Aptamer functionalized sheet-like α-Fe2O3/Fe3O4 magnetic nanocomposite-based electrochemical nano-biosensor for the accurate detection of nucleolin

Min Sun; Boyi Jiang; Jiawei Wang; Zhou Wang; Xinyan Gao; Xiaoting Yi

doi:10.25259/AJC_455_2025

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Original Article

:19;

4552025

doi:

10.25259/AJC_455_2025

Aptamer functionalized sheet-like α-Fe₂O₃/Fe₃O₄ magnetic nanocomposite-based electrochemical nano-biosensor for the accurate detection of nucleolin

Min Sun^a, Boyi Jiang^b, Jiawei Wang^a, Zhou Wang^c, Xinyan Gao^d, Xiaoting Yi^a,*

aZhenjiang 359 Hospital, Zhenjiang, P.R. China

bThe People’s Hospital of Danyang, Affiliated Danyang Hospital of Nantong University, Zhenjiang, P.R. China

cCollege of Vanadium and Titanium, Panzhihua University, Panzhihua, China

dShanghai Southwest Weiyu Secondary School International Division, Shanghai, P.R. China

*Corresponding author: E-mail address: yixiaoting359@163.com (X. Yi)

Received: 2025-04-30, Accepted: 2025-09-23, Epub ahead of print: 2026-01-09, Published: 2026-02-02

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This is an open-access article distributed under the terms of the Creative Commons Attribution-Non Commercial-Share Alike 4.0 License, which allows others to remix, transform, and build upon the work non-commercially, as long as the author is credited and the new creations are licensed under the identical terms.

Abstract

The detection of tumor biomarkers is critical in diagnostics, prevention, and therapeutics. As a significant biomarker of renal carcinoma, breast cancer, lymphocytic leukemia, and so on, the accurate detection of nucleolin (NCL) is of great significance in clinical examination. Due to the inherent limitations of complex operation, time consumption, and low sensitivity of traditional methods, there is an urgent need to develop rapid, sensitive, and simple detection strategies. In this project, α-Fe₂O₃/Fe₃O₄@Au magnetic nanocomposites (MNCs) were fabricated via a hydrothermal and calcination process with the reduction of chloroauric acid. Further, depending on the facile combination of Au and sulfur, α-Fe₂O₃/Fe₃O₄@Au-Apt/BSA probes were constructed. Subsequently, the aptamer on the probes could specifically recognize and bind to NCL through its high affinity for the target. The magnetic probes, after capturing the target, could self-assemble on the surface of a magnetic glassy carbon electrode (MGCE) under magnetic induction for electrochemical detection, which improved the operability and avoided possible bio-coating contamination. The nano-biosensors revealed favorable linear relationship (R² = 0.995) of the current signal and NCL concentration within 0.1 pg/mL-10 ng/mL, a low limit of detection (LOD) of 0.24 pg/mL, high stability with RSD of 1.04%, satisfactory guarantee period of 20 days, strong specificity, and favorable recoveries in human serum samples (98.09-105.65%), which outperformed conventional EC methods, suggesting the promising prospect of nano-biosensors for NCL detection in clinical examination.

Keywords

Detection

Label-free

Magnetically induced self-assembly

Nano-biosensor

Nucleolin

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1. Introduction

The prevention and treatment of malignant tumors has become a topic of concern due to the high associated mortality rate and increasing incidence rate [1,2]. Detection of tumor markers has proven to be an effective adjunctive prevention for malignant tumors [3,4]. Already, many tumor markers have been discovered and are used in clinical practice. Nucleolin (NCL) protein is one such important tumor marker. NCL is primarily located in the nucleolus, but is also distributed in the cytoplasm and on the cell surface [5,6]. In rapidly proliferating tumor cells, expression and localization of NCL often deviate from normal, and its level is dramatically higher than that in normal cells. This is especially correlated with malignant cancers [7,8]. In addition, NCL is crucial for the initiation and activation of multiple signaling pathways that affect the growth, survival, migration, and invasiveness of tumor cells [9,10]. NCL has also been proven to be overexpressed on the surfaces of tumor cells, including lymphoblastic leukemia cells, breast cancer cells, renal cell carcinoma, etc [11].

Several assays can be used for measuring NCL concentrations, including enzyme-linked immunosorbent assay (ELISA) [12], immunohistochemistry [13], immunofluorescence [14], fluorescence resonance energy transfer (FRET) [15], plasmon enhanced Raman scattering (PERS) [16], fluorescence (FL) [17], surface-enhanced Raman scattering (SERS) [18], electrochemiluminescence (ECL) technology [19], electrochemical (EC) [20], and so on. As other methods suffer from the disadvantages of high cost, time-consuming, and low sensitivity, EC technology is highly favored due to its low cost, facile operation, small size, and integrated design. However, the traditional EC methods remain complicated with excessive time-consumption, need for pre-processing, longer detection periods, etc. Therefore, the development of a novel, rapid, simple, and reliable EC method has become a research hotspot.

