003cem\u003eS/N\u003c/em\u003e\u0026thinsp;=\u0026thinsp;3).\u003c/p\u003e\n \u003cp\u003eCompared with many previously reported detection methods (Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e), the electrochemical biosensor developed in this study demonstrated superior sensitivity and an ultra-low detection limit.\u003c/p\u003e\n \u003cdiv class=\"gridtable\"\u003e\n \u003ctable id=\"Tab1\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eComparison with other nucleic acid detection schemes\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eDetection technique\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eAnalytes\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eLinear detection range\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eDetection limit\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eReference\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePhoto-ATRP\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003emiRNA-21\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e10fM\u0026thinsp;~\u0026thinsp;1nM\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.35fM\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e31\u003c/span\u003e]\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePET-RAFT\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003emiRNA-21\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.1nM\u0026thinsp;~\u0026thinsp;10aM\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4.48aM\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e32\u003c/span\u003e]\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePET-RAFT\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eKRAS G12C-ssDNA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0fM\u0026thinsp;~\u0026thinsp;10nM\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e8fM\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e33\u003c/span\u003e]\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCHA-HCR\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003emiRNA 21\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e10fM\u0026thinsp;~\u0026thinsp;100pM\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.52fM\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e34\u003c/span\u003e]\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMNAzyme-CHA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003emiRNA-21\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1pM\u0026thinsp;~\u0026thinsp;100nM\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3.2fM\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e35\u003c/span\u003e]\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePhoto-Fenton-RAFT\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003emiRNA-144\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.01fM\u0026thinsp;~\u0026thinsp;10pM\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4.44aM\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eThis work\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\n \u003ch2\u003e3.6. Electrochemical biosensor selectivity\u003c/h2\u003e\n \u003cp\u003eSelectivity is a critical factor influencing the overall performance of a biosensor. To assess the specificity of the fabricated biosensor, its electrochemical responses to various RNA sequences were tested, including the target tRNA, a single-base mismatch (smRNA), a three-base mismatch (tmRNA), and a fully mismatched control (cRNA), each at a fixed concentration of 10 pM. As shown in \u003cstrong\u003eFig. S3 B\u003c/strong\u003e, the signal responses of smRNA, tmRNA, and cRNA were 23%, 16% and 10.6% of the tRNA signal, respectively. These results demonstrate that the biosensor is capable of effectively distinguishing the target tRNA from non-complementary or mismatched sequences. The high selectivity can be attributed to the molecular recognition capability of the polyA-DNA probe, which enables efficient hybridization with fully complementary targets while discriminating against mismatched bases.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\n \u003ch2\u003e3.7. Anti-interference and detection of RNA targets in human serum\u003c/h2\u003e\n \u003cp\u003eAnti-interference capability is a critical performance indicator for assessing the applicability of biosensors in real biological matrices. To systematically evaluate the anti-interference performance of the developed sensor in complex serum environments, normal human serum (NHS) at varying dilution ratios (5%, 10%, 15%, 20%, and 30%) was used as the test medium, while phosphate-buffered saline (PBS) served as the control. The detection signals for tRNA were measured using SWV.