2D Nanoflower-like MnO₂-Modified Flexible Electrode with Compressive Strain-Enhanced Sensitivity for Reliable Cholesterol Monitoring

Highlights

What are the main findings?

• 2D flower-like MnO₂ improves interfacial charge transfer in cholesterol sensing.
• Sensitivity reaches 0.2497 μA·μM⁻¹·cm⁻² under −1% compressive strain.
• The sensor achieves a 1–160 μM linear range and a 0.16 μM detection limit.

What are the implications of the main findings?

• Compressive strain offers a route to tune flexible electrochemical sensor performance.
• Flexoelectric polarization contributes to strain-enhanced cholesterol oxidation.
• MnO₂/SPE shows promise for wearable and on-site cholesterol monitoring.

Abstract

The development of flexible, high-performance electrochemical sensors for continuous and on-site monitoring of cholesterol is critical for advancing cardiovascular health diagnostics and personalized medicine. Here, we present a flexible MnO₂-modified screen-printed electrode (MnO₂/SPE) for cholesterol detection, where two-dimensional flower-like MnO₂ nanomaterials are used to functionalize the electrode surface. The MnO₂ nanocomposite significantly improves the electrochemical performance of the SPE by reducing interfacial impedance, optimizing charge transfer kinetics, and providing abundant active sites for electrocatalytic oxidation. The MnO₂/SPE sensor exhibits excellent analytical performance, including a wide linear range (1–160 μM), a low limit of detection (0.16 μM), and a high sensitivity of 0.197 μA·μM⁻¹·cm⁻². Notably, compressive mechanical strain (−1%) is demonstrated to further boost the sensing performance via the flexoelectric polarization effect, increasing the sensitivity to 0.2497 μA·μM⁻¹·cm⁻² and accelerating the electrocatalytic oxidation of cholesterol. The MnO₂/SPE sensor also shows good stability, reproducibility, and anti-interference capability, with all relative deviations below 5%. This work provides a new strategy to enhance the performance of flexible electrochemical sensors through strain engineering and demonstrates great potential for wearable cholesterol monitoring.

1. Introduction

Cardiovascular diseases represent the major global public health burden, and real-time monitoring of blood cholesterol concentration is pivotal for the early diagnosis, risk stratification, and clinical management of these conditions [1,2,3]. To date, various analytical strategies have been developed for cholesterol detection, including fluorescence spectroscopy [4], high-performance liquid chromatography [5,6], and chemiluminescence [7]. However, these conventional methods suffer from inherent drawbacks such as complicated operation, time-consuming procedures, tedious sample pretreatment, high instrumental costs, and requirements for skilled personnel, which severely restrict their practical applications in point-of-care settings. Therefore, the development of simple, portable, highly sensitive, and cost-effective cholesterol sensors is urgently needed to enable real-time detection and wearable health monitoring.

Electrochemical sensing has emerged as a highly promising alternative [8,9], owing to its inherent advantages including simple operation, high sensitivity, rapid response. In particular, flexible screen-printed electrodes (SPEs) represent an ideal platform for constructing clinical point-of-care cholesterol sensors, distinguished by their simplicity, disposability, biocompatibility, low cost, and scalability for large-scale production [10,11,12]. Electrochemical sensors constructed on flexible screen-printed electrodes (SPEs) show excellent compatibility with portable and miniaturized electrochemical workstations, and have attracted extensive attention owing to their compact size, rapid response, and suitability for home-based testing [13,14]. Among nanomaterials explored for electrochemical cholesterol detection, metal oxides [15,16,17] and carbon-based materials [18,19,20] have been extensively employed in nonenzymatic electrochemical sensors owing to their unique physicochemical properties. As a typical transition metal oxide, MnO₂ exhibits distinctive advantages in electrochemical cholesterol sensing due to its multivalent redox behavior and tunable electronic structure [21]. MnO₂ nanomaterials exhibit outstanding enzyme-mimetic activity and are thus regarded as ideal substitutes for natural enzymes [22]. Carbon-based electrodes modified with MnO₂ nanoclusters were applied for non-enzymatic cholesterol detection. The incorporated MnO₂ improves electron-transfer capability and yields a prominent enhancement in the anodic oxidation peak current of cholesterol. The electro-oxidation proceeds via a surface-controlled two-electron-transfer pathway, directly converting cholesterol to cholestenone [15]. Furthermore, the micro-morphology of MnO₂ nanostructures plays a decisive role in determining their electrocatalytic performance [23,24]. Two-dimensional MnO₂ nanosheets, featuring ultrahigh specific surface area, efficient surface reactivity, excellent electron transport capability, non-toxic nature, and low cost, are widely recognized as promising candidates for the electrochemical detection of biomolecules [25,26,27]. While various morphologies of MnO₂-based electrochemical sensors have been developed in previous reports [22,23,24,25,26], research on fabrication of flexible MnO₂-based cholesterol sensors remains scarce, with obvious limitations. Mechanical strain can regulate the surface electronic structure of electrocatalysts and affect their mechanical stability. Therefore, strain engineering still needs to be further investigated for the sensing properties and mechanical stability of flexible manganese dioxide cholesterol sensors.