In this work, sheet-like α-Fe₂O₃/Fe₃O₄@Au magnetic nanocomposites (MNCs) were prepared to develop an electrochemical nano-biosensor for NCL determination. The magnetic property of α-Fe₂O₃/Fe₃O₄@Au MNCs has been applied for the magnetic self-assembly. With said application, the construction of the sensor no longer remains confined to layer-by-layer solid phase incubation, but is completed in a centrifuge tube, which drastically shortens the pretreatment time. In addition, composite materials could significantly enhance the electrochemical performance through the synergistic effect between materials [21-24]. Through the incorporation of highly conductive materials such as Au nanoparticles (AuNPs) [25-27] and benefiting from the abundant electrochemical active sites provided by the high specific surface area of Fe₂O₃/Fe₃O₄ [28,29], the sensor achieved significant signal amplification and dramatically improved sensitivity. According to the specific recognition ability of aptamers and the formation characteristic of Au-S bonds, we designed an aptamer containing sulfhydryl to recognize NC, and attached it to the gold-coated MNCs to form the capture probes. The bovine serum albumin (BSA) was employed to block the adsorption sites of the nanocomposites’ surface to improve specificity. Finally, the capture probes were magnetically self-assembled onto the surface of a magnetic glassy carbon electrode (MGCE) for detection. The form and detection process have been displayed in Figure 1. Such a biosensor provided a robust and adaptable quantitative analysis method of trace amount NCL without any tedious amplification steps, which was expected to provide an early intervention basis for a variety of tumors in clinical practice.

Flow diagram for the construction and detection principle of the nano-biosensors for NCL detection.

2. Materials and Methods

2.1. Materials and reagents

Ferric chloride hexahydrate (FeCl₃·6H₂O), sodium dihydrogen phosphate (NaH₂PO₄), potassium ferricyanide (K₃Fe(CN)₆), potassium ferrocyanide trihydrate (K₄Fe(CN)₆·3H₂O), tetrachloroaurate(III) trihydrate (HAuCl₄·4H₂O), sodium borohydride (NaBH₄), potassium chloride (KCl), hydrochloric acid (HCl), tris(hydroxymethyl)aminomethane (Tris-base), and ethylenediaminetetraacetic acid (EDTA, purity ≥ 99.0% for most, 47.8% for HAuCl₄·4H₂O, 36.0-38.0% for HCl, and 99.9% for EDTA) were purchased from Sinopharm Chemical Reagent Co., Ltd. Polyethylenimine (PEI, purity ≥ 99.0%) was purchased from Macklin Biochemical Co., Ltd. Tris(2-carboxyethyl)phosphine (TCEP, purity ≥ 98.0%) was purchased from Aladdin Reagent Co., Ltd. Bovine serum albumin (BSA, ≥ 98.0%) and phosphate-buffered saline (PBS, 1×, pH 7.2) were purchased from Sigo Biotechnology Co., Ltd. The aptamer (Apt: 5′-SH-C6-TTGGTGGTGGTGGTTGTGGTGGTGGTGG-3′) was synthesized by Sangon Biotech Co. Ltd, while NCL was purchased from MedChemExpress Co. Ltd. The actual serum sample was provided by the Danyang People’s Hospital.

2.2. Preparation and characterization of sheet-like α-Fe₂O₃/Fe₃O₄@Au MNCs

Firstly, Ferric chloride hexahydrate and sodium dihydrogen phosphate dihydrate were dissolved in deionized water of 80 mL to form a homogeneous solution containing their concentrations of 25 mM and 7.5 mM. The solution was put into a hydrothermal reactor, transferred into a muffle furnace, and heated at 220°C for 24 h with a heating rate of 3°C/min. When the heat was over, the hydrothermal reactor was naturally cooled to indoor temperature, and the suspension was separated by centrifugation at 10,000 rpm. The solid fraction was then collected, washed, and dried in a vacuum oven at 60°C for 12 h. After ground, the α-Fe₂O₃ nanosheets (NSs) were obtained. Subsequently, 50 mg of α-Fe₂O₃ NSs were fully mixed with 0.6 g of glucose. The mixture was transferred to a crucible, after which the crucible, together with the mixture, was calcined at 600°C for 4 h. The setup was naturally cooled, the solid was ground, and the sheet-like α-Fe₂O₃/Fe₃O₄ MNCs were obtained.

Then, 50 mg of α-Fe₂O₃/Fe₃O₄ MNCs were added to a 150 mL polymine (PEI) solution of 10 g/L and ultrasonically dispersed for 30 min. The suspension was heated to react for 2 h in a water bath at 90°C. Once the reaction ceased, the solid was separated by centrifugation and dried at 60°C. The α-Fe₂O₃/Fe₃O₄-PEI nanocomposites were obtained. Then, 15 mg of α-Fe₂O₃/Fe₃O₄-PEI nanocomposites were put into 150 mL double double-distilled water (DDW) and ultrasonically dispersed for 10 min. Then, 1 mL of chloroauric acid solution (20 mg/mL) was poured into the above-mentioned suspension, and ultrasonically reacted for 30 min; 3 mL trisodium citrate dihydrate solution of 38 mM was added and mechanically stirred for 1 min, and 9 mL sodium borohydride solution of 0.075 wt% was instilled and sequentially stirred for 15 min till the suspension turned black. The suspension was separated by centrifugation, washed, dried, and ground. The sheet-like α-Fe₂O₃/Fe₃O₄@Au MNCs were obtained. Finally, the morphologies of α-Fe₂O₃/Fe₃O₄ and α-Fe₂O₃/Fe₃O₄@Au MNCs were observed by scanning electron microscopy (SEM) and transmission electron microscopy (TEM) determinations, respectively. The X-ray diffraction (XRD) pattern of α-Fe₂O₃/Fe₃O₄ MNCs was characterized by the XRD technique, and their hysteresis loops were examined by the vibrating sample magnetometer (VSM) technique.