\u003c/p\u003e\n \u003cp\u003eAs shown in \u003cstrong\u003eFig. S3 A\u003c/strong\u003e, the recovery rates, calculated by comparing the current responses at each serum concentration to that of the PBS control, were 96.5% (5% NHS), 83.8% (10% NHS), 76.1% (15% NHS), 53.99% (20% NHS), and 51.6% (30% NHS), respectively. These results indicate that the sensor maintains excellent anti-interference performance at serum concentrations up to 15%, with signal recovery rates exceeding 70%. Even under higher serum concentrations (15\u0026ndash;30%), the detection signal remained at approximately 50%, demonstrating good tolerance to matrix interference and supporting its potential use in clinical sample analysis.\u003c/p\u003e\n \u003cp\u003eTo further assess the sensor\u0026rsquo;s accuracy in complex biological matrices, the standard addition method was employed for the quantitative detection of target tRNA in serum samples. NHS was diluted five-fold (5%, v/v) before analysis to mitigate matrix effects. The endogenous tRNA background level was first measured, followed by spike-recovery experiments in which known concentrations of tRNA standards (1 fM, 0.1 pM, and 10 pM) were added to the serum samples. As summarized in \u003cstrong\u003eTable S2\u003c/strong\u003e, the recovery rates ranged from 93.1\u0026ndash;98.2%, meeting the acceptable criteria for bioanalytical methods (typically 80\u0026ndash;120%). These results confirm that the biosensor maintains high detection accuracy and reliability even in complex serum matrices, highlighting its strong potential for practical application in clinical tRNA diagnostics.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"4. Conclusion","content":"\u003cp\u003eThis study reports the development of a tRNA electrochemical biosensor based on\u0026nbsp;α-Fe\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e3\u003c/sub\u003e/Ti\u003csub\u003e3\u003c/sub\u003eC\u003csub\u003e2\u003c/sub\u003eT\u003csub\u003ex\u003c/sub\u003e MXene photo-Fenton catalysts for RAFT polymerization. From a material design perspective, the construction of α-Fe\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e3\u003c/sub\u003e/Ti\u003csub\u003e3\u003c/sub\u003eC\u003csub\u003e2\u003c/sub\u003eT\u003csub\u003ex\u003c/sub\u003e MXene heterostructures significantly enhanced photocatalytic activity under visible light, thereby generating robust electrochemical signals for sensing. The biosensor demonstrated outstanding analytical performance, including an exceptionally broad linear range spanning five orders of magnitude (0.01 fM-10 pM), an ultralow detection limit of 4.44 aM for tRNA, and a selectivity coefficient above 90% in complex biological matrices. These results underscore the potential of this α-Fe\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e3\u003c/sub\u003e/Ti\u003csub\u003e3\u003c/sub\u003eC\u003csub\u003e2\u003c/sub\u003eT\u003csub\u003ex\u003c/sub\u003e MXene -based platform for translation into clinical diagnostics, offering a promising strategy for ultrasensitive and reliable detection of disease-associated biomarkers.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eCredit author statement\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eThao Thi Nguyen:\u003c/strong\u003e Original Draft, Investigation, Methodology, Formal analysis.\u003cstrong\u003e\u0026nbsp;Zhidan\u003c/strong\u003e \u003cstrong\u003eTian:\u003c/strong\u003e Review \u0026amp; Editing. \u003cstrong\u003eWeibo Huang:\u003c/strong\u003e Review \u0026amp; Editing. \u003cstrong\u003eQinyuan Xu:\u003c/strong\u003e Data curation. \u003cstrong\u003eShuaibing Yu:\u003c/strong\u003e Data curation. \u003cstrong\u003eGengzhi Sun:\u003c/strong\u003e Review \u0026amp; Editing. \u003cstrong\u003eJinming Kong:\u003c/strong\u003e Conceptualization, Review \u0026amp; Editing, Funding acquisition, Resources.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration of competing interest\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData will be made available on request.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was financially by the National Natural Science Foundation of China (Nos. 21974068).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eQiang F, Qiushi R, Thomas G, Amin R, Junwang T, Greg G (2017)Development of a robust PET-RAFT polymerization using graphitic carbon nitride (g-C\u003csub\u003e3\u003c/sub\u003eN\u003csub\u003e4\u003c/sub\u003e). 