Oxidase Activity-Enhanced Manganese (III) Oxide Nanozyme Grown in Dendritic Mesoporous Silica Nanoparticles for Sensitive Lateral Flow Immunoassay of C-Reactive Protein
JiaqiFang1
FangGao2✉Emailfgao@usst.edu.cn
YeZhang1
YiningYao3
ShaonianYe2
ChaoLiu1✉,2Emailcliu@chem.ecnu.edu.cn
ChengzhongYu1✉,2,3Phone/0000-0003- 3707-0785Phone+61 7 33463283Emailc.yu@uq.edu.au
1School of Chemistry and Molecular EngineeringEast China Normal University200241ShanghaiP. R. China
2Institute of Energy Materials ScienceUniversity of Shanghai for Science and Technology200093ShanghaiPR China
3Australian Institute for Bioengineering and NanotechnologyThe University of Queensland Brisbane4072BrisbaneQueenslandAustralia
Jiaqi Fang1, Fang Gao2, Ye Zhang1, Yining Yao3, Shaonian Ye2, Chao Liu1, Chengzhong Yu1, 3
1School of Chemistry and Molecular Engineering, East China Normal University, Shanghai, 200241, PR China
2Institute of Energy Materials Science, University of Shanghai for Science and Technology, Shanghai, 200093, PR China
3Australian Institute for Bioengineering and Nanotechnology, The University of Queensland Brisbane, Queensland 4072, Australia
Corresponding Authors
*Fang Gao − Institute of Energy Materials Science, University of Shanghai for Science and Technology, Shanghai, 200093, PR China; orcid.org/0000-0002-5224-3843; Email: fgao@usst.edu.cn
*Chao Liu − School of Chemistry and Molecular Engineering, East China Normal University, Shanghai, 200241, PR China; orcid.org/0000-0003-0316-4994; Email: cliu@chem.ecnu.edu.cn
*Chengzhong Yu − Australian Institute for Bioengineering and Nanotechnology, The University of Queensland, Brisbane, Queensland 4072, Australia; School of Chemistry and Molecular Engineering, East China Normal University, Shanghai 200241, P. R. China; orcid.org/0000-0003-3707-0785; Email: c.yu@uq.edu.au; Tel: +61 7 33463283
Abstract
The development of cost-effective nanozymes with signal amplification function to overcome the limited sensitivity of lateral flow immunoassays (LFIAs) is of great significance. Here, we introduce the first Mn2O3-based colorimetric LFIA platform for sensitive C-reactive protein (CRP) detection. By confining Mn2O3 within dendritic mesoporous silica nanoparticles (DMSNs), DMSN-Mn2O3 nanozymes have been synthesized with enhanced active site exposure and uniform particle sizes. It is demonstrated that DMSN-Mn2O3 exhibits better catalytic performance compared to DMSN-MnO2 and pure Mn2O3, showing the importance of control over both composition and nanostructure in enhancing the nanozyme functions. In the built H2O2-free colorimetric LFIA platform, DMSN-Mn2O3 demonstrates rapid catalytic amplification (30 s) for CRP detection, achieving a limit of detection of 0.25 ng mL− 1, a 6-fold improvement over conventional colloidal gold-based LFIAs. This work contributes to the rational design of high-performance nanozymes and provides a platform technology for point-of-care diagnostics with enhanced sensitivity.
Keywords:
Mesoporous silica nanoparticle
Manganese oxide
Oxidase-like nanozyme
Lateral flow immunoassay
C-reactive protein
Introduction
The paper-based lateral flow immunoassay (LFIA) strip is a crucial point-of-care testing (POCT) device designed for rapid detection of analytes [1]. Renowned for its cost-effectiveness, ease of production, rapidity, and portability, LFIA aligns with the World Health Organization (WHO)’s ASSURED criteria (affordable, sensitive, specific, user-friendly, rapid, robust, equipment-free, and deliverable to end-users), making it widely used in clinical diagnostics [2, 3]. Conventional colorimetric LFIAs predominantly rely on colloidal gold nanoparticles (AuNPs), but its sensitivity is not satisfactory, particularly for low-concentration analytes within complex biological matrices [4]. To improve the detection sensitivity, nanomaterials capable of signal amplification (e.g., fluorescence [5], photothermal [6], and Raman [7] signals) have been developed. However, such approaches typically necessitate specialized instrumentation, undermining the practicality of POCT applications [8]. Consequently, colorimetric signal amplification strategies that enable visual readout with enhanced sensitivity have garnered significant attention [9].
Nanozymes, which are nanoscale materials that mimic the catalytic activity of natural enzymes, offer promising alternatives due to their good stability and tunable functionalities [10]. Among various nanozymes, peroxidase (POD)-like and oxidase (OXD)-like nanozymes have been extensively integrated into LFIAs to catalyze chromogenic reactions for colorimetric signal amplification [11]. POD-like nanozymes require hydrogen peroxide (H2O2) in the catalytic reaction to generate hydroxyl radicals (·OH), which subsequently oxidize an organic substrate (such as tetramethylbenzidine, TMB) from colorless to blue (the color of oxidized TMB, ox-TMB) [12]. However, the reliance on H2O2 reactant introduces problems such as reagent instability, complicated H2O2 concentration optimization process and cost inefficiency [13]. In contrast, OXD-like nanozymes offer advantages by catalyzing TMB oxidation using dissolved oxygen (O2) [12, 14], thus the problems associated with H2O2 can be alleviated. In this regard, there is growing interest in developing LFIA platforms based on OXD-like nanozymes to enhance operational simplicity.
To date, numerous nanomaterials exhibiting OXD-like activity have been identified [12]. Among them, manganese dioxide (MnO2) has been applied in LFIA due to its good OXD-like catalytic activity and low cost [15]. For example, Cai et al. utilized MnO2 nanosheets to detect aflatoxin B1 in LFIA, achieving a limit of detection (LoD) of 15 pg mL− 1 and a broad detection range of 0.01–150 ng mL− 1 [16]. Dong et al. employed PDA@MnO2 nanozymes to quantify SARS-CoV-2 S antigen in LFIA, achieving a sensitivity 18.7 times higher than that of conventional AuNP-based LFIA [17]. It is noted that manganese oxides (MnOx) exist in multiple oxidation states [18], their catalytic activity is highly dependent on the manganese oxidation states [19]. In a study comparing the OXD-like activity of MnOx with different oxidation states, Chen et al. demonstrated that Mn2O3 exhibited the highest OXD-like activity [20]. However, the application of Mn2O3 as an OXD-like nanozyme in LFIA application has not been reported.
In LFIA applications, the performance of nanozymes depends critically on both their composition and nanostructural properties. While the intrinsic activity of nanozymes is related to composition, the catalytic performance can be further improved through nanostructure engineering that increases active site exposure. For instance, the in situ growth of Prussian blue within dendritic mesoporous silica nanoparticles (DMSNs) has been demonstrated to enhance the catalytic efficiency by increasing the number of accessible active sites, thereby enhancing the LFIA sensitivity [21]. DMSNs, characterized by their uniform particle sizes, large pore channels and highly accessible surface areas, serve as ideal carriers for signal amplification specifically for LFIA applications [22, 23]. The uniform particle sizes ensure assay’s reproducibility. Moreover, the excellent dispersibility of DMSNs facilitates the migration of labelled particles, and the ease of functionalization allows for the conjugation of recognition elements, such as antibodies, which are crucial for the assay’s functionality [24, 25]. It is noted that Mn2O3 with OXD-like activity (e.g. synthesized by the calcination of MnCO3) usually has small specific surface area, non-uniform particle size, poor water dispersibility and difficulty in antibody conjugation [20], thus has limited applicability in LFIA. In this regard, it is hypothesized that by dispersing Mn2O3 within DMSNs, the resultant nanozyme may have enhanced both catalytic efficiency and applicability in LFIA.