2.3. Construction and evaluation of nano-biosensors

Firstly, the aptamer was dissolved in DDW to form a 20 μM Apt solution, and Tris(2-carboxyethyl) phosphine (TCEP) was added into the solution in the ratio of 1:100 for TCEP and Apt to selectively reduce probable disulfide bonds. Subsequently, 15 μL of α-Fe₂O₃-Fe₃O₄@Au suspension (20 mg/mL) was mixed with 15 μL TCEP-reduced Apt solution, the mixture was incubated overnight at 4°C for the links of Au and Apt and centrifuged. The supernatant was discarded, and the phosphate buffer saline (PBS) was used to remove the uncombined Apt. The α-Fe₂O₃-Fe₃O₄@Au-Apt MNCs were obtained. For blocking the nonspecific sites to accurately detect NCL, 30 μL of bovine serum albumin (BSA, 0.25%) was mixed with the as-prepared α-Fe₂O₃-Fe₃O₄@Au-Apt MNCs and incubated for 1 h at indoor temperature and centrifuged. The supernatant was discarded and washed with PBS to remove the uncombined BSA; the α-Fe₂O₃-Fe₃O₄@Au-Apt/BSA probes were obtained. The α-Fe₂O₃-Fe₃O₄@Au-Apt/BSA probes were dispersed in 30 μL of NCL solution (10 ng/mL) and incubated for 30 min at 37°C. They were then centrifuged, the supernatant was discarded, and PBS was used to remove the uncombined NCL. The α-Fe₂O₃-Fe₃O₄@Au-Apt-NCL nano-biosensors were obtained. Above all, the intermediates and the product were dispersed in 30 μL DDW, 9 μL suspensions were dropped onto MGCE, and the corresponding capacities, including the cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS), and differential pulse voltammetry (DPV), were conducted thrice on a CHI-660E electrochemical workstation with MGCE, Ag/AgCl electrode, and Pt electrode as the working, reference, and counter electrodes, respectively. Therein, the concentration of the KCl solution was 3 M. CV was carried out with the scan voltage of -0.1-0.7 V and scan rate of 100 mV/s, EIS was investigated with the frequency range of 0.1-10 kHz and signal amplitude of 5 mV, and DPV analysis was set at 0.2 V (vs. Ag/AgCl).

3. Results and Discussion

3.1. Characteristics of sheet-like α-Fe₂O₃/Fe₃O₄ and α-Fe₂O₃/Fe₃O₄@Au MNCs

The characteristics of α-Fe₂O₃/Fe₃O₄ and α-Fe₂O₃/Fe₃O₄@Au MNCs have been exhibited in Figure 2. From the SEM morphology assessment (Figure 2a), α-Fe₂O₃/Fe₃O₄ MNCs had a round nanosheet structure, and their average diameter and thickness were approximately 147.3 nm and 29.5 nm, respectively. The oblate morphology contributed to the formation of a uniform thin film on the MGCE, which helped to improve the electroconductibility of the nano-biosensor. After surface modification, AuNPs were distributed on the surface of the sheet-like α-Fe₂O₃/Fe₃O₄ MNCs. Figure 2(b) represents the TEM image of α-Fe₂O₃/Fe₃O₄@Au MNCs; obviously, AuNPs adhered onto the surfaces of α-Fe₂O₃/Fe₃O₄ MNCs were around 12 nm from the dark spots. Figure 2(c) showed the XRD pattern of α-Fe₂O₃/Fe₃O₄ MNCs, the diffraction peaks at 33.2°, 35.6°, 40.9°, 49.5°, 54.1°, 62.4°, and 64.0° corresponded to the α-Fe₂O₃ standard PDF card (JCPDS No. 33-0664), revealing the presence of α-Fe₂O₃ in the system. However, the ratio of diffraction peaks at 33.152° and 35.611° almost equaled 1:1, which was inconsistent with that of 10:7 for the α-Fe₂O₃ standard PDF card. Obviously, the diffraction peak at 35.6° enhanced the diffraction intensity, corresponding to Fe₃O₄ standard PDF card (JCPDS No. 03-0863), which suggested the presence of Fe₃O₄ in the system. Additionally, the crystallite size of MNCs could be calculated to be 24.02 nm according to the Scherrer formula (Eq. 1) [30]:

(1)

D = 0.89 λ / β \cos θ

(a) SEM morphology of α-Fe2O3/Fe3O4 MNCs, (b) TEM image of α-Fe2O3/Fe3O4@Au MNCs, (c) XRD of α-Fe2O3/Fe3O4 MNCs, and (d) hysteresis loops of α-Fe2O3/Fe3O4 MNCs (red curves) and α-Fe2O3/Fe3O4@Au MNCs (blue curves)

Where D was the crystallite size, λ was the wavelength of Cu-Kα radiation, β represented the full width at half maximum of diffraction peaks, and θ was the Bragg diffraction angle. The crystallite size measured by XRD was much smaller than the particle size in the SEM morphology, indicating that the particle was composed of multiple crystallites.

