Ultrasensitive sensing platform based on Au 0.1 FeCoNiCu high-entropy alloy nanoparticle-DNA walker dual signal amplification for electrochemical detection of miRNA-21

Ultrasensitive sensing platform based on Au 0.1 FeCoNiCu high-entropy alloy nanoparticle-DNA walker dual signal amplification for electrochemical detection of miRNA-21 | 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 Ultrasensitive sensing platform based on Au 0.1 FeCoNiCu high-entropy alloy nanoparticle-DNA walker dual signal amplification for electrochemical detection of miRNA-21 Li Ruiyi, Li Mingyao, Wang Miao, Li Zaijun This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6946913/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 9 You are reading this latest preprint version Abstract Overcoming the critical limitation of poor sensitivity in electrochemical biosensors for early breast cancer diagnosis, this study presents an innovative approach. We developed a robust coordination strategy, utilizing serine-functionalized graphene quantum dot (SGQD), to synthesize Au 0.1 FeCoNiCu high-entropy alloy nanoparticles (HEA NPs). The resulting Au 0.1 FeCoNiCu HEA NPs (32 ± 1.7 nm) exhibit a single FCC phase, elemental homogeneity, and graphene surface modification. This unique structure confers significantly enhanced catalytic activity (> 12-fold higher than pure Au NPs) and superior affinity towards polar electrolytes. Furthermore, we integrated these HEA NPs with a DNA walker circuit to construct a novel electrochemical biosensor for ultrasensitive miRNA-21 detection. Target miRNA-21 triggers the DNA walker, immobilizing ferrocene molecules on the electrode surface and generating a measurable electrochemical signal. This dual-amplification strategy (HEA NP catalysis + DNA walker) achieved unprecedented sensitivity: a linear current response (at 0.22 V) over an extraordinary range (1×10⁻²⁰ M to 1×10⁻¹⁵ M) and a record-low detection limit of 3.4×10⁻²¹ M. This represents a 2–3 orders of magnitude sensitivity improvement over state-of-the-art sensors, successfully demonstrated in serum analysis. High entropy alloy Strong coordination route Early cancer diagnosis Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 1 Introduction Increasing evidence shows dysregulated expression of MiRNA-21 strongly correlates with formation, invasion and metastasis of breast cancer [ 1 ]. Development of sensing technique for precise analysis of miRNA-21 provided strong impetus to real need for early diagnosis of breast cancer [ 2 ]. The main analytical techniques for miRNA-21 are fluorescence [ 3 ], surface-enhanced Raman scattering [ 4 ], electrochemiluminescence [ 5 ] and electrochemical biosensor [ 6 ]. Electrochemical biosensor received a more attention because of high sensitivity, low cost and rapid response. However, classical electrochemical biosensor is far from meeting the need for high sensitivity of early breast cancer diagnosis [ 7 ]. Many metal materials were investigated for construction of electrochemical detection of miRNA-21. Au is commonly used electrochemical sensing material due to excellent electrical conductivity, catalytic activity and chemical inertness [ 8 ]. However, high-cost, poor functional diversity and resource scarcity limit its wide use. Graphene is a rising star of sensing materials owing to large specific surface, high electrical conductivity and good mechanical property, but it cannot provide special catalytic capacity [ 9 ]. To resolve the problem, hybrid of Au with graphene become one new research hotspot [ 10 ]. All Au nanoparticles are connected into one whole by graphene, accelerating the electron transfer between different Au nanoparticles. This results in an improved catalytic activity. Heterojunction sensing materials caused great research interest in analytical chemistry [ 11 ]. Formation of heterojunction narrows bandgap and leads to an improved catalytic activity. Despite these advances, design and synthesis of sensing material for electrochemical detection of miRNA-21 in early diagnosis of breast cancer is still challenging. High entropy alloy (HEA) is one single-phase solid solution composing of five or more metal elements [ 12 ]. Mixing of all metal elements in HEA brings multi-element active sites. Interestingly, the active sites can be optimized by adjusting element configuration and composition [ 13 ]. HEA nanoparticles become an ideal candidate of electrochemical sensing materials due to good multifunctionality, catalytic activity and selectivity [ 14 ]. However, following three problems restrict its application in electrochemical detection of miRNA-21. (1) Current synthesis is difficult to be used for large-scale production of HEA nanoparticles due to the need of special equipment and severe reaction conditions. (2) Current HEA nanoparticles offer a lower catalytic activity compared with noble metal nanoparticle. (3) Current HEA nanoparticles offer a poor affinity with polar analyte and redox probe due to their hydrophobic characteristics. In the study, we reported one strong coordination route for synthesis of Au 0.1 FeCoNiCu high-entropy alloy nanoparticles by introducing serine-functionalized graphene quantum dot (SGQD). The resulting Au 0.1 FeCoNiCu exhibits catalytic activity of more than 12-fold that of sole Au nanoparticle. The biosensor with Au 0.1 FeCoNiCu and DNA walker exhibits ultrahigh sensitivity for electrochemical detection of miRNA-21. 2 Experimental 2.1 Au 0.1 FeCoNiCu synthesis FeCl 3 (0.8 mmol), CoCl 2 (0.8 mmol), NiCl 2 (0.8 mmol), CuCl 2 (0.8 mmol) and HAuCl 4 (0.08 mmol) were dissolved in a Ser-GQD aqueous solution (50 mL,100 mg mL − 1 ) witht stirring to form a purple-black clear solution. Followed by spray drying and annealing in N 2 at 600℃ for 2 h with a heating rate of 1℃ min − 1 to obtain Au 0.1 FeCoNiCu. The procedure was also used for synthesis of sole Au nanoparticle unless no addition of FeCl 3 , CoCl 2 , NiCl 2 and CuCl 2 . 2.2 Biosensor construction H1 and H2-Fc were heated at 95℃ for 5 min and then cooled to room temperature to form hairpin structure. The pre-treated H1 was mixed with an equal volume of 1 mM TCEP solution and incubated for 1 h to form an activated H1; The walker solution (10 µM) was mixed with an equal volume of 10 µM aptamer, heated at 95℃ for 5 min, and cooled to room temperature to form double-stranded walker-Apt. The resulting walker-Apt was mixed with an equal volume of 1 mM TCEP and incubated for 1 h to form activated walker-Apt; Firstly, equal volume of Au 0.1 FeCoNiCu dispersion and chitosan solution were mixed to form Au 0.1 FeCoNiCu/chitosan dispersion, in which the mass ratio of Au 0.1 FeCoNiCu and chitosan is 1:5. Its 5 µL was dropped on the surface of glass carbon electrode (GCE, 2 mm in diameter) and dried. Then, 5 µL of activated H1 and 5 L of walker-Apt solution were dropped on the surface of Au 0.1 FeCoNiCu/GCE. Followed incubation for 2 h, washing with ultrapure water and drying. Finally, 5 µL of 1 mM mercaptohexanol (MCH) was dropped, incubated for 2 h, washed with ultrapure water and dried to obtain a biosensor. The as-prepared biosensor was stored at 4 ℃ in refrigerator before use. 2.3 Electrochemical detection of miRNA-21 Equal volume of 10 µM pre-treated H2-Fc solution and MiRNA-21standard solution with different concentration (or sample solution) was mixed. Its 100 µL was dropped on the biosensor, then incubated at 37℃ for 1 h, washed with ultrapure water, and dried with N 2 flow. The incubated biosensor was given to the DPV measurement in a PBS of pH7. 3 Results and Discussions 3.1 Design and synthesis Figure 1 outlines the design and synthesis of SGQD and Au 0.1 FeCoNiCu high-entropy alloy (HEA). SGQD synthesis employs citric acid as a carbon source to form graphene sheets, while serine introduces functional groups via amide bonds between its amine and graphene carboxyl groups, enhancing metal coordination through hydroxyl and carboxyl moieties. The HEA integrates Au (enabling biomolecule immobilization via Au–S bonds) with Fe, Co, Ni, and Cu—adjacent fourth-period transition metals with sequentially varying electronic structures. Their low-energy-barrier orbital hybridization facilitates electronic reconstruction, boosting catalytic activity and versatility. Cu partially substitutes Au due to electronic similarity. Synthesis involves a strong coordination route: metal ions first form homogeneous Me-SGQD complexes ( Fig. S1 ), ensuring atomic dispersion. Subsequent thermal decomposition of SGQD generates reducing gases that reduce metal ions to atoms, bypassing vapor-phase processes (unlike carbothermal shock [ 15 ]). Gas-stream-driven collisions under high-entropy conditions enable alloying at sub-melting temperatures, yielding single-phase Au 0.1 FeCoNiCu nanocrystals. This method avoids metal vaporization, leveraging gas-phase energy for controlled nanocrystal formation. (199 words) 3.2 Material characterization Morphology and element dispersion of Au 0.1 FeCoNiCu were characterized by SEM, TEM, HAADF-STEM and element mapping technique (Fig. 2 ). Three-dimensional structure composing of graphene sheets appears on the SEM image. Small Au 0.1 FeCoNiCu nanoparticles are dispersed on graphene sheets. Au 0.1 FeCoNiCu nanoparticle offers a sphere-like morphology with an average particle size of 32.5±1.4 nm (Fig. s2). There are many light and dark dots on the HAADF-STEM image, corresponding to different metal atoms. Among Au, Fe, Co, Ni and Cu, Au atom indicates the best brightness, because of the biggest atomic weight. Atomic strain map reveals that the stress is dispersed anywhere in the lattice of Au 0.1 FeCoNiCu nanoparticles. The mixing of Au, Fe, Co, Ni and Cu causes an uneven stress distribution. Based on the electron diffraction pattern, live profiles of IFFT of the (200) and (220) faces were obtained. Average spacings (220) and (200) faces are 0.308 nm and 0.342 nm, respectively. However, an obvious difference in the spacing of different metal elements can be observed from Fig. 2 F and G. This is because mixing of Au, Fe, Co, Ni and Cu leads to disorder and twisting of lattice. The results of element mapping analysis confirm the spatial distribution of Au, Fe, Co, Ni and Cu, showing an excellent elemental homogeneity Crystal structure of Au 0.1 FeCoNiCu nanoparticle was characterized by XRD technique. Figure 3 A shows XRD patterns of Au 0.1 FeCoNiCu mainly includes four diffraction peaks at 41.47 o , 45.76 o , 75.51 o and 84.25 o , corresponding to (111), (200), (220) and (311) panes of FCC phase [ 16 ]. Further, Fig. 3 B depicts crystal structures of Au 0.1 FeCoNiCu is formed by octahedron being combined face to face. Chemical environment of Au 0.1 FeCoNiCu was characterized by FTIR and XPS spectra. The FTIR spectrum includes five IR