Herein, we report the first example of a Mn2O3-based colorimetric LFIA platform and demonstrate its application potential in the detection of C-reactive protein (CRP), a crucial biomarker for diagnosing and monitoring inflammatory conditions, infections tissue damage, and assessing cardiovascular risk and treatment efficacy [26]. In the Mn2O3-based colorimetric LFIA platform, DMSNs with large pores, high specific surface area, uniform size, good dispersibility, and ease of functionalization are introduced for label signal amplification in LFIA (Scheme 1). By dispersing Mn2O3 nanoparticles within the large pores of DMSNs, DMSN-Mn2O3 (denoted S-Mn2O3) can be obtained with better OXD-like activity compared to MnO2 dispersed in DMSNs (named as S-MnO2). When used as a label material in LFIA after functionalization, the S-Mn2O3-based LFIA platform achieved a detection range of 10–1000 ng mL− 1 and a LoD of 0.25 ng mL− 1 in CRP detection without using H2O2, surpassing that of previously reported LFIAs, including Au@PdNP-based LFIA (0.32 ng mL− 1 [27]) and CeO2-based LFIA (117 ng mL− 1 [28]). This work provides a cost-effective and high-performance LFIA platform for future clinical diagnostic applications.
Scheme 1
Schematic illustration of enhancing the oxidase activity of manganese oxide through a valence-specific nanoarchitectonic strategy, and its application in catalyzing TMB oxidation to enable immuno-carboxyl-S-Mn2O3 (I-CS-Mn2O3)-based colorimetric LFIA signal amplification for CRP detection.
Materials and methods
Materials
All chemicals were used as received without further purification. Triethanolamine (TEA), cetyltrimethylammonium bromide (CTAB), manganese(II) nitrate tetrahydrate (Mn(NO3)2⋅4H2O), manganese(II) chloride tetrahydrate (MnCl2⋅4H2O), 3-aminopropyltriethoxysilane (APTES), succinic anhydride (SA), TMB, acetic acid (HAc), D(+)-sucrose, polyvinylpyrrolidone (PVP, Mw ≈ 30,000), and bovine serum albumin (BSA) were obtained from Adamas-beta. Phosphate-buffered saline (PBS) was received from Sigma-Aldrich. Poly(ethylene glycol) (PEG, Mw ≈ 1,500), tetraethyl orthosilicate (TEOS), sodium salicylate (NaSal), ammonium bicarbonate (NH4HCO3), sodium acetate (NaAc), methanol, N,N-Dimethylformamide (DMF) and ethanol were obtained from General-Regent, Tween 20 was purchased from TCI (Shanghai) Development Co., Ltd. Monoclonal mouse anti-human CRP antibodies (ABCRP0801 and ABCRP0802, represented with anti-CRP 1st mAbs and anti-CRP 2nd mAbs, respectively), human serum albumin (HSA), and goat anti-mouse (GAM) IgG were purchased from Changsha Bioadvantage Co. Ltd. Carcinoembryonic antigen (CEA), human alpha-fetoprotein (AFP), prostate-specific antigen (PSA), and CRP antigen were obtained from Shanghai Linc-Bio Science Co., Ltd. Human serum was obtained from Shanghai Jianglai Biotechnology Co., Ltd. Nitrocellulose (NC) membrane (CN140) was purchased from Sartorius. Absorbent pad, sample pad, conjugate pad and PVC backing card were purchased from Shanghai KinbioTech Co, Ltd. Deionized water (DI water) used for all experiments was obtained from a Milli-Q system (Millipore, Bedford, MA).
Characterization
Transmission electron microscopy (TEM) and high-resolution TEM (HRTEM) imaging, coupled with energy-dispersive X-ray spectroscopy (EDS) elemental mapping, were performed with a Thermo Talos F200X instrument. For TEM sample preparation, powder specimens were dispersed in ethanol via sonication and deposited onto holey carbon films supported by copper grids. Field-emission scanning electron microscopy (FESEM) was performed on a Hitachi S-4800 system to characterize sample morphology. SEM samples were prepared by dispersing powder samples in ethanol, then depositing them onto the aluminum foil pieces, which were subsequently attached to a conductive carbon film on the SEM mount. X-ray diffraction (XRD) patterns were collected using a Rigaku SmartLab SE (Cu-Kα) diffractometer. Metal ion concentrations was determined by inductively coupled plasma optical emission spectroscopy (ICP-OES) using an Agilent 5800 ICP-OES. X-ray photoelectron spectroscopy (XPS) tests were carried out using a PHI Quantera II ESCA system using Al Kα radiation at 1486.8 V with He I excitation (21.22 eV) as the monochromatic light source, with C 1s binding energy set at 284.8 eV as the reference. Fourier transform infrared (FTIR) spectra were acquired using a Thermal-Nicolet iS50 spectrometer over the range of 4000 ~ 500 cm− 1. The specific surface area was determined by the Brunauer-Emmett-Teller (BET) method, and the pore size distribution was analyzed using the Barrett-Joyner-Halenda (BJH) method derived from the adsorption branch of the nitrogen isotherms. Prior to analysis, samples were degassed under vacuum at 453 K for 12 h. Ultraviolet-visible (UV-vis) absorption spectra were acquired in the 550–750 nm range using a Lambda 950 UV-vis spectrophotometer. Catalytic kinetics were recorded via absorption measurements on a BioTek Synergy H1 multimode microplate reader. Zeta potential and dynamic light scattering (DLS) measurements were carried out in DI water at 25°C using a NanoBrook 90Plus PALS from Brookhaven Instruments. Electron spin resonance (ESR, BRUKER EMX plus-6/1. Germany) was used to detect the reactive oxygen species (ROS) by applying 5,5-dimethyl-1-pyrroline-1-oxide (DMPO) and 2,2,6,6-tetramethylpiperidinyl-1-oxyl (TEMP) as the spin trapping agent.
Synthesis of DMSNs
DMSNs were prepared according to a reported method with modification [5]. In a typical experiment, 408 mg of TEA was dissolved int 150 mL of DI water and stirred at 80°C for 30 min. Afterwards, 2.28 g of CTAB and 1.008 g of NaSal were added to the solution, and the mixture was stirred for an additional hour. 24 mL of TEOS and 1.5 mL of ethanol were then introduced, and the reaction proceeded with stirring for another 3 h. The resulting DMSNs were isolated by centrifugation, washed thoroughly with ethanol to remove the residual reactants, and calcined in the air atmosphere at 550°C for 5 h with a heating rate of 2°C min− 1.