Herein, we develop a flexible MnO₂/SPE cholesterol sensor based on flexible screen-printed electrodes and enzyme-like MnO₂ nanosheets. The sensor exhibits a favorable linear response and outstanding sensitivity, stability, and selectivity toward cholesterol. The sensing performance of the MnO₂/SPE sensor under a range of mechanical strains is systematically investigated, and the effect of strain engineering on the sensor sensitivity as well as the corresponding strain-tuning mechanism is analyzed. The scalable screen-printing process enables low-cost mass production of the sensor, and its single-use disposable design avoids complex regeneration procedures and cross-contamination risks, further favoring miniaturization, system integration, and portable point-of-care applications. This work provides a high-performance, low-cost flexible electrochemical strategy for cholesterol detection, offering a reference for the subsequent development of portable and wearable sensing platforms

2. Experimental Section

2.1. Synthesis of MnO₂ Nanoflowers

MnO₂ nanosheets were first synthesized via a hydrothermal method [25]. Briefly, KMnO₄ and KCl powders with a molar ratio of 3:1 were dissolved in deionized water. After magnetic stirring, the mixture was transferred into a Teflon-lined autoclave and heated at 180 °C for 13 h. The resultant product was collected by centrifugation, washed thoroughly with deionized water, and dried to obtain MnO₂ powder with a nanoflower-like structure (Figure 1a). KMnO₄ and KCl were purchased from Shanghai Macklin Biochemical Co., Ltd., Shanghai, China. Detailed synthetic procedures are provided in Supplementary Note S1.

Figure 1. (a) Schematic illustration of the synthesis of δ-MnO₂ nanoflower structures and the fabrication of the MnO₂-SPE sensor. (b) Schematic exploded view of the layered stacking architecture of the MnO₂-SPE sensor. (c–e) SEM of δ-MnO₂ nanosheets; (f) TEM image of MnO₂ nanosheet; (g) high-magnification TEM image of MnO₂ nanosheet. (h) Magnified HRTEM image of the region labeled by the wathet square in (d).

2.2. Fabrication of the MnO₂/SPE Sensor

The electrochemical cholesterol sensors were fabricated on commercial screen-printed electrodes (SPEs) and they were purchased from Zensor R&D Co., Ltd., Taipei, Taiwan. Benefiting from the low-cost commercial SPE substrate and the simple drop-casting modification process, the as-fabricated MnO₂/SPE sensor is intentionally designed as a single-use disposable device, which is compatible with large-scale batch production and point-of-care application demands. Prior to modification, the SPEs were activated in phosphate buffer solution (PBS, pH = 7.4). Then, 5 mg of as-prepared MnO₂ nanopowders was dispersed in 5 mL of deionized water via ultrasonication to form a homogeneous suspension, followed by drop-casting onto the activated SPE. The as-prepared two-dimensional MnO₂ nanoflower dispersion is shown in Figure S1. Detailed fabrication protocols are provided in Supplementary Note S2.

2.3. Materials Characterizations

The morphologies and structures were characterized by scanning electron microscopy (SEM, SU5000: Hitachi, Tokyo, Japan) and transmission electron microscopy (TEM, JEOL 2100F: JEOL, Tokyo, Japan). X-ray diffraction (XRD, SmartLab: Rigaku, Tokyo, Japan) was utilized to investigate the crystalline characteristics and clarify the crystal structure of the synthesized materials. X-ray photoelectron spectroscopy (XPS, ESCALAB 250Xi: Thermo Fisher Scientific, Waltham, MA, USA) was applied to analyze elemental components and determine the chemical valence states of the samples.

2.4. Preparation of Cholesterol Solution

The 20.0 mg of cholesterol standard was accurately weighed and transferred into a clean dry beaker. After adding 20 mL of Triton X-100 surfactant solution (1%, BIOISCO, Lianyungang, China), the mixture was heated in a water bath at a constant temperature of 65 °C and magnetically stirred continuously at 500 rpm until a transparent solution was obtained. Subsequently, heating was terminated, and 0.1 M PBS electrolyte was added under continuous stirring to make the final volume up to 50 mL. The mixed solution was stirred until homogeneous, then sealed and stored in a refrigerator at refrigeration temperature for subsequent electrochemical measurements. Further details are provided in Supplementary Note S3.

