Dual-Mode Photothermal and Colorimetric Immunosensor Based on Polydopamine@Prussian Blue Nanocomposite for Sensitive Detection of Benzocaine

Dual-Mode Photothermal and Colorimetric Immunosensor Based on Polydopamine@Prussian Blue Nanocomposite for Sensitive Detection of Benzocaine | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Dual-Mode Photothermal and Colorimetric Immunosensor Based on Polydopamine@Prussian Blue Nanocomposite for Sensitive Detection of Benzocaine Bao-Zhu Jia, Wen-Feng Zhang, Qing-Chun Yin, Xue-Ying Rui, Lin Luo, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7663415/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 07 Feb, 2026 Read the published version in Microchimica Acta → Version 1 posted 4 You are reading this latest preprint version Abstract A dual-mode immunosensor based on polydopamine@Prussian blue nanoparticles (PDA@PB NPs) was developed for rapid and sensitive detection of benzocaine (BZC) in aquatic food. PDA@PB NPs possess excellent peroxidase-like activity, photothermal conversion efficiency, and antibody-binding capacity, enabling simple, crosslinker-free antibody immobilization. Upon target recognition, the sensor produces a distinct blue color via TMB–H 2 O 2 oxidation and a robust near-infrared photothermal signal, allowing dual-mode quantitative analysis. Under optimized conditions, the sensor showed a linear range of 0.01–1000 ng/mL, with detection limits of 0.33 ng/mL (colorimetric) and 0.82 ng/mL (photothermal). The integration of colorimetric and photothermal outputs improves detection accuracy, reduces matrix interference, and eliminates the need for complex instrumentation. Validation with spiked real samples confirmed its reliability and applicability. This portable, cost-effective immunosensor offers a promising tool for on-site monitoring of BZC residues in aquatic products, contributing to enhanced food safety surveillance. Polydopamine Prussian Blue Nanozyme Benzocaine Dual-Mode Assay Photothermal Immunoassay Colorimetric Immunoassay Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 1. Introduction Ensuring food safety requires accurate and rapid monitoring of chemical residues, including veterinary drugs, pesticides, and anesthetics, that may remain in edible animal products. Among these contaminants, benzocaine (BZC), a widely used local anesthetic in aquaculture, is often applied to sedate fish during transportation and spawning operations [ 1 , 2 ]. However, excessive or unregulated use can lead to residues in fish tissues, posing health risks such as methemoglobinemia and allergic reactions in humans [ 3 ]. Consequently, sensitive and reliable analytical methods for BZC detection are essential for food safety control and regulatory supervision. Conventional enzyme-linked immunosorbent assays (ELISA) have become indispensable tools in food residue monitoring because of their high specificity, simplicity, and suitability for large-scale screening [ 4 – 7 ]. Nevertheless, typical colorimetric ELISA systems rely on natural enzymes such as horseradish peroxidase (HRP), which suffer from poor stability and single-signal output [ 8 , 9 ]. These limitations can result in false-positive or false-negative outcomes, especially in complex food matrices [ 10 ]. To overcome these challenges, researchers have incorporated nanomaterials into ELISA to enhance the sensitivity and reliability. In recent years, numerous nanozyme-based immunosensing platforms have been developed to enhance analytical reliability by integrating colorimetric and photothermal dual readouts. Wei et al. first established a Prussian Blue (PB)-based multicolor and photothermal dual-readout immunosensor for prostate specific antigen detection [ 11 ], demonstrating the feasibility of using PB nanozymes for visual and thermal quantification. Building upon this concept, Ding et al. constructed a black phosphorus–gold nanohybrid (BP/Au)-based dual-mode immunoassay for diethylstilbestrol determination [ 12 ], further confirming the effectiveness of combining peroxidase-like activity with photothermal effects. Subsequently, Huang et al. reported a Ti₃C₂Tₓ/AuNP-based colorimetric–photothermal immunosensor for zearalenone detection [ 13 ], achieving improved signal stability and sensitivity through MXene–metal synergy. Most recently, Gong et al. introduced a switchable colorimetric–photothermal lateral flow immunoassay that allowed flexible transition between the two detection modes, illustrating the versatility of dual-mode strategies for on-site immunoassays [ 14 ]. Collectively, these studies confirm that integrating colorimetric and photothermal outputs can significantly enhance the accuracy and adaptability of immunoassays. Nevertheless, most of these systems still require complex antibody crosslinking or involve nanomaterials with limited biocompatibility, underscoring the need for a facile and stable dual-mode sensing platform. Prussian Blue (PB), a classical iron-based nanozyme, is particularly attractive owing to its strong peroxidase-like activity and high photothermal efficiency under near-infrared (NIR) irradiation [ 15 , 16 ]. However, practical application is often hindered by limited antibody binding efficiency and aggregation during bioconjugation [ 17 ]. Polydopamine (PDA), inspired by mussel adhesive proteins, offers an elegant solution due to its excellent adhesion, biocompatibility, and ability to immobilize biomolecules without additional crosslinkers [ 18 ]. In recent years, PDA has found growing applications in biosensor development for the detection of hazardous substances, benefiting from its superior biocompatibility, adhesion, and functional versatility [ 19 ]. Integrating PDA with PB into a PDA@PB nanocomposite can therefore synergistically combine catalytic, photothermal, and bioconjugation properties—ideal for constructing novel dual-mode immunosensors. In this work, we synthesized PDA@PB nanocomposites through a facile in situ deposition of PB on Fe(III)-coordinated PDA nanospheres. The resulting materials exhibited strong peroxidase-like activity, efficient photothermal conversion, and stable antibody immobilization. A colorimetric–photothermal dual-mode immunosensor was thus developed for the sensitive detection of BZC residues. In this sensing system, the oxidation of 3,3′,5,5′-tetramethylbenzidine (TMB) by PDA@PB produced both a visible color change and a corresponding temperature rise under 808 nm laser irradiation, enabling dual quantitative readouts. This strategy not only ensures high sensitivity and accuracy, but also provides portable and visual detection, offering great potential for on-site monitoring of BZC residues in aquatic food safety surveillance. 2. Experimental 2.1 Materials and reagents Benzocaine, propofol, quinaldine, eugenol, phenoxyethanol, lidocaine and tricaine standards were purchased from Tan-Mo Technology Co., Ltd. (Beijing, China). Dopamine hydrochloride, 3,3’,5,5’-tetramethylbenzidine (TMB) were sourced from Macklin (Shanghai, China). FeCl 3 ·6H 2 O and potassium ferrocyanide were supplied by Aladdin Industrial Corporation (Shanghai, China). The antigen and monoclonal antibodies against benzocaine (anti-BZC mAbs) were prepared previously in our laboratory [ 20 ]. Unless otherwise noted, all chemicals employed were of analytical grade and used as received. Ultrapure water was used to prepare all aqueous solutions throughout the study. 2.2 Apparatus UV absorption spectra were acquired with a SpectraMax i3x multifunctional microplate reader (Molecular Devices, USA). Elemental composition and surface states were analyzed via XPS on an ESCALAB 250Xi spectrometer (Thermo Scientific, USA). TEM imaging was performed using a Tecnai G2 F20 instrument (FEI, USA). XRD patterns were recorded using a D8 Advance system (Bruker, Germany), while FT-IR spectra were collected with a Nicolet iS10 analyzer (Thermo Scientific, USA). ELISA plate washing was conducted using a Multiskan MK2 washer (Thermo Scientific, MA, USA). 2.3 Synthesis of PDA@PB nanocomposite Fe(III)-polydopamine (PDA-Fe) nanospheres were fabricated based on a modified literature method [ 21 ]. In brief, 60 mg of dopamine and 8.3 mg of FeCl 3 ·6H 2 O were fully solubilized in 170 mL of ultrapure water and stirred at room temperature for 1 h. Then, 30 mL of Tris buffer (22.5 mg/mL) was added, and the mixture was continuously stirred for an additional 2 h. The resulting PDA-Fe nanospheres were collected by centrifugation and thoroughly washed five times with ultrapure water. Finally, the precipitate was dispersed in 10 mL of ultrapure water for subsequent experiments. For PDA@PB NP preparation, 5 mL of 2 mM K 4 [Fe(CN) 6 ]and 5 mL of 0.6 M HCl were gradually added to the PDA-Fe suspension under constant stirring. After 20 min of reaction, Prussian blue formed on the PDA-Fe nanospheres. The resulting PDA@PB NPs were separated by centrifugation, washed thoroughly with deionized water (five times), and then freeze-dried to obtain PDA@PB NP powder. 2.4 Fabrication of antibody-conjugated PDA@PB NP immunoprobe The antibody-conjugated PDA@PB NP immunoprobe was synthesized via a straightforward mixing and adsorption approach [ 22 ]. Briefly, 0.2 mg of PDA@PB NP was dispersed in 1 mL phosphate-buffered saline (PBS, 0.01 M, pH 7.4). Subsequently, 20 µL of anti-BZC monoclonal antibody (1 mg mL − 1 )) was introduced, and the mixture was gently agitated at 4°C for 2 h. Following incubation, 100 µL of 10% BSA was added to block nonspecific adsorption sites. Afterward, the solution was centrifuged and rinsed with ultrapure water to remove unbound antibodies and excess BSA. The resulting PDA@PB@Ab immunoprobe was re-suspended in 1 mL of borate buffer containing BSA (BB-BSA, pH 6.4) for storage and subsequent use. The efficiency of antibody conjugation was quantified through indirect ELISA. In brief, 4, 8, 12, 16, 20, and 24 µg of anti-BZC mAb were each added to 1 mL of PDA-PB nanosphere suspension. After incubation and centrifugation, the supernatants were collected and diluted 4-fold to measure the remaining unbound antibodies. The coupling efficiency was determined using the equation: Coupling efficiency = [(total antibodies) – (supernatant antibodies)]/ (total antibodies) × 100% [ 23 ]. 