Figure 2(d) revealed the hysteresis loops of α-Fe₂O₃/Fe₃O₄ and α-Fe₂O₃/Fe₃O₄@Au MNCs; they both displayed soft magnetism performances, and their saturation magnetizations, respectively, achieved 27.9 emu/g and 9.9 emu/g. The saturation magnetization of α-Fe₂O₃/Fe₃O₄@Au MNCs decreased against that of α-Fe₂O₃/Fe₃O₄ MNCs, because AuNPs remedied many lattice imperfections of α-Fe₂O₃/Fe₃O₄ MNCs, resulting in the decrease of saturation magnetization for α-Fe₂O₃/Fe₃O₄@Au MNCs.

3.2. Feasibility of nano-biosensors for NCL detection

CV and EIS analyses for the construction process of the nano-biosensor have been shown in Figure 3. As shown in Figure 3(a), bare MGCE revealed a large oxidation and reduction peak of currents (curve a), which established the basics of electrochemical detection. The current peak (curve b) declined as the sheet-like α-Fe₂O₃/Fe₃O₄ MNCs assembled onto the surface of MGCE, owing to their strong magnetism, which was due to the spatial potential resistance generated by α-Fe₂O₃/Fe₃O₄ MNCs of impeding [Fe(CN)₆]^3-/4- to arrive at MGCE. The current peak (curve c) for MGCE/α-Fe₂O₃/Fe₃O₄@Au MNCs drastically increased, because Au possesses excellent electroconductibility. AuNPs adhered onto the surfaces of α-Fe₂O₃/Fe₃O₄ MNCs significantly improved their electron transfer ability in the solution, resulting in a larger current signal, indicating that the material could effectively achieve signal amplification. When α-Fe₂O₃/Fe₃O₄@Au-Apt was assembled onto MGCE, the current peak (curve d) decreased, which originated from the electronegativity of the DNA phosphate backbone. This property induced a local high potential region on the surface of the nanomaterials, which hindered the electron transport path. This result, in turn, verified the successful loading of aptamers on magnetic α-Fe₂O₃/Fe₃O₄@Au. When α-Fe₂O₃/Fe₃O₄@Au-Apt/BSA was assembled onto the surface of MGCE, the current peak (curve e) continued to decrease, revealing that BSA decreased the electroconductibility. When α-Fe₂O₃/Fe₃O₄@Au-Apt/BSA probes recognized NCL and assembled onto the surface of MGCE, the current peak (curve f) further decreased, since the NCL protein itself was not conductive; the further decrease of the current indicated that the probes successfully captured the NCL.

(a) CV and (b) EIS analyses of bare MGCE (curve a), MGCE/α-Fe2O3/Fe3O4 (curve b), MGCE/α-Fe2O3/Fe3O4@Au (curve c), MGCE/α-Fe2O3/Fe3O4@Au-Apt (curve d), MGCE/α-Fe2O3/Fe3O4@Au-Apt/BSA (curve e), MGCE/α-Fe2O3/Fe3O4@Au-Apt-NCL (curve f).

Accordingly, EIS curves of bare MGCE, α-Fe₂O₃/Fe₃O₄, α-Fe₂O₃/Fe₃O₄@Au, α-Fe₂O₃/Fe₃O₄@Au-Apt, α-Fe₂O₃/Fe₃O₄@Au-Apt/BSA, and α-Fe₂O₃/Fe₃O₄@Au-Apt-NCL have been displayed in Figure 3(b). As was well-known, the larger the diameter of the EIS curve was, the lower the electroconductibility was, i.e., a smaller current peak [31]. Therefore, the resistance of EIS curve (b) for α-Fe₂O₃/Fe₃O₄ was larger than that (a) for bare MGCE, while the resistance of EIS curve (c) for α-Fe₂O₃/Fe₃O₄@Au immensely decreased. Subsequently, with the loading of Apt, BSA, and NCL, the resistance of the EIS curve (d-f) increased stepwise. The variation trend of this process was consistent with the change of resistance caused by the principle we assumed and with the results of CV.

Afterwards, the ESI data were analyzed by ZView2 software through the corresponding Randles equivalent circuit for Nyquist plots, The fitted parameters, including electrolyte solution resistance (R_s), charge transfer resistance (R_ct), double layer capacitance (C_d), and Warburg’s impedance due to ion diffusion (W₀) [32,33], have been listed in Table 1, and the time constant (τ) for the response speed in the electrochemical system was calculated by Eq (2).

(2)

R_{c t} \times C_{d} = \frac{1}{2} π f_{\max} = τ

Table 1. The fitting parameters with different modified MGCEs in Nyquist plots.