absorption bands and absorption peaks. The band at about 3421 cm -1 is IR absorption of stretching vibration of O-H, N-H and = C-H bonds. The band at about 1636 is IR absorption of stretching vibration of C = O and C = C bonds. The peak at 1424 cm -1 is IR absorption of stretching vibration of C-O bond. The peak at 1084 cm -1 is IR absorption of stretching vibration of C-O-C bond. The peak at 610 cm -1 is IR absorption of out-of-plane bending vibration of -CH 3 group. Total XPS spectrum includes the XPS peaks of Au4f, C1s, O1s, Fe2p, Co2p, N2p and Cu2p, verifying the existence of gold (Au), carbon (C), oxygen (O), iron (Fe), cobalt (Co), nickel (Ni) and copper (Cu) (Fig. s3). High-resolution XPS spectrum of C1s includes three peaks at 284.80, 287.59 and 289.97 eV, which correspond to C-C, C = O and O-C = O bonds. High-resolution XPS spectrum of Au4f includes four peaks at 83.99, 87.99, 85.46 and 89.75 eV. Two peaks at 83.99 and 85.46 eV correspond to Au4f 7/2 and Au4f 5/2 of Au 0 , showing the existence of Au 0 . Two peaks at 87.99 and 89.75 eV correspond to Au4f 7/2 and Au4f 5/2 of Au + , showing the existence of Au + [ 17 ]. High-resolution XPS spectrum of Fe2p includes six peaks at 708.92, 711.69, 716.46, 722.02, 724.79 and 729.62 eV. Two peaks at 708.92 and 722.02 eV correspond to Fe2p 3/2 and Fe2p 1/2 of Fe 0 , showing the existence of Fe 0 . Two peaks at 711.69 and 724.79 eV correspond to Fe2p 3/2 and Fe2p 1/2 of Fe 2+ , showing the existence of Fe 2+ . Two peaks at 716.46 and 729.62 eV correspond to Fe2p 3/2 and Fe2p 1/2 of Fe 3+ , showing the existence of Fe 3+ . High-resolution XPS spectrum of Co2p includes six peaks at 779.68, 781.12, 785.23, 794.60, 796.05 and 800.59 eV. Two peaks at 779.68 and 794.60 eV correspond to Co2p 3/2 and Co2p 1/2 of Co 3+ , showing the existence of Co 3+ . Two peaks at 781.12 and 796.05 eV correspond to Co2p 3/2 and Co2p 1/2 of Co 2+ , showing the existence of Co 2+ . Two peaks at 785.23 and 800.59 eV correspond to Co2p 3/2 and Co2p 1/2 of satellite Co2p (Sat.). High-resolution XPS spectrum of Ni2p includes four peaks at 851.47, 857.10, 867.58 and 872.68 eV. Two peaks at 851.57 and 867.58 eV correspond to Ni2p 3/2 and Ni2p 1/2 of Ni 0 , showing the existence of Ni 0 . Two peaks at 857.10 and 872.68 eV correspond to Ni2p 3/2 and Ni2p 1/2 of Ni 2+ , showing the existence of Ni 2+ . High-resolution XPS spectrum of Cu2p includes six peaks at 932.16, 933.75, 942.87, 951.65, 953.55 and 960.21 eV. Two peaks at 932.16 and 951.65 eV correspond to Cu2p 3/2 and Cu2p 1/2 of Cu 0 , showing the existence of Cu 0 . Two peaks at 933.75 and 953.55 eV correspond to Cu2p 3/2 and Cu2p 1/2 of Cu 2+ , showing the existence of Cu 2+ . Two peaks at 942.87 and 960.21 eV correspond to Cu2p 3/2 and Cu2p 1/2 of satellite Cu2p (Sat.), showing the existence of Sat. The above results confirm the presence of high valent Au, Fe, Co, Ni and Cu species, which can combine with SGQD by coordination bonds to form SGQD layer on the surface of Au 0.1 FeCoNiCu nanoparticle [ 18 ]. 3.3 Catalytic activity evaluation To evaluate catalytic activity of Au 0.1 FeCoNiCu, CV and DPV curves of Au 0.1 FeCoNiCu and Au-control electrodes were measured in 1 mM K 4 Fe(CN) 6 solution. Fig. s4 shows CV curves of Au 0.1 FeCoNiCu and Au-control electrodes offer one pair of redox peaks, showing Au 0.1 FeCoNiCu and Au-control can catalyze oxidation and reduction of Fe(CN) 6 4− . However, the oxidation potential and reduction potentials of Au 0.1 FeCoNiCu electrode are lower than that of Au-control electrode, verifying Au 0.1 FeCoNiCu has a better catalytic activity. Further, the DPV curves indicate that Au 0.1 FeCoNiCu has a more sensitive current response. The peak current is more than 12-fold that of Au-control electrode, showing a significantly enhanced catalytic activity. To understand the improvement in catalytic activity, CV behavior of Au 0.1 FeCoNiCu and Au-control electrodes were studied by measuring the electrochemical surface areas (ECSAs). Fig. s5 shows Au 0.1 FeCoNiCu offers a higher CV current density, verifying the presence of more active sites. Further, the relationships between change in the current density (ΔJ) and scan rates for Au 0.1 FeCoNiCu and Au-control electrodes were shown in Fig. s5B. There is one good linear relationship between ΔJ and scan rate. The corresponding slopes (C dl ) are 70 mF cm − 2 of Au 0.1 FeCoNiCu and 1 mF cm − 2 of Au-control. For similar nanomaterials, the C dl value can represent the ECSA of catalyst. Thus, the above data demonstrate the ECSA of Au 0.1 FeCoNiCu is more than 70-fold that of Au-control. Based on the result, we suggest excellent catalytic activity of Au 0.1 FeCoNiCu is attributed to a more ECSA produced by mixing of Au, Fe, Co, Ni and Cu. 3.4 Biosensor construction Biosensor construction includes immobilization of Au 0.1 FeCoNiCu (HEA), H1, walker-Apt and MCH on GCE (Fig. 4 A). HEA, H1, walker-Apt and MCH are orderly designed to catalyze redox of Fc, capture H2-Fc, identify miRNA-21 and block residual active sites. MiRNA-21 specifically binds with Apt in walker-Apt to release free walker. The released walker hybridizes with H1 on the biosensor to open harpin structure of H1. The formed walker-H1 combines with H2-Fc to release one free walker. Meanwhile, one H2-Fc was immobilized on the surface of HEA by hybridization of H1 with H2. The redox of Fc in H2-Fc under catalysis of HEA produces a sensitive electrochemical response. The above procedure achieves to one-step walking of DNA walker. The released walker binds with another H1 on the biosensor to trigger next walking of DNA walker. By the walking, one walker can bring many H2-Fc probes to the biosensor. The redox of Fc molecules in these H2-Fc probes produces a significant signal amplification. The sensing principle was tested by native PAGE analysis (Fig. 4 B). One walker-Apt band and one H2 band appear on electrophoregram of walker-Apt + H2 (lane 6). No walker band and Apt band can be seen from lane 6, verifying H2 cannot hydridize with walker-Apt to release free walker. One walker-H1 band and one Apt-miRNA-21 band appear on electrophoregram of walker-Apt + H1 + miRNA-21 (lane 7). This confirms miRNA-21 can bind with Apt in walker-Apt to form Apt-miRNA-21. The released walker can bind with H1 to form walker-H1. One H1-H2 band and one walker-Apt band appear on electrophoregram of walker-Apt + H1 + H2 + miRNA-21 (lane 8). In the presence of miRNA-21, H1 can combine with H2 to form H1-H2. The above results demonstrate that the sensing principle is of high reliability. CV and EIS behaviours of different electrodes were examined in 1 mM K 4 Fe(CN) 6 in PBS of pH 7.4 to understand electrochemical property of biosensor. Figure 4 C and D show the modification of Au 0.1 FeCoNiCu causes an increased CV current and a reduced charge transfer impedance (R ct ) (Table s1 ). Due to excellent conductivity and catalytic activity, the introduction of Au 0.1 FeCoNiCu accelerates redox of Fe(CN) 6 4- on electrode surface, leading to an increased CV current and a reduced R ct . Different from Au 0.1 FeCoNiCu, the modification of H1, walker-Apt and MCH causes an obviously decreased CV current and an increasd R ct . This is because H1, walker-Apt and MCH are non-conductive. Their introduction partly blocks the electron transfer pathways between electrode and electrolyte, reducing the CV current. However, the incubation in a mixed H2-Fc solution with miRNA-21 results in an increased CV current with a reduced R ct . This is because the presence of miRNA-21 triggers the DNA walker. By the walking, one walker-Apt molecule brings many H2-Fc probes to the biosensor. The redox of Fc molecules in these H2-Fc probes achieves to significant signal amplification, indicating an obviously increased CV current. The CV behaviour was investigated by varying scan rate to study on the electrode reaction kinetics (Fig. 4 E and F). The increase of scan rate brings an increase in the CV peak current. There is a good relationship between CV peak current and square root of scan rate, verifying the electrode reaction is controlled by electrolyte diffusion. Such a fast electrode reaction process is attributed to excellent conductivity and catalytic activity of Au 0.1 FeCoNiCu. Further, the CV measurement was repeated for 100 times. Figure 4 G indicates 100-cycle didn’t cause significant change in CV curve, showing that the omission of electroactive ingredients can be ignored. 3.5 Electrochemical detection of miRNA CV and DPV curves of the biosensor in the PBS of pH 7.4 were measured in the absence and the presence of 1×10 − 15 M miRNA-21. Figure 5 A and B shows the introduction of miRNA-21 brings significant increase in CV and DPV peak currents, producing sensitive electrochemical response. This is because the presence of miRNA-21 triggers the DNA walker, bringing H2-Fc probe to the biosensor. By DNA walking, one miRNA-21 molecule can bring many H2-Fc probes to the biosensor. Their redox causes significant signal amplification, indicating an increased CV and DPV current. To obtain high sensitivity, DPV technique was selected for electrochemical detection of miRNA-21. The influence of incubation time and H2-Fc concentration on DPV current was studied to optimize detection conditions. Figure 5 C shows increasing incubation time brings an increase in DPV peak current when the time is less than 40 min. This is because more time produces more steps of DNA walking, increasing number of H2-Fc probes on the biosensor. The redox of these probes produces an obviously increased DPV peak current. However, the DPV peak current tends to stable when the incubation time is more than 60 min. This is because the immobilization and removal of H2-Fc probes on the biosensor reaches a dynamic equilibrium state. At the state, number of H2-Fc probes on the biosensor keeps almost unchanged, indicating a stable DPV current. Figure 5 D shows the increase of H2-Fc concentration produces an increased DPV peak current when the volume is less than 10 µL. However, the DPV peak current tends to stable when the volume is more than 10 µL. This is because the H2-Fc solution of less than 10 µL cannot provide enough number of H2-Fc probes needed for the DNA walker. A more volume of H2-Fc solution results in a more H2-Fc probes on the biosensor, increasing the DPV peak current. When the volume of H2-Fc solution reaches to 10 µL, the need can can be met. Thus, the increase of H2-Fc volume cannot bring more H2-Fc probes to the biosensor, showing a stable DPV peak current. Figure 5 E presents the DPV curves in the presence of different concentration of miRNA-21. The DPV current increases with increasing miRNA-21 concentration. This is because a higher concentration of miRNA-21 brings a faster DNA walking proceess. Accordingly, within