2.5 Protocol of the dual-mode immunosensor The dual-mode immunoassay protocol is illustrated in Scheme 1 . Briefly, 100 µL of BZC-OVA (used as the coating antigen) in 0.05 M carbonate buffer (pH 9.5) was dispensed into individual wells of a 96-well plate and kept at 37 ℃ overnight for coating. Afterward, the wells were rinsed twice with PBST solution (0.01 M phosphate-buffered saline containing 0.05% Tween-20, pH 7.4). To block nonspecific adsorption, 120 µL of blocking solution was added, followed by incubation at 37 ℃ for 3 h. Finally, the plate was dried at 37 ℃ for 1 h. Next, 50 µL of either BZC standard solution or sample extract and 50 µL of PDA@PB NP@Ab immunoprobe were added to each well and incubated at 37 ℃ for 30 min. After five washes, 100 µL of TMB-H 2 O 2 substrate (v/v = 1:1) was introduced into each well, followed by a 10-min incubation at 37 ℃. For photothermal test, the microplate strips were placed into a custom-built 3D-printed holder (Fig. S1 ) and subjected to 808 nm near-infrared (NIR) laser exposure at 1.5 W/cm² for 150 s. Thermal responses were captured and tracked using a handheld NIR imaging camera. A calibration curve was then established by associating the change in temperature with the logarithmic concentration of BZC, enabling determination of its level in unknown samples. For colorimetric analysis, optical density at 652 nm was promptly detected via SpectraMax i3x multifunctional microplate reader. The antibody binding rate, expressed as the ratio B/B 0 , was determined, where B 0 denotes the signal without BZC and B corresponds to the signal in its presence. To quantify BZC, a calibration curve was established by fitting the log-transformed concentration values against the measured B/B 0 values using a sigmoidal four-parameter logistic fitting method. This fitted model was subsequently employed to calculate BZC content in tested samples. 2.6 Sample preparation Aquaculture water samples underwent filtration using a 0.22 µm membrane, followed by a 5-fold dilution with 0.01 M PBS buffer (pH 7.4) prior to analysis by the immunoassay. For shrimp and tilapia, 1.0 g of homogenized tissue was mixed with 1.0 mL acetonitrile and vigorously agitated for 2.5 minutes. The upper layer was then collected into a clean tube preloaded with a sorbent mixture (75 mg MgSO 4 , 30 mg PSA, 15 mg NaCl, and 30 mg C18), shaken for another 2 min, and subjected to centrifugation at 7000 rpm. The extract was subsequently diluted tenfold in PBS and used for subsequent detection by the immunoassay. 2.7 Method validation To verify the reliability of the immunosensor, fortified aquaculture water, shrimp, and carp samples at 5.0, 50.0, and 100.0 ng/g (or ng/mL for water) of BZC were analyzed by both the immunosensor and LC-MS/MS. Detailed instrumental parameters for LC-MS/MS are provided in the Supplementary Material (Fig. S2). The quantitative consistency of the immunosensor and LC-MS/MS was verified by regression analysis. 3. Results and discussion 3.1 Characterization of PDA@PB NP The synthesis of PDA@PB NPs involved two sequential steps. First, dopamine and Fe 3+ ions acted as building blocks for the formation of PDA-Fe nanospheres via a pre-polymerization doping strategy. Subsequently, surface-anchored Fe 3+ ions underwent in situ coordination with potassium ferrocyanide (K 4 [Fe(CN) 6 ]), yielding the final PDA@PB NP (Scheme 1 ). TEM imaging (Fig. S3) verified the uniform morphology of the PDA-Fe spheres with an average size of ~ 160 nm. As shown in Fig. S4, at acidic conditions, PB was effectively formed through the reaction between Fe 3+ ions and K 4 [Fe(CN) 6 ], characterized by a prominent absorption band around 710 nm. The optimal synthesis conditions for PDA@PB NP were identified when a mixture containing 2 mM K 4 [Fe(CN) 6 ]and 0.6 M HCl resulted in the highest absorbance, suggesting their suitability for PDA@PB NP formation. Under these optimized parameters, PB was evenly deposited on PDA-Fe spheres. Characterization was performed using TEM, FT-IR, XRD, and XPS. Figure 1 A and S3 illustrate that both PDA-Fe and PDA@PB NP maintained spherical shapes, whereas the latter showed a rougher surface texture. Elemental mapping and EDS spectra (Fig. S5 and Fig. 1 A) demonstrated that C, N, O, Fe, and K elements were evenly distributed throughout the PDA@PB NP. The FT-IR spectrum (Fig. 1 D) exhibited characteristic C ≡ N stretching bands at 2080 cm⁻¹, indicating successful integration of PB. XRD analysis (Fig. 1 C) showed that PDA-Fe exhibited an amorphous structure without noticeable diffraction signals [ 24 ]. In contrast, PDA@PB NP displayed peaks at 17.465°, 24.578°, 35.277°, 39.351°, 43.716°, 50.635°, 54.066°, and 57.491°, which were indexed to the (200), (220), (400), (420), (422), (440), (600), and (620) planes of PB (JCPDS #73–0687). XPS data (Fig. 1 D) further validated the composition, with strong signals from Fe, C, and N. High-resolution spectra (Fig. 1 E–G) provided detailed chemical state information. In the Fe 2p spectrum (Fig. 1 E), peaks at 708.4 and 721.5 eV indicated Fe²⁺ (2p₃/₂ and 2p₁/₂), while signals at 709.1, 712.6, and 725.1 eV reflected Fe³⁺ states. The C 1s spectrum (Fig. 1 F) exhibited binding energy peaks at 284.7, 285.6, and 288.5 eV, corresponding to C–C, C ≡ N, and C–N bonds. N 1s spectra (Fig. 1 G) showed signals at 397.6 and 399.5 eV, consistent with cyanide groups coordinated with Fe²⁺ and Fe 3+ within the PB lattice, in accordance with previous studies [ 25 , 26 ]. 3.2 Evaluation of the peroxidase-like performance of PDA@PB NP The property of the PDA@PB NP to mimic peroxidase activity was assessed using TMB as substrate. Figure 2 A reveals that a distinct signal at 652 nm—corresponding to oxidized TMB (ox-TMB)—was only generated when both the PDA@PB NP and H 2 O 2 coexisted, whereas negligible response was detected in the absence of either component. To comprehensively assess their catalytic capability, kinetic experiments were performed by varying the concentrations of TMB or H 2 O 2 . The reaction velocity data were fitted to the Michaelis-Menten model to extract the key kinetic constants, namely the maximum catalytic rate (Vmax) and the substrate affinity constant (Km). the Michaelis-Menten is as follow: V= \(\:\frac{{V}_{max}\:\left[S\right]}{{K}_{m}+\left[S\right]}\) The reaction velocity (V) corresponds to the initial rate of the colorimetric reaction, while [S]denotes the substrate concentration. Nonlinear regression of the data (Fig. 2 B-E) was employed to extract kinetic constants, confirming that the catalytic activity of PDA@PB NP aligns with Michaelis-Menten behavior. The calculated apparent Michaelis constants (Km) for TMB and hydrogen peroxide were 0.41 mM and 2.29 mM, respectively. Corresponding maximum velocities (Vmax) were determined to be 1.44×10 − 8 mM s − 1 for TMB and 5.91 × 10 − 8 mM s − 1 for H 2 O 2 . To further evaluate the catalytic performance of PDA@PB NPs, their apparent kinetic parameters (Km and Vmax) toward TMB and H₂O₂ were compared with those of other photothermal-active nanozymes reported in recent dual- or multi-mode sensing studies (Table S1 ). The Km (TMB) of PDA@PB NPs (0.41 mM) is close to or lower than that of most representative systems such as Cu–N/O single-atom, CoFe PBAs/WS₂, and Ti₃C₂Tₓ/AuNPs nanozymes (Table S1 ), indicating a strong affinity for the substrate. In addition, the moderate Km (H₂O₂) (2.29 mM) and relatively low Vmax values confirm efficient catalytic turnover under mild conditions. These results demonstrate that PDA@PB NPs possess high substrate affinity and catalytic efficiency, making them promising candidates for subsequent dual-mode immunoassay applications. To assess the application potential of PDA@PB NP in immunoassay, their stability under varying conditions was assessed. The effect of pH on the peroxidase-like activity was first evaluated by monitoring the TMB catalytic oxidation reaction initiated by H 2 O 2 over a pH range from 2.4 to 10.4. Fig. S6 illustrates that the PDA@PB NP displayed evident pH-dependent behavior, showing markedly increased catalytic performance in acidic environments. Notably, the highest activity was achieved at pH 6.4, and this condition was consequently chosen for the following tests. In addition, the storage stability of PDA@PB NP was evaluated to further verify their practical applicability. As shown in Fig. 2 F, over 87% of its initial catalytic performance was preserved after 20 days at room temperature, reflecting excellent storage stability. Overall, the findings confirm that PDA@PB NPs exhibit strong peroxidase-mimicking activity and favorable stability during storage underscoring their reliability and potential as promising artificial nanozymes for immunoassay applications. 3.3 Characterization of the photothermal performance of PDA@PB NP Prussian blue-based nanostructures have been previously identified as excellent agents for photothermal conversion. Meanwhile, oxidized TMB (ox-TMB) shows a prominent absorbance peak near 750 nm in the NIR region, supporting its role in light-to-heat transformation [ 27 , 28 ]. As presented in Fig. 2 G, when irradiated with an 808 nm NIR laser, different reaction mixtures exhibited varied thermal responses. A notable temperature elevation was observed with PDA@PB NPs alone, while the highest thermal response appeared when PDA@PB NP were combined with H 2 O 2 and TMB, likely due to the formation of oxidized TMB (ox-TMB). In comparison, negligible heat generation was detected in other control groups. This verify that PDA@PB NPs possess both enzyme-like catalytic performance and efficient photothermal capability. Additionally, temperature elevations were measured for dispersions containing PDA@PB NP at concentrations of 50, 125, 250, 500, and 750 µg/mL after 300 s of laser exposure. The thermal response showed a direct correlation with nanomaterial concentration, with the optimal irradiation time determined to be 150 s (Fig. 2 H). To further assess the photothermal durability, three successive irradiation cycles were performed (Fig. 2 I). The reproducible heating profiles over repeated cycles indicate strong thermal robustness of the synthesized PDA@PB NPs. These results highlight the suitability of PDA@PB NP for potential implementation in photothermal-based immunoassay platforms. 3.4 Evaluation of antibody-conjugated PDA@PB NP immunoprobe Mussel-inspired polydopamine exhibits strong adhesion to a wide range of organic and inorganic substrates, attributed to its high binding strength and rich surface chemistry [ 29 ]. In particular, the abundant quinone groups in PDA can react with amino groups on antibodies via Michael addition, forming stable covalent bonds. This conjugation strategy has been demonstrated to be more efficient and biocompatible than conventional chemical methods [ 30 ]. To verify the successful conjugation of anti-BZC monoclonal antibodies (Ab) to the PDA@PB NPs, changes in zeta potential and UV–Vis absorption spectra were analyzed. As shown in Fig. 3 A, the zeta potential of PDA@PB NP increased from − 14.67 mV to − 5.77 mV after antibody modification, indicating surface charge alteration due to Ab attachment. Moreover, a characteristic protein absorption peak at 280 nm was observed in the UV–Vis spectrum of the immunoprobe, which was absent in the spectrum of unmodified PDA@PB NP (Fig. 3 B), further confirming successful antibody-PDA@PB NP conjugation. The performance of the PDA@PB NP@Ab immunoprobe was evaluated in terms of catalytic activity and conjugation efficiency, with PB serving as a control. As shown in Fig. 3 C, after antibody conjugation and BSA blocking, the catalytic activity of PB NPs toward TMB decreased by 41.7%, likely due to the obstruction of catalytic sites. In contrast, PDA@PB NP exhibited a smaller reduction of 21.7%, suggesting superior biocompatibility and improved preservation of catalytic activity. Additionally, conjugation efficiencies were assessed across a range of antibody concentrations. PB NPs showed efficiencies ranging from 44.7% to 63.8%, which decreased as the antibody dosage increased. In comparison, PDA@PB NP demonstrated consistently higher efficiencies, ranging from 64.9% to 77.3% (Fig. 3 D). This enhancement can be attributed to the PDA layer, which facilitates strong and stable antibody conjugation through multiple interactions, including electrostatic attraction, Schiff base formation, and Michael addition [ 31 ]. 