Synthesis of S-Mn2O3
150 mg of DMSNs was dispersed into 10 mL of ethanol containing x mmol of Mn(NO3)2⋅4H2O, where x = 0.75, 1.00, or 1.25. The suspension was stirred at room temperature for 12 h, followed by ethanol evaporation at 40°C to yield a solid residue. The product was ground into a homogeneous powder and evenly spread at the bottom of a glass bottle. The glass bottle was then placed inside a larger sealed glass bottle containing saturated NH4HCO3. After heating at 35°C for 24 h, the obtained DMSN-MnCO3 composite was washed three times with a mixture of ethanol and water. Finally, the S-Mn2O3 was synthesized by calcining the DMSN-MnCO3 in the air atmosphere at 600°C for 2 h with a heating rate of 5°C min− 1.
Synthesis of S-MnO2
The DMSN-MnCO3 composite was calcined in the air atmosphere at 400°C for 2 h with a heating rate of 5°C min− 1, yielding DMSN-MnO2 (S-MnO2).
Synthesis of Mn2O3
An aqueous NH4HCO3 solution (30 mL) was mixed with 30 mL of MnCl2⋅4H2O aqueous solution under vigorous stirring at room temperature for 10 min. The mixture was hydrothermally treated at 80 ℃ for 30 min in a Teflon-lined stainless-steel autoclave. After cooling to room temperature, the precipitate MnCO3 was collected by centrifugation and washed three times with ethanol. Mn2O3 was obtained by calcining of MnCO3 in the air atmosphere at 600°C for 2 h with a heating rate of 5°C min− 1.
Synthesis of CS-Mn2O3
A two-step approach was used to graft carboxyl (-COOH) groups onto S-Mn2O3 [29]. First, 100 mg S-Mn2O3 was dispersed in 30 mL of toluene, followed by the addition of 90 µL of APTES. The mixture was stirred at 110°C for 12 h, then the solid was collected by centrifugation and washed with ethanol to remove the unreacted APTES, yielding AS-Mn2O3. Subsequently, AS-Mn2O3 was dispersed in 20 mL of DMF containing 0.1g SA and stirred at room temperature for 12 h. The product was washed three times with ethanol, resulting in CS-Mn2O3.
Synthesis of I-CS-Mn2O3
CS-Mn2O3 nanoparticles were functionalized with anti-CRP 2nd mAbs via physical adsorption. Briefly, CS-Mn2O3 was suspended in 1 mL of PBS buffer (10 mM, pH 7.4), followed by the addition of 75 µL of anti-CRP 2nd mAbs (1 mg mL− 1). After 2 h of incubation at room temperature with gentle agitation, the I-CS-Mn2O3 was centrifuged and washed with PBS-Tween 20 (PBST) wash buffer (10 mM, pH 7.4, 0.05% v/v) to remove unbound antibodies. To minimize nonspecific binding, I-CS-Mn2O3 was treated with a blocking buffer containing 10% (w/v) BSA, 0.5% (v/v) Tween 20 for 2 h. Finally, the I-CS-Mn2O3 was resuspended in a preservation medium (20 mM PBS, pH 7.4; 1% (w/v) sucrose, 2.5% (w/v) BSA, 2% (w/v) PEG-1,500) and stored at 4°C.
Determination of OXD-like activity of nanozymes
The catalytic activity of nanozymes (S-Mn2O3, S-Mn2O3-1, S-Mn2O3-2, S-MnO2 and Mn2O3) was evaluated using a colorimetric method with TMB as the substrate. For each sample (DMSN and the listed nanozymes), 40 µL of sample solution (500 µg mL− 1) was mixed with a TMB working solution comprising 10 µL TMB (12 mM) and 150 µL of 0.2 M NaAc-HAc buffer (pH 4). Following a 3-minute incubation period, UV-vis absorption spectra were recorded between 550–750 nm. The OXD-like activity was quantified based on the absorbance values at 652 nm. To assess pH effects, the absorbance at 652 nm was measured for S-Mn2O3 in TMB working solutions buffered at different pH values under otherwise identical conditions.
Determination of catalytic kinetics
The kinetics of OXD-like activity were determined by measuring the absorbance at 652 nm at 1-min intervals for 30 min in TMB working solutions with varying TMB concentrations (2, 4, 6, 8, 12, 16, and 40 mM) using a microplate reader. The kinetic parameters were derived from Lineweaver-Burk plots.
Fabrication of LFIA
Sample pads and conjugate pads were treated with a solution containing 20 mM PBS buffer (pH 7.4), 1% (w/v) BSA, 1% (v/v) Tween 20, 2.5% (w/v) sucrose, and 0.3% (w/v) PVP 30,000 for 1 minute, followed by drying at 37°C. The anti-CRP 1st mAbs (1 mg mL− 1) and GAM IgG (1 mg mL− 1) in PBS were dispensed onto the designated capture zone of the NC membrane at a rate of 1.0 µL cm− 1. The membrane was then incubated at 37°C for 12 h. Finally, the pre-treated sample pad, conjugate pad, NC membrane, and absorbent pad were laminated onto a plastic backing card. The assembled card was then cut into 3-mm-wide strips.
Detection of CRP using I-CS-Mn2O3-based LFIA
For CRP detection, 100 µL of a mixture containing 95 µL CRP standard solution and 5 µL I-CS-Mn2O3 was applied to the sample pad of the LFIA. After 15 min of capillary-driven migration, semi-quantitative results were obtained by visual inspection of color intensity at T line and C line. Quantitative analysis was conducted by measuring grayscale values of both lines using ImageJ software. Subsequently, 15 µL of 10 mM TMB solution was dropped on the membrane. After 30 s, the semi-quantitative and quantitative results were re-evaluated as described. Strip images were taken by an iPhone 14, and analyzed with ImageJ software. The signal intensity ratios (IT/IC) were calibrated against CRP concentrations. All groups were performed in triplicate.
Results and Discussion
Characterization of S-Mn2O3
DMSNs were synthesized using a reported method [5]. SEM image (Fig. S1A) reveals spherical DMSNs with an average particle size of ~ 200 nm and large open pores. TEM analysis further reveals their radially aligned large pores (Fig. S1B). The preparation process of S-Mn2O3 is illustrated in Fig. 1A. The prepared DMSNs were immersed in a Mn(NO3)2 solution. The DMSN-Mn2+ composites were obtained via solvent evaporation. A solid-ammonia gas reaction process [30] led to the growth of MnCO3 within the pores of DMSN and the formation of DMSN-MnCO3 composite. TEM analysis of the resultant DMSN-MnCO3 (Fig. S2A) indicates that the dendritic structure is retained. XRD patterns (Fig. S2B) reveal that DMSNs have an amorphous nature, while diffractions matching the MnCO3 reference (PDF#44-1472) are observed in DMSN-MnCO3, indicating the successful growth of MnCO3 within the large pores of DMSNs.
Calcination of DMSN-MnCO3 at 600 ℃ induced the transformation of MnCO3 in the composite to Mn2O3, yielding the product S-Mn2O3. SEM and TEM images of S-Mn2O3 (Fig. 1B and C) display a well-preserved dendritic mesoporous structure with minimal changes in particle uniformity. High-angle annular dark-field scanning TEM (HAADF-STEM) coupled with EDS elemental mapping images (Fig. 1D) show the homogeneous distribution of Si, O and Mn signals in one nanoparticle. XRD analysis (Fig. 1E) shows that the diffraction pattern of S-Mn2O3 aligns with the standard Mn2O3 reference pattern (PDF#41-1442). Collectively, the above characterization results indicate the successful synthesis of S-Mn2O3.