2.5. Electrochemical Measurements

All electrochemical characterizations in this work were carried out on an electrochemical workstation using a standard three-electrode configuration, consisting of a carbon-based working electrode, a reference electrode and a counter electrode. A 0.1 M phosphate-buffered saline (PBS) solution was used as the supporting electrolyte. For cyclic voltammetry (CV) measurements, the potential window was set from −1.0 V to 1.0 V (vs. Ag/AgCl) at a scan rate of 100 mV s⁻¹. Differential pulse voltammetry (DPV) was performed over the potential range of 0.37–0.8 V (vs. Ag/AgCl). Electrochemical impedance spectroscopy (EIS) was acquired at an open-circuit potential of 0.4 V (vs. Ag/AgCl) with the frequency range from 1000 kHz down to 0.01 Hz. All electrochemical measurements were conducted at room temperature under constant ambient conditions to guarantee experimental repeatability and reliability. All electrochemical experiments were independently repeated at least 3 times, and the standard deviation (SD) was calculated for statistical analysis. Detailed information is provided in Supplementary Note S4. The strain on MnO₂ was introduced through a two-point bending apparatus. The bent PET substrate was considered as a circular arc for computing the applied strain.

3. Results and Discussion

The fabrication process of the MnO₂/SPE sensor is schematically illustrated in Figure 1a. Through a simple hydrothermal method, δ-MnO₂ nanosheets were prepared successfully. The SPE adopts an integrated three-electrode configuration on a flexible PET substrate. Both the working electrode (WE) and counter electrode (CE) are fabricated with carbon ink, and the integrated reference electrode (RE) is Ag/AgCl. The nominal geometrical surface area of the working electrode is 0.126 cm², which is a standard value for this commercial SPE model and widely adopted in peer-reviewed studies using the same platform. All sensitivity values in this work are normalized to this geometrical surface area. As shown in Figure 1b, the as-prepared MnO₂/SPE sensor adopts a multilayer architecture on a flexible PET substrate: carbon ink is screen-printed to form the working and counter electrodes with an integrated Ag/AgCl reference electrode, and δ-MnO₂ nanoflowers are further drop-cast as the functional sensing layer. The as-fabricated SPE exhibits a compact structure and excellent mechanical flexibility, rendering it highly suitable for portable, point-of-care, and wearable sensing applications. An aqueous suspension of MnO₂ was drop-cast onto the PBS-preactivated working electrode surface of the SPE to construct the MnO₂-SPE electrochemical sensor for cholesterol detection.

The structure and morphology of the as-prepared MnO₂ were systematically characterized using SEM, TEM, XRD, and XPS. As revealed by the scanning electron microscopy (SEM) images (Figure 1c–e), the MnO₂ nanomaterial exhibits a unique nanoflower-like architecture assembled from two-dimensional (2D) centrosymmetric nanosheets. Transmission electron microscopy (TEM) analysis further verifies that MnO₂ presents a sheet-like structure with a smooth and intact surface without obvious pores. Figure 1h displays the high-resolution TEM (HRTEM) image with clear lattice fringes. The measured lattice spacing of 0.347 nm corresponds to the (002) plane of MnO₂, which is consistent with the standard JCPDS card No. 80-1098 [25]. This value corresponds to the (002) crystal plane of MnO₂, which is in good agreement with the standard lattice parameter documented in the PDF card (JCPDS No. 80-1098). The clear lattice fringes further verify the high crystallinity of the MnO₂ nanomaterial. The X-ray diffraction (XRD) pattern of the as-prepared MnO₂ exhibits distinct diffraction peaks at 2θ = 12.30°, 25.12°, 36.65°, and 66.96°, which are well indexed to the (001), (002), (111), and (114) crystal planes of standard MnO₂ (JCPDS No. 80-1098). This confirms the formation of pure crystalline MnO₂ with no detectable impurity phases. The fundamental structural unit of MnO₂ is the MnO₆ octahedron, which possesses a twofold rotation axis and a perpendicular mirror plane (inset in Figure 2a). X-ray photoelectron spectroscopy (XPS) was employed to characterize the chemical composition and surface electronic structure of the MnO₂ nanomaterial. As illustrated in Figure 2b, Mn and O are the predominant elements, consistent with the expected composition of MnO₂. The high-resolution Mn 2p₃/₂ spectrum (Figure 2c) displays two distinct peaks at 642.0 eV and 643.9 eV, which are assigned to Mn⁴⁺ and Mn³⁺ species, respectively. The O 1s spectrum (Figure 2d) can be deconvoluted into two peaks at 529.4 eV and 531.5 eV, corresponding to lattice oxygen (O_lat) and adsorbed oxygen species (O_ads), respectively. The presence of O_ads is attributed to oxygen vacancies on the MnO₂ surface, which can capture external oxygen molecules.

Figure 2. Characterizations of the MnO₂ nanosheets. (a) XRD pattern; (b) XPS survey spectrum; (c) high-resolution Mn 2p_3/2 XPS spectrum; (d) high-resolution O1s XPS spectrum.