3.5 Performance of the immunosensor To achieve the optimal detection performance of the dual-mode immunosensor, a series of critical parameters were methodically investigated. These included the buffer type and its pH used for reconstituting the PDA@PB NP@Ab immunoprobe, the dosage of anti-BZC monoclonal antibodies employed during probe fabrication, as well as the concentrations of both the coating antigen and the antibody, alongside the incubation time required for effective antigen-antibody binding. As shown in Fig. S7, the assay yielded its best performance under the following optimized conditions: (1) borate buffer at pH 6.4 was adopted for probe dispersion; (2) 4.0 µg of anti-BZC monoclonal antibody was utilized for coupling with PDA@PB NP; (3) the working concentration of the antibody was adjusted to 1.0 µg/mL; (4) the antigen for coating was employed at 1.0 µg/mL A reaction time of 30 min was selected based on the maximum detection response. Under these optimized conditions, both colorimetric and thermal readouts were recorded across a gradient of BZC concentrations. Final solution temperatures were measured using a customized infrared imaging setup following 808 nm NIR laser irradiation (Fig. S1 ). As depicted in Fig. 4 A, the thermal output declined progressively with increased levels of BZC. The change in temperature (Y) exhibited good linear relationship with the logarithmic value of BZC concentration (X) in the range from 0.01 to 1000 ng/mL. The linear equation was Y = − 2.01X + 21.06. The detection limit (LOD) was 0.82 ng/mL (S/N = 3). In the colorimetric assay, the antibody binding rate B/B 0 exhibited a gradual decline as the BZC concentration increased (Fig. 4 B). A good linear relationship was identified between B/B 0 and the logarithmic value of BZC concentration, modeled by the equation: Y = − 0.011X + 0.68. The LOD of the colorimetric assay was 0.33/ng mL (S/N = 3). To evaluate specificity, several structurally or functionally related fish anesthetics—including procaine, propofol, quinaldine, eugenol, phenoxyethanol, lidocaine, and tricaine—were tested. As shown in Fig. 4 C, these analogs produced responses similar to the blank, markedly lower than the signal observed for BZC. These results demonstrate that the dual-mode immunoassay offers excellent selectivity for BZC detection. 3.6 Detection of BZC in real samples The analytical performance of the proposed dual-mode immunosensor was further verified by detecting benzocaine in spiked aquaculture water, fish, and shrimp samples. As shown in Table S2, the concentrations obtained by the colorimetric and photothermal modes were in close agreement with those determined by the reference LC–MS/MS method at spiking levels of 5.0, 50.0, and 100.0 ng/mL (or ng/g). The recoveries ranged from 81.3% to 98.5%, with relative standard deviations below 11.5%, indicating good accuracy and precision for complex matrices. To statistically assess the consistency between the two methods, paired t-tests were performed for each matrix type (Fig. 4 D–F). No significant difference (p > 0.05) was observed between the immunosensor results (both detection modes) and LC–MS/MS, confirming that the proposed assay provides quantitative accuracy comparable to the instrumental standard method. From a regulatory perspective, the European Commission Regulation (EU) No 37/2010 classifies benzocaine as “not applicable,” meaning that no maximum residue limit (MRL) is established for food-producing animals such as finfish. In contrast, the U.S. FDA has proposed an import tolerance limit of 50 ng/g for benzocaine residues in fish muscle [ 32 ]. This regulatory discrepancy highlights the need for sensitive analytical techniques capable of monitoring trace-level residues in aquatic products. Table 1 summarizes representative nanomaterial-based methods for benzocaine determination, with all linear ranges and LODs converted to mass-concentration units (ng/mL or ng/g) for direct comparison. Electrochemical sensors based on hollow carbon nanobowls [ 1 ] and boron-doped diamond electrodes [ 3 ] achieve good sensitivity (LOD = 0.66 and 8.25 ng/mL, respectively) but provide single-signal outputs, making them more susceptible to complex matrix interferences. A Fe₃O₄@polydopamine molecularly imprinted nanozyme [ 33 ] exhibits high selectivity (LOD 0.66 ng/mL) yet offers single-mode colorimetric output. The indirect SERRS–Ag NPs assay [ 34 ] allows rapid, broad-range measurement (100–10 4 ng/mL) but involves derivatization and requires specialized Raman equipment. Table 1 An overview on recently reported nanomaterial-based methods for the determination of benzocaine Method Nanomaterial Linear range (ng/mL or g) LOD (ng/mL or g) Sample type Reference Electrochem- ical Hollow carbon nanobowl modified electrode 1.65–330 0.66 Human saliva [ 1 ] Electrochem- ical Miniaturized boron-doped diamond electrode 16.5– 6.6×10 4 8.25 Pharmaceutical products, human urine [ 3 ] Colorimetric assay Fe₃O₄@polydopamine molecularly imprinted nanozyme 5.0–65 0.66 Ear drops, tap and Nile water [ 33 ] Indirect SERS (azo coupling) Ag nanoparticles 100 − 10000 78 Pharmaceutical gels, sprays, enemas [ 34 ] Dual-mode colorimetric– photothermal immunoassay PDA@PB nanocomposite 0.01–1000 3.3 (colorimetric) / 8.2 (photothermal) Water, fish, shrimp This work In contrast, the PDA@PB-based dual-mode colorimetric–photothermal immunosensor developed in this study combines the excellent specificity of immunoassays with simple optical detection and photothermal analysis, achieving a ultra-low detection limit of 3.3 ng/g for the colorimetric mode and 8.2 ng/g for the photothermal mode (obtained by multiplying the LOD for buffer solution with a dilution factor 10-fold involved in sample preparation). The method also demonstrated high recoveries and good reproducibility in spiked samples, confirming its suitability for routine food-safety surveillance. However, the two signal outputs in this work cannot be acquired simultaneously. Developing sensing apparatuses capable of detecting multiple signals in real time would be an effective strategy to achieve in situ detection and accelerate data processing in future studies. 4. Conclusion In this study, a multifunctional nanocomposite, PDA@PB NP, was successfully synthesized through a two-step process involving the formation of PDA-Fe nanospheres followed by in situ deposition of Prussian blue. Benefiting from the synergistic properties of polydopamine and Prussian blue, PDA@PB NP demonstrated excellent peroxidase-mimicking catalytic activity, robust photothermal conversion efficiency, and good stability. Moreover, the PDA shell enabled efficient and stable antibody conjugation, preserving enzymatic activity and enhancing conjugation efficiency, which facilitated the construction of a bifunctional immunoprobe. Based on this, a dual-mode immunosensor was developed for sensitive and selective detection of BZC. The assay showed high specificity and satisfactory recovery rates in real samples including fish, shrimp, and aquaculture water. Notably, the method exhibited good agreement with LC-MS/MS, demonstrating its reliability. In summary, the proposed PDA@PB NP-based dual-mode immunosensor offers a simple, sensitive, and robust strategy for trace-level detection of BZC, providing promising prospects for food safety monitoring and regulatory compliance in aquaculture product testing. However, the current reader cannot simultaneously acquire the two signal outputs, integrating real-time multi-signal detection in future sensing devices will further enhance its applicability for in situ monitoring and high-throughput analysis. Declarations Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Funding This work was supported by the Guangdong Basic and Applied Basic Research Foundation (2023A1515030011). Author Contribution Bao-Zhu Jia: Data curation, Formal analysis, Investigation, Methodology, Writing – original draft; Wen-Feng Zhang: Data curation, Investigation, Methodology; Qing-Chun Yin: Data curation, Investigation, Methodology; Xue-Ying Rui: Data curation, Methodology, Investigation. Lin Luo: Conceptualization, Writing – review & editing, Formal analysis, Funding acquisition; Zhen-Lin Xu: Investigation, Resources, Funding acquisition; Data Availability Data will be made available on request. References Vandervelde, E M, (2005). Enzyme‐linked immunoassay (ELISA): Its practical application to the diagnosis of hepatitis B. Journal of Medical Virology 3, 17-18. https://doi.org/10.1002/jmv.1890030105 Bolarinwa, I F, Orfila, C, Morgan, M R A, (2014). Development and Application of an Enzyme-Linked Immunosorbent Assay (ELISA) for the Quantification of Amygdalin, a Cyanogenic Glycoside, in Food. Journal of Agricultural and Food Chemistry 62, 6299-6305. https://doi.org/10.1021/jf501978d Liu, Z, Zhang, B, Sun, J, Yi, Y, Li, M, Du, D, Zhu, F, Luan, J, (2018). Highly efficient detection of salbutamol in environmental water samples by an enzyme immunoassay. Science of The Total Environment, 861-865. https://doi.org/https://doi.org/10.1016/j.scitotenv.2017.08.324 Charernchai, S, Chikae, M, Phan, T T, Wonsawat, W, Hirose, D, Takamura, Y, (2022). Automated Paper-Based Femtogram Sensing Device for Competitive Enzyme-Linked Immunosorbent Assay of Aflatoxin B1 Using Submicroliter Samples. Analytical Chemistry 94, 5099-5105. https://doi.org/10.1021/acs.analchem.1c05401 Li, Y, Wang, L, Cui, Z, Liu, S, Wang, S, Ren, J, Tian, Y, Shu, R, Luo, X, Liao, Y, Wang, J, Zhang, D, (2022). White peroxidase-mimicking nanozyme˗nanocarrier of enzyme labeled antibody to enhance catalytic performance and relieve color interference of immunoassay. Sensors and Actuators B: Chemical 364. https://doi.org/10.1016/j.snb.2022.131909 Wang, Y, Bu, T, Cao, Y, Wu, H, Xi, J, Feng, Q, Xuan, C, Wang, L, (2023). A Versatile PdRu Bimetallic Nanoenzyme-Integrated Enzyme-Linked Immunosorbent Assay for Highly Sensitive Escherichia coli O157:H7 Detection. Analytical Chemistry 95, 