To further elucidate the chemical states of S-Mn2O3, XPS analysis was employed. The Mn 3s spectrum (Fig. 1F) exhibits a spin-energy splitting of 5.2 eV for S-Mn2O3, which is close to the theoretical value for Mn2O3 (5.4 eV) [31]. The Mn 2p spectrum (Fig. S3A) displays characteristic peaks at 641.0 eV (Mn 2p3/2) and 652.4 eV (Mn 2p1/2). Deconvolution of the Mn 2p3/2 peak reveals contributions from Mn3+ (640.8 eV) and Mn4+ (643.0 eV) species [32], with a spin-orbit splitting of 11.6 eV, consistent with Mn2O3 [33]. The O 1s spectrum (Fig. 1G) shows a shoulder at 529.0 eV corresponding to Mn-O-Mn bonds [34], and a main peak at 532.5 eV assigned to Si-O bonds [29]. These findings substantiate the successful synthesis of the S-Mn2O3. Nitrogen adsorption-desorption isotherms of DMSNs and S-Mn2O3 are presented in Fig. S3B. Compared to DMSNs, there is a reduction in BET specific surface area from 464.8 m2 g− 1 (DMSNs) to 162.2 m2 g− 1 (S-Mn2O3), accompanied by a decrease in average pore size (from 47.4 to 30.4 nm) and total pore volume (from 1.46 to 0.65 cm3 g− 1). These changes are attributed to the partial pore occupation by Mn2O3 nanoparticles.
A
To study the impact of the structure of nanozymes on their OXD-like activity, four control samples were prepared. Firstly, S-Mn2O3-1 (0.75 mmol Mn precursor) and S-Mn2O3-2 (1.25 mmol Mn precursor) were synthesized. SEM and TEM images (Fig. S4A–D) indicate that Mn2O3 is confined within the pores of S-Mn2O3-1, similar to S-Mn2O3. Nevertheless, nanoparticles grown outside the pores (indicated by arrows) are observed in S-Mn2O3-2 due to Mn precursor overloading. XRD analysis (Fig. S5) confirm the Mn2O3 phase (PDF#41-1442) in the two composites. The ICP-OES quantified Mn loading (Table S1), demonstrating increased Mn incorporation with higher precursor loading amounts. Secondly, S-MnO2 (calcined at 400°C) and Mn2O3 were prepared. TEM and XRD analyses reveal that S-MnO2 maintains the dendritic mesoporous structure (Fig. S6A) and has a MnO2 phase (PDF#30–0820; Fig. S6B). In contrast, Mn2O3 forms aggregate (Fig. S7A) with reduced surface accessibility, and its XRD pattern corresponds to the Mn2O3 phase (Fig. S7B). XPS analysis of the Mn 3s spectra reveal a spin-energy separation of 4.83 eV for S-MnO2 (Fig. S6C), consistent with the standard value for MnO2 (4.78 eV) [35]. For Mn2O3, the separation is 5.45 eV, aligning with the expected value for Mn2O3 [31].Fig. 1
Characterization of S-Mn2O3. (A) Schematic illustration of the preparation process for S-Mn2O3. (B) SEM image, (C) TEM image, and (D) HAADF-STEM image with EDS-elemental mapping of S-Mn2O3. (E) XRD patterns of DMSNs and S-Mn2O3. (F) Mn 3s and (G) O 1s XPS spectra of S-Mn2O3.
OXD-like Activity of S-Mn2O3
The OXD-like catalytic performance of S-Mn2O3 against control samples was systematically evaluated. The catalytic activity was determined by monitoring the oxidation reaction of TMB in the absence of H2O2, which generates a blue oxidized product (ox-TMB) with an absorbance maximum at 652 nm. The reaction conditions were firstly optimized by investigating the pH-dependent catalytic activity, with pH 4 identified as the optimal condition (Fig. S8). To investigate the impact of Mn content on OXD-like activity, the UV–vis spectra of S-Mn2O3, S-Mn2O3-1 and S-Mn2O3-2 were measured (Fig. S9). No absorbance peak is observed for TMB alone in the absence of the catalyst or the catalyst solution without TMB (Fig. S9). The nanozyme S-Mn2O3 prepared with 1 mmol Mn precursor exhibits the highest absorption intensity at 652 nm (Fig. S9), due to its higher Mn2O3 content than S-Mn2O3-1 and non-aggregated nature compared to S-Mn2O3-2.
The OXD-like activity of S-Mn2O3 is also higher than Mn2O3 and S-MnO2 (Fig. S10). To gain more quantitative information, the steady-state kinetics of the OXD-like catalytic activity were analyzed using the Michaelis-Menten model with TMB as the substrate to determine substrate affinity and catalytic efficiency. The initial velocity (ν) was calculated from absorbance changes at 652 nm using the Lambert-Beer law (Eqs. 1 and 2):
(Eq. 1);
(Eq. 2);
where A is the absorption at 652 nm, ε is the molar absorption coefficient (3.9 × 104 M− 1⋅cm− 1), b is the optical path length (0.625 cm), c is the ox-TMB concentration, and t is the reaction time. By varying the concentration of TMB, the steady-state kinetic parameters, including the Michaelis constant (Km) and the maximum reaction velocity (Vmax) were determined using the Lineweaver-Burk equation (Eq. 3).
(Eq. 3);
A
where V and Vmax represent the initial velocity and the maximal reaction velocity, respectively. [S] is the concentration of substrate, and Km is the Michaelis constant [36]. Michaelis-Menten curves (Fig. 2A–C) and Lineweaver-Burk plots (Fig. 2D–F) for S-MnO2, Mn2O3 and S-Mn2O3 reveal Km values of 0.318 mM for S-MnO2, 0.293 mM for Mn2O3, and 0.184 mM for S-Mn2O3, demonstrating the highest substrate affinity for S-Mn2O3 [37]. Moreover, the Km value was determined to be 0.232 mM for S-Mn2O3-1 and 0.243 mM for S-Mn2O3-2 (Table S2), underscoring the critical role of Mn dispersion state within the DMSN framework. Compared to Mn(IV)-based S-MnO2, the Mn(III) oxidation state in S-Mn2O3 favors reactive oxygen species (ROS) generation, thereby facilitating TMB oxidation [38]. In addition, the confinement of Mn2O3 within DMSN increases the active site exposure than bare Mn2O3, leading to enhanced activity.To elucidate the catalytic mechanisms of the three nanozymes, we analyzed the types of ROS generated during catalysis. Within the S-Mn2O3 catalytic system, ESR spectroscopy using DMPO and TEMP as spin-trapping agents detected ·OH and superoxide anion radicals (O2·⁻), but failed to detect singlet oxygen (1O2) (Fig. 2G). These results indicate a reaction pathway of O2 → O2·⁻ → H2O2 →·OH [39]. Subsequently, the capabilities of the three nanozymes (S-MnO2, Mn2O3, and S-Mn2O3) to generate O2·⁻ and ·OH were compared using ESR spectroscopy (Fig. 2H–I). The results revealed that S-Mn2O3 exhibited stronger signal intensities than both S-MnO2 and Mn2O3, demonstrating that S-Mn2O3 possesses the highest ROS generation capacity among the tested three catalysts, in accordance with literature [20, 21] and the conclusion from steady-state kinetic studies. Overall, the enhanced OXD-like activity of S-Mn2O3 can be attributed to its controlled composition and nanostructure, thus this nanozyme will be selected to develop LFIA platform.