To explore the effects of MnO₂ modification on the interfacial behavior of screen-printed electrodes (SPEs) and the cholesterol sensing performance of MnO₂/SPE sensors, we performed systematic electrochemical characterizations on the MnO₂/SPE electrode, focusing on interfacial kinetics, electrocatalytic activity, and quantitative detection capability. Electrochemical impedance spectroscopy (EIS) was employed to characterize charge transfer and ion diffusion kinetics at the interfaces of bare SPEs and MnO₂/SPEs. EIS measurements were performed over a frequency range from 1000 kHz to 0.01 Hz at an open circuit potential of 0.4 V. The corresponding Nyquist plots and equivalent circuit are presented in Figure 3a. Both electrodes exhibit typical Nyquist characteristics, comprising a semicircle in the high-frequency region and a linear diffusion tail in the low-frequency region. The Nyquist plots were fitted using the equivalent circuit model Z [28],

Z = R₁ + (R₂ + W₀₁)‖CPE₁

(1)

where R₁ represents the ohmic series resistance, R₂ denotes the charge transfer resistance, CPE₁ is the constant phase element, and W₀₁ corresponds to the Warburg impedance (reflecting the ion diffusion process at low frequencies). Similar R₁ values for the bare SPE and MnO₂/SPE indicate that MnO₂ modification does not significantly alter the intrinsic conductivity of the electrode system. The semicircle in the high-frequency region is associated with the charge-transfer process, whose diameter reflects the charge-transfer resistance (R₂). Fitting results show that the R₂ value of the bare SPE is approximately 270 Ω, which is significantly reduced to 130 Ω following MnO₂ modification. This confirms that the introduction of MnO₂ provides abundant electroactive sites at the electrode/electrolyte interface, effectively lowering the interfacial charge-transfer resistance and accelerating electron transfer kinetics. In the low-frequency region, the Nyquist curve exhibits a typical Warburg diffusion slope, and the slope of the MnO₂/SPE is notably larger than that of the bare SPE. This observation indicates that MnO₂ modification substantially reduces ion diffusion resistance, optimizes the transport and diffusion behavior of electrolyte ions on the electrode surface, and thereby lays a favorable foundation for constructing a high-performance electrochemical sensing interface. To explore the electrocatalytic activity of MnO₂ nanomaterials, cyclic voltammetry (CV) was first employed to compare the electrochemical response characteristics of the different electrodes. As illustrated in Figure 3b, no obvious redox peaks are observed for the bare SPE in pure phosphate-buffered saline (PBS) buffer. Upon the addition of 100 μM cholesterol, only a weak oxidative response is detected, with an oxidation peak current of merely 3.7 μA-indicating that the bare SPE exhibits negligible electrocatalytic activity toward cholesterol oxidation. In contrast, the MnO₂/SPE electrode shows a significantly enhanced electrochemical response in 100 μM cholesterol solution, with an oxidation peak current of up to 12.3 μA (approximately 3.3 times that of the bare SPE) and distinct redox peaks. This arises from the large specific surface area and superior catalytic activity of MnO₂ nanosheets, which facilitate efficient electron extraction for the oxidation of cholesterol.

Figure 3. Electrochemical Characterization of the MnO₂/SPE sensors. (a) EIS curves of the bare SPE and MnO₂/SPE sensors, with the corresponding equivalent circuit shown in the inset. (b) Cyclic voltammetry (CV) curves of the MnO₂/SPE sensor and bare SPE in phosphate-buffered saline (PBS) solution and in a solution containing 100 μM cholesterol, respectively; (c) CV curves of MnO₂/SPE at various scan rates, and (d) the corresponding linear relationship plot. (e) DPV curves of the MnO₂/SPE sensor in cholesterol solutions with different concentrations. (f) Linear relationship between the oxidation peak current and cholesterol concentration.

To further elucidate the reaction kinetics of cholesterol at the electrode surface, cyclic voltammetry (CV) measurements were performed on the MnO₂/SPE electrode at scan rates ranging from 60 to 160 mV/s. As shown in Figure 3c, the oxidation peak current of cholesterol increased proportionally with the scan rate, while the peak potential remained nearly unchanged, indicating good reversibility of the electrochemical process. A linear fit of the oxidation peak current versus scan rate yielded the equation I_p = 0.054ν + 4.827 with a linear regression coefficient R² = 0.995 (Figure 3d). The linear relationship between peak current and scan rate verifies that the electro-oxidation of cholesterol on the MnO₂/SPE surface follows a typical surface-controlled process, in which the reaction rate is dominated by adsorption and catalytic steps at the electrode surface rather than diffusion in the bulk solution. MnO₂ exhibits strong affinity and high catalytic activity toward the hydroxyl group (–OH) at the C3 position of cholesterol. The electrocatalytic oxidation of cholesterol on MnO₂/SPE proceeds via a proton-coupled electron transfer process, in which the C3 hydroxyl group of cholesterol is dehydrogenated to form cholestenone with the release of two protons and two electrons. Mn⁴⁺ in MnO₂ acts as the electrocatalytic center, accepting electrons to form low-valence Mn species (Mn³⁺/Mn²⁺), which can be electrochemically regenerated to Mn⁴⁺ during the anodic scan, enabling continuous catalytic turnover [29]. The whole reaction may be written as