9237-9243. https://doi.org/10.1021/acs.analchem.3c00743 Yu, Z, Gong, H, Li, M, Tang, D, (2022). Hollow prussian blue nanozyme-richened liposome for artificial neural network-assisted multimodal colorimetric-photothermal immunoassay on smartphone. Biosensors and Bioelectronics 218. https://doi.org/10.1016/j.bios.2022.114751 Gao, Y, Zhou, Y, Chandrawati, R, (2019). Metal and Metal Oxide Nanoparticles to Enhance the Performance of Enzyme-Linked Immunosorbent Assay (ELISA). ACS Applied Nano Materials 3, 1-21. https://doi.org/10.1021/acsanm.9b02003 Song, M, Xing, J, Cai, H, Gao, X, Li, C, Liu, C, Li, X, Fu, X, Ding, S, Cheng, W, Chen, R, (2023). Pomegranate-Bionic Encapsulating Horseradish Peroxidase Using Dopamine Flexible Scaffold-Coated Multishell Porous ZIF-8 To Enhance Immunochromatographic Diagnosis. ACS Nano 17, 10748-10759. https://doi.org/10.1021/acsnano.3c02164 Chen, Y, Ma, J, Yin, X, Deng, Z, Liu, X, Yang, D, Zhao, L, Sun, J, Wang, J, Zhang, D, (2023). Joint-detection of Salmonella typhimurium and Escherichia coli O157:H7 by an immersible amplification dip-stick immunoassay. Biosensors and Bioelectronics 224, 115075. https://doi.org/https://doi.org/10.1016/j.bios.2023.115075 Zhao, Y, Wang, X, Pan, S, Hong, F, Lu, P, Hu, X, Jiang, F, Wu, L, Chen, Y, (2024). Bimetallic nanozyme–bioenzyme hybrid material-mediated ultrasensitive and automatic immunoassay for the detection of aflatoxin B1 in food. Biosensors and Bioelectronics 248. https://doi.org/10.1016/j.bios.2023.115992 Zhang, X, Wu, D, Wu, Y, Li, G, (2021). Bioinspired nanozyme for portable immunoassay of allergenic proteins based on A smartphone. Biosensors and Bioelectronics 172. https://doi.org/10.1016/j.bios.2020.112776 Dou, L, Bai, Y, Liu, M, Shao, S, Yang, H, Yu, X, Wen, K, Wang, Z, Shen, J, Yu, W, (2022). ‘Three-To-One’ multi-functional nanocomposite-based lateral flow immunoassay for label-free and dual-readout detection of pathogenic bacteria. Biosensors and Bioelectronics 204. https://doi.org/10.1016/j.bios.2022.114093 Wu, H, Bu, T, Cao, Y, Wang, Y, Xi, J, Li, M, Li, R, Jia, P, Wang, L, (2023). Double-Enzyme Active Vanadium Nanospheres-Mediated Ratiometric Multicolor Immunosensors for Sensitive Detection of the T-2 Toxin. Analytical Chemistry 95(12), 5275-5284. https://doi.org/10.1021/acs.analchem.2c05197 Liu, Z, Xianyu, Y, Zheng, W, Zhang, J, Luo, Y, Chen, Y, Dong, M, Wu, J, Jiang, X, (2018). T-1-Mediated Nanosensor for Immunoassay Based on an Activatable MnO2 Nanoassembly. Analytical chemistry 90, 2765-2771. https://doi.org/10.1021/acs.analchem.7b04817 Zhu, X, Shan, J, Dai, L, Shi, F, Wang, J, Wang, H, Li, Y, Wu, D, Ma, H, Wei, Q, Ju, H, (2023). PB@PDA nanocomposites as nanolabels and signal reporters for separate-type cathodic photoelectrochemical immunosensors in the detection of carcinoembryonic antigens. Talanta 254. https://doi.org/10.1016/j.talanta.2022.124134 Liu, S, Dou, L, Yao, X, Zhang, W, Zhao, B, Wang, Z, Ji, Y, Sun, J, Xu, B, Zhang, D, Wang, J, (2020). Polydopamine nanospheres as high-affinity signal tag towards lateral flow immunoassay for sensitive furazolidone detection. Food Chemistry 315. https://doi.org/10.1016/j.foodchem.2020.126310 Lai, X, Zhang, G, Zeng, L, Xiao, X, Peng, J, Guo, P, Zhang, W, Lai, W, (2020). Synthesis of PDA-Mediated Magnetic Bimetallic Nanozyme and Its Application in Immunochromatographic Assay. ACS Applied Materials & Interfaces 13, 1413-1423. https://doi.org/10.1021/acsami.0c17957 Su, L, Chen, Y, Wang, L, Zhang, H, Sun, J, Wang, J, Zhang, D, (2021). Dual-signal based immunoassay for colorimetric and photothermal detection of furazolidone. Sensors and Actuators B: Chemical 331. https://doi.org/10.1016/j.snb.2020.129431 Lu, D, Jiang, H, Zhang, G, Luo, Q, Zhao, Q, Shi, X, (2021). An In Situ Generated Prussian Blue Nanoparticle-Mediated Multimode Nanozyme-Linked Immunosorbent Assay for the Detection of Aflatoxin B1. ACS Applied Materials & Interfaces 13, 25738-25747. https://doi.org/10.1021/acsami.1c04751 Guo, L, Zhang, Y-J, Yu, Y-L, Wang, J-H, (2020). In Situ Generation of Prussian Blue by MIL-53 (Fe) for Point-of-Care Testing of Butyrylcholinesterase Activity Using a Portable High-Throughput Photothermal Device. Analytical Chemistry 92, 14806-14813. https://doi.org/10.1021/acs.analchem.0c03575 Fu, G, Sanjay, S T, Zhou, W, Brekken, R A, Kirken, R A, Li, X, (2018). Exploration of Nanoparticle-Mediated Photothermal Effect of TMB-H 2 O 2 Colorimetric System and Its Application in a Visual Quantitative Photothermal Immunoassay. Analytical Chemistry 90, 5930-5937. https://doi.org/10.1021/acs.analchem.8b00842 Wang, J, Xu, X, Li, Z, Li, M, Qiu, B, (2024). Sensitive electrochemical detection of benzocaine based on hollow carbon nanobowl modified electrode. Journal of Electroanalytical Chemistry 952. https://doi.org/10.1016/j.jelechem.2023.117893 Uehara, S A, Andrade, D R, Takata, R, Gomes Júnior, A V, Vidal, M V, (2019). The effectiveness of tricaine, benzocaine, clove oil, and menthol as anesthetics for lambari-bocarra Oligosarcus argenteus. Aquaculture 502, 326-331. https://doi.org/10.1016/j.aquaculture.2018.12.054 Pysarevska, S, Dubenska, L, Plotycya, S, Švorc, Ľ, (2018). A state-of-the-art approach for facile and reliable determination of benzocaine in pharmaceuticals and biological samples based on the use of miniaturized boron-doped diamond electrochemical sensor. Sensors and Actuators B: Chemical 270, 9-17. https://doi.org/https://doi.org/10.1016/j.snb.2018.05.012 Lin, S-Q, Jia, B-Z, Luo, W, Wang, H, Lei, H-T, Zhang, W-F, Xu, Z-L, Luo, L, (2023). Controllable formation of polydopamine on carbon dots for ultrasensitive detection of alkaline phosphatase and ratiometric fluorescence immunoassay of benzocaine. Food Chemistry 426. https://doi.org/10.1016/j.foodchem.2023.136582 Li, Y, Xie, Y, Wang, Z, Zang, N, Carniato, F, Huang, Y, Andolina, C M, Parent, L R, Ditri, T B, Walter, E D, Botta, M, Rinehart, J D, Gianneschi, N C, (2016). Structure and Function of Iron-Loaded Synthetic Melanin. ACS Nano 10, 10186-10194. https://doi.org/10.1021/acsnano.6b05502 Chen, Y, Ren, J, Yin, X, Li, Y, Shu, R, Wang, J, Zhang, D, (2022). Vanadium Disulfide Nanosheet Boosts Optical Signal Brightness as a Superior Enzyme Label to Improve the Sensitivity of Lateral Flow Immunoassay. Analytical Chemistry 94, 8693-8703. https://doi.org/10.1021/acs.analchem.2c01008 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 Ma, H, He, Y, Liu, H, Xu, L, Li, J, Huang, M, Wei, Y, (2019). Anchoring of Prussian blue nanoparticles on polydopamine nanospheres as an efficient peroxidase mimetic for colorimetric sensing. Colloids and Surfaces A: Physicochemical and Engineering Aspects 577, 622-629. https://doi.org/https://doi.org/10.1016/j.colsurfa.2019.06.035 Zhou, L, Chen, J, Li, R, Wei, L, Xiong, H, Wang, C, Chai, K, Chen, M, Zhu, Z, Yao, T, Lin, Y, Dong, C, Shi, S, (2021). Metal‐Polyphenol‐Network Coated Prussian Blue Nanoparticles for Synergistic Ferroptosis and Apoptosis via Triggered GPX4 Inhibition and Concurrent In Situ Bleomycin Toxification. Small 17. https://doi.org/10.1002/smll.202103919 Liang, H, Liu, Y, Qileng, A, Shen, H, Liu, W, Xu, Z, Liu, Y, (2023). PEI-coated Prussian blue nanocubes as pH-Switchable nanozyme: Broad-pH-responsive immunoassay for illegal additive. Biosensors and Bioelectronics 219. https://doi.org/10.1016/j.bios.2022.114797 Hao, W, Huang, Y, Wang, L, Liang, J, Yang, S, Su, L, Zhang, X, (2023). Smartphone-Based Photothermal Lateral Flow Immunoassay Using Rhenium Diselenide Nanosheet. ACS Applied Materials & Interfaces 15, 9665-9674. https://doi.org/10.1021/acsami.2c22616 Zhang, G, Hu, H, Deng, S, Xiao, X, Xiong, Y, Peng, J, Lai, W, (2023). An integrated colorimetric and photothermal lateral flow immunoassay based on bimetallic Ag–Au urchin-like hollow structures for the sensitive detection of E. coli O157:H7. Biosensors and Bioelectronics 225. https://doi.org/10.1016/j.bios.2023.115090 Lee, H, Dellatore, S M, Miller, W M, Messersmith, P B, (2007). Mussel-Inspired Surface Chemistry for Multifunctional Coatings. Science 318, 426-430. https://doi.org/doi:10.1126/science.1147241 Wang, Z, Xing, K, Ding, N, Wang, S, Zhang, G, Lai, W, (2022). Lateral flow immunoassay based on dual spectral-overlapped fluorescence quenching of polydopamine nanospheres for sensitive detection of sulfamethazine. Journal of hazardous materials 423, 127204. https://doi.org/10.1016/j.jhazmat.2021.127204 Meng, Y, Liu, P, Zhou, W, Ding, J, Liu, J, (2018). Bioorthogonal DNA Adsorption on Polydopamine Nanoparticles Mediated by Metal Coordination for Highly Robust Sensing in Serum and Living Cells. ACS nano 12, 9070-9080. https://doi.org/10.1021/acsnano.8b03019 Yan, X, Song, Y, Wu, X, Zhu, C, Su, X, Du, D, Lin, Y, (2017). Oxidase-mimicking activity of ultrathin MnO 2 nanosheets in colorimetric assay of acetylcholinesterase activity. Nanoscale 9, 2317-2323. https://doi.org/10.1039/C6NR08473G ACD Pharmaceuticals, AS (2017) Environmental Assessment in Support of an Import Tolerance for Benzocaine in Food Derived from Atlantic Salmon and Rainbow Trout. ACD Pharmaceuticals. https://www.fda.gov/files/animal%20%26%20veterinary/published/Environmental-Assessment-for-Benzocaine-Import-Tolerance.pdf. Accessed 19 September 2025. Additional Declarations No competing interests reported. Supplementary Files Graphical.tif lSupplementaryMaterials.docx Appendix A. Supplementary Materials Supplementary Materials associated with this article can be found, in the online version, at http://dx.doi.org/xxx. Cite Share Download PDF Status: Published Journal Publication published 07 Feb, 2026 Read the published version in Microchimica Acta → Version 1 posted Editorial decision: Revision requested 27 Sep, 2025 Editor assigned by journal 23 Sep, 2025 Submission checks completed at journal 22 Sep, 2025 First submitted to journal 20 Sep, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7663415","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":521586725,"identity":"83fd9f7c-a529-4c99-8938-78dbf8cea84a","order_by":0,"name":"Bao-Zhu Jia","email":"","orcid":"","institution":"Guangdong University of Education","correspondingAuthor":false,"prefix":"","firstName":"Bao-Zhu","middleName":"","lastName":"Jia","suffix":""},{"id":521586726,"identity":"ee31abfa-3ccc-474a-ab39-973b5f317431","order_by":1,"name":"Wen-Feng Zhang","email":"","orcid":"","institution":"Guangdong Provincial Key Laboratory of Chemical Measurement and Emergency Test Technology","correspondingAuthor":false,"prefix":"","firstName":"Wen-Feng","middleName":"","lastName":"Zhang","suffix":""},{"id":521586727,"identity":"fc888ac0-27cd-492d-aaa6-ac4d7354345d","order_by":2,"name":"Qing-Chun Yin","email":"","orcid":"","institution":"Hainan Institute for Food Control","correspondingAuthor":false,"prefix":"","firstName":"Qing-Chun","middleName":"","lastName":"Yin","suffix":""},{"id":521586728,"identity":"cfa2580e-8369-4807-beae-314c0dd7ad84","order_by":3,"name":"Xue-Ying