Fig. 2
Catalytic kinetics and ROS analysis of the three nanozymes. Michaelis-Menten curves of (A) S-MnO2, (B) Mn2O3 and (C) S-Mn2O3 at varying concentrations of TMB. The Lineweaver-Burk plots of (D) S-MnO2, (E) Mn2O3 and (F) S-Mn2O3 calculated from the corresponding Michaelis − Menten curves. (G) ESR spectra identifying ROS (1O2, ·OH and O2.−) generated during S-Mn2O3 catalysis at pH 4.0. ESR spectra comparing the generation capacities of (H) O2.− and (I) ·OH among S-MnO2, Mn2O3, and S-Mn2O3.
Water Dispersibility and Stability of CS-Mn2O3
Good water dispersibility and stability of label materials are critical for accurate and reliable detection of target analytes in LFIA [5]. To improve the water dispersibility of S-Mn2O3, a two-step surface functionalization strategy was employed to introduce carboxyl groups [29]. As illustrated in Fig. 3A, the silica surface of S-Mn2O3 was first treated with APTES to introduce amino groups, followed by reacting with SA to generate carboxyl-functionalized S-Mn2O3 (CS-Mn2O3). The FTIR spectra of S-Mn2O3 and CS-Mn2O3 (Fig. 3B) were recorded in the range of 4000–550 cm− 1. For S-Mn2O3, prominent peaks at 1062–1054 cm− 1 and 804–799 cm− 1 are attributed to asymmetrical and symmetrical stretching vibration of Si–O–Si, respectively [40], while peaks at 672 cm− 1 and 579 cm− 1 correspond to Mn–O stretching modes [41, 42]. CS-Mn2O3 exhibits new peaks at 2919 cm− 1 and 2850 cm− 1 (C–H stretching vibration) and characteristic carboxyl group signals at 1723 cm− 1 (C = O stretching vibration), 1641 cm− 1 (C–O deformation) and 1409 cm− 1 (O–H in-plane deformation vibration) [40]. Additionally, the characteristic peak at 1557 cm− 1 associated with C–N stretching and N–H deformation vibration [29] suggests the formation of amide bonds during the sequential functionalization process, indicating successful carboxyl group modification.
Zeta potential measurements (Fig. 3C) were further conducted to characterize the modification process. The surface charge shifted from − 24.32 ± 0.40 mV (S-Mn2O3) to + 24.69 ± 1.39 mV after amine functionalization (denoted as AS-Mn2O3) and finally to − 27.83 ± 0.48 mV for CS-Mn2O3, consistent with the sequential grafting of amino and carboxyl groups. The zeta potential of CS-Mn2O3 ( < − 25 mV) indicates strong colloidal stability [43]. DLS analysis (Fig. 3D) indicates a hydrodynamic size of 221.3 ± 6.7 nm and a polydispersity index (PDI) of 0.11 ± 0.01 for CS-Mn2O3, reflecting excellent monodispersity. The pronounced Tyndall effect observed in the inset of Fig. 3D further shows the colloidal stability of CS-Mn2O3 in aqueous solution.
The structural integrity of CS-Mn2O3 was also assessed by XRD. As shown in Fig. 3E, the Mn2O3 crystalline phase remains intact after carboxyl group modification. Given the susceptibility of Mn(III) to disproportionation in aqueous environments, long-term stability of CS-Mn2O3 was assessed by storing it in PBS for six months. Post-storage XRD patterns (Fig. 3E) detected little change in the crystalline structure, indicating robust structural stability. After carboxyl group modification, the OXD-like activity of S-Mn2O3 slightly decreased (Fig. 3F). This phenomenon may be attributed to the occupation of partial active sites by the carboxyl functional groups. Additionally, the OXD-like activity of CS-Mn2O3 is largely retained, as evidenced by comparable Kₘ values for fresh (0.237 mM) and stored (0.251 mM) samples (Fig. 3G). This stability surpasses that of natural OXD enzymes [12], highlighting the material’s promising potential in bioanalytical applications.
Fig. 3
Water Dispersibility and Stability of CS-Mn2O3. (A) Schematic illustration of the surface functionalization process from S-Mn2O3 to CS-Mn2O3 for LFIA application. (B) FTIR spectra of S-Mn2O3 and CS-Mn2O3. (C) Zeta potential measurements of S-Mn2O3, AS-Mn2O3 and CS-Mn2O3. (D) Hydrodynamic diameter distribution of CS-Mn2O3 in DI water, with the inset displaying the Tyndall effect shown by the CS-Mn2O3 dispersion. (E) XRD patterns of CS-Mn2O3 before and after storage in PBS for 6 months. The Lineweaver-Burk plots of (F) CS-Mn2O3 and (G) CS-Mn2O3 after 6 months of storage in PBS.
Analytical Performance of I-CS-Mn2O3
CS-Mn2O3 with excellent OXD-like activity and excellent aqueous dispersibility, serves as an ideal labeling material for developing cost-effective and sensitive LFIA platforms. To evaluate its efficacy as an LFIA label, CS-Mn2O3 was conjugated with anti-CRP 2nd mAbs to form I-CS-Mn2O3, then applied for CRP detection (Scheme 1). The sensitive and accurate on-site detection of CRP is critical for the rapid diagnosis and monitoring of inflammatory conditions, guiding treatment decisions, and assessing cardiovascular risk [44]. The assay was conducted by dropping 100 µL of a premixed solution containing the CRP standard and I-CS-Mn2O3 onto the sample pad. Capillary forces [45] drove the migration of the CRP/I-CS-Mn2O3 immunocomplex along the strip, where it was captured by pre-immobilized 1st mAbs on the test line (T line), forming a gray band via specific antigen–antibody interactions. Excess I-CS-Mn2O3 migrated to the control line (C line), where it was captured by pre-immobilized GAM, producing a second gray band to confirm test validity. Subsequently, a TMB substrate solution was applied to the NC membrane. The OXD-like activity of I-CS-Mn2O3 catalyzed the oxidation of TMB, converting it from colorless into blue ox-TMB, thereby significantly enhancing signal visibility by transforming the faint gray color of nanoparticles to a vivid blue color. LFIA strip images were photographed, and the intensity ratio of the T line to the C line (IT/IC) was quantified.