Cholesterol (C₂₇H₄₆O) + MnO₂ + 2H⁺ ⟶ Cholestenone (C₂₇H₄₄O) + Mn²⁺ + 2H₂O

(2)

Mn²⁺ + 2H₂O ⟶ MnO₂ + 4H⁺ + 2e⁻

(3)

Differential pulse voltammetry (DPV) was employed to evaluate the quantitative detection performance of the MnO₂/SPE sensor for cholesterol. As shown in Figure 3e, the oxidation peak current increased linearly with cholesterol concentration across the range of 1–160 μM, while the peak potential remained nearly unchanged, confirming a stable electrode reaction process. Linear fitting of the peak current versus concentration yielded the calibration curve I_p = 0.014c + 1.38 with a correlation coefficient of R² = 0.9959, indicating an excellent linear relationship. The limit of detection (LOD) was calculated to be 0.16 μM using the 3σ/s method, where σ is the standard deviation of blank measurements and s is the slope of the calibration curve. The sensor exhibited a linear detection range of 1–160 μM, and its sensitivity, normalized to the electrode’s geometric area, reached 0.197 μA·μM⁻¹·cm⁻². These results demonstrate that the MnO₂/SPE sensor possesses outstanding linearity and high sensitivity toward cholesterol, making it a promising candidate for practical sensing applications. Comprehensive comparisons of the linear range and limit of detection (LOD) with previously reported cholesterol sensors are summarized in Table 1.

Table 1. Performance comparison of cholesterol sensors.

Electrode Materials	Linear Range (μM)	Detection Limit (μM)	Sample Matrix	Reference
MnO2 nanoclusters/graphene	0.0012–0.024	0.00042	Diluted human serum	[29]
MnO2NS	1–200	0.5	Diluted human serum	[30]
β-Cyclodextrin/Fe3O4	2.88–150	2.88	PBS buffer	[31]
CNT-Pt/ChOx/Sol-Gel	4–100	1.4	Diluted human serum	[32]
NiO/CVD-grown graphene	2–40	0.13	PBS buffer	[33]
β-CD/PNAANI/rGO	1–50	0.5	Diluted human serum	[34]
MnO2 NS-FAM	30–200	0.33	Diluted human serum	[35]
ZGCY@MnO2	8–200	1.7	PBS buffer	[36]
MnO2/SPE	1–160	0.16	PBS buffer	This work

The as-fabricated MnO₂/SPE electrochemical sensor exhibits a LOD of 0.16 μM and a linear detection range of 1–160 μM for cholesterol. Notably, the LOD of this sensor is significantly lower than that of the β-cyclodextrin/Fe₃O₄ composite reported by Willyam et al. [31] (LOD = 2.88 μM). Furthermore, compared with the NiO/CVD-grown graphene sensor (linear range: 2–40 μM) reported by Rengaraj et al. [33]. and the β-D/PNAANI/rGO sensor (linear range: 1–50 μM) reported by Yang et al. [34], the MnO₂/SPE electrode presents a distinctly broader linear detection window. Zhu developed a fluorescence sensing platform based on MnO₂ nanosheets for cholesterol detection, achieving a linear range of 30–200 μM with a detection limit of 0.33 μM in diluted human serum [35], and Zhang et al. constructed an “Off-On” selective cholesterol sensor based on phosphorescence resonance energy transfer using ZGCY@MnO₂, exhibiting a linear response of 8–200 μM and a detection limit of 1.7 μM in PBS buffer [36]. As shown in Table 1, for studies involving biological matrices, the samples were generally prepared by heavily diluting plasma or serum with buffer and spiking exogenous cholesterol. Such a sample preparation protocol greatly reduces matrix complexity and weakens the interference from endogenous components such as proteins and lipoproteins, and the spiked free cholesterol differs from the native lipoprotein-bound cholesterol in real blood. The present work was also conducted entirely in PBS buffer to investigate the fundamental strain-regulation mechanism, and thus shares this common limitation. Systematic validation in minimally diluted or native biological samples will be carried out in future work to advance the practical translation of the sensor.