Rui","email":"","orcid":"","institution":"South China Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Xue-Ying","middleName":"","lastName":"Rui","suffix":""},{"id":521586729,"identity":"052df993-c59b-4e7b-b1d6-759aeca9cf0b","order_by":4,"name":"Lin Luo","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAw0lEQVRIiWNgGAWjYBACPmYg8YCBQQ7KZyashQ2kJoGBwZgELQwQLYkNxGth5z3AkNhmkz6//fAzCYYK68QG9rMHCDiMLwGoJS13w5k0MwmGM+mJDTx5CQS08BgAtRzO3SDBYCbB2HY4sUECKEKMlnT5GezfJBj/kaAlgeEGD9CWBiK1HEg4l2a44UxOsUXCsXTjNp4c/Fr4+c8YPvhQZiMv3358440PNday/exn8GsBgQNwVgIDNKZGwSgYBaNgFFAGAEkXOTvVt6A8AAAAAElFTkSuQmCC","orcid":"","institution":"South China Agricultural University","correspondingAuthor":true,"prefix":"","firstName":"Lin","middleName":"","lastName":"Luo","suffix":""},{"id":521586730,"identity":"7a85cf88-b240-40dc-abac-0ca471d604f7","order_by":5,"name":"Zhen-Lin Xu","email":"","orcid":"","institution":"South China Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Zhen-Lin","middleName":"","lastName":"Xu","suffix":""}],"badges":[],"createdAt":"2025-09-20 08:08:11","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7663415/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7663415/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s00604-026-07881-3","type":"published","date":"2026-02-07T15:59:37+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":95231031,"identity":"b1ad3df3-e39f-41c4-9a45-1e1ca2e3de54","added_by":"auto","created_at":"2025-11-05 16:39:38","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":628982,"visible":true,"origin":"","legend":"\u003cp\u003e(A) TEM image of PDA@PB NPsand EDS element mapping images of C, N, O, Fe, K and HAADF in PDA-Fe@PB NPs; (B) FTIR sepctra of PDA-Fe, and PDA-Fe@PB NPs,; (C) XRD pattern of PDA-Fe, PDA-Fe@PB NPs; (D) Full-range XPS spectra of PDA@PB NPs; High-resolution XPS spectra of Fe 2p (E), C 1s (F), N 1s (G).\u003c/p\u003e","description":"","filename":"18.png","url":"https://assets-eu.researchsquare.com/files/rs-7663415/v1/462ce737ee599cf1dbc56ec8.png"},{"id":95231280,"identity":"35392131-59d8-4f2d-bcaa-667500e56005","added_by":"auto","created_at":"2025-11-05 16:39:48","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":510071,"visible":true,"origin":"","legend":"\u003cp\u003e(A) UV-vis spectra of the catalytic oxidation of TMB by PDA@PB NPs; Michaelis-Menten model of PDA@PB NPs with TMB (B) and H2O2 (D); Lineweaver-Burk double-reciprocal model of PDA@PB NPs with TMB (C) and H2O2 (E); Storage stability of PDA@PB NPs (F); Photothermal performance of PDA@PB NPs in a TMB-H2O2 system (G); Thermal heating curves of different concentrations of PDA@PB NPs under irradiated by an 808 nm laser (H); Photothermal stability of PDA@PB NPs upon three cycles of the on/off NIR laser (I).\u003c/p\u003e","description":"","filename":"27.png","url":"https://assets-eu.researchsquare.com/files/rs-7663415/v1/bcc22218b430a16637e0cb52.png"},{"id":95231017,"identity":"5ea3329d-76ac-47b4-ab1a-3c70a99c20b1","added_by":"auto","created_at":"2025-11-05 16:39:34","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":332121,"visible":true,"origin":"","legend":"\u003cp\u003e(A) Zeta potential of PB, PB@Ab, PDA@PB NPs and PDA@PB NP@Ab; (B); (C) Catalytic activity of PDA@PB NPs and PB before and after coupling with antibody; (D) Coupling efficiency of PB and PDA@PB NPs with different amounts of anti-BZC antibody (group 1, 2, 3, 4, 5, 6 corresponding to 4、8、12、16、20、24 μg)\u003c/p\u003e","description":"","filename":"35.png","url":"https://assets-eu.researchsquare.com/files/rs-7663415/v1/ba80663389ec3e473fc24a4a.png"},{"id":95231034,"identity":"8229fe75-0574-44be-b6cb-8694a3ba8603","added_by":"auto","created_at":"2025-11-05 16:39:38","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":194938,"visible":true,"origin":"","legend":"\u003cp\u003eOptimization of the immunoassay working parameters: type (A) and pH (B) of reconstitution buffer for PDA@PB NPs@Ab immunoprobe; (C) working concentration of anti-BZC mAb; (D) incubation time for immunoreaction.\u003c/p\u003e","description":"","filename":"44.png","url":"https://assets-eu.researchsquare.com/files/rs-7663415/v1/f591b08464a84740a82782fc.png"},{"id":95231037,"identity":"6f6cc252-9ef7-454e-8f2d-b56b5ab6ba2a","added_by":"auto","created_at":"2025-11-05 16:39:39","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":483632,"visible":true,"origin":"","legend":"\u003cp\u003e(A) Photothermometric calibration curve for BZC detection, inset: linear range; (B) Colorimetric calibration curve, inset: linear range; Photothermal (C) and colorimetric (D) response of the dual-mode immunoassay against benzocaine (100 ng mL-1), procaine, propofol, quinaldine, eugenol, phenoxyethanol, lidocaine and tricaine (1.0 μg mL-1).\u003c/p\u003e","description":"","filename":"53.png","url":"https://assets-eu.researchsquare.com/files/rs-7663415/v1/1416549c209cf0c8a6c623bd.png"},{"id":102234167,"identity":"6cd6528e-2e4f-4ce1-b18b-220f6d40abe0","added_by":"auto","created_at":"2026-02-09 16:07:00","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2721001,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7663415/v1/cbe0e9f2-810b-4b2c-b46b-91b9bde2694e.pdf"},{"id":95188715,"identity":"cbf1500d-fa94-4c8d-9d59-148bf81dd2d7","added_by":"auto","created_at":"2025-11-05 09:47:31","extension":"tif","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":1958322,"visible":true,"origin":"","legend":"","description":"","filename":"Graphical.tif","url":"https://assets-eu.researchsquare.com/files/rs-7663415/v1/2452f86094cd42851a73b8eb.tif"},{"id":95188716,"identity":"32df7c2a-ec49-4006-a943-416cf43765b3","added_by":"auto","created_at":"2025-11-05 09:47:31","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":5997784,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAppendix A. Supplementary Materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSupplementary Materials associated with this article can be found, in the online version, at http://dx.doi.org/xxx.\u003c/p\u003e","description":"","filename":"lSupplementaryMaterials.docx","url":"https://assets-eu.researchsquare.com/files/rs-7663415/v1/00891778ab27a86c5a2ef9d2.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Dual-Mode Photothermal and Colorimetric Immunosensor Based on Polydopamine@Prussian Blue Nanocomposite for Sensitive Detection of Benzocaine","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eEnsuring food safety requires accurate and rapid monitoring of chemical residues, including veterinary drugs, pesticides, and anesthetics, that may remain in edible animal products. Among these contaminants, benzocaine (BZC), a widely used local anesthetic in aquaculture, is often applied to sedate fish during transportation and spawning operations [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. However, excessive or unregulated use can lead to residues in fish tissues, posing health risks such as methemoglobinemia and allergic reactions in humans [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Consequently, sensitive and reliable analytical methods for BZC detection are essential for food safety control and regulatory supervision.\u003c/p\u003e\u003cp\u003eConventional enzyme-linked immunosorbent assays (ELISA) have become indispensable tools in food residue monitoring because of their high specificity, simplicity, and suitability for large-scale screening [\u003cspan additionalcitationids=\"CR5 CR6\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Nevertheless, typical colorimetric ELISA systems rely on natural enzymes such as horseradish peroxidase (HRP), which suffer from poor stability and single-signal output [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. These limitations can result in false-positive or false-negative outcomes, especially in complex food matrices [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. To overcome these challenges, researchers have incorporated nanomaterials into ELISA to enhance the sensitivity and reliability.\u003c/p\u003e\u003cp\u003eIn recent years, numerous nanozyme-based immunosensing platforms have been developed to enhance analytical reliability by integrating colorimetric and photothermal dual readouts. Wei et al. first established a Prussian Blue (PB)-based multicolor and photothermal dual-readout immunosensor for prostate specific antigen detection [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e], demonstrating the feasibility of using PB nanozymes for visual and thermal quantification. Building upon this concept, Ding et al. constructed a black phosphorus\u0026ndash;gold nanohybrid (BP/Au)-based dual-mode immunoassay for diethylstilbestrol determination [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e], further confirming the effectiveness of combining peroxidase-like activity with photothermal effects. Subsequently, Huang et al. reported a Ti₃C₂Tₓ/AuNP-based colorimetric\u0026ndash;photothermal immunosensor for zearalenone detection [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e], achieving improved signal stability and sensitivity through MXene\u0026ndash;metal synergy. Most recently, Gong et al. introduced a switchable colorimetric\u0026ndash;photothermal lateral flow immunoassay that allowed flexible transition between the two detection modes, illustrating the versatility of dual-mode strategies for on-site immunoassays [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Collectively, these studies confirm that integrating colorimetric and photothermal outputs can significantly enhance the accuracy and adaptability of immunoassays. Nevertheless, most of these systems still require complex antibody crosslinking or involve nanomaterials with limited biocompatibility, underscoring the need for a facile and stable dual-mode sensing platform.\u003c/p\u003e\u003cp\u003ePrussian Blue (PB), a classical iron-based nanozyme, is particularly attractive owing to its strong peroxidase-like activity and high photothermal efficiency under near-infrared (NIR) irradiation [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. However, practical application is often hindered by limited antibody binding efficiency and aggregation during bioconjugation [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Polydopamine (PDA), inspired by mussel adhesive proteins, offers an elegant solution due to its excellent adhesion, biocompatibility, and ability to immobilize biomolecules without additional crosslinkers [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. In recent years, PDA has found growing applications in biosensor development for the detection of hazardous substances, benefiting from its superior biocompatibility, adhesion, and functional versatility [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Integrating PDA with PB into a PDA@PB nanocomposite can therefore synergistically combine catalytic, photothermal, and bioconjugation properties\u0026mdash;ideal for constructing novel dual-mode immunosensors.