an incubation of 60 min more H2-Fc probes were brought to the biosensor, providing an increased DPV current. Further, the relationship between DPV peak current and the logarithmic value of miRNA-21 concentration (LOG(C miRNA -21)) was shown in Fig. 5 F. When the miRNA-21 concentration is in the range of 10 − 15 ~10 − 20 M, there is one good linear relationship between the DPV peak current (I p , µA) and the LOG(C miRNA−21 , M). The corresponding linear equation is : I p =76.433×LOG(C miRNA−21 ) + 1560.8, affording R 2 of 0.997 and detection limit of 3.4×10 − 21 M (S/N = 3). The sensitivity is higher than that of other biosensors for miRNA-21 in Table s2. The biosensor was used for consecutive DPV detection of 1×10 − 15 M miRNA-21. The RSD of 100 consecutive detection is 2.1%, showing a good repeatability. Ten biosensors were constructed by the same method and used for DPV detection of 1×10 − 15 miRNA-21. The RSD of fifty biosensors is 1.9%, showing a good reproducibility. The biosensor was stored at 4°C and then remove it every other week, and return to room temperature for DPV detection of 1×10 − 15 miRNA-21. The RSD of 8-week storage is 2.7%, showing a desirable storage stability. The interference effects of diverse species (inorganic ions, organic molecules, bovine serum albumin) on the DPV peak current of 1 × 10⁻¹⁵ M miRNA-21 were systematically investigated. Experimental analyses demonstrated that each interferent (1 mg) induced a current variation of < 1.2%, attributed to their inability to bind the aptamer in the walker-Apt complex, thereby failing to activate the DNA walker process or translocate H₂-Fc probes. Specificity assessments via hybridization with single-base mismatch (miRNA-21-1M), two-base mismatch (miRNA-21-2M), three-base mismatch (miRNA-21-3M), miRNA-210, or miRNA-221 revealed peak currents reduced to 2.78% of the intact miRNA-21 signal ( Fig. S6 ), confirming single-nucleotide discrimination. These results highlight two critical merits: (1) robust anti-interference performance in complex matrices due to selective Apt-target binding, and (2) single-base resolution for miRNA-21 detection. The sensor thus enables precise quantification in serum despite coexisting biomolecular interferents and structurally similar miRNAs, underscoring its reliability for real-sample applications. 3.6 Sample analysis To validate the reliability of the proposed sensor in real-sample analysis, recovery tests were systematically conducted using human serum. Two serum samples were diluted 10,000-fold and spiked with standard miRNA-21 concentrations spanning five orders of magnitude (0.05, 0.5, 5, 50, and 500 aM) for parallel detection. Each concentration level underwent a minimum of five replicate measurements, with mean recovery rates and relative standard deviations (RSDs) calculated to assess precision and accuracy. As detailed in Table 1 , the method exhibited robust performance, with spiked recoveries ranging from 95.0–102.0% and RSDs between 1.4% and 4.99%, confirming its reproducibility in complex biological matrices. Subsequently, the sensor was applied to quantify endogenous miRNA-21 levels in normal human serum. As summarized in Table S3 , baseline miRNA-21 concentrations in healthy individuals were determined to be 0.015, 0.084, 0.13, and 0.78 fM, values substantially lower than those reported in serum from breast cancer patients [ 19 ]. This stark contrast underscores the method’s sensitivity in distinguishing pathological from physiological miRNA-21 expression. Notably, recovery tests for endogenous analytes yielded 96.5–104.4% recoveries with RSDs of 1.2–2.77%, further validating the method’s accuracy under clinically relevant conditions. Table 1 Results of the miRNA-21 standard added serum recovery test by the proposed method (N = 5) Samples miRNA-21 added (aM) miRNA-21 found by proposed method (aM) Recovery (%) RSD (%) Serum sample 1 0.05 0.048 ± 0.004 96.0 4.99 0.5 0.498 ± 0.05 99.6 3.75 5 5.03 ± 0.17 100.6 2.44 50 50.14 ± 1.22 100.3 2.32 500 499.6 ± 4.4 99.9 1.45 Serum sample 2 0.05 0.051 ± 0.008 102.0 3.54 0.5 0.475 ± 0.08 95.0 3.31 5 5.04 ± 0.63 100.8 1.99 50 49.81 ± 1.01 99.6 2.73 500 502.3 ± 10.23 100.5 1.40 Conclusions The study presents one scalable metal-SGQD strong coordination route for synthesizing single-phase Au 0.1 FeCoNiCu high-entropy alloy nanoparticle. The synthesis realizes impressive catalytic activity and affinity with polar redox probe, addressing key challenges in the development of high-entropy alloy nanoparticles. The electrocatalytic activity is more than 12-fold than that of sole Au nanoparticle. The biosensor based on Au 0.1 FeCoNiCu, coupled with the DNA walker, exhibits an ultralow detection limit of 3.4×10 -21 M, outperforming all previously reported biosensors. This work also offers a promising strategy for large-scale production of high-entropy alloy nanoparticles in sensing, biomedicine, catalysis, and energy storage and conversion. Declarations Data availability Data will be made available from the corresponding author on reasonable request. Conflicts of interest There are no conflicts to declare. Acknowledgements The authors acknowledge the Chemical Testing Center of the School of Chemistry and Materials Engineering, Jiangnan University for support. Author contributions Li Ruiyi : Investigation. Li Mingyao : Investigation. Wang Miao: Investigation. Li Zaijun : Conceptualization, Methodology. Funding This work was supported by The National Key Research and Development Program of China (No. 2021YFA0910200). References Li H, Tie XJ (2024) Exploring research progress in studying serum exosomal miRNA-21 as a molecular diagnostic marker for breast cancer, Clin. Transl. Oncol. 26: 2166-2171. https://doi.org/10.1007/s12094-024-03454-z Zoughi S, Faridbod F, S. Moradi S (2024) Rapid enzyme-free detection of miRNA-21 in human ovarian cancerous cells using a fluorescent nanobiosensor designed based on hairpin DNA-templated silver nanoclusters, Anal. Chim. Acta 1320: 342968. https://doi.org/10.1016/j.aca.2024.342968 He M, Hou Z, Yin F, Cheng W, Xiang Y, Wang Z (2024) Simultaneous detection of breast cancer biomarkers HER2 and miRNA-21 based on duplex-specific nuclease signal amplification, J. Mater. Chem. B 12: 9930-9937. https://doi.org/10.1039/d4tb01845a Tan HS, Wang T, Han JM, Liu M, Li SS (2024) Dual-signal SERS biosensor based on spindle-shaped gold array for sensitive and accurate detection of miRNA 21, Sensor. Actuat. B-Chem. 403: 135157. https://doi.org/10.1016/j.snb.2023.135157 Zheng K, Zheng Q, Mu X, Li MJ, Yi C (2024) A smartphone‑assisted electrochemiluminescent biosensor for highly sensitive detection of miRNA‑21 based on Ru(bpy)2(L) 4+ @MOF‑5 Microchim. Acta 191: 596. https://doi.org/10.1007/s00604-024-06675-9 Zhang LN, He YB (2024) A label-free and ultrasensitive electrochemical biosensor using hybrid polypyrrole/gold nanoelectrocatalyst mediated signal amplification for the detection of miRNA-21, Anal. Methods 16: 7567-7575. https://doi.org/10.1039/d4ay01215a Cimminol W, Migliorelli D, Singh S, Miglione A, Generelli S, Cinti1S (2023) Design of a printed electrochemical strip towards miRNA‑21 detection in urine samples: optimization of the experimental procedures for real sample application, Anal. Bioanal. Chem. 415: 4511-4520. https://doi.org/10.1007/s00216-023-04659-x Hou Z, Zheng J, Zhang C, Li L, Chen D, Hu L, Xiong B, Ye H, Jaffrezic-Renault N, Guo Z (2022) Direct ultrasensitive electrochemical detection of breast cancer biomarker-miRNA-21 employing an aptasensor based on a microgel nanoparticle composite, Sensor. Actuat. B-Chem. 367: 132067. https://doi.org/10.1016/j.snb.2022.132067 Huang MS, Govindasamy M, Chinnapaiyan S, Lin YT, Lu SY, Samukawa S, Huang CH (2024) Electrochemical biosensor based on an atomic layered composite of graphene oxide/graphene as an electrode material towards selective and sensitive detection of miRNA-21, Microchem. J. 199 : 110112. https://doi.org/10.1016/j.microc.2024.110112 Khodadoust A, Nasirizadeh N, Seyfati SM, Taheri RA, Ghanei M, Bagheri H (2023) High-performance strategy for the construction of electrochemical biosensor for simultaneous detection of miRNA-141 and miRNA-21 as lung cancer biomarkers, Talanta 252: 123863. https://doi.org/10.1016/j.talanta.2022.123863 Wang X, Li H, Jian H, Liu L, Zeng H, Ai J, Tang J (2025) Robust nCuO modulated by defect engineering enhanced photoelectrochemical biosensor for the detection of miRNA-21, Biosens. Bioelectron. 271: 117014. https://doi.org/10.1016/j.bios.2024.117014 Tang C, Lvm CL, Chena P, Wang AJ, Feng JJ, Cheang TY, Xia H (2024) Dendritic quinary PtRhMoCoFe high-entropy alloy as a robust immunosensing nanoplatform for ultrasensitive detection of biomarker, Bioelectrochemistry 157: 108639. https://doi.org/10.1016/j.bioelechem.2024.108639 Raj G, Nandan R, Gakhad P, Kumar K, Singh AK, Nanda KK (2024) A novel electrochemical high-performance non-enzymatic glucose sensing based on face-centred cubic FeCoNiMnCr high entropy nano alloys encapsulated in NCNTs, Chem. Eng. J. 503: 158041. https://doi.org/10.1016/j.cej.2024.158041 Song D, Huang X, Liu Q, Li G, Xu X, Wang X, Wang J, Lu X, Gao F (2024) Rational construction of ultrathin PtPdRhCuM (M=Mn, Co or Ni) high entropy alloy nanotubes with rich defects for enhanced electrochemical activity: Electrochemical aldicarb sulfone sensing, Sensor. Actuat. B-Chem. 405: 135337. https://doi.org/10.1016/j.snb.2024.135337 Wu Y, Lei Z, Liu X, Wang H, Lu Z (2018) Eight in one: high-entropy-alloy nanoparticles synthesized by carbothermal shock, Sci. Bull. 63: 737-738. https://doi.org/10.1016/j.addma.2023.103716 Bajaj D, Chen Z, Qu SJ, Feng AH, Li DY, Chen DL (2023) Distinct origins of deformation twinning in an additively-manufactured high-entropy alloy, Additive Manufacturing 74: 103716. https://doi.org/10.1016/j.addma.2023.103716 Pan P, Li RY, Wang X, Shen YR, Li ZJ (2025) Electrochemical biosensor based on Au 5 Ir@graphenequantumdot nanocomposite and DNA walker for detection of atrazine in environmental water with extremely high sensitivity and selectivity, Sensor. Actuat. B-Chem. 423: 136873. https://doi.org/10.1016/j.snb.2024.136873 Zhang Q, Li RY, Li ZJ, Yang YQ, Liu XB (2024) Mild and scalable synthesis of a high performance CrFeCoNiRu0.05 high entropy nano-alloy/carbon electrocatalyst for efficient urea production with a chelate-based ionic liquid, New J. Chem. 48: 9738-9747. https://doi.org/10.1039/D4NJ00835A Fu S, Xie C, Yang Z, Jiang M, Cheng J, Zhu C, Wu K, Ye H, Xia W, Jaffrezic-Renault N, Guo Z (2023) Electrochemical signal amplification strategy based on trace metal ion modified WS 2 for ultra-sensitive detection of miRNA-21, Talanta 260: 124552. https://doi.org/10.1016/j.talanta.2023.124552 Additional Declarations No competing interests reported. Supplementary Files Electronicsupportinginformation.docx Graphiticabstract.tif Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 12 Jul, 2025 Reviews received at journal 12 Jul, 2025 Reviews received at journal 08 Jul, 2025 Reviewers agreed at journal 03 Jul, 2025 Reviewers agreed at journal 02 Jul, 2025 Reviewers invited by journal 02 Jul, 2025 Editor assigned by journal 25 Jun, 2025 Submission checks completed at journal 25 Jun, 2025 First submitted to journal 21 Jun, 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. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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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-6946913","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":479435308,"identity":"654bc8a8-5990-4032-a25b-4e7562b14b5d","order_by":0,"name":"Li Ruiyi","email":"","orcid":"","institution":"Jiangnan University","correspondingAuthor":false,"prefix":"","firstName":"Li","middleName":"","lastName":"Ruiyi","suffix":""},{"id":479435310,"identity":"5392fe4e-da2a-4286-9eb6-b8d8cc9e414e","order_by":1,"name":"Li Mingyao","email":"","orcid":"","institution":"Jiangnan University","correspondingAuthor":false,"prefix":"","firstName":"Li","middleName":"","lastName":"Mingyao","suffix":""},{"id":479435311,"identity":"5b7cbf88-4a9f-4f29-ba89-20911d19cec0","order_by":2,"name":"Wang Miao","email":"","orcid":"","institution":"Wuxi Hospital of Traditional Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Wang","middleName":"","lastName":"Miao","suffix":""},{"id":479435312,"identity":"64ec5a18-8448-46cf-9070-62884c18ceb4","order_by":3,"name":"Li Zaijun","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABCUlEQVRIiWNgGAWjYFACxgYDBgYLHn6Ggw1wMQkitEjwSDYQrwWqxuAAChcPkG8/3FDMu0NCxvjg4cbPBb/uyJszMB+8zcNgl4fTWT2JDca8ZyR4zA4cbJae2ffMcGcDW7I1D0NyMS4tzAwgLW1gLQ3SvD2HGTcc4DGT5mE4kNiAQwsb/0OIFuOGg82/gVrsNxzg/4ZXC48E1BYDhoNt0jw/DicCbWHDq0VC4mGD4VygFokDB9useRsOJ284zGZsOccgGacW+f70ZwZv22zs+Wccf3yb589h2w3Hmx/eeFNhh1MLyDsGEPsOAMOvDRwiQGCAWz1IyQMwxQ8y9Q9elaNgFIyCUTBCAQAJe1eA5JCXlAAAAABJRU5ErkJggg==","orcid":"","institution":"Jiangnan University","correspondingAuthor":true,"prefix":"","firstName":"Li","middleName":"","lastName":"Zaijun","suffix":""}],"badges":[],"createdAt":"2025-06-21 21:53:12","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6946913/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6946913/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":85962121,"identity":"dd0cac6e-437d-4733-b51f-ce2da86c8a25","added_by":"auto","created_at":"2025-07-03 15:54:19","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":470399,"visible":true,"origin":"","legend":"\u003cp\u003eDesign and synthesis of SGQD and Au\u003csub\u003e0.1\u003c/sub\u003eFeCoNiCu\u003c/p\u003e","description":"","filename":"Fig.1.png","url":"https://assets-eu.researchsquare.com/files/rs-6946913/v1/1e2873f04fb53a74e88e8dd5.png"},{"id":85963618,"identity":"1e928de1-f531-4e5e-8270-5a47deea9fb9","added_by":"auto","created_at":"2025-07-03 16:10:19","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":10195896,"visible":true,"origin":"","legend":"\u003cp\u003eSEM (A), TEM (B)\u003cstrong\u003e, \u003c/strong\u003eHAADF-STEM images (C), atomic strain map (D) and electron diffraction pattern (E) of the selected area in Fig. 2C, live profiles of IFFT of the (200) (F) and (220) (C) (G), elemental mappings of Au (H), Co (I), Cu (J), Fe (K), Ni (L) and merged elements (M)\u003c/p\u003e","description":"","filename":"Fig.2.png","url":"https://assets-eu.researchsquare.com/files/rs-6946913/v1/e39081f773d6290646682d36.png"},{"id":85962698,"identity":"9f805d26-ea6d-4c0b-93d6-528fab426afd","added_by":"auto","created_at":"2025-07-03 16:02:19","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":4421837,"visible":true,"origin":"","legend":"\u003cp\u003eXRD pattern (A), schematic structure (B), FTIR spectrum (C), and high-resolution XPS spectra of C1s (D), Au4f (E), Fe2p (F), Co2p (G), Ni2p (H) and Cu2p (I)\u003c/p\u003e","description":"","filename":"Fig.3.png","url":"https://assets-eu.researchsquare.com/files/rs-6946913/v1/e585ed2a7fc71a55c2edc186.png"},{"id":85962700,"identity":"c3a39da9-7a52-427b-9132-142a1bbc2b34","added_by":"auto","created_at":"2025-07-03 16:02:19","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":2714267,"visible":true,"origin":"","legend":"\u003cp\u003eBiosensor construction scheme (A), electrophoregrams (B) of miRNA-21 (lane 1), Apt (lane 2), walker (lane 3), H1 (lane 4), H2 (lane 5), warker-Apt+H2 (lane 6), H1+ walker-Apt+ miRNA-21 (lane 7), and H1+ walker-Apt+ miRNA-21+H2 (lane 8), CV curves (C) and Nyquist plots (Inert: equivalent circuit) (D) of bare GCE (a), HEA/GCE (b), walker-Apt/H1/HEA/GCE (c), MCH/walker-Apt/H1/GCE before (d) and after incubated at 37 °C for 60 min in a mixed H2-Fc solutioon with miRNA-21, CV curves (E) of the biosensor at scan rates of 10, 20, 30, 40, 50, 60,70, 80, 90 and 100 mV s\u003csup\u003e-1\u003c/sup\u003e, plots (F) of the CV peak current \u003cem\u003evs\u003c/em\u003e. the scan rate, and CV cures of biosensor at scan rate of 10 mV s\u003csup\u003e-1\u003c/sup\u003e for 1st and 100\u003csup\u003eth\u003c/sup\u003e cycles. The electrolyte: 1 mM K\u003csub\u003e4\u003c/sub\u003eFe(CN)\u003csub\u003e6\u003c/sub\u003e in the PBS of pH 7.4\u003c/p\u003e","description":"","filename":"Fig.4.png","url":"https://assets-eu.researchsquare.com/files/rs-6946913/v1/35043d67ede0f7a364b6bcc9.png"},{"id":85963619,"identity":"6bdb30f4-9c7e-487c-b5c0-3300bb651d79","added_by":"auto","created_at":"2025-07-03 16:10:19","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1948842,"visible":true,"origin":"","legend":"\u003cp\u003eCV (A) and DPV curves (B) in the absence (a) and the presence of 1´10\u003csup\u003e−15\u003c/sup\u003e M miRNA-21 (b), plots of the DPV peak current vs. the incubation time (C) and H2-Fc volume (D), DPV curves in the presence of 0.0, 1×10\u003csup\u003e-20\u003c/sup\u003e, 5×10\u003csup\u003e-20\u003c/sup\u003e, 1×10\u003csup\u003e-19\u003c/sup\u003e, 5×10\u003csup\u003e-19\u003c/sup\u003e, 1×10\u003csup\u003e-18\u003c/sup\u003e, 5×10\u003csup\u003e-18\u003c/sup\u003e, 1×10\u003csup\u003e-17\u003c/sup\u003e, 5×10\u003csup\u003e-17\u003c/sup\u003e, 1×10\u003csup\u003e-16\u003c/sup\u003e, 5×10\u003csup\u003e-16\u003c/sup\u003e, and 1×10\u003csup\u003e-15\u003c/sup\u003e M miRNA-21 (E), and linear relationship curve of the DPV peak current with the LOG(C\u003csub\u003emiRNA-21\u003c/sub\u003e) (F), and the DPV peak current at different repeated detection times (G), different biosensors (H) and different storage time (I)\u003c/p\u003e","description":"","filename":"Fig.5.png","url":"https://assets-eu.researchsquare.com/files/rs-6946913/v1/41a9aad09ecff86131afb0de.png"},{"id":85963853,"identity":"db2d7bb1-2bd3-401e-accc-10def7e0e1bd","added_by":"auto","created_at":"2025-07-03 16:18:29","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":20634753,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6946913/v1/8965c694-0b27-493a-84a8-3911c1ab1a9b.pdf"},{"id":85962128,"identity":"2f76f5de-0761-4076-ba8c-549adc8acaeb","added_by":"auto","created_at":"2025-07-03 15:54:19","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":1101908,"visible":true,"origin":"","legend":"","description":"","filename":"Electronicsupportinginformation.docx","url":"https://assets-eu.researchsquare.com/files/rs-6946913/v1/9f668113ff5ef62f89b0d9bc.docx"},{"id":85962701,"identity":"b5426549-80fa-4cb9-b8f7-d0f524b8edaa","added_by":"auto","created_at":"2025-07-03 16:02:19","extension":"tif","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":559136,"visible":true,"origin":"","legend":"","description":"","filename":"Graphiticabstract.tif","url":"https://assets-eu.researchsquare.com/files/rs-6946913/v1/d31b4fc288c818b01fdc1525.tif"}],"financialInterests":"No competing interests reported.","formattedTitle":"Ultrasensitive sensing platform based on Au 0.1 FeCoNiCu high-entropy alloy nanoparticle-DNA walker dual signal amplification for electrochemical detection of miRNA-21","fulltext":[{"header":"1 Introduction","content":"\u003cp\u003eIncreasing evidence shows dysregulated expression of MiRNA-21 strongly correlates with formation, invasion and metastasis of breast cancer [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Development of sensing technique for precise analysis of miRNA-21 provided strong impetus to real need for early diagnosis of breast cancer [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. The main analytical techniques for miRNA-21 are fluorescence [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e], surface-enhanced Raman scattering [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e], electrochemiluminescence [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e] and electrochemical biosensor [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Electrochemical biosensor received a more attention because of high sensitivity, low cost and rapid response. However, classical electrochemical biosensor is far from meeting the need for high sensitivity of early breast cancer diagnosis [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eMany metal materials were investigated for construction of electrochemical detection of miRNA-21. Au is commonly used electrochemical sensing material due to excellent electrical conductivity, catalytic activity and chemical inertness [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. However, high-cost, poor functional diversity and resource scarcity limit its wide use. Graphene is a rising star of sensing materials owing to large specific surface, high electrical conductivity and good mechanical property, but it cannot provide special catalytic capacity [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. To resolve the problem, hybrid of Au with graphene become one new research hotspot [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. All Au nanoparticles are connected into one whole by graphene, accelerating the electron transfer between different Au nanoparticles. This results in an improved catalytic activity. Heterojunction sensing materials caused great research interest in analytical chemistry [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Formation of heterojunction narrows bandgap and leads to an improved catalytic activity. Despite these advances, design and synthesis of sensing material for electrochemical detection of miRNA-21 in early diagnosis of breast cancer is still challenging.