Reliability is an essential prerequisite for the practical application of electrochemical sensors. Herein, the anti-interference capability, reproducibility, and long-term stability of the MnO₂/SPE sensor were systematically evaluated. First, differential pulse voltammetry (DPV) measurements were performed in a 100 μM cholesterol (CHO) solution to assess the anti-interference performance. Equal concentrations of potential interferents, including glutamic acid (Glu), ascorbic acid (AA), uric acid (UA), NaCl, and KCl, were individually added to the detection system. The corresponding DPV curves and current response histograms are shown in Figure 4a,b. The relative current variations induced by AA, Glu, NaCl, UA and KCl were calculated to be −1.19%, −0.35%, +2.77%, +0.77% and +2.16%, respectively. All deviations were within ±3%, well below the 5% interference threshold, verifying the excellent anti-interference capability of the MnO₂/SPE sensor. Subsequently, storage stability and continuous operational stability were evaluated. The MnO₂/SPE sensor maintained a stable cholesterol response after 7-day storage and delivered consistent signals in repeated DPV scanning cycles. As illustrated in Figure 4c, the oxidation peak current of a single electrode slightly decreased from 3.096 μA to 2.971 μA after seven days, with a relative attenuation of only 4%-well below the 5% stability threshold. This demonstrates that the MnO₂/SPE sensor maintains stable electrochemical performance during continuous operation and possesses desirable long-term stability.

Figure 4. (a) DPV curves of the MnO₂/SPE electrode in the presence of different interfering substances for anti-interference performance evaluation; (b) Interference Study of the MnO₂/SPE Sensor for Cholesterol Detection; (c) DPV curves of the same electrode for consecutive 7-day stability test; (d) DPV curves of five independent electrodes toward the same concentration of cholesterol solution for reproducibility assessment.

Furthermore, five independently fabricated MnO₂/SPE electrodes were used for parallel detection of cholesterol at the same concentration to evaluate reproducibility. As presented in Figure 4d, the DPV curves of the different electrodes nearly overlapped, with no distinct differences in oxidation peak current. Thus, the MnO₂/SPE sensor exhibits excellent fabrication reproducibility and favorable batch-to-batch consistency. From a practical application perspective, the proposed MnO₂/SPE sensor is intentionally designed for single-use disposable operation. On one hand, repeated electrochemical cycling and buffer rinsing may induce partial detachment of the drop-cast MnO₂ nanoflower layer and deteriorate sensing performance. On the other hand, the sensor features an extremely low fabrication cost owing to the mass-producible SPE substrate and simple modification process, making a discard-after-use mode economically feasible. This disposable configuration completely eliminates the risk of cross-contamination between samples and avoids cumbersome electrode regeneration and cleaning steps, which is highly compatible with the demands of point-of-care testing and large-scale batch application scenarios. Given the single-use design of the sensor, repeated-use stability is not a core performance metric for its intended application mode. This methodological choice is aligned with the practical positioning of low-cost disposable sensors, and systematic investigation of reusable protocols will be included in future work targeting long-term monitoring scenarios.

Flexible MnO₂/SPE sensors require excellent bending strain stability to meet reliability demands during production, transportation and practical utilization. Moreover, strain engineering can modulate the surface electronic structure of materials, thereby regulating the surface electrochemical activity of sensing materials and ultimately tuning the analytical performance of electrochemical sensors [37,38,39]. For instance, Ling et al. [38] demonstrated that surface strain engineering could boost the alkaline hydrogen evolution reaction (HER) activity of CoO nanorods (NRs). Tensile strain effectively couples the atomic and electronic structures of CoO NRs with HER catalytic activity and induces the formation of abundant oxygen vacancies. Similarly, Wei et al. [40] found that the application of tensile strain to zero-valent iron promotes sufficient electron filling at ohmic heterojunctions, thereby greatly improving the removal efficiency of heavy metal contaminants. Zhu et al. [41] demonstrated that localized compressive lattice strain in Pd nanofilms could effectively downshift the d-band center of Pd(111) facets and weaken Pd–H adsorption energy, achieving an 11.9-fold enhancement in hydrogen sensing response [41]. Inspired by this, this work explores strain engineering as a route to optimize the cholesterol sensing performance of MnO₂/SPE cholesterol sensors. Multiple approaches are available for strain introduction, mainly including substrate deformation [42], lattice mismatch [43], thermal expansion mismatch [44], and laser shocking [45]. Among these techniques, the substrate deformation strategy presents prominent advantages in strain controllability, continuity and material compatibility, and has been widely applied in diverse research fields.

A clamping-assisted method was adopted to introduce mechanical strain into MnO₂ film (Figure 5a). The MnO₂-modified SPE was firmly fixed, and mechanical strain was precisely applied via an adjustable clamp. Considering that the thickness of the MnO₂ film is far lower than that of the screen-printed electrode and the applied strain magnitude is limited to a low level, synchronous deformation between the MnO₂ film and the underlying electrode can be reasonably approximated. The corresponding strain structural model is displayed in Figure 5b. Figure 5c presents the strain calculation model of the flexible electrode. The corresponding Equations (4)–(6) were adopted to quantitatively calculate the tensile or compressive strain borne by the electrode during bending [46].

$$\epsilon ={\displaystyle \frac{S}{2R}}$$

(4)

$$\sin \theta ={\displaystyle \frac{b}{D}}$$

(5)

$${\displaystyle \frac{2\theta }{2\pi }}={\displaystyle \frac{L}{2\mathit{\pi D}}}$$

(6)

Figure 5. (a) Optical image of the two-point bending apparatus for applying strain on MnO₂. (b) Schematic illustration of the flexible strain model; (c) Schematic diagram of strain calculation. The two arrows mark the electrode thickness region. S: electrode thickness, D: radius of curvature, θ: half central angle, L: original substrate length, 2b: substrate arc length.