Modified MGCEs	R_ct (Ω)	R_s (Ω)	C_dl (μF)	W_o (σ)	τ (s)
Non-modified	147.5	102.4	0.8102	0.4351	119.5045
α-Fe₂O₃/Fe₃O₄	326.8	132.6	0.8194	0.4035	267.7799
α-Fe₂O₃/Fe₃O₄@Au	125.1	69.03	0.9205	0.4401	115.1545
α-Fe₂O₃-Fe₃O₄@Au-Apt	162.6	145.0	0.8280	0.4511	134.6312
α-Fe₂O₃-Fe₃O₄@Au-Apt/BSA	361.3	115.9	0.8280	0.3733	299.1564
α-Fe₂O₃-Fe₃O₄@Au-Apt-NCL	491.8	116.1	0.7588	0.1793	373.1778

The τ of bare MGCE was only 119.5045 s, revealing better electroconductibility; while, the τ of α-Fe₂O₃/Fe₃O₄ increased to 267.7799 s due to their effects of steric hindrance; however, when α-Fe₂O₃/Fe₃O₄@Au MNCs were loaded, τ decreased to 115.1545 s, owing to the excellent electroconductibility of Au, and when α-Fe₂O₃-Fe₃O₄@Au-Apt were continually loaded, τ increased to 134.6312 s, reflecting the attenuated electroconductibility. As α-Fe₂O₃-Fe₃O₄@Au-Apt/BSA were loaded, the τ increased to 299.1564 s, suggesting that BSA increased the steric hindrance of α-Fe₂O₃-Fe₃O₄@Au-Apt. Subsequently, when α-Fe₂O₃-Fe₃O₄@Au-Apt-NCL were loaded, τ tremendously increased to 373.1778 s, which demonstrated the superiority of the detection according to the large change. As demonstrated by the preceding results, the temporal trends of R_ct and τ exhibited a consistent pattern and were inversely proportional to the change of current.

According to above analyses, it was found that, in the construction process, each step for the loading of various substances could cause the change of current peak, which could effectively reflect the feasibility of nano-biosensors for NCL detection.

3.3. Optimization of construction conditions for nano-biosensors

For obtaining the best detection accuracy, the construction conditions for the nano-biosensors, including the concentrations of α-Fe₂O₃/Fe₃O₄@Au and Apt, as well as the incubation temperature and the incubation time of NCL have been optimized in Figure 4. The effect of the concentration for α-Fe₂O₃/Fe₃O₄@Au on the NCL detection capacity has been shown in Figure 4(a), with the concentration of α-Fe₂O₃/Fe₃O₄@Au increasing form 5 mg/mL to 15 mg/mL, the current peak increase; however, after the concentration of α-Fe₂O₃/Fe₃O₄@Au exceeded 15 mg/mL, the current peak decreased with the increase of the concentration suggesting that in the lower concentration, the increase of α-Fe₂O₃/Fe₃O₄@Au concentration resulted in the increase of Au content, the electroconductibility had been improved. However, with the continue increase of α-Fe₂O₃/Fe₃O₄@Au concentration, the steric hindrance of α-Fe₂O₃/Fe₃O₄@Au obviously increased, resulting the decrease of current peak. So, 15 mg/mL of α-Fe₂O₃/Fe₃O₄@Au concentration was selected for the construction of the nano-biosensors. The effect of Apt concentration on the detection has been displayed in Figure 4(b), obviously, with the increase of Apt concentration, the current peak dramatically decreased, and after the Apt concentration achieved 10 μM, the current peak remained almost changed value, revealing that the loaded Apt on the surfaces of α-Fe₂O₃/Fe₃O₄@Au received saturation state, more Apt also could not be loaded onto the surface, these superfluous Aptamers were washed and removed. So, 10 μM of Apt concentration was employed for the construction of the nano-biosensors. In the last incubation process of α-Fe₂O₃/Fe₃O₄@Au-Apt/BSA and NCL, the incubation temperature and incubation time have been optimized in Figures 4(c, d), respectively. It could be seen that the current peak of α-Fe₂O₃/Fe₃O₄@Au-Apt-NCL decreased with the incubation temperature rising form 25°C to 45°C, and reached the minimum when the incubation temperature was 45°C. On the contrary, with the continued rise in incubation temperature, the current peak increased, which revealed that the rise of incubation temperature in the lower temperature would benefit for the combination of α-Fe₂O₃/Fe₃O₄@Au-Apt/BSA and NCL, but with the continued rise of incubation temperature, Apt, BSA, and NCL might be destroyed, even lost activities, so their combination amounts might decrease, resulting in the increase of current peaks. Therefore, 45°C of the incubation temperature was applied. Similarly, their current peak decreased with the incubation time rising from 20 min to 30 min, and achieved the minimum at 30 min, revealing that their combination at 45°C increased with the extension of incubation time. However, with the continuous lengthening of incubation time, Apt, BSA, and NCL might be destroyed, so the current peaks increased again. Therefore, 30 minutes of incubation time was selected for the construction of the nano-biosensors.

Optimized results of the concentrations for (a) α-Fe2O3/Fe3O4@Au and (b) Apt, as well as the (c) incubation temperature and the (d) incubation time of NCL.