\u003c/p\u003e \u003cp\u003eHigh entropy alloy (HEA) is one single-phase solid solution composing of five or more metal elements [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Mixing of all metal elements in HEA brings multi-element active sites. Interestingly, the active sites can be optimized by adjusting element configuration and composition [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. HEA nanoparticles become an ideal candidate of electrochemical sensing materials due to good multifunctionality, catalytic activity and selectivity [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. However, following three problems restrict its application in electrochemical detection of miRNA-21. (1) Current synthesis is difficult to be used for large-scale production of HEA nanoparticles due to the need of special equipment and severe reaction conditions. (2) Current HEA nanoparticles offer a lower catalytic activity compared with noble metal nanoparticle. (3) Current HEA nanoparticles offer a poor affinity with polar analyte and redox probe due to their hydrophobic characteristics.\u003c/p\u003e \u003cp\u003eIn the study, we reported one strong coordination route for synthesis of Au\u003csub\u003e0.1\u003c/sub\u003eFeCoNiCu high-entropy alloy nanoparticles by introducing serine-functionalized graphene quantum dot (SGQD). The resulting Au\u003csub\u003e0.1\u003c/sub\u003eFeCoNiCu exhibits catalytic activity of more than 12-fold that of sole Au nanoparticle. The biosensor with Au\u003csub\u003e0.1\u003c/sub\u003eFeCoNiCu and DNA walker exhibits ultrahigh sensitivity for electrochemical detection of miRNA-21.\u003c/p\u003e"},{"header":"2 Experimental","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Au\u003csub\u003e0.1\u003c/sub\u003eFeCoNiCu synthesis\u003c/h2\u003e \u003cp\u003eFeCl\u003csub\u003e3\u003c/sub\u003e (0.8 mmol), CoCl\u003csub\u003e2\u003c/sub\u003e (0.8 mmol), NiCl\u003csub\u003e2\u003c/sub\u003e (0.8 mmol), CuCl\u003csub\u003e2\u003c/sub\u003e (0.8 mmol) and HAuCl\u003csub\u003e4\u003c/sub\u003e (0.08 mmol) were dissolved in a Ser-GQD aqueous solution (50 mL,100 mg mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) witht stirring to form a purple-black clear solution. Followed by spray drying and annealing in N\u003csub\u003e2\u003c/sub\u003e at 600℃ for 2 h with a heating rate of 1℃ min\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e to obtain Au\u003csub\u003e0.1\u003c/sub\u003eFeCoNiCu. The procedure was also used for synthesis of sole Au nanoparticle unless no addition of FeCl\u003csub\u003e3\u003c/sub\u003e, CoCl\u003csub\u003e2\u003c/sub\u003e, NiCl\u003csub\u003e2\u003c/sub\u003e and CuCl\u003csub\u003e2\u003c/sub\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Biosensor construction\u003c/h2\u003e \u003cp\u003eH1 and H2-Fc were heated at 95℃ for 5 min and then cooled to room temperature to form hairpin structure. The pre-treated H1 was mixed with an equal volume of 1 mM TCEP solution and incubated for 1 h to form an activated H1; The walker solution (10 \u0026micro;M) was mixed with an equal volume of 10 \u0026micro;M aptamer, heated at 95℃ for 5 min, and cooled to room temperature to form double-stranded walker-Apt. The resulting walker-Apt was mixed with an equal volume of 1 mM TCEP and incubated for 1 h to form activated walker-Apt; Firstly, equal volume of Au\u003csub\u003e0.1\u003c/sub\u003eFeCoNiCu dispersion and chitosan solution were mixed to form Au\u003csub\u003e0.1\u003c/sub\u003eFeCoNiCu/chitosan dispersion, in which the mass ratio of Au\u003csub\u003e0.1\u003c/sub\u003eFeCoNiCu and chitosan is 1:5. Its 5 \u0026micro;L was dropped on the surface of glass carbon electrode (GCE, 2 mm in diameter) and dried. Then, 5 \u0026micro;L of activated H1 and 5 L of walker-Apt solution were dropped on the surface of Au\u003csub\u003e0.1\u003c/sub\u003eFeCoNiCu/GCE. Followed incubation for 2 h, washing with ultrapure water and drying. Finally, 5 \u0026micro;L of 1 mM mercaptohexanol (MCH) was dropped, incubated for 2 h, washed with ultrapure water and dried to obtain a biosensor. The as-prepared biosensor was stored at 4 ℃ in refrigerator before use.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Electrochemical detection of miRNA-21\u003c/h2\u003e \u003cp\u003eEqual volume of 10 \u0026micro;M pre-treated H2-Fc solution and MiRNA-21standard solution with different concentration (or sample solution) was mixed. Its 100 \u0026micro;L was dropped on the biosensor, then incubated at 37℃ for 1 h, washed with ultrapure water, and dried with N\u003csub\u003e2\u003c/sub\u003e flow. The incubated biosensor was given to the DPV measurement in a PBS of pH7.\u003c/p\u003e \u003c/div\u003e"},{"header":"3 Results and Discussions","content":"\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\n \u003ch2\u003e3.1 Design and synthesis\u003c/h2\u003e\n \u003cp\u003eFigure \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e outlines the design and synthesis of SGQD and Au\u003csub\u003e0.1\u003c/sub\u003eFeCoNiCu high-entropy alloy (HEA). SGQD synthesis employs citric acid as a carbon source to form graphene sheets, while serine introduces functional groups via amide bonds between its amine and graphene carboxyl groups, enhancing metal coordination through hydroxyl and carboxyl moieties. The HEA integrates Au (enabling biomolecule immobilization via Au\u0026ndash;S bonds) with Fe, Co, Ni, and Cu\u0026mdash;adjacent fourth-period transition metals with sequentially varying electronic structures. Their low-energy-barrier orbital hybridization facilitates electronic reconstruction, boosting catalytic activity and versatility. Cu partially substitutes Au due to electronic similarity. Synthesis involves a strong coordination route: metal ions first form homogeneous Me-SGQD complexes (\u003cstrong\u003eFig. \u003cspan class=\"InternalRef\"\u003eS1\u003c/span\u003e\u003c/strong\u003e), ensuring atomic dispersion. Subsequent thermal decomposition of SGQD generates reducing gases that reduce metal ions to atoms, bypassing vapor-phase processes (unlike carbothermal shock [\u003cspan class=\"CitationRef\"\u003e15\u003c/span\u003e]). Gas-stream-driven collisions under high-entropy conditions enable alloying at sub-melting temperatures, yielding single-phase Au\u003csub\u003e0.1\u003c/sub\u003eFeCoNiCu nanocrystals. This method avoids metal vaporization, leveraging gas-phase energy for controlled nanocrystal formation. (199 words)\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\n \u003ch2\u003e3.2 Material characterization\u003c/h2\u003e\n \u003cp\u003eMorphology and element dispersion of Au\u003csub\u003e0.1\u003c/sub\u003eFeCoNiCu were characterized by SEM, TEM, HAADF-STEM and element mapping technique (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e). Three-dimensional structure composing of graphene sheets appears on the SEM image. Small Au\u003csub\u003e0.1\u003c/sub\u003eFeCoNiCu nanoparticles are dispersed on graphene sheets. Au\u003csub\u003e0.1\u003c/sub\u003eFeCoNiCu nanoparticle offers a sphere-like morphology with an average particle size of 32.5\u0026plusmn;1.4 nm (Fig. s2). There are many light and dark dots on the HAADF-STEM image, corresponding to different metal atoms. Among Au, Fe, Co, Ni and Cu, Au atom indicates the best brightness, because of the biggest atomic weight. Atomic strain map reveals that the stress is dispersed anywhere in the lattice of Au\u003csub\u003e0.1\u003c/sub\u003eFeCoNiCu nanoparticles. The mixing of Au, Fe, Co, Ni and Cu causes an uneven stress distribution. Based on the electron diffraction pattern, live profiles of IFFT of the (200) and (220) faces were obtained. Average spacings (220) and (200) faces are 0.308 nm and 0.342 nm, respectively. However, an obvious difference in the spacing of different metal elements can be observed from Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eF and G. This is because mixing of Au, Fe, Co, Ni and Cu leads to disorder and twisting of lattice. The results of element mapping analysis confirm the spatial distribution of Au, Fe, Co, Ni and Cu, showing an excellent elemental homogeneity\u003c/p\u003e\n \u003cp\u003eCrystal structure of Au\u003csub\u003e0.1\u003c/sub\u003eFeCoNiCu nanoparticle was characterized by XRD technique. Figure \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eA shows XRD patterns of Au\u003csub\u003e0.1\u003c/sub\u003eFeCoNiCu mainly includes four diffraction peaks at 41.47\u003csup\u003eo\u003c/sup\u003e, 45.76\u003csup\u003eo\u003c/sup\u003e, 75.51\u003csup\u003eo\u003c/sup\u003e and 84.25\u003csup\u003eo\u003c/sup\u003e, corresponding to (111), (200), (220) and (311) panes of FCC phase [\u003cspan class=\"CitationRef\"\u003e16\u003c/span\u003e]. Further, Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eB depicts crystal structures of Au\u003csub\u003e0.1\u003c/sub\u003eFeCoNiCu is formed by octahedron being combined face to face.\u003c/p\u003e\n \u003cp\u003eChemical environment of Au\u003csub\u003e0.1\u003c/sub\u003eFeCoNiCu was characterized by FTIR and XPS spectra. The FTIR spectrum includes five IR absorption bands and absorption peaks. The band at about 3421 cm\u003csup\u003e-1\u003c/sup\u003e is IR absorption of stretching vibration of O-H, N-H and =\u0026thinsp;C-H bonds. The band at about 1636 is IR absorption of stretching vibration of C\u0026thinsp;=\u0026thinsp;O and C\u0026thinsp;=\u0026thinsp;C bonds. The peak at 1424 cm\u003csup\u003e-1\u003c/sup\u003e is IR absorption of stretching vibration of C-O bond. The peak at 1084 cm\u003csup\u003e-1\u003c/sup\u003e is IR absorption of stretching vibration of C-O-C bond. The peak at 610 cm\u003csup\u003e-1\u003c/sup\u003e is IR absorption of out-of-plane bending vibration of -CH\u003csub\u003e3\u003c/sub\u003e group.