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Biosensors \u0026amp; Bioelectronics 80: 98-104. https://doi.org/10.1016/j.bios.2015.09.069\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"microchimica-acta","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"miac","sideBox":"Learn more about [Microchimica Acta](https://link.springer.com/journal/604)","snPcode":"604","submissionUrl":"https://submission.springernature.com/new-submission/604/3","title":"Microchimica Acta","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"α-Fe2O3/Ti3C2TxMXene, heterojunction photocatalysis, biosensor, Photo-Fenton Catalysts, RNA detection, signal amplification","lastPublishedDoi":"10.21203/rs.3.rs-7482836/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7482836/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eα-Fe\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e3\u003c/sub\u003e nanoparticles possess a narrow band gap (~2.1 eV) and exhibit strong absorption in the visible light range, making them promising candidates for photocatalytic applications. However, their poor electrical conductivity, high electron–hole recombination rate, and short charge diffusion length limit their practical performance. To address these limitations, α-Fe\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e3\u003c/sub\u003e was integrated with Ti\u003csub\u003e3\u003c/sub\u003eC\u003csub\u003e2\u003c/sub\u003eT\u003csub\u003ex\u003c/sub\u003e MXene to construct a composite photo-Fenton catalyst that drives RAFT polymerization. In this system, the MXene substrate not only disperses α-Fe\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e3\u003c/sub\u003e nanoparticles efficiently but also facilitates the generation of abundant photoinduced electrons under visible-light excitation. This enhances the Fe\u003csup\u003e3+\u003c/sup\u003e/Fe\u003csup\u003e2+\u003c/sup\u003e redox cycling and accelerates H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e decomposition, yielding a high concentration of hydroxyl radicals (•OH). To further explore the functional applicability of this catalyst, it was employed in a biosensing platform for the ultrasensitive detection of microRNA-144. In this design, the •OH radicals initiate RAFT polymerization, allowing for a significant amplification of the electrochemical signal. The resulting sensor exhibits a wide detection range (0.01 fM to 10 pM) and an ultralow detection limit of 4.44 aM. These findings highlight the potential of α-Fe\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e3\u003c/sub\u003e/Ti\u003csub\u003e3\u003c/sub\u003eC\u003csub\u003e2\u003c/sub\u003eT\u003csub\u003ex\u003c/sub\u003e MXene composites in photocatalysis, polymer chemistry, and biomedical sensing, offering new insights for future technological innovations.\u003c/p\u003e","manuscriptTitle":"α-Fe 2 O 3 /Ti 3 C 2 T x MXene Heterostructures as Photo-Fenton Catalysts Driving RAFT Polymerization for Ultrasensitive Electrochemical microRNA Sensing","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-09-12 09:18:57","doi":"10.21203/rs.3.rs-7482836/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-09-25T14:22:58+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-09-22T03:37:40+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-09-19T07:33:26+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-09-16T18:08:04+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-09-16T15:09:31+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-09-16T02:31:11+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"110555915104743686438584008048092308788","date":"2025-09-12T00:33:57+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"319911310801519653656533981099675933220","date":"2025-09-09T07:33:49+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"169159993300199895635071219776806304864","date":"2025-09-07T08:43:10+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"201115918693899473067819828502538048561","date":"2025-09-07T00:48:39+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"315299758962011221819229176795062052762","date":"2025-09-06T23:13:01+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-09-06T22:35:05+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-09-01T11:32:26+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-08-31T23:20:21+00:00","index":"","fulltext":""},{"type":"submitted","content":"Microchimica Acta","date":"2025-08-28T18:46:29+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"microchimica-acta","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"miac","sideBox":"Learn more about [Microchimica Acta](https://link.springer.com/journal/604)","snPcode":"604","submissionUrl":"https://submission.springernature.com/new-submission/604/3","title":"Microchimica Acta","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"9bd68c25-c554-4d08-a40a-ed5307f33d1a","owner":[],"postedDate":"September 12th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2026-01-26T16:05:43+00:00","versionOfRecord":{"articleIdentity":"rs-7482836","link":"https://doi.org/10.1007/s00604-025-07669-x","journal":{"identity":"microchimica-acta","isVorOnly":false,"title":"Microchimica Acta"},"publishedOn":"2026-01-22 15:58:52","publishedOnDateReadable":"January 22nd, 2026"},"versionCreatedAt":"2025-09-12 09:18:57","video":"","vorDoi":"10.1007/s00604-025-07669-x","vorDoiUrl":"https://doi.org/10.1007/s00604-025-07669-x","workflowStages":[]},"version":"v1","identity":"rs-7482836","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7482836","identity":"rs-7482836","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}