3.4. Linear range of nano-biosensors for NCL detection

The determination signal of DPV in electrochemical detection was more stable, so DPV detection was employed to investigate the linear range of nano-biosensors for NCL detection. Various concentrations of NCL (0.01-100 ng/mL) were detected by the nano-biosensors, and the DPV signals have been shown in Figure 5(a). Obviously, with the NCL concentration increasing from 0.1 pg/mL to 10 ng/mL, the peak value of the DPV signal decreased. Their linear relationship of DPV signal peak and logarithmic value of NCL concentration has been revealed in Figure 5(b). The linear equation was I=-6.638lgC+128.287 with the regression variance (R²) of 0.995. It must be particularly noted that the DPV signal peaks deviated from the linear at NCL concentrations of 0.01 pg/mL and 100 ng/mL, revealing that the linear range of the nano-biosensors for NCL detection was 0.1-10 ng/mL. The LOD was calculated as 0.24 pg/mL according to LOD=3σ/slope, while the LOQ was also calculated as 0.79 pg/mL according to LOQ=10σ/slope (The σ value was calculated from the standard deviation of five independent measurements of blank samples (without target) analyzed under identical conditions). The nano-biosensors displayed lower LOD and LOQ values for NCL detection, revealing the higher sensitivity of the nano-biosensors. The capacities of the related literature on NCL detection have been listed in Table 2. Compared with the detection approaches, the nano-biosensors possessed a wider linear range and lower LOD, which prompted the possibility of NCL detection at lower concentrations. In addition, in terms of practicality, this method has strong anti-matrix interference ability, compared with other methods (such as FL required pretreatment to avoid photobleaching or background fluorescence interference; SERS was susceptible to impurities covering the active sites, and the samples often needed to be purified by extraction; FRET suffered from light interference and required to be purified), it offered distinct advantages in practical applications, which demonstrated the significant prospect of the nano-biosensors in clinical examination.

DPV curves of (a) the nano-biosenser for the detection of different concentrations of NCL, and (b) the linear relationship between I% and the logarithm of NCL concentration.

Table 2. Capacities of the related methods for NCL detection.

Method	Linear range	LOD	Reference
FRET	8.1 pM–359 pM	0.05 pM	[15]
PERS	50 pM–100 nM	71 pM	[16]
EC	0.5 nM–1.0 μM	0.16 nM	[20]
FL	0–50 nM	2.2 nM	[34]
SERS	0.1 nM–1.0 μM	0.068 nM	[35]
EC	0.1 pM–10 nM	0.034 pM	[36]
EC	0.1 pg/mL–10 ng/mL	0.24 pg/mL	This work

3.5. Stability, specificity, and guarantee period of nano-biosensors for NCL detection

Firstly, to ensure the reproducibility of electrode manufacturing and to minimize sensor variability, all electrode preparation processes were based on liquid phase incubation to avoid the biofouling that might be generated by multiple incubations of the solid phase layer on the electrode surface, and all electrochemical measurement processes were conducted under controlled environmental conditions. To verify the reproducibility of the sensor, five groups of α-Fe₂O₃/Fe₃O₄@Au-Apt-NCL probes prepared under the same conditions were dropped onto MGCE, and the current signals were determined as Figure 6(a). The Relative Standard Deviation (RSD) of five detection results was only 1.04%, revealing the high accuracy of NCL detection for the nano-biosensors and the stability of the nano-biosensors for NCL detection. To verify the storage stability, the α-Fe₂O₃/Fe₃O₄@Au-Apt-NCL probes were stored at 4°C in the dark for 5, 10, 15, and 20 days, respectively. The RSD of the detection results received 1.44%, and in 20 days, the current peaks remained 97.3-101.3% of detection accuracy compared with the first detection result for unsaved probes (Figure 6b), suggesting a satisfactory guarantee period, which could meet the needs of clinical examination. Keeping the same concentration of α-Fe₂O₃/Fe₃O₄@Au-Apt/BSA, 1 µg/mL of HER2, VEGF, PSA, MUC1, and EGFR solutions. Additionally, 10 ng/mL of NCL solution, and mixture solution containing NCL of 10 ng/mL and all above substances with each concentration of 1 µg/mL were detected with the same approach, the current peak values have been shown in Figure 6(c). The concentrations of HER2, VEGF, PSA, MUC1, and EGFR were 100 times of NCL concentration; however, the signal changes were small, while 1 µg/mL of NCL in the mixture solution and individual solution tremendously decreased the signals, which certified the excellent specificity of nano-biosensors for NCL detection. At the same time, the detection result of the mixture solution with large concentrations of the other substances wasn’t affected; a large current change was obtained, which proved the superior selectivity of the nano-biosensors.

(a) Stability, (b) guarantee period, and (c) selectivity with HER2 (1 µg/mL), VEGF (1 µg/mL), PSA (1 µg/mL), MUC1 (1 µg/mL), EGFR (1 µg/mL), NCL (10 ng/mL), and mixture solution (containing NCL (10 ng/mL) and all above substances with each concentration of 1 µg/mL)

To investigate the clinical application potential of biosensors, human serum samples were employed for verification. A standard addition approach was employed to introduce varying concentrations of NCL (0.1, 10, and 100 pg/mL) into the treated serum. The experimental results showed that the recoveries of 98.09-105.65% with RSD ≤ 2.66% (Table 3).

Table 3. Determination of NCL in spiked actual samples (n = 3).