To optimize assay performance and minimize false signals, several critical parameters were systematically optimized (Fig. S11). These parameters included the ratio of 2nd mAb to CS-Mn2O3, the quantity of I-CS-Mn2O3, the catalytic reaction time, and the concentration of TMB. The optimal conditions were determined to be a 2:1 ratio of 2nd mAb to CS-Mn2O3, 5 µg of I-CS-Mn2O3, a catalytic reaction time of 30 seconds, and a TMB solution of 10 mM. The sensitivity of the I-CS-Mn2O3-based LFIA was determined across a range of CRP concentrations. Figure 4A displays the LFIA strips exhibiting gray bands due to the intrinsic colorimetric signals of the label, with T line intensity increasing proportionally to CRP concentration. To improve sensitivity, a post-amplification step was introduced by adding TMB, which resulted in enhanced blue colorimetric signals, as depicted in Fig. 4B. The correlation between IT/IC ratio and CRP concentration is presented in Fig. 4C, demonstrating a strong linear relationship (R2 = 0.998) over the CRP range of 25 − 1000 ng mL− 1, with a LoD of 25 ng mL− 1 (inset of Fig. 4C). Figure 4D demonstrates the IT/IC ratio following amplification as a function of CRP concentration, extending the visible detection range to 10 − 1000 ng mL− 1 (R2 = 0.991) and achieving a lower LoD of 0.25ng mL− 1 (inset of Fig. 4D).
To assess the specificity of the assay, several interference proteins such as CEA, AFP, PSA, and HSA were tested. As revealed in Fig. 4E, these proteins do not generate detectable signals, whereas a low CRP concentration (5 ng mL− 1) generates a robust signal, even in the presence of mixed interference proteins, suggesting the high specificity of the colorimetric-catalytic LFIA for CRP detection. Compared with reported CRP detection methods (Fig. 4F), our approach exhibits enhanced sensitivity.
Fig. 4
Colorimetric I-CS-Mn2O3-labelled LFIA test strips for CRP detection. Photographs of LFIA test strips for CRP detection at concentrations of 0–1000 ng mL− 1 under (A) unamplified and (B) amplified detection modes. Calibration curves correlate the colorimetric signal intensity ratio of test line to control line (IT/IC) with CRP concentration for (C) unamplified and (D) amplified LFIA systems. Insets in (C) and (D) show linear response ranges of 25–1000 ng mL− 1 (unamplified LFIA) and 10–1000 ng mL− 1 (amplified LFIA) in human serum, respectively. Error bars indicate the standard errors of three independent experiments. (E) Specificity evaluation of the I-SC-Mn2O3-labelled LFIA system against potential interferents (cross-reactive proteins at 1 µg mL− 1). (F) Comparison of CRP detection performance across different detection methods: enzyme-linked immunosorbent assay (ELISA) [46], Au@Pd NP-based LFIA [27]; Gold nanorod (AuNR)-based LFIA [47], electrochemical sensor [48], and quantum dot (QD)-based LFIA [49].
Conclusion
In summary, this study successfully developed a Mn2O3-based colorimetric LFIA platform that achieves rapid and sensitive CRP detection without using H2O2. By controlling the composition and nanostructure, the S-Mn2O3 nanozymes demonstrate enhanced OXD-mimicking activity. Our developed LFIA achieved a LoD of 0.25 ng mL− 1 for CRP, surpassing that of previously reported LFIAs [27, 28]. Our strategy sheds insights in the development of cost-effective and highly sensitive POCT tools, which holds significant promises for early disease detection and monitoring.
Supplementary Information
The online version contains supplementary material available at
Authorship contributions
Jiaqi Fang: Conceptualization, Data curation, Formal analysis, Methodology, Investigation, Validation, Visualization, Writing–original draft. Fang Gao: Conceptualization, Formal analysis, Writing–review & editing. Ye Zhang: Data curation, Methodology, Investigation. Yining Yao: Data curation, Methodology, Investigation, Software. Shaonian Ye: Data curation, Investigation, Software. Chao Liu: Conceptualization, Supervision, Project administration, Funding acquisition. Chengzhong Yu: Conceptualization, Supervision, Project administration, Funding acquisition, Writing-review & editing.
A
Acknowledgement
This work is supported by the Multifunctional Platform for Innovation (004), East China Normal University and the Center for Instrumental Analysis, University of Shanghai for Science and Technology.
A
Funding
This work was supported by the National Natural Science Foundation of China (NSFC 21905092, 22075085, and 22475072), Natural Science Foundation of Shanghai Municipality (25ZR1402379), and the Fundamental Research Funds for the Central Universities.
Data availability
No datasets were generated or analyzed during the current study.
Declarations
Competing interests
The authors declare no competing interests
Electronic Supplementary Material
Below is the link to the electronic supplementary material
A
Author Contribution
Jiaqi Fang: Conceptualization, Data curation, Formal analysis, Methodology, Investigation, Validation, Visualization, Writing–original draft. Fang Gao: Conceptualization, Formal analysis, Writing–review & editing. Ye Zhang: Data curation, Methodology, Investigation. Yining Yao: Data curation, Methodology, Investigation, Software. Shaonian Ye: Data curation, Investigation, Software. Chao Liu: Conceptualization, Supervision, Project administration, Funding acquisition. Chengzhong Yu: Conceptualization, Supervision, Project administration, Funding acquisition, Writing-review & editing.
References
1.
Ye H, Liu Y, Zhan L, Liu Y, Qin Z (2020) Signal amplification and quantification on lateral flow assays by laser excitation of plasmonic nanomaterials. Theranostics 10:4359–4373. https://doi.org/10.7150/thno.44298
2.
Land KJ, Boeras DI, Chen X-S, Ramsay AR, Peeling RW (2018) REASSURED diagnostics to inform disease control strategies, strengthen health systems and improve patient outcomes. Nat Microbiol 4:46–54. https://doi.org/10.1038/s41564-018-0295-3
3.
Truong PL, Yin Y, Lee D, Ko SH (2023) Advancement in COVID-19 detection using nanomaterial‐based biosensors. Exploration 3:20210232. https://doi.org/10.1002/exp.20210232
4.
Chen R, Chen X, Zhou Y, Lin T, Leng Y, Huang X, Xiong Y (2022) Three-in-One Multifunctional Nanohybrids with Colorimetric Magnetic Catalytic Activities to Enhance Immunochromatographic Diagnosis. ACS Nano 16:3351–3361. https://doi.org/10.1021/acsnano.2c00008
5.
Gao F, Liu Y, Lei C, Liu C, Song H, Gu Z, Jiang P, Jing S, Wan J, Yu C (2021) The Role of Dendritic Mesoporous Silica Nanoparticles’ Size for Quantum Dots Enrichment and Lateral Flow Immunoassay Performance. Small Methods 5:2000924. https://doi.org/10.1002/smtd.202000924
6.
Hu H, Zhang G, Liu J, Liu D, Deng S, Peng J, Lai W (2024) Development of High-Performance and Multifunctional Nanoparticles Powered the Integrated Diagnosis and Treatment of Escherichia coli O157:H7. Anal Chem 96:5205–5214. https://doi.org/10.1021/acs.analchem.3c05519
7.
Sánchez-Purrà M, Carré-Camps M, de Puig H, Bosch I, Gehrke L, Hamad-Schifferli K (2017) Surface-Enhanced Raman Spectroscopy-Based Sandwich Immunoassays for Multiplexed Detection of Zika and Dengue Viral Biomarkers. ACS Infect Dis 3:767–776. https://doi.org/10.1021/acsinfecdis.7b00110
8.