In the above equations, ε represents the strain, s is the electrode thickness (0.07 cm), D denotes the curvature radius, θ refers to the central half-angle, L is the original substrate length (5 cm), and b corresponds to the half-arc length of the bent substrate. Interfacial slippage may occur between the MnO₂ active layer and PET substrate during flexible strain testing, which impedes the complete transfer of mechanical strain from the substrate to the MnO₂ film and further impairs the accuracy of strain performance evaluation. To assess the mechanical stability of the MnO₂/SPE sensor under mechanical strain, the strain response of the bare screen-printed electrode (SPE) was first investigated. This step eliminates interference arising from the intrinsic strain sensitivity of the substrate electrode, which could otherwise compromise the performance analysis of MnO₂-modified electrodes. Differential pulse voltammetry (DPV) measurements were performed in cholesterol solutions with a concentration gradient of 20–100 μM under various applied strains (−1%, −0.5%, 0%, +0.5%, and +1%), and the corresponding results are illustrated in Figure 6. As reflected in the DPV curves (Figure 6a–e), the oxidation peak current of the bare SPE toward identical cholesterol concentrations exhibited negligible variations across different strain states. Combined with the peak current plots in Figure 6f, the current signals under diverse strain conditions were highly overlapped without an obvious changing trend, and the relative current variation remained far below the acceptable error threshold of 5%. These findings demonstrate that the bare SPE possesses outstanding electrochemical stability and extremely low intrinsic strain sensitivity within the strain range of −1% to +1%. Consequently, the substrate electrode exerts a negligible influence on subsequent strain measurements of modified electrodes, thereby providing a solid and reliable experimental foundation for further evaluating the strain-regulated sensing behavior of MnO₂/SPE.

Figure 6. (a–e) DPV curves of the bare electrode under applied strains of −1%, −0.5%, 0%, 0.5%, and 1%, respectively. (f) Oxidation peak current vs. cholesterol concentration at different strains.

To elucidate how mechanical strain modulates the cholesterol-sensing performance of the MnO₂/SPE flexible sensor, we systematically characterized its response to varying cholesterol concentrations using differential pulse voltammetry (DPV) over a strain range of −1% to +1%. Figure 7a–e presents the DPV curves of the MnO₂/SPE electrode under applied strains of −1%, −0.5%, 0%, 0.5%, and 1%, respectively. The corresponding linear relationships between the oxidation peak current and cholesterol concentration are plotted in Figure 7f. Electrochemical measurements reveal that, within the strain range of −1% to +1%, the oxidation peak current exhibits a strong linear correlation with cholesterol concentration under all applied strains, demonstrating the sensor’s reliable quantitative detection capability even under mechanical deformation. Notably, strain exerts a pronounced modulatory effect on the sensor’s response current: the oxidation peak currents under compressive strains (−1% and −0.5%) are significantly higher than those under the strain-free condition (0%), whereas tensile strains (0.5% and 1%) yield lower currents. These results indicate that compressive strain enhances the sensor’s response signal, while tensile strain leads to signal attenuation. This phenomenon can be well rationalized by the flexocatalysis mechanism of MnO₂ nanosheets, as illustrated in Figure 8. When the flexible MnO₂/SPE electrode undergoes mechanical bending, the two-dimensional MnO₂ nanosheets experience non-uniform strain, breaking the inversion symmetry of the crystal lattice and inducing flexoelectric polarization (P_flexo) within the nanosheets (Figure 8a,b). This effect generates a built-in electric field that drives efficient separation of photogenerated electron–hole pairs and suppresses their recombination, thereby significantly improving the interfacial electron transfer efficiency and electrocatalytic activity [25]. Specifically, under compressive (negative) strain, the bending-induced strain gradient gives rise to a stronger flexoelectric polarization, resulting in a more intense built-in electric field. This field enhances charge separation, as visualized by the asymmetric distribution of electrons and holes across the potential energy landscape (Figure 8c). The resulting charge distribution modulates the Mn⁴⁺/Mn²⁺ redox pathway and enables synergistic catalysis: electrons enriched at the cathode side promote Mn⁴⁺ reduction and cholesterol oxidation, while holes at the anode side facilitate Mn²⁺ regeneration.

Figure 7. (a–e) Differential pulse voltammetry (DPV) curves of the MnO₂/SPE sensor under applied strains of −1%, −0.5%, 0%, 0.5%, and 1%, respectively; (f) linear plots of oxidation peak current versus cholesterol concentration.