NCL spiked concentration (pM)	I (μA)	Detection value (pM)	RSD (%)	Recovery rate (%)
0.1	134.77	0.1057	2.66	105.65
10	121.63	10.054	1.01	100.54
100	115.07	98.087	0.93	98.09

4. Conclusions

α-Fe₂O₃ NSs were fabricated via the hydrothermal process, and with α-Fe₂O₃ nanosheets as precursors, sheet-like α-Fe₂O₃/Fe₃O₄ MNCs were prepared via the calcination process with a diameter of 147.3 nm, a thickness of 29.5 nm, and a saturation magnetization of 27.9 emu/g. Subsequently, with PEI as a bridging agent, α-Fe₂O₃/Fe₃O₄@Au MNCs were prepared via the reduction of trisodium citrate dihydrate with the saturation magnetizations of 9.9 emu/g and AuNPs of 12 nm. The as-prepared α-Fe₂O₃/Fe₃O₄@Au MNCs had abundant electrochemical active sites due to their high specific surface area, and exhibited good biocompatibility and easy functionalization due to the presence of Au. Moreover, the distinctive magnetic properties enabled significant simplification of electrochemical detection procedures through magnetic self-assembly and separation technologies.

Utilizing the peculiarity of easy combination for Au and sulfur, the α-Fe₂O₃-Fe₃O₄@Au-Apt MNCs probes were constructed for NCL detection. The nano-biosensors revealed favorable linear relationship of the current signal and NCL concentration with R² of 0.995, wonderful LOD of 0.24 pg/mL and LOQ of 0.79 pg/mL, excellent stability with RSD of 1.04%, satisfactory guarantee period of 20 days, superior specificity, as well as satisfactory recoveries (98.09-105.65%) in human serum samples, offering a promising tool for early cancer diagnosis and treatment monitoring. Future efforts should focus on accelerating its translational application through miniaturization and promoting its clinical deployment through rigorous clinical validation.

Acknowledgment

This work was supported by the Zhenjiang Science & Technology Program (Grant No. SH2023086 and SH2024041).

CRediT authorship contribution statement

Min Sun: Design, Literature search, Experimental studies, Data acquisition, Data analysis, Manuscript preparation. Boyi Jiang: Literature search, Experimental studies, Data analysis, Manuscript preparation. Jiawei Wang: Design, Clinical studies, Data acquisition. Zhou Wang: Concepts. Xinyan Gao: Manuscript editing and review. Xiaoting Yi: Concepts, Definition intrllectual content, Manuscript editing and review.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Declaration of generative AI and AI-assisted technologies in the writing process

The authors confirm that there was no use of artificial intelligence (AI)-assisted technology for assisting in the writing or editing of the manuscript and no images were manipulated using AI.

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1. Introduction

2. Materials and Methods

2.1. Materials and reagents
2.2. Preparation and characterization of sheet-like α-Fe2O3/Fe3O4@Au MNCs
2.3. Construction and evaluation of nano-biosensors

3. Results and Discussion

3.1. Characteristics of sheet-like α-Fe2O3/Fe3O4 and α-Fe2O3/Fe3O4@Au MNCs
3.2. Feasibility of nano-biosensors for NCL detection
3.3. Optimization of construction conditions for nano-biosensors
3.4. Linear range of nano-biosensors for NCL detection
3.5. Stability, specificity, and guarantee period of nano-biosensors for NCL detection

4. Conclusions

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[1] Feng L., Chen Y., Ren J., Qu X.. A graphene functionalized electrochemical aptasensor for selective label-free detection of cancer cells. Biomaterials. 2011;32:2930-2937. https://doi.org/10.1016/j.biomaterials.2011.01.002
[Google Scholar]

[2] Zhang H., Lv Z., Zhang H., Zhang Y., He D., Liu R.. Ultrasensitive detection of PrPC in human serum using label-free electrochemical biosensor based on α-iron trioxide/ferriferrous oxide magnetic nanocomposites. Microchemical Journal. 2025;212:113369. https://doi.org/10.1016/j.microc.2025.113369
[Google Scholar]

[3] Ma M., Zhang X., Zhang H., Xu Z., Li S., Liu R.. Ultrasensitive detection of CEA in human serum using label-free electrochemical biosensor with magnetic self-assembly based on α-Fe₂O₃/Fe₃O₄ nanorods. Vacuum. 2024;227:113433. https://doi.org/10.1016/j.vacuum.2024.113433
[Google Scholar]

[4] Yue Y., Liu M., Ma M., Xu Z., Zhang H., Wang Q., Liu R.. CRISPR/Cas14a integrated with DNA walker based on magnetic self-assembly for human papillomavirus type 16 oncoprotein E7 ultrasensitive detection. Biosensors & Bioelectronics. 2025;272:117135. https://doi.org/10.1016/j.bios.2025.117135
[Google Scholar]

[5] Zhang R., Zhang Z., Du H., Wang J., Wang X., Zhou T., Wang F.. A nucleolin-activated polyvalent aptamer nanoprobe for the detection of cancer cells. Analytical and Bioanalytical Chemistry. 2023;415:2217-2226. https://doi.org/10.1007/s00216-023-04629-3
[Google Scholar]

[6] Tajrishi M.M., Tuteja R., Tuteja N.. Nucleolin: The most abundant multifunctional phosphoprotein of nucleolus. Communicative & Integrative Biology. 2011;4:267-275. https://doi.org/10.4161/cib.4.3.14884
[Google Scholar]