Meng X, Zuo W, Wu P, Song Y, Yang G-J, Zhang S, Yang J, Zou X, Wei W, Zhang D et al (2024) Bimetallic Nanozyme: A Credible Tag for In Situ-Catalyzed Reporter Deposition in the Lateral Flow Immunoassay for Ultrasensitive Cancer Diagnosis. Nano Lett 24:51–60. https://doi.org/10.1021/acs.nanolett.3c03118
9.
Zhou Y, Ding L, Wu Y, Huang X, Lai W, Xiong Y (2019) Emerging strategies to develop sensitive AuNP-based ICTS nanosensors. TrAC. Trends Anal Chem 112:147–160. https://doi.org/10.1016/j.trac.2019.01.006
10.
Wang Z, Zhang R, Yan X, Fan K (2020) Structure and activity of nanozymes: Inspirations for de novo design of nanozymes. Mater Today 41:81–119. https://doi.org/10.1016/j.mattod.2020.08.020
11.
Loynachan CN, Thomas MR, Gray ER, Richards DA, Kim J, Miller BS, Brookes JC, Agarwal S, Chudasama V, McKendry RA, Stevens MM (2018) Platinum Nanocatalyst Amplification: Redefining the Gold Standard for Lateral Flow Immunoassays with Ultrabroad Dynamic Range. ACS Nano 12:279–288. https://doi.org/10.1021/acsnano.7b06229
12.
Huang Y, Ren J, Qu X (2019) Nanozymes: Classification, Catalytic Mechanisms, Activity Regulation, and Applications. Chem Rev 119:4357–4412. https://doi.org/10.1021/acs.chemrev.8b00672
13.
Huang L, Niu Y, Li R, Liu H, Wang Y, Xu G, Li Y, Xu Y (2019) VOx Quantum Dots with Multienzyme-Mimic Activities and the Application in Constructing a Three-Dimensional (3D) Coordinate System for Accurate Discrimination of the Hydrogen Peroxide over a Broad Concentration Range. Anal Chem 91:5753–5761. https://doi.org/10.1021/acs.analchem.8b05923
14.
Wu J, Wang X, Wang Q, Lou Z, Li S, Zhu Y, Qin L, Wei H (2019) Nanomaterials with enzyme-like characteristics (nanozymes): next-generation artificial enzymes (II). Chem Soc Rev 48:1004–1076. https://doi.org/10.1039/c8cs00457a
15.
Li H, Peng Y, Huang X, Wan R, Zhang L, Wang X, Han L, Li L, Wang C, Chen J (2024) Advances in design and preparation of nanozymes and their applications for constructing higher sensitive lateral flow assays. Coord Chem Rev 510:215797. https://doi.org/10.1016/j.ccr.2024.215797
16.
Cai X, Liang M, Ma F, Zhang Z, Tang X, Jiang J, Guo C, Ramzy Mohamed S, Abdel Goda A, Dawood DH et al (2022) Nanozyme-strip based on MnO2 nanosheets as a catalytic label for multi-scale detection of aflatoxin B1 with an ultrabroad working range. Food Chem 377:131965. https://doi.org/10.1016/j.foodchem.2021.131965
17.
Dong T, Zhang X, Yuan J, Lin Z, Yin P, Yu H, Wang M, Liu A (2023) Sensitive Lateral Flow Immunoassay Based on Specific Peptide and Superior Oxidase Mimics with a Universal Dual-Mode Significant Signal Amplification. Anal Chem 95:12532–12540. https://doi.org/10.1021/acs.analchem.3c02821
18.
Tang M, Zhang Z, Sun T, Li B, Wu Z (2022) Manganese-Based Nanozymes: Preparation, Catalytic Mechanisms, and Biomedical Applications. Adv Healthc Mater 11:2201733. https://doi.org/10.1002/adhm.202201733
19.
Jiang X, Gray P, Patel M, Zheng J, Yin J-J (2020) Crossover between anti- and pro-oxidant activities of different manganese oxide nanoparticles and their biological implications. J Mater Chem B 8:1191–1201. https://doi.org/10.1039/c9tb02524c
20.
Chen ZJ, Huang Z, Sun YM, Xu ZL, Liu J (2021) The Most Active Oxidase-Mimicking Mn2O3 Nanozyme for Biosensor Signal Generation. Chem – Eur J 27:9597–9604. https://doi.org/10.1002/chem.202100567
21.
Gong H, Gai S, Tao Y, Du Y, Wang Q, Ansari AA, Ding H, Wang Q, Yang P (2024) Colorimetric and Photothermal Dual-Modal Switching Lateral Flow Immunoassay Based on a Forced Dispersion Prussian Blue Nanocomposite for the Sensitive Detection of Prostate-Specific Antigen. Anal Chem 96:8665–8673. https://doi.org/10.1021/acs.analchem.4c00862
22.
Gao F, Lei C, Liu Y, Song H, Kong Y, Wan J, Yu C (2021) Rational Design of Dendritic Mesoporous Silica Nanoparticles’ Surface Chemistry for Quantum Dot Enrichment and an Ultrasensitive Lateral Flow Immunoassay. ACS Appl Mater Interfaces 13:21507–21515. https://doi.org/10.1021/acsami.1c02149
23.
Gao F, Liu C, Yao Y, Lei C, Li S, Yuan L, Song H, Yang Y, Wan J, Yu C (2022) Quantum dots’ size matters for balancing their quantity and quality in label materials to improve lateral flow immunoassay performance for C-reactive protein determination. Biosens Bioelectron 199:113892. https://doi.org/10.1016/j.bios.2021.113892
24.
Gao F, Ye S, Huang L, Gu Z (2024) A nanoparticle-assisted signal-enhancement technique for lateral flow immunoassays. J Mater Chem B 12:6735–6756. https://doi.org/10.1039/d4tb00865k
25.
Zhuang H, Xu C, Gao F, Li Y, Lei C, Yu C (2022) Recent Advances in Silica-Nanomaterial-Assisted Lateral Flow Assay. Bioengineering 9:266. https://doi.org/10.3390/bioengineering9070266
26.
Vashist SK, Venkatesh AG, Marion Schneider E, Beaudoin C, Luppa PB, Luong JHT (2016) Bioanalytical advances in assays for C-reactive protein. Biotechnol Adv 34:272–290. https://doi.org/10.1016/j.biotechadv.2015.12.010
27.
Yao N, Li X, Tian Y, Huang Z, Duan Y (2023) Core-shell Au@PdNPs based colorimetric enhanced lateral flow immunoassay for C-reactive protein detection. Sens Actuators B 379:133247. https://doi.org/10.1016/j.snb.2022.133247
28.
Kong DY, Heo NS, Kang JW, Lee JB, Kim HJ, Kim MI (2022) Nanoceria-based lateral flow immunoassay for hydrogen peroxide-free colorimetric biosensing for C-reactive protein. Anal Bioanal Chem 414:3257–3265. https://doi.org/10.1007/s00216-022-03877-z
29.
Zhang C, Li X, Jiang Z, Zhang Y, Wen T, Fang M, Tan X, Alsaedi A, Hayat T, Wang X Selective Immobilization of Highly Valent Radionuclides by Carboxyl Functionalized Mesoporous Silica Microspheres: Batch, XPS, and, Analyses EXAFS (2018) ACS Sustainable Chem Eng 6:15644–15652.https://doi.org/10.1021/acssuschemeng.8b04146
30.