\u003c/p\u003e\u003cp\u003eIn this work, we synthesized PDA@PB nanocomposites through a facile in situ deposition of PB on Fe(III)-coordinated PDA nanospheres. The resulting materials exhibited strong peroxidase-like activity, efficient photothermal conversion, and stable antibody immobilization. A colorimetric\u0026ndash;photothermal dual-mode immunosensor was thus developed for the sensitive detection of BZC residues. In this sensing system, the oxidation of 3,3\u0026prime;,5,5\u0026prime;-tetramethylbenzidine (TMB) by PDA@PB produced both a visible color change and a corresponding temperature rise under 808 nm laser irradiation, enabling dual quantitative readouts. This strategy not only ensures high sensitivity and accuracy, but also provides portable and visual detection, offering great potential for on-site monitoring of BZC residues in aquatic food safety surveillance.\u003c/p\u003e"},{"header":"2. Experimental","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1 Materials and reagents\u003c/h2\u003e\u003cp\u003eBenzocaine, propofol, quinaldine, eugenol, phenoxyethanol, lidocaine and tricaine standards were purchased from Tan-Mo Technology Co., Ltd. (Beijing, China). Dopamine hydrochloride, 3,3\u0026rsquo;,5,5\u0026rsquo;-tetramethylbenzidine (TMB) were sourced from Macklin (Shanghai, China). FeCl\u003csub\u003e3\u003c/sub\u003e\u0026middot;6H\u003csub\u003e2\u003c/sub\u003eO and potassium ferrocyanide were supplied by Aladdin Industrial Corporation (Shanghai, China). The antigen and monoclonal antibodies against benzocaine (anti-BZC mAbs) were prepared previously in our laboratory [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Unless otherwise noted, all chemicals employed were of analytical grade and used as received. Ultrapure water was used to prepare all aqueous solutions throughout the study.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e2.2 Apparatus\u003c/h2\u003e\u003cp\u003eUV absorption spectra were acquired with a SpectraMax i3x multifunctional microplate reader (Molecular Devices, USA). Elemental composition and surface states were analyzed via XPS on an ESCALAB 250Xi spectrometer (Thermo Scientific, USA). TEM imaging was performed using a Tecnai G2 F20 instrument (FEI, USA). XRD patterns were recorded using a D8 Advance system (Bruker, Germany), while FT-IR spectra were collected with a Nicolet iS10 analyzer (Thermo Scientific, USA). ELISA plate washing was conducted using a Multiskan MK2 washer (Thermo Scientific, MA, USA).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003e2.3 Synthesis of PDA@PB nanocomposite\u003c/h2\u003e\u003cp\u003eFe(III)-polydopamine (PDA-Fe) nanospheres were fabricated based on a modified literature method [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. In brief, 60 mg of dopamine and 8.3 mg of FeCl\u003csub\u003e3\u003c/sub\u003e\u0026middot;6H\u003csub\u003e2\u003c/sub\u003eO were fully solubilized in 170 mL of ultrapure water and stirred at room temperature for 1 h. Then, 30 mL of Tris buffer (22.5 mg/mL) was added, and the mixture was continuously stirred for an additional 2 h. The resulting PDA-Fe nanospheres were collected by centrifugation and thoroughly washed five times with ultrapure water. Finally, the precipitate was dispersed in 10 mL of ultrapure water for subsequent experiments.\u003c/p\u003e\u003cp\u003eFor PDA@PB NP preparation, 5 mL of 2 mM K\u003csub\u003e4\u003c/sub\u003e[Fe(CN)\u003csub\u003e6\u003c/sub\u003e]and 5 mL of 0.6 M HCl were gradually added to the PDA-Fe suspension under constant stirring. After 20 min of reaction, Prussian blue formed on the PDA-Fe nanospheres. The resulting PDA@PB NPs were separated by centrifugation, washed thoroughly with deionized water (five times), and then freeze-dried to obtain PDA@PB NP powder.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003e2.4 Fabrication of antibody-conjugated PDA@PB NP immunoprobe\u003c/h2\u003e\u003cp\u003eThe antibody-conjugated PDA@PB NP immunoprobe was synthesized via a straightforward mixing and adsorption approach [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Briefly, 0.2 mg of PDA@PB NP was dispersed in 1 mL phosphate-buffered saline (PBS, 0.01 M, pH 7.4). Subsequently, 20 \u0026micro;L of anti-BZC monoclonal antibody (1 mg mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)) was introduced, and the mixture was gently agitated at 4\u0026deg;C for 2 h. Following incubation, 100 \u0026micro;L of 10% BSA was added to block nonspecific adsorption sites. Afterward, the solution was centrifuged and rinsed with ultrapure water to remove unbound antibodies and excess BSA. The resulting PDA@PB@Ab immunoprobe was re-suspended in 1 mL of borate buffer containing BSA (BB-BSA, pH 6.4) for storage and subsequent use. The efficiency of antibody conjugation was quantified through indirect ELISA. In brief, 4, 8, 12, 16, 20, and 24 \u0026micro;g of anti-BZC mAb were each added to 1 mL of PDA-PB nanosphere suspension. After incubation and centrifugation, the supernatants were collected and diluted 4-fold to measure the remaining unbound antibodies. The coupling efficiency was determined using the equation: Coupling efficiency = [(total antibodies) \u0026ndash; (supernatant antibodies)]/ (total antibodies) \u0026times; 100% [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e].\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\u003ch2\u003e2.5 Protocol of the dual-mode immunosensor\u003c/h2\u003e\u003cp\u003eThe dual-mode immunoassay protocol is illustrated in Scheme \u003cspan refid=\"Sch1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. Briefly, 100 \u0026micro;L of BZC-OVA (used as the coating antigen) in 0.05 M carbonate buffer (pH 9.5) was dispensed into individual wells of a 96-well plate and kept at 37 ℃ overnight for coating. Afterward, the wells were rinsed twice with PBST solution (0.01 M phosphate-buffered saline containing 0.05% Tween-20, pH 7.4). To block nonspecific adsorption, 120 \u0026micro;L of blocking solution was added, followed by incubation at 37 ℃ for 3 h. Finally, the plate was dried at 37 ℃ for 1 h. Next, 50 \u0026micro;L of either BZC standard solution or sample extract and 50 \u0026micro;L of PDA@PB NP@Ab immunoprobe were added to each well and incubated at 37 ℃ for 30 min. After five washes, 100 \u0026micro;L of TMB-H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e substrate (v/v\u0026thinsp;=\u0026thinsp;1:1) was introduced into each well, followed by a 10-min incubation at 37 ℃.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eFor photothermal test, the microplate strips were placed into a custom-built 3D-printed holder (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e) and subjected to 808 nm near-infrared (NIR) laser exposure at 1.5 W/cm\u0026sup2; for 150 s. Thermal responses were captured and tracked using a handheld NIR imaging camera. A calibration curve was then established by associating the change in temperature with the logarithmic concentration of BZC, enabling determination of its level in unknown samples.\u003c/p\u003e\u003cp\u003eFor colorimetric analysis, optical density at 652 nm was promptly detected via SpectraMax i3x multifunctional microplate reader. The antibody binding rate, expressed as the ratio B/B\u003csub\u003e0\u003c/sub\u003e, was determined, where B\u003csub\u003e0\u003c/sub\u003e denotes the signal without BZC and B corresponds to the signal in its presence. To quantify BZC, a calibration curve was established by fitting the log-transformed concentration values against the measured B/B\u003csub\u003e0\u003c/sub\u003e values using a sigmoidal four-parameter logistic fitting method. This fitted model was subsequently employed to calculate BZC content in tested samples.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003e2.6 Sample preparation\u003c/h2\u003e\u003cp\u003eAquaculture water samples underwent filtration using a 0.22 \u0026micro;m membrane, followed by a 5-fold dilution with 0.01 M PBS buffer (pH 7.4) prior to analysis by the immunoassay. For shrimp and tilapia, 1.0 g of homogenized tissue was mixed with 1.0 mL acetonitrile and vigorously agitated for 2.5 minutes. The upper layer was then collected into a clean tube preloaded with a sorbent mixture (75 mg MgSO\u003csub\u003e4\u003c/sub\u003e, 30 mg PSA, 15 mg NaCl, and 30 mg C18), shaken for another 2 min, and subjected to centrifugation at 7000 rpm. The extract was subsequently diluted tenfold in PBS and used for subsequent detection by the immunoassay.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\u003ch2\u003e2.7 Method validation\u003c/h2\u003e\u003cp\u003eTo verify the reliability of the immunosensor, fortified aquaculture water, shrimp, and carp samples at 5.0, 50.0, and 100.0 ng/g (or ng/mL for water) of BZC were analyzed by both the immunosensor and LC-MS/MS. Detailed instrumental parameters for LC-MS/MS are provided in the Supplementary Material (Fig. S2). The quantitative consistency of the immunosensor and LC-MS/MS was verified by regression analysis.\u003c/p\u003e\u003c/div\u003e"},{"header":"3. Results and discussion","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003e3.1 Characterization of PDA@PB NP\u003c/h2\u003e\u003cp\u003eThe synthesis of PDA@PB NPs involved two sequential steps. First, dopamine and Fe\u003csup\u003e3+\u003c/sup\u003e ions acted as building blocks for the formation of PDA-Fe nanospheres via a pre-polymerization doping strategy. Subsequently, surface-anchored Fe\u003csup\u003e3+\u003c/sup\u003e ions underwent in situ coordination with potassium ferrocyanide (K\u003csub\u003e4\u003c/sub\u003e[Fe(CN)\u003csub\u003e6\u003c/sub\u003e]), yielding the final PDA@PB NP (Scheme \u003cspan refid=\"Sch1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). TEM imaging (Fig. S3) verified the uniform morphology of the PDA-Fe spheres with an average size of ~\u0026thinsp;160 nm. As shown in Fig. S4, at acidic conditions, PB was effectively formed through the reaction between Fe\u003csup\u003e3+\u003c/sup\u003e ions and K\u003csub\u003e4\u003c/sub\u003e[Fe(CN)\u003csub\u003e6\u003c/sub\u003e], characterized by a prominent absorption band around 710 nm. The optimal synthesis conditions for PDA@PB NP were identified when a mixture containing 2 mM K\u003csub\u003e4\u003c/sub\u003e[Fe(CN)\u003csub\u003e6\u003c/sub\u003e]and 0.6 M HCl resulted in the highest absorbance, suggesting their suitability for PDA@PB NP formation.