\u003c/p\u003e\n \u003cp\u003eTotal XPS spectrum includes the XPS peaks of Au4f, C1s, O1s, Fe2p, Co2p, N2p and Cu2p, verifying the existence of gold (Au), carbon (C), oxygen (O), iron (Fe), cobalt (Co), nickel (Ni) and copper (Cu) (Fig. s3). High-resolution XPS spectrum of C1s includes three peaks at 284.80, 287.59 and 289.97 eV, which correspond to C-C, C\u0026thinsp;=\u0026thinsp;O and O-C\u0026thinsp;=\u0026thinsp;O bonds. High-resolution XPS spectrum of Au4f includes four peaks at 83.99, 87.99, 85.46 and 89.75 eV. Two peaks at 83.99 and 85.46 eV correspond to Au4f\u003csub\u003e7/2\u003c/sub\u003e and Au4f\u003csub\u003e5/2\u003c/sub\u003e of Au\u003csup\u003e0\u003c/sup\u003e, showing the existence of Au\u003csup\u003e0\u003c/sup\u003e. Two peaks at 87.99 and 89.75 eV correspond to Au4f\u003csub\u003e7/2\u003c/sub\u003e and Au4f\u003csub\u003e5/2\u003c/sub\u003e of Au\u003csup\u003e+\u003c/sup\u003e, showing the existence of Au\u003csup\u003e+\u003c/sup\u003e [\u003cspan class=\"CitationRef\"\u003e17\u003c/span\u003e]. High-resolution XPS spectrum of Fe2p includes six peaks at 708.92, 711.69, 716.46, 722.02, 724.79 and 729.62 eV. Two peaks at 708.92 and 722.02 eV correspond to Fe2p\u003csub\u003e3/2\u003c/sub\u003e and Fe2p\u003csub\u003e1/2\u003c/sub\u003e of Fe\u003csup\u003e0\u003c/sup\u003e, showing the existence of Fe\u003csup\u003e0\u003c/sup\u003e. Two peaks at 711.69 and 724.79 eV correspond to Fe2p\u003csub\u003e3/2\u003c/sub\u003e and Fe2p\u003csub\u003e1/2\u003c/sub\u003e of Fe\u003csup\u003e2+\u003c/sup\u003e, showing the existence of Fe\u003csup\u003e2+\u003c/sup\u003e. Two peaks at 716.46 and 729.62 eV correspond to Fe2p\u003csub\u003e3/2\u003c/sub\u003e and Fe2p\u003csub\u003e1/2\u003c/sub\u003e of Fe\u003csup\u003e3+\u003c/sup\u003e, showing the existence of Fe\u003csup\u003e3+\u003c/sup\u003e. High-resolution XPS spectrum of Co2p includes six peaks at 779.68, 781.12, 785.23, 794.60, 796.05 and 800.59 eV. Two peaks at 779.68 and 794.60 eV correspond to Co2p\u003csub\u003e3/2\u003c/sub\u003e and Co2p\u003csub\u003e1/2\u003c/sub\u003e of Co\u003csup\u003e3+\u003c/sup\u003e, showing the existence of Co\u003csup\u003e3+\u003c/sup\u003e. Two peaks at 781.12 and 796.05 eV correspond to Co2p\u003csub\u003e3/2\u003c/sub\u003e and Co2p\u003csub\u003e1/2\u003c/sub\u003e of Co\u003csup\u003e2+\u003c/sup\u003e, showing the existence of Co\u003csup\u003e2+\u003c/sup\u003e. Two peaks at 785.23 and 800.59 eV correspond to Co2p\u003csub\u003e3/2\u003c/sub\u003e and Co2p\u003csub\u003e1/2\u003c/sub\u003e of satellite Co2p (Sat.). High-resolution XPS spectrum of Ni2p includes four peaks at 851.47, 857.10, 867.58 and 872.68 eV. Two peaks at 851.57 and 867.58 eV correspond to Ni2p\u003csub\u003e3/2\u003c/sub\u003e and Ni2p\u003csub\u003e1/2\u003c/sub\u003e of Ni\u003csup\u003e0\u003c/sup\u003e, showing the existence of Ni\u003csup\u003e0\u003c/sup\u003e. Two peaks at 857.10 and 872.68 eV correspond to Ni2p\u003csub\u003e3/2\u003c/sub\u003e and Ni2p\u003csub\u003e1/2\u003c/sub\u003e of Ni\u003csup\u003e2+\u003c/sup\u003e, showing the existence of Ni\u003csup\u003e2+\u003c/sup\u003e. High-resolution XPS spectrum of Cu2p includes six peaks at 932.16, 933.75, 942.87, 951.65, 953.55 and 960.21 eV. Two peaks at 932.16 and 951.65 eV correspond to Cu2p\u003csub\u003e3/2\u003c/sub\u003e and Cu2p\u003csub\u003e1/2\u003c/sub\u003e of Cu\u003csup\u003e0\u003c/sup\u003e, showing the existence of Cu\u003csup\u003e0\u003c/sup\u003e. Two peaks at 933.75 and 953.55 eV correspond to Cu2p\u003csub\u003e3/2\u003c/sub\u003e and Cu2p\u003csub\u003e1/2\u003c/sub\u003e of Cu\u003csup\u003e2+\u003c/sup\u003e, showing the existence of Cu\u003csup\u003e2+\u003c/sup\u003e. Two peaks at 942.87 and 960.21 eV correspond to Cu2p\u003csub\u003e3/2\u003c/sub\u003e and Cu2p\u003csub\u003e1/2\u003c/sub\u003e of satellite Cu2p (Sat.), showing the existence of Sat. The above results confirm the presence of high valent Au, Fe, Co, Ni and Cu species, which can combine with SGQD by coordination bonds to form SGQD layer on the surface of Au\u003csub\u003e0.1\u003c/sub\u003eFeCoNiCu nanoparticle [\u003cspan class=\"CitationRef\"\u003e18\u003c/span\u003e].\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\n \u003ch2\u003e3.3 Catalytic activity evaluation\u003c/h2\u003e\n \u003cp\u003eTo evaluate catalytic activity of Au\u003csub\u003e0.1\u003c/sub\u003eFeCoNiCu, CV and DPV curves of Au\u003csub\u003e0.1\u003c/sub\u003eFeCoNiCu and Au-control electrodes were measured in 1 mM K\u003csub\u003e4\u003c/sub\u003eFe(CN)\u003csub\u003e6\u003c/sub\u003e solution. Fig. s4 shows CV curves of Au\u003csub\u003e0.1\u003c/sub\u003eFeCoNiCu and Au-control electrodes offer one pair of redox peaks, showing Au\u003csub\u003e0.1\u003c/sub\u003eFeCoNiCu and Au-control can catalyze oxidation and reduction of Fe(CN)\u003csub\u003e6\u003c/sub\u003e\u003csup\u003e4\u0026minus;\u003c/sup\u003e. However, the oxidation potential and reduction potentials of Au\u003csub\u003e0.1\u003c/sub\u003eFeCoNiCu electrode are lower than that of Au-control electrode, verifying Au\u003csub\u003e0.1\u003c/sub\u003eFeCoNiCu has a better catalytic activity. Further, the DPV curves indicate that Au\u003csub\u003e0.1\u003c/sub\u003eFeCoNiCu has a more sensitive current response. The peak current is more than 12-fold that of Au-control electrode, showing a significantly enhanced catalytic activity. To understand the improvement in catalytic activity, CV behavior of Au\u003csub\u003e0.1\u003c/sub\u003eFeCoNiCu and Au-control electrodes were studied by measuring the electrochemical surface areas (ECSAs). Fig. s5 shows Au\u003csub\u003e0.1\u003c/sub\u003eFeCoNiCu offers a higher CV current density, verifying the presence of more active sites. Further, the relationships between change in the current density (\u0026Delta;J) and scan rates for Au\u003csub\u003e0.1\u003c/sub\u003eFeCoNiCu and Au-control electrodes were shown in Fig. s5B. There is one good linear relationship between \u0026Delta;J and scan rate. The corresponding slopes (C\u003csub\u003edl\u003c/sub\u003e) are 70 mF cm\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e of Au\u003csub\u003e0.1\u003c/sub\u003eFeCoNiCu and 1 mF cm\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e of Au-control. For similar nanomaterials, the C\u003csub\u003edl\u003c/sub\u003e value can represent the ECSA of catalyst. Thus, the above data demonstrate the ECSA of Au\u003csub\u003e0.1\u003c/sub\u003eFeCoNiCu is more than 70-fold that of Au-control. Based on the result, we suggest excellent catalytic activity of Au\u003csub\u003e0.1\u003c/sub\u003eFeCoNiCu is attributed to a more ECSA produced by mixing of Au, Fe, Co, Ni and Cu.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\n \u003ch2\u003e3.4 Biosensor construction\u003c/h2\u003e\n \u003cp\u003eBiosensor construction includes immobilization of Au\u003csub\u003e0.1\u003c/sub\u003eFeCoNiCu (HEA), H1, walker-Apt and MCH on GCE (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eA). HEA, H1, walker-Apt and MCH are orderly designed to catalyze redox of Fc, capture H2-Fc, identify miRNA-21 and block residual active sites. MiRNA-21 specifically binds with Apt in walker-Apt to release free walker. The released walker hybridizes with H1 on the biosensor to open harpin structure of H1. The formed walker-H1 combines with H2-Fc to release one free walker. Meanwhile, one H2-Fc was immobilized on the surface of HEA by hybridization of H1 with H2. The redox of Fc in H2-Fc under catalysis of HEA produces a sensitive electrochemical response. The above procedure achieves to one-step walking of DNA walker. The released walker binds with another H1 on the biosensor to trigger next walking of DNA walker. By the walking, one walker can bring many H2-Fc probes to the biosensor. The redox of Fc molecules in these H2-Fc probes produces a significant signal amplification.\u003c/p\u003e\n \u003cp\u003eThe sensing principle was tested by native PAGE analysis (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eB). One walker-Apt band and one H2 band appear on electrophoregram of walker-Apt\u0026thinsp;+\u0026thinsp;H2 (lane 6). No walker band and Apt band can be seen from lane 6, verifying H2 cannot hydridize with walker-Apt to release free walker. One walker-H1 band and one Apt-miRNA-21 band appear on electrophoregram of walker-Apt\u0026thinsp;+\u0026thinsp;H1\u0026thinsp;+\u0026thinsp;miRNA-21 (lane 7). This confirms miRNA-21 can bind with Apt in walker-Apt to form Apt-miRNA-21. The released walker can bind with H1 to form walker-H1. One H1-H2 band and one walker-Apt band appear on electrophoregram of walker-Apt\u0026thinsp;+\u0026thinsp;H1\u0026thinsp;+\u0026thinsp;H2\u0026thinsp;+\u0026thinsp;miRNA-21 (lane 8). In the presence of miRNA-21, H1 can combine with H2 to form H1-H2. The above results demonstrate that the sensing principle is of high reliability.\u003c/p\u003e\n \u003cp\u003eCV and EIS behaviours of different electrodes were examined in 1 mM K\u003csub\u003e4\u003c/sub\u003eFe(CN)\u003csub\u003e6\u003c/sub\u003e in PBS of pH 7.4 to understand electrochemical property of biosensor. Figure \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eC and D show the modification of Au\u003csub\u003e0.1\u003c/sub\u003eFeCoNiCu causes an increased CV current and a reduced charge transfer impedance (R\u003csub\u003ect\u003c/sub\u003e) (Table \u003cspan class=\"InternalRef\"\u003es1\u003c/span\u003e). Due to excellent conductivity and catalytic activity, the introduction of Au\u003csub\u003e0.1\u003c/sub\u003eFeCoNiCu accelerates redox of Fe(CN)\u003csub\u003e6\u003c/sub\u003e\u003csup\u003e4-\u003c/sup\u003e on electrode surface, leading to an increased CV current and a reduced R\u003csub\u003ect\u003c/sub\u003e. Different from Au\u003csub\u003e0.1\u003c/sub\u003eFeCoNiCu, the modification of H1, walker-Apt and MCH causes an obviously decreased CV current and an increasd R\u003csub\u003ect\u003c/sub\u003e. This is because H1, walker-Apt and MCH are non-conductive. Their introduction partly blocks the electron transfer pathways between electrode and electrolyte, reducing the CV current. However, the incubation in a mixed H2-Fc solution with miRNA-21 results in an increased CV current with a reduced R\u003csub\u003ect\u003c/sub\u003e. This is because the presence of miRNA-21 triggers the DNA walker. By the walking, one walker-Apt molecule brings many H2-Fc probes to the biosensor. The redox of Fc molecules in these H2-Fc probes achieves to significant signal amplification, indicating an obviously increased CV current.\u003c/p\u003e\n \u003cp\u003eThe CV behaviour was investigated by varying scan rate to study on the electrode reaction kinetics (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eE and F). The increase of scan rate brings an increase in the CV peak current. There is a good relationship between CV peak current and square root of scan rate, verifying the electrode reaction is controlled by electrolyte diffusion. Such a fast electrode reaction process is attributed to excellent conductivity and catalytic activity of Au\u003csub\u003e0.1\u003c/sub\u003eFeCoNiCu. Further, the CV measurement was repeated for 100 times. Figure \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eG indicates 100-cycle didn\u0026rsquo;t cause significant change in CV curve, showing that the omission of electroactive ingredients can be ignored.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\n \u003ch2\u003e3.5 Electrochemical detection of miRNA\u003c/h2\u003e\n \u003cp\u003eCV and DPV curves of the biosensor in the PBS of pH 7.4 were measured in the absence and the presence of 1\u0026times;10\u003csup\u003e\u0026minus;\u0026thinsp;15\u003c/sup\u003e M miRNA-21. Figure \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eA and B shows the introduction of miRNA-21 brings significant increase in CV and DPV peak currents, producing sensitive electrochemical response. This is because the presence of miRNA-21 triggers the DNA walker, bringing H2-Fc probe to the biosensor. By DNA walking, one miRNA-21 molecule can bring many H2-Fc probes to the biosensor. Their redox causes significant signal amplification, indicating an increased CV and DPV current. To obtain high sensitivity, DPV technique was selected for electrochemical detection of miRNA-21.