[7] Hovanessian A.G., Soundaramourty C., El Khoury D., Nondier I., Svab J., Krust B.. Surface expressed nucleolin is constantly induced in tumor cells to mediate calcium-dependent ligand internalization. PloS One. 2010;5:e15787. https://doi.org/10.1371/journal.pone.0015787
[Google Scholar]

[8] Shaw P., Doonan J.. The nucleolus: Playing by different rules? Cell Cycle. 2005;4:102-105. https://doi.org/10.4161/cc.4.1.1467
[Google Scholar]

[9] Soundararajan S., Wang L., Sridharan V., Chen W., Courtenay-Luck N., Jones D., Spicer E.K., Fernandes D.J.. Plasma membrane nucleolin is a receptor for the anticancer aptamer AS1411 in MV4-11 leukemia cells. Molecular Pharmacology. 2009;76:984-991. https://doi.org/10.1124/mol.109.055947
[Google Scholar]

[10] Ugrinova I., Petrova M., Chalabi-Dchar M., Bouvet P.. Multifaceted nucleolin protein and its molecular partners in oncogenesis. Advances in Protein Chemistry and Structural Biology. 2018;111:133-164. https://doi.org/10.1016/bs.apcsb.2017.08.001
[Google Scholar]

[11] Li H., Bai X., Wang N., Chen X., Li J., Zhang Z., Tang J.. Aptamer-based microcantilever biosensor for ultrasensitive detection of tumor marker nucleolin. Talanta. 2016;146:727-731. https://doi.org/10.1016/j.talanta.2015.06.034
[Google Scholar]

[12] Ezzatifar F., Rafiei A., Valadan R., Asgarian-Omran H., Jeddi-Tehrani M.. Detection of novel autoantibodies to nucleolin’s RNA-binding domains as a Serum Tumor Biomarker Through ELISA. Iranian Journal of Allergy Asthma and Immunology. 2022;21:616629. https://doi.org/10.18502/ijaai.v21i6.11520
[Google Scholar]

[13] Paris E.A., Bahr J.M., Basu S., Barua A.. Changes in nucleolin expression during malignant transformation leading to ovarian high-grade serous carcinoma. Cancers. 2023;15:661. https://doi.org/10.3390/cancers15030661
[Google Scholar]

[14] Tang Y., Lin X., Chen C., Tong Z., Sun H., Li Y., Liang P., Jiang B.. Nucleolin improves heart function during recovery from myocardial infarction by modulating macrophage polarization. Journal of Cardiovascular Pharmacology and Therapeutics. 2021;26:386-395. https://doi.org/10.1177/1074248421989570
[Google Scholar]

[15] Li Y.R., Liu Q., Hong Z., Wang H.F.. Magnetic separation-assistant fluorescence resonance energy transfer inhibition for highly sensitive probing of nucleolin. Analytical Chemistry. 2015;87:12183-12189. https://doi.org/10.1021/acs.analchem.5b03064
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[16] Shi C.F., Li Z.Q., Wang C., Li J., Xia X.H.. Ultrasensitive plasmon enhanced Raman scattering detection of nucleolin using nanochannels of 3D hybrid plasmonic metamaterial. Biosensors & Bioelectronics. 2021;178:113040. https://doi.org/10.1016/j.bios.2021.113040
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[17] Hwang D.W., Ko H.Y., Lee J.H., Kang H., Ryu S.H., Song I.C., Lee D.S., Kim S.. A nucleolin-targeted multimodal nanoparticle imaging probe for tracking cancer cells using an aptamer. Journal of Nuclear Medicine : Official Publication, Society of Nuclear Medicine. 2010;51:98-105. https://doi.org/10.2967/jnumed.109.069880
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[18] Hanif S., Liu H.L., Ahmed S.A., Yang J.M., Zhou Y., Pang J., Ji L.N., Xia X.H., Wang K.. Nanopipette-based SERS aptasensor for subcellular localization of cancer biomarker in single cells. Analytical Chemistry. 2017;89:9911-9917. https://doi.org/10.1021/acs.analchem.7b02147
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[19] Gao W., Liu Y., Zhang H., Wang Z.. Electrochemiluminescence biosensor for nucleolin imaging in a single tumor cell combined with synergetic therapy of tumor. ACS Sensors. 2020;5:1216-1222. https://doi.org/10.1021/acssensors.0c00292
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[20] Su X., Han Y., Liu Z., Fan L., Guo Y.. One-pot synthesized AuNPs/MoS₂/rGO nanocomposite as sensitive electrochemical aptasensing platform for nucleolin detection. Journal of Electroanalytical Chemistry. 2020;859:113868. https://doi.org/10.1016/j.jelechem.2020.113868
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[23] Qayyum A., Rehman M.O., Ahmad F., Khan M.A., Ramay S.M., Atiq S.. Performance optimization of Nd-doped LaNiO₃ as an electrode material in supercapacitors. Solid State Ionics. 2023;395:116227. https://doi.org/10.1016/j.ssi.2023.116227
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[26] Danish M., Islam M., Ahmad F., Madni M.N., Jahangeer M.. Synthesis of M-type hexaferrite reinforced graphene oxide composites for electromagnetic interference shielding. Journal of Physics and Chemistry of Solids. 2024;185:111783. https://doi.org/10.1016/j.jpcs.2023.111783
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