\u003c/p\u003e\u003cp\u003eUnder these optimized parameters, PB was evenly deposited on PDA-Fe spheres. Characterization was performed using TEM, FT-IR, XRD, and XPS. Figure\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA and S3 illustrate that both PDA-Fe and PDA@PB NP maintained spherical shapes, whereas the latter showed a rougher surface texture. Elemental mapping and EDS spectra (Fig. S5 and Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA) demonstrated that C, N, O, Fe, and K elements were evenly distributed throughout the PDA@PB NP. The FT-IR spectrum (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD) exhibited characteristic C\u0026thinsp;\u0026equiv;\u0026thinsp;N stretching bands at 2080 cm⁻\u0026sup1;, indicating successful integration of PB. XRD analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC) showed that PDA-Fe exhibited an amorphous structure without noticeable diffraction signals [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. In contrast, PDA@PB NP displayed peaks at 17.465\u0026deg;, 24.578\u0026deg;, 35.277\u0026deg;, 39.351\u0026deg;, 43.716\u0026deg;, 50.635\u0026deg;, 54.066\u0026deg;, and 57.491\u0026deg;, which were indexed to the (200), (220), (400), (420), (422), (440), (600), and (620) planes of PB (JCPDS #73\u0026ndash;0687). XPS data (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD) further validated the composition, with strong signals from Fe, C, and N. High-resolution spectra (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE\u0026ndash;G) provided detailed chemical state information. In the Fe 2p spectrum (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE), peaks at 708.4 and 721.5 eV indicated Fe\u0026sup2;⁺ (2p₃/₂ and 2p₁/₂), while signals at 709.1, 712.6, and 725.1 eV reflected Fe\u0026sup3;⁺ states. The C 1s spectrum (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF) exhibited binding energy peaks at 284.7, 285.6, and 288.5 eV, corresponding to C\u0026ndash;C, C\u0026thinsp;\u0026equiv;\u0026thinsp;N, and C\u0026ndash;N bonds. N 1s spectra (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eG) showed signals at 397.6 and 399.5 eV, consistent with cyanide groups coordinated with Fe\u0026sup2;⁺ and Fe\u003csup\u003e3+\u003c/sup\u003e within the PB lattice, in accordance with previous studies [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e].\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003e3.2 Evaluation of the peroxidase-like performance of PDA@PB NP\u003c/h2\u003e\u003cp\u003eThe property of the PDA@PB NP to mimic peroxidase activity was assessed using TMB as substrate. Figure\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA reveals that a distinct signal at 652 nm\u0026mdash;corresponding to oxidized TMB (ox-TMB)\u0026mdash;was only generated when both the PDA@PB NP and H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e coexisted, whereas negligible response was detected in the absence of either component. To comprehensively assess their catalytic capability, kinetic experiments were performed by varying the concentrations of TMB or H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e. The reaction velocity data were fitted to the Michaelis-Menten model to extract the key kinetic constants, namely the maximum catalytic rate (Vmax) and the substrate affinity constant (Km). the Michaelis-Menten is as follow:\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eV=\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\frac{{V}_{max}\\:\\left[S\\right]}{{K}_{m}+\\left[S\\right]}\\)\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\u003cp\u003eThe reaction velocity (V) corresponds to the initial rate of the colorimetric reaction, while [S]denotes the substrate concentration. Nonlinear regression of the data (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB-E) was employed to extract kinetic constants, confirming that the catalytic activity of PDA@PB NP aligns with Michaelis-Menten behavior. The calculated apparent Michaelis constants (Km) for TMB and hydrogen peroxide were 0.41 mM and 2.29 mM, respectively. Corresponding maximum velocities (Vmax) were determined to be 1.44\u0026times;10\u003csup\u003e\u0026minus;\u0026thinsp;8\u003c/sup\u003e mM s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e for TMB and 5.91 \u0026times; 10\u003csup\u003e\u0026minus;\u0026thinsp;8\u003c/sup\u003e mM s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e for H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e. To further evaluate the catalytic performance of PDA@PB NPs, their apparent kinetic parameters (Km and Vmax) toward TMB and H₂O₂ were compared with those of other photothermal-active nanozymes reported in recent dual- or multi-mode sensing studies (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). The Km (TMB) of PDA@PB NPs (0.41 mM) is close to or lower than that of most representative systems such as Cu\u0026ndash;N/O single-atom, CoFe PBAs/WS₂, and Ti₃C₂Tₓ/AuNPs nanozymes (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e), indicating a strong affinity for the substrate. In addition, the moderate Km (H₂O₂) (2.29 mM) and relatively low Vmax values confirm efficient catalytic turnover under mild conditions. These results demonstrate that PDA@PB NPs possess high substrate affinity and catalytic efficiency, making them promising candidates for subsequent dual-mode immunoassay applications.\u003c/p\u003e\u003cp\u003eTo assess the application potential of PDA@PB NP in immunoassay, their stability under varying conditions was assessed. The effect of pH on the peroxidase-like activity was first evaluated by monitoring the TMB catalytic oxidation reaction initiated by H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e over a pH range from 2.4 to 10.4. Fig. S6 illustrates that the PDA@PB NP displayed evident pH-dependent behavior, showing markedly increased catalytic performance in acidic environments. Notably, the highest activity was achieved at pH 6.4, and this condition was consequently chosen for the following tests. In addition, the storage stability of PDA@PB NP was evaluated to further verify their practical applicability. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF, over 87% of its initial catalytic performance was preserved after 20 days at room temperature, reflecting excellent storage stability. Overall, the findings confirm that PDA@PB NPs exhibit strong peroxidase-mimicking activity and favorable stability during storage underscoring their reliability and potential as promising artificial nanozymes for immunoassay applications.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003e3.3 Characterization of the photothermal performance of PDA@PB NP\u003c/h2\u003e\u003cp\u003ePrussian blue-based nanostructures have been previously identified as excellent agents for photothermal conversion. Meanwhile, oxidized TMB (ox-TMB) shows a prominent absorbance peak near 750 nm in the NIR region, supporting its role in light-to-heat transformation [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. As presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eG, when irradiated with an 808 nm NIR laser, different reaction mixtures exhibited varied thermal responses. A notable temperature elevation was observed with PDA@PB NPs alone, while the highest thermal response appeared when PDA@PB NP were combined with H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e and TMB, likely due to the formation of oxidized TMB (ox-TMB). In comparison, negligible heat generation was detected in other control groups. This verify that PDA@PB NPs possess both enzyme-like catalytic performance and efficient photothermal capability. Additionally, temperature elevations were measured for dispersions containing PDA@PB NP at concentrations of 50, 125, 250, 500, and 750 \u0026micro;g/mL after 300 s of laser exposure. The thermal response showed a direct correlation with nanomaterial concentration, with the optimal irradiation time determined to be 150 s (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eH). To further assess the photothermal durability, three successive irradiation cycles were performed (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eI). The reproducible heating profiles over repeated cycles indicate strong thermal robustness of the synthesized PDA@PB NPs. These results highlight the suitability of PDA@PB NP for potential implementation in photothermal-based immunoassay platforms.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003e3.4 Evaluation of antibody-conjugated PDA@PB NP immunoprobe\u003c/h2\u003e\u003cp\u003eMussel-inspired polydopamine exhibits strong adhesion to a wide range of organic and inorganic substrates, attributed to its high binding strength and rich surface chemistry [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. In particular, the abundant quinone groups in PDA can react with amino groups on antibodies via Michael addition, forming stable covalent bonds. This conjugation strategy has been demonstrated to be more efficient and biocompatible than conventional chemical methods [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eTo verify the successful conjugation of anti-BZC monoclonal antibodies (Ab) to the PDA@PB NPs, changes in zeta potential and UV\u0026ndash;Vis absorption spectra were analyzed. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, the zeta potential of PDA@PB NP increased from \u0026minus;\u0026thinsp;14.67 mV to \u0026minus;\u0026thinsp;5.77 mV after antibody modification, indicating surface charge alteration due to Ab attachment. Moreover, a characteristic protein absorption peak at 280 nm was observed in the UV\u0026ndash;Vis spectrum of the immunoprobe, which was absent in the spectrum of unmodified PDA@PB NP (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB), further confirming successful antibody-PDA@PB NP conjugation.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThe performance of the PDA@PB NP@Ab immunoprobe was evaluated in terms of catalytic activity and conjugation efficiency, with PB serving as a control. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC, after antibody conjugation and BSA blocking, the catalytic activity of PB NPs toward TMB decreased by 41.7%, likely due to the obstruction of catalytic sites. In contrast, PDA@PB NP exhibited a smaller reduction of 21.7%, suggesting superior biocompatibility and improved preservation of catalytic activity. Additionally, conjugation efficiencies were assessed across a range of antibody concentrations. PB NPs showed efficiencies ranging from 44.7% to 63.8%, which decreased as the antibody dosage increased. In comparison, PDA@PB NP demonstrated consistently higher efficiencies, ranging from 64.9% to 77.3% (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). This enhancement can be attributed to the PDA layer, which facilitates strong and stable antibody conjugation through multiple interactions, including electrostatic attraction, Schiff base formation, and Michael addition [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e].