\u003c/p\u003e\n \u003cp\u003eThe influence of incubation time and H2-Fc concentration on DPV current was studied to optimize detection conditions. Figure \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eC shows increasing incubation time brings an increase in DPV peak current when the time is less than 40 min. This is because more time produces more steps of DNA walking, increasing number of H2-Fc probes on the biosensor. The redox of these probes produces an obviously increased DPV peak current. However, the DPV peak current tends to stable when the incubation time is more than 60 min. This is because the immobilization and removal of H2-Fc probes on the biosensor reaches a dynamic equilibrium state. At the state, number of H2-Fc probes on the biosensor keeps almost unchanged, indicating a stable DPV current. Figure \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eD shows the increase of H2-Fc concentration produces an increased DPV peak current when the volume is less than 10 \u0026micro;L. However, the DPV peak current tends to stable when the volume is more than 10 \u0026micro;L. This is because the H2-Fc solution of less than 10 \u0026micro;L cannot provide enough number of H2-Fc probes needed for the DNA walker. A more volume of H2-Fc solution results in a more H2-Fc probes on the biosensor, increasing the DPV peak current. When the volume of H2-Fc solution reaches to 10 \u0026micro;L, the need can can be met. Thus, the increase of H2-Fc volume cannot bring more H2-Fc probes to the biosensor, showing a stable DPV peak current.\u003c/p\u003e\n \u003cp\u003eFigure \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eE presents the DPV curves in the presence of different concentration of miRNA-21. The DPV current increases with increasing miRNA-21 concentration. This is because a higher concentration of miRNA-21 brings a faster DNA walking proceess. Accordingly, within an incubation of 60 min more H2-Fc probes were brought to the biosensor, providing an increased DPV current. Further, the relationship between DPV peak current and the logarithmic value of miRNA-21 concentration (LOG(C\u003csub\u003emiRNA\u003c/sub\u003e-21)) was shown in Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eF. When the miRNA-21 concentration is in the range of 10\u003csup\u003e\u0026minus;\u0026thinsp;15\u003c/sup\u003e~10\u003csup\u003e\u0026minus;\u0026thinsp;20\u003c/sup\u003e M, there is one good linear relationship between the DPV peak current (I\u003csub\u003ep\u003c/sub\u003e, \u0026micro;A) and the LOG(C\u003csub\u003emiRNA\u0026minus;21\u003c/sub\u003e, M). The corresponding linear equation is : I\u003csub\u003ep\u003c/sub\u003e=76.433\u0026times;LOG(C\u003csub\u003emiRNA\u0026minus;21\u003c/sub\u003e)\u0026thinsp;+\u0026thinsp;1560.8, affording R\u003csup\u003e2\u003c/sup\u003e of 0.997 and detection limit of 3.4\u0026times;10\u003csup\u003e\u0026minus;\u0026thinsp;21\u003c/sup\u003e M (S/N\u0026thinsp;=\u0026thinsp;3). The sensitivity is higher than that of other biosensors for miRNA-21 in Table s2.\u003c/p\u003e\n \u003cp\u003eThe biosensor was used for consecutive DPV detection of 1\u0026times;10\u003csup\u003e\u0026minus;\u0026thinsp;15\u003c/sup\u003e M miRNA-21. The RSD of 100 consecutive detection is 2.1%, showing a good repeatability. Ten biosensors were constructed by the same method and used for DPV detection of 1\u0026times;10\u003csup\u003e\u0026minus;\u0026thinsp;15\u003c/sup\u003e miRNA-21. The RSD of fifty biosensors is 1.9%, showing a good reproducibility. The biosensor was stored at 4\u0026deg;C and then remove it every other week, and return to room temperature for DPV detection of 1\u0026times;10\u003csup\u003e\u0026minus;\u0026thinsp;15\u003c/sup\u003e miRNA-21. The RSD of 8-week storage is 2.7%, showing a desirable storage stability.\u003c/p\u003e\n \u003cp\u003eThe interference effects of diverse species (inorganic ions, organic molecules, bovine serum albumin) on the DPV peak current of 1 \u0026times; 10⁻\u0026sup1;⁵ M miRNA-21 were systematically investigated. Experimental analyses demonstrated that each interferent (1 mg) induced a current variation of \u0026lt;\u0026thinsp;1.2%, attributed to their inability to bind the aptamer in the walker-Apt complex, thereby failing to activate the DNA walker process or translocate H₂-Fc probes. Specificity assessments via hybridization with single-base mismatch (miRNA-21-1M), two-base mismatch (miRNA-21-2M), three-base mismatch (miRNA-21-3M), miRNA-210, or miRNA-221 revealed peak currents reduced to 2.78% of the intact miRNA-21 signal (\u003cstrong\u003eFig. S6\u003c/strong\u003e), confirming single-nucleotide discrimination. These results highlight two critical merits: (1) robust anti-interference performance in complex matrices due to selective Apt-target binding, and (2) single-base resolution for miRNA-21 detection. The sensor thus enables precise quantification in serum despite coexisting biomolecular interferents and structurally similar miRNAs, underscoring its reliability for real-sample applications.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\n \u003ch2\u003e3.6 Sample analysis\u003c/h2\u003e\n \u003cp\u003eTo validate the reliability of the proposed sensor in real-sample analysis, recovery tests were systematically conducted using human serum. Two serum samples were diluted 10,000-fold and spiked with standard miRNA-21 concentrations spanning five orders of magnitude (0.05, 0.5, 5, 50, and 500 aM) for parallel detection. Each concentration level underwent a minimum of five replicate measurements, with mean recovery rates and relative standard deviations (RSDs) calculated to assess precision and accuracy. As detailed in Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e, the method exhibited robust performance, with spiked recoveries ranging from 95.0\u0026ndash;102.0% and RSDs between 1.4% and 4.99%, confirming its reproducibility in complex biological matrices.\u003c/p\u003e\n \u003cp\u003eSubsequently, the sensor was applied to quantify endogenous miRNA-21 levels in normal human serum. As summarized in \u003cstrong\u003eTable S3\u003c/strong\u003e, baseline miRNA-21 concentrations in healthy individuals were determined to be 0.015, 0.084, 0.13, and 0.78 fM, values substantially lower than those reported in serum from breast cancer patients [\u003cspan class=\"CitationRef\"\u003e19\u003c/span\u003e]. This stark contrast underscores the method\u0026rsquo;s sensitivity in distinguishing pathological from physiological miRNA-21 expression. Notably, recovery tests for endogenous analytes yielded 96.5\u0026ndash;104.4% recoveries with RSDs of 1.2\u0026ndash;2.77%, further validating the method\u0026rsquo;s accuracy under clinically relevant conditions.\u0026nbsp;\u003c/p\u003e\n \u003ctable id=\"Tab2\" border=\"1\" class=\"fr-table-selection-hover\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eResults of the miRNA-21 standard added serum recovery test by the proposed method (N\u0026thinsp;=\u0026thinsp;5)\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eSamples\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003emiRNA-21 added\u003c/p\u003e\n \u003cp\u003e(aM)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003emiRNA-21 found by proposed method\u003c/p\u003e\n \u003cp\u003e(aM)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eRecovery\u003c/p\u003e\n \u003cp\u003e(%)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eRSD\u003c/p\u003e\n \u003cp\u003e(%)\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\" rowspan=\"5\"\u003e\n \u003cp\u003eSerum sample 1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.048\u0026thinsp;\u0026plusmn;\u0026thinsp;0.004\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e96.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e4.99\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.498\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e99.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e3.75\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e5.03\u0026thinsp;\u0026plusmn;\u0026thinsp;0.17\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e100.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e2.44\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e50\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e50.14\u0026thinsp;\u0026plusmn;\u0026thinsp;1.22\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e100.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e2.32\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e500\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e499.6\u0026thinsp;\u0026plusmn;\u0026thinsp;4.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e99.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.45\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"5\"\u003e\n \u003cp\u003eSerum sample 2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.051\u0026thinsp;\u0026plusmn;\u0026thinsp;0.008\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e102.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e3.54\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.475\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e95.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e3.31\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e5.04\u0026thinsp;\u0026plusmn;\u0026thinsp;0.63\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e100.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.99\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e50\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e49.81\u0026thinsp;\u0026plusmn;\u0026thinsp;1.01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e99.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e2.73\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e500\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e502.3\u0026thinsp;\u0026plusmn;\u0026thinsp;10.23\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e100.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.40\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003cp\u003e\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Conclusions","content":"\u003cp\u003eThe study presents one scalable metal-SGQD strong coordination route for synthesizing single-phase\u0026nbsp;Au\u003csub\u003e0.1\u003c/sub\u003eFeCoNiCu\u0026nbsp;high-entropy alloy nanoparticle. The\u0026nbsp;synthesis\u0026nbsp;realizes\u0026nbsp;impressive\u0026nbsp;catalytic activity and affinity with polar redox probe, addressing key challenges in the development of\u0026nbsp;high-entropy alloy nanoparticles.