Figure 8. (a) Band structure engineering under flexoelectric polarization (P_flexo). The built-in electric field induced by mechanical strain leads to the tilting of the conduction band (CB) and valence band (VB), with E_F denoting the Fermi level. (b) Schematic of the synergistic catalysis driven by flexoelectric polarization. The polarization-induced charge separation spatially partitions the Mn⁴⁺/Mn²⁺ redox cycle: electrons (black spheres) accumulate on one side to drive Mn⁴⁺ reduction and cholesterol oxidation, while holes (open circles) are enriched on the opposite side to promote Mn²⁺ regeneration. (c) Potential energy diagram of charge separation and Mn⁴⁺/Mn²⁺ redox catalysis. (d) Sensitivity of the MnO₂/SPE sensor under applied strains of −1%, −0.5%, 0%, 0.5%, and 1%.

Sensitivity serves as a core Fig.-of-merit to evaluate the comprehensive performance of electrochemical sensors, and it is defined by the formula [47]:

$$S={\displaystyle \frac{K}{M}}$$

(7)

where K is the slope of the linear calibration plot between current response and analyte concentration, M represents the geometric area of the electrode, and S denotes the sensor sensitivity. The sensitivity values under various strain conditions were calculated based on the current–concentration relationships under different strains in Figure 7f, and the results are summarized in Figure 8d. Under the strain-free state, the sensor delivers a baseline sensitivity of 0.197 μA⋅μM⁻¹⋅cm⁻². When tensile strains of +0.5% and +1% are applied, the sensitivity declines to 0.1387 μA⋅μM⁻¹⋅cm⁻² and 0.1199 μA⋅μM⁻¹⋅cm⁻², with respective reductions of 29.5% and 39.1%. This phenomenon primarily stems from the effect of tensile (positive) strain. When the nanosheets are subjected to tensile bending, the resulting strain distribution induces a weaker enhancement of flexoelectric polarization relative to the compressive state. Meanwhile, excessive tensile deformation weakens the interlayer contact of the nanosheets and increases the impedance of electron transport pathways, ultimately leading to a decline in the sensing response current. In contrast, the oxidation peak current increases monotonically with the elevation of compressive strain. At compressive strains of −0.5% and −1%, the sensitivity is enhanced to 0.2235 μA⋅μM⁻¹⋅cm⁻² and 0.2497 μA⋅μM⁻¹⋅cm⁻², with increments of 13.5% and 26.7%, respectively. Under compressive (negative) strain, the bending-induced strain gradient readily triggers strengthened flexoelectric polarization, which further improves the separation efficiency of electron–hole pairs. Consequently, the oxidation peak current is boosted, and the overall sensing performance is effectively optimized. Strain engineering enables precise regulation of the electrochemically active interface of electrodes, thereby optimizing the electrochemical sensing performance of the sensor.

It should be noted that all sensing performance evaluations in this work were conducted in standard PBS buffer solutions with spiked cholesterol, and verification in real biological matrices (e.g., serum or plasma) has not been performed. Therefore, the practical applicability of the sensor in complex clinical samples remains to be further validated. In addition, only individual interfering substances were evaluated in the anti-interference tests, and the mutual interaction effects among multiple coexisting interferents have not been statistically analyzed via ANOVA. These aspects will be systematically investigated in our future work to further improve the reliability and practical value of the sensor.

4. Conclusions

In summary, a flexible MnO₂/SPE sensor was fabricated using two-dimensional flower-like MnO₂ nanomaterials. EIS results revealed that MnO₂ modification reduced the electrode impedance from 270 Ω of bare SPE to 130 Ω. CV measurements demonstrated a linear correlation between oxidation peak current and scan rate. DPV tests confirmed that the sensor achieved favorable sensing performance within the cholesterol concentration range of 1–160 μM, with a low detection limit of 0.16 μM and a sensitivity of 0.197 μA·μM⁻¹·cm⁻². Moreover, the MnO₂/SPE sensor exhibited excellent stability, reproducibility and anti-interference capability; both the current decay rate over seven-day continuous detection and the relative standard deviation of five repeated measurements were below 5%. Mechanical strain presents a remarkable modulation effect on the sensing performance. Under 1% tensile strain, the sensitivity decreased to 0.1199 μA·μM⁻¹·cm⁻², accompanied by an obvious decline in oxidation peak current. By contrast, −1% compressive strain improved the sensitivity up to 0.2497 μA·μM⁻¹·cm⁻² and optimized the overall sensing behavior. This strain-dependent behavior can be rationally explained by the flexocatalysis mechanism. Under compressive strain, the enhanced flexoelectric polarization of MnO₂ nanosheets facilitates the separation of electron–hole pairs, which acts synergistically with the intrinsic redox cycling catalytic activity of Mn valence states. Such synergism further boosts the electrocatalytic oxidation of cholesterol and elevates the response signal. Strain engineering strategy enables the precise modulation of the electrochemically active interface of the electrode, thereby optimizing its electrochemical sensing performance. This proposed mechanism offers a new insight into the performance optimization of flexible electrochemical sensors.