Zhang Y, Yao Y, Xie F, Hu Wl, Zou Y, Zhao Q, Li S, Yang Y, Gu Z, Yu C (2024) Iron consumption strengthens anti-tumoral STING activation mediated by manganese-based nanoparticles. Nano Today 58:102446. https://doi.org/10.1016/j.nantod.2024.102446
31.
Chen N, Wang W, Ma Y, Chuai M, Zheng X, Wang M, Xu Y, Yuan Y, Sun J, Li K et al (2023) Aqueous Zinc–Chlorine Battery Modulated by a MnO2 Redox Adsorbent. Small Methods 8:2201553. https://doi.org/10.1002/smtd.202201553
32.
Long X, Zhang H, Cao X, Wang H, Shimokawa K, Chi H, Zhang C, Okamoto A, Li X (2022) Biogenic Mn2O3 via the redox of Shewanella oneidensis MR-1 for peroxymonosulfate advanced oxidation. Sep Purif Technol 303:122144. https://doi.org/10.1016/j.seppur.2022.122144
33.
Salavati-Niasari M, Mohandes F, Davar F, Saberyan K (2009) Fabrication of chain-like Mn2O3 nanostructures via thermal decomposition of manganese phthalate coordination polymers. Appl Surf Sci 256:1476–1480. https://doi.org/10.1016/j.apsusc.2009.09.006
34.
Dai L, Sun Q, Guo J, Cheng J, Xu X, Guo H, Li D, Chen L, Si P, Lou J, Ci L (2019) Mesoporous Mn2O3 rods as a highly efficient catalyst for Li-O2 battery. J Power Sources 435:226833. https://doi.org/10.1016/j.jpowsour.2019.226833
35.
Yang W, Zhu Y, You F, Yan L, Ma Y, Lu C, Gao P, Hao Q, Li W (2018) Insights into the surface-defect dependence of molecular oxygen activation over birnessite-type MnO2. Appl Catal B 233:184–193. https://doi.org/10.1016/j.apcatb.2018.03.107
36.
Liu YL, Zhao XJ, Yang XX, Li YF (2013) A nanosized metal–organic framework of Fe-MIL-88NH2 as a novel peroxidase mimic used for colorimetric detection of glucose. Analyst 138:4526–4531. https://doi.org/10.1039/c3an00560g
37.
Qiao W, He B, Yang J, Ren W, Zhao R, Zhang Y, Bai C, Suo Z, Xu Y, Wei M, Jin H (2024) Pt@AuNF nanozyme and horseradish peroxidase-based lateral flow immunoassay dual enzymes signal amplification strategy for sensitive detection of zearalenone. Int J Biol Macromol 254:127746. https://doi.org/10.1016/j.ijbiomac.2023.127746
38.
Wang Z, Shen X, Gao X (2021) Density Functional Theory Mechanistic Insight into the Peroxidase- and Oxidase-like Activities of Nanoceria. J Phys Chem C 125:23098–23104. https://doi.org/10.1021/acs.jpcc.1c04878
39.
Lu W, Chen J, Kong L, Zhu F, Feng Z, Zhan J (2021) Oxygen vacancies modulation Mn3O4 nanozyme with enhanced oxidase-mimicking performance for l-cysteine detection. Sens Actuators B. 333.https://doi.org/10.1016/j.snb.2021.129560
40.
Zhao J, Bu Dy, Zhang N, Tian Dn M, Ly Y, Hf (2021) Cytotoxicity of mesoporous silica modified by amino and carboxyl groups on vascular endothelial cells. Environ Toxicol 36:1422–1433. https://doi.org/10.1002/tox.23138
41.
Najjar R, Awad R, Abdel-Gaber AM (2019) Physical Properties of Mn2O3 Nanoparticles Synthesized by Co-precipitation Method at Different pH Values. J Supercond Novel Magn 32:885–892. https://doi.org/10.1007/s10948-018-4765-x
42.
Umar A, Jung I, Ibrahim AA, Akhtar MS, Kumar SA, Alhamami MAM, Almas T, Almehbad N, Baskoutas S (2024) Porous Mn2O3 nanorods-based electrode for high-performance electrochemical supercapacitor. J Energy Storage 81:110305. https://doi.org/10.1016/j.est.2023.110305
43.
Shnoudeh AJ, Hamad I, Abdo RW, Qadumii L, Jaber AY, Surchi HS, Alkelany SZ (2019) Synthesis, Characterization, and Applications of Metal Nanoparticles. In Biomaterials and Bionanotechnology. 527–612
44.
Huang S, Liu Z, Yan Y, Chen J, Yang R, Huang Q, Jin M, Shui L (2022) Triple signal-enhancing electrochemical aptasensor based on rhomboid dodecahedra carbonized-ZIF67 for ultrasensitive CRP detection. Biosens Bioelectron 207:114129. https://doi.org/10.1016/j.bios.2022.114129
45.
Mahmoudi T, de la Guardia M, Shirdel B, Mokhtarzadeh A, Baradaran B (2019) Recent advancements in structural improvements of lateral flow assays towards point-of-care testing. TrAC. Trends Anal Chem 116:13–30. https://doi.org/10.1016/j.trac.2019.04.016
46.
Vashist SK, Czilwik G, van Oordt T, von Stetten F, Zengerle R, Marion Schneider E, Luong JHT (2014) One-step kinetics-based immunoassay for the highly sensitive detection of C-reactive protein in less than 30min. Anal Biochem 456:32–37. https://doi.org/10.1016/j.ab.2014.04.004
47.
Pang R, Zhu Q, Wei J, Wang Y, Xu F, Meng X, Wang Z (2021) Development of a gold-nanorod-based lateral flow immunoassay for a fast and dual-modal detection of C-reactive protein in clinical plasma samples. RSC Adv 11:28388–28394. https://doi.org/10.1039/d1ra04404d
48.
Pinyorospathum C, Chaiyo S, Sae-ung P, Hoven VP, Damsongsang P, Siangproh W, Chailapakul O (2019) Disposable paper-based electrochemical sensor using thiol-terminated poly(2-methacryloyloxyethyl phosphorylcholine) for the label-free detection of C-reactive protein. Microchim Acta 186:472. https://doi.org/10.1007/s00604-019-3559-6
49.
Lv Y, Wang F, Li N, Wu R, Li J, Shen H, Li LS, Guo F (2019) Development of dual quantum dots-based fluorescence-linked immunosorbent assay for simultaneous detection on inflammation biomarkers. Sens Actuators B 301:127118. https://doi.org/10.1016/j.snb.2019.127118
Table of Content
This study optimized nanozyme catalytic performance by tuning material composition and nanostructure, constructing a H₂O₂-free colorimetric LFIA platform based on DMSN-confined Mn₂O₃. The platform achieved a CRP detection limit of 0.25 ng/mL, lower than those of previously reported nanozyme-based colorimetric LFIA methods.
Jiaqi Fang 1, Fang Gao 2* Ye Zhang 1, Yining Yao 3, Shaonian Ye 2, Chao Liu 1*, Chengzhong Yu 1 3*
The material composition and nanostructure of nanozymes matters for Improving Lateral Flow Immunoassay Performance