\u0026nbsp;The electrocatalytic activity\u0026nbsp;is more than 12-fold than that of sole Au nanoparticle. The\u0026nbsp;biosensor based on\u0026nbsp;Au\u003csub\u003e0.1\u003c/sub\u003eFeCoNiCu, coupled with the DNA walker,\u0026nbsp;exhibits an ultralow detection limit of 3.4\u0026times;10\u003csup\u003e-21\u003c/sup\u003e M, outperforming all previously reported biosensors. This work also offers a promising strategy for large-scale production of high-entropy alloy nanoparticles in sensing, biomedicine, catalysis, and energy storage and conversion.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eData availability\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData will be made available from the corresponding author on reasonable request.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflicts of interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThere are no conflicts to declare.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors acknowledge the Chemical Testing Center of the School of Chemistry and Materials Engineering, Jiangnan University for support.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eLi Ruiyi\u003c/strong\u003e: Investigation. \u003cstrong\u003eLi Mingyao\u003c/strong\u003e: Investigation.\u0026nbsp;\u003cstrong\u003eWang Miao:\u0026nbsp;\u003c/strong\u003eInvestigation. \u003cstrong\u003eLi Zaijun\u003c/strong\u003e: Conceptualization, Methodology.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by The National Key Research and Development Program of China (No. 2021YFA0910200).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eLi H, Tie XJ (2024) Exploring research progress in studying serum exosomal miRNA-21 as a molecular diagnostic marker for breast cancer, Clin. Transl. Oncol. 26: 2166-2171. https://doi.org/10.1007/s12094-024-03454-z\u003c/li\u003e\n \u003cli\u003eZoughi S, Faridbod F, S. Moradi S (2024) Rapid enzyme-free detection of miRNA-21 in human ovarian cancerous cells using a fluorescent nanobiosensor designed based on hairpin DNA-templated silver nanoclusters, Anal. Chim. Acta 1320: 342968. https://doi.org/10.1016/j.aca.2024.342968\u003c/li\u003e\n \u003cli\u003eHe M, Hou Z, Yin F, Cheng W, Xiang Y, Wang Z (2024) Simultaneous detection of breast cancer biomarkers HER2 and miRNA-21 based on duplex-specific nuclease signal amplification, J. Mater. Chem. B 12: 9930-9937. https://doi.org/10.1039/d4tb01845a\u003c/li\u003e\n \u003cli\u003eTan HS, Wang T, Han JM, Liu M, Li SS (2024) Dual-signal SERS biosensor based on spindle-shaped gold array for sensitive and accurate detection of miRNA 21, Sensor. Actuat. B-Chem. 403: 135157. https://doi.org/10.1016/j.snb.2023.135157\u003c/li\u003e\n \u003cli\u003eZheng K, Zheng Q, Mu X, Li MJ, Yi C (2024) A smartphone‑assisted electrochemiluminescent biosensor for highly sensitive detection of miRNA‑21 based on Ru(bpy)2(L)\u003csup\u003e4+\u003c/sup\u003e@MOF‑5 Microchim. Acta 191: 596. https://doi.org/10.1007/s00604-024-06675-9\u003c/li\u003e\n \u003cli\u003eZhang LN, He YB (2024) A label-free and ultrasensitive electrochemical biosensor using hybrid polypyrrole/gold nanoelectrocatalyst mediated signal amplification for the detection of miRNA-21, Anal. Methods 16: 7567-7575. https://doi.org/10.1039/d4ay01215a\u003c/li\u003e\n \u003cli\u003eCimminol W, Migliorelli D, Singh S, Miglione A, Generelli S, Cinti1S (2023) Design of a printed electrochemical strip towards miRNA‑21 detection in urine samples: optimization of the experimental procedures for real sample application, Anal. Bioanal. Chem. 415: 4511-4520. https://doi.org/10.1007/s00216-023-04659-x\u003c/li\u003e\n \u003cli\u003eHou Z, Zheng J, Zhang C, Li L, Chen D, Hu L, Xiong B, Ye H, Jaffrezic-Renault N, Guo Z (2022) Direct ultrasensitive electrochemical detection of breast cancer biomarker-miRNA-21 employing an aptasensor based on a microgel nanoparticle composite, Sensor. Actuat. B-Chem. 367: 132067. https://doi.org/10.1016/j.snb.2022.132067\u003c/li\u003e\n \u003cli\u003eHuang MS, Govindasamy M, Chinnapaiyan S, Lin YT, Lu SY, Samukawa S, Huang CH (2024) Electrochemical biosensor based on an atomic layered composite of graphene oxide/graphene as an electrode material towards selective and sensitive detection of miRNA-21, Microchem. J. 199 : 110112. https://doi.org/10.1016/j.microc.2024.110112\u003c/li\u003e\n \u003cli\u003eKhodadoust A, Nasirizadeh N, Seyfati SM, Taheri RA, Ghanei M, Bagheri H (2023) High-performance strategy for the construction of electrochemical biosensor for simultaneous detection of miRNA-141 and miRNA-21 as lung cancer biomarkers, Talanta 252: 123863. https://doi.org/10.1016/j.talanta.2022.123863\u003c/li\u003e\n \u003cli\u003eWang X, Li H, Jian H, Liu L, Zeng H, Ai J, Tang J (2025) Robust nCuO modulated by defect engineering enhanced photoelectrochemical biosensor for the detection of miRNA-21, Biosens. Bioelectron. 271: 117014. https://doi.org/10.1016/j.bios.2024.117014\u003c/li\u003e\n \u003cli\u003eTang C, Lvm CL, Chena P, Wang AJ, Feng JJ, Cheang TY, Xia H (2024) Dendritic quinary PtRhMoCoFe high-entropy alloy as a robust immunosensing nanoplatform for ultrasensitive detection of biomarker, Bioelectrochemistry 157: 108639. https://doi.org/10.1016/j.bioelechem.2024.108639\u003c/li\u003e\n \u003cli\u003eRaj G, Nandan R, Gakhad P, Kumar K, Singh AK, Nanda KK (2024) A novel electrochemical high-performance non-enzymatic glucose sensing based on face-centred cubic FeCoNiMnCr high entropy nano alloys encapsulated in NCNTs, Chem. Eng. J. 503: 158041. https://doi.org/10.1016/j.cej.2024.158041\u003c/li\u003e\n \u003cli\u003eSong D, Huang X, Liu Q, Li G, Xu X, Wang X, Wang J, Lu X, Gao F (2024) Rational construction of ultrathin PtPdRhCuM (M=Mn, Co or Ni) high entropy alloy nanotubes with rich defects for enhanced electrochemical activity: Electrochemical aldicarb sulfone sensing, Sensor. Actuat. B-Chem. 405: 135337. https://doi.org/10.1016/j.snb.2024.135337\u003c/li\u003e\n \u003cli\u003eWu Y, Lei Z, Liu X, Wang H, Lu Z (2018) Eight in one: high-entropy-alloy nanoparticles synthesized by carbothermal shock, Sci. Bull. 63: 737-738. https://doi.org/10.1016/j.addma.2023.103716\u003c/li\u003e\n \u003cli\u003eBajaj D, Chen Z, Qu SJ, Feng AH, Li DY, Chen DL (2023) Distinct origins of deformation twinning in an additively-manufactured high-entropy alloy, Additive Manufacturing 74: 103716. https://doi.org/10.1016/j.addma.2023.103716\u003c/li\u003e\n \u003cli\u003ePan P, Li RY, Wang X, Shen YR, Li ZJ (2025) Electrochemical biosensor based on Au\u003csub\u003e5\u003c/sub\u003eIr@graphenequantumdot nanocomposite and DNA walker for detection of atrazine in environmental water with extremely high sensitivity and selectivity, Sensor. Actuat. B-Chem. 423: 136873. https://doi.org/10.1016/j.snb.2024.136873\u003c/li\u003e\n \u003cli\u003eZhang Q, Li RY, Li ZJ, Yang YQ, Liu XB (2024) Mild and scalable synthesis of a high performance CrFeCoNiRu0.05 high entropy nano-alloy/carbon electrocatalyst for efficient urea production with a chelate-based ionic liquid, New J. Chem. 48: 9738-9747. https://doi.org/10.1039/D4NJ00835A\u003c/li\u003e\n \u003cli\u003eFu S, Xie C, Yang Z, Jiang M, Cheng J, Zhu C, Wu K, Ye H, Xia W, Jaffrezic-Renault N, Guo Z (2023) Electrochemical signal amplification strategy based on trace metal ion modified WS\u003csub\u003e2\u003c/sub\u003e for ultra-sensitive detection of miRNA-21, Talanta 260: 124552. https://doi.org/10.1016/j.talanta.2023.124552\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":"High entropy alloy, Strong coordination route, Early cancer diagnosis","lastPublishedDoi":"10.21203/rs.3.rs-6946913/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6946913/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eOvercoming the critical limitation of poor sensitivity in electrochemical biosensors for early breast cancer diagnosis, this study presents an innovative approach. We developed a robust coordination strategy, utilizing serine-functionalized graphene quantum dot (SGQD), to synthesize Au\u003csub\u003e0.1\u003c/sub\u003eFeCoNiCu high-entropy alloy nanoparticles (HEA NPs). The resulting Au\u003csub\u003e0.1\u003c/sub\u003eFeCoNiCu HEA NPs (32\u0026thinsp;\u0026plusmn;\u0026thinsp;1.7 nm) exhibit a single FCC phase, elemental homogeneity, and graphene surface modification. This unique structure confers significantly enhanced catalytic activity (\u0026gt;\u0026thinsp;12-fold higher than pure Au NPs) and superior affinity towards polar electrolytes. Furthermore, we integrated these HEA NPs with a DNA walker circuit to construct a novel electrochemical biosensor for ultrasensitive miRNA-21 detection. Target miRNA-21 triggers the DNA walker, immobilizing ferrocene molecules on the electrode surface and generating a measurable electrochemical signal. This dual-amplification strategy (HEA NP catalysis\u0026thinsp;+\u0026thinsp;DNA walker) achieved unprecedented sensitivity: a linear current response (at 0.22 V) over an extraordinary range (1\u0026times;10⁻\u0026sup2;⁰ M to 1\u0026times;10⁻\u0026sup1;⁵ M) and a record-low detection limit of 3.4\u0026times;10⁻\u0026sup2;\u0026sup1; M. This represents a 2\u0026ndash;3 orders of magnitude sensitivity improvement over state-of-the-art sensors, successfully demonstrated in serum analysis.\u003c/p\u003e","manuscriptTitle":"Ultrasensitive sensing platform based on Au 0.1 FeCoNiCu high-entropy alloy nanoparticle-DNA walker dual signal amplification for electrochemical detection of miRNA-21","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-07-03 15:54:14","doi":"10.21203/rs.3.rs-6946913/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-07-12T18:33:10+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-07-12T15:04:12+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-07-08T10:16:03+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"235931262017128333209233546978193849264","date":"2025-07-04T00:36:48+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"35819993826117220552111273294572876712","date":"2025-07-02T06:56:27+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-07-02T06:17:01+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-06-25T13:32:50+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-06-25T08:17:57+00:00","index":"","fulltext":""},{"type":"submitted","content":"Microchimica Acta","date":"2025-06-21T21:49:03+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":"f1a5c2c6-8c80-44e8-bc0e-0282cee42b52","owner":[],"postedDate":"July 3rd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2025-07-18T11:38:38+00:00","versionOfRecord":[],"versionCreatedAt":"2025-07-03 15:54:14","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6946913","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6946913","identity":"rs-6946913","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}