\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003e3.5 Performance of the immunosensor\u003c/h2\u003e\u003cp\u003eTo achieve the optimal detection performance of the dual-mode immunosensor, a series of critical parameters were methodically investigated. These included the buffer type and its pH used for reconstituting the PDA@PB NP@Ab immunoprobe, the dosage of anti-BZC monoclonal antibodies employed during probe fabrication, as well as the concentrations of both the coating antigen and the antibody, alongside the incubation time required for effective antigen-antibody binding. As shown in Fig. S7, the assay yielded its best performance under the following optimized conditions: (1) borate buffer at pH 6.4 was adopted for probe dispersion; (2) 4.0 \u0026micro;g of anti-BZC monoclonal antibody was utilized for coupling with PDA@PB NP; (3) the working concentration of the antibody was adjusted to 1.0 \u0026micro;g/mL; (4) the antigen for coating was employed at 1.0 \u0026micro;g/mL A reaction time of 30 min was selected based on the maximum detection response.\u003c/p\u003e\u003cp\u003eUnder these optimized conditions, both colorimetric and thermal readouts were recorded across a gradient of BZC concentrations. Final solution temperatures were measured using a customized infrared imaging setup following 808 nm NIR laser irradiation (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). As depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA, the thermal output declined progressively with increased levels of BZC. The change in temperature (Y) exhibited good linear relationship with the logarithmic value of BZC concentration (X) in the range from 0.01 to 1000 ng/mL. The linear equation was Y = \u0026minus;\u0026thinsp;2.01X\u0026thinsp;+\u0026thinsp;21.06. The detection limit (LOD) was 0.82 ng/mL (S/N\u0026thinsp;=\u0026thinsp;3). In the colorimetric assay, the antibody binding rate B/B\u003csub\u003e0\u003c/sub\u003e exhibited a gradual decline as the BZC concentration increased (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). A good linear relationship was identified between B/B\u003csub\u003e0\u003c/sub\u003e and the logarithmic value of BZC concentration, modeled by the equation: Y = \u0026minus;\u0026thinsp;0.011X\u0026thinsp;+\u0026thinsp;0.68. The LOD of the colorimetric assay was 0.33/ng mL (S/N\u0026thinsp;=\u0026thinsp;3).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eTo evaluate specificity, several structurally or functionally related fish anesthetics\u0026mdash;including procaine, propofol, quinaldine, eugenol, phenoxyethanol, lidocaine, and tricaine\u0026mdash;were tested. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC, these analogs produced responses similar to the blank, markedly lower than the signal observed for BZC. These results demonstrate that the dual-mode immunoassay offers excellent selectivity for BZC detection.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003e3.6 Detection of BZC in real samples\u003c/h2\u003e\u003cp\u003eThe analytical performance of the proposed dual-mode immunosensor was further verified by detecting benzocaine in spiked aquaculture water, fish, and shrimp samples. As shown in Table S2, the concentrations obtained by the colorimetric and photothermal modes were in close agreement with those determined by the reference LC\u0026ndash;MS/MS method at spiking levels of 5.0, 50.0, and 100.0 ng/mL (or ng/g). The recoveries ranged from 81.3% to 98.5%, with relative standard deviations below 11.5%, indicating good accuracy and precision for complex matrices. To statistically assess the consistency between the two methods, paired t-tests were performed for each matrix type (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD\u0026ndash;F). No significant difference (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05) was observed between the immunosensor results (both detection modes) and LC\u0026ndash;MS/MS, confirming that the proposed assay provides quantitative accuracy comparable to the instrumental standard method.\u003c/p\u003e\u003cp\u003eFrom a regulatory perspective, the European Commission Regulation (EU) No 37/2010 classifies benzocaine as \u0026ldquo;not applicable,\u0026rdquo; meaning that no maximum residue limit (MRL) is established for food-producing animals such as finfish. In contrast, the U.S. FDA has proposed an import tolerance limit of 50 ng/g for benzocaine residues in fish muscle [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. This regulatory discrepancy highlights the need for sensitive analytical techniques capable of monitoring trace-level residues in aquatic products.\u003c/p\u003e\u003cp\u003eTable\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e summarizes representative nanomaterial-based methods for benzocaine determination, with all linear ranges and LODs converted to mass-concentration units (ng/mL or ng/g) for direct comparison. Electrochemical sensors based on hollow carbon nanobowls [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e] and boron-doped diamond electrodes [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e] achieve good sensitivity (LOD\u0026thinsp;=\u0026thinsp;0.66 and 8.25 ng/mL, respectively) but provide single-signal outputs, making them more susceptible to complex matrix interferences. A Fe₃O₄@polydopamine molecularly imprinted nanozyme [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e] exhibits high selectivity (LOD 0.66 ng/mL) yet offers single-mode colorimetric output. The indirect SERRS\u0026ndash;Ag NPs assay [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e] allows rapid, broad-range measurement (100\u0026ndash;10\u003csup\u003e4\u003c/sup\u003e ng/mL) but involves derivatization and requires specialized Raman equipment.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eAn overview on recently reported nanomaterial-based methods for the determination of benzocaine\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"6\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eMethod\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eNanomaterial\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eLinear range (ng/mL or g)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eLOD\u003c/p\u003e\u003cp\u003e(ng/mL or g)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eSample type\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003eReference\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eElectrochem-\u003c/p\u003e\u003cp\u003eical\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eHollow carbon nanobowl modified electrode\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1.65\u0026ndash;330\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.66\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eHuman saliva\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eElectrochem-\u003c/p\u003e\u003cp\u003eical\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eMiniaturized boron-doped diamond electrode\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e16.5\u0026ndash; 6.6\u0026times;10\u003csup\u003e4\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e8.25\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003ePharmaceutical products, human urine\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eColorimetric assay\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eFe₃O₄@polydopamine molecularly imprinted nanozyme\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e5.0\u0026ndash;65\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.66\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eEar drops, tap and Nile water\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e[\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eIndirect SERS (azo coupling)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eAg nanoparticles\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e100 \u0026minus;\u0026thinsp;10000\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e78\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003ePharmaceutical gels, sprays, enemas\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e[\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eDual-mode colorimetric\u0026ndash;\u003c/p\u003e\u003cp\u003ephotothermal immunoassay\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003ePDA@PB nanocomposite\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.01\u0026ndash;1000\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e3.3 (colorimetric) / 8.2 (photothermal)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eWater, fish, shrimp\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eThis work\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eIn contrast, the PDA@PB-based dual-mode colorimetric\u0026ndash;photothermal immunosensor developed in this study combines the excellent specificity of immunoassays with simple optical detection and photothermal analysis, achieving a ultra-low detection limit of 3.3 ng/g for the colorimetric mode and 8.2 ng/g for the photothermal mode (obtained by multiplying the LOD for buffer solution with a dilution factor 10-fold involved in sample preparation). The method also demonstrated high recoveries and good reproducibility in spiked samples, confirming its suitability for routine food-safety surveillance. However, the two signal outputs in this work cannot be acquired simultaneously. Developing sensing apparatuses capable of detecting multiple signals in real time would be an effective strategy to achieve in situ detection and accelerate data processing in future studies.\u003c/p\u003e\u003c/div\u003e"},{"header":"4. Conclusion","content":"\u003cp\u003eIn this study, a multifunctional nanocomposite, PDA@PB NP, was successfully synthesized through a two-step process involving the formation of PDA-Fe nanospheres followed by in situ deposition of Prussian blue. Benefiting from the synergistic properties of polydopamine and Prussian blue, PDA@PB NP demonstrated excellent peroxidase-mimicking catalytic activity, robust photothermal conversion efficiency, and good stability. Moreover, the PDA shell enabled efficient and stable antibody conjugation, preserving enzymatic activity and enhancing conjugation efficiency, which facilitated the construction of a bifunctional immunoprobe. Based on this, a dual-mode immunosensor was developed for sensitive and selective detection of BZC. The assay showed high specificity and satisfactory recovery rates in real samples including fish, shrimp, and aquaculture water. Notably, the method exhibited good agreement with LC-MS/MS, demonstrating its reliability. In summary, the proposed PDA@PB NP-based dual-mode immunosensor offers a simple, sensitive, and robust strategy for trace-level detection of BZC, providing promising prospects for food safety monitoring and regulatory compliance in aquaculture product testing. However, the current reader cannot simultaneously acquire the two signal outputs, integrating real-time multi-signal detection in future sensing devices will further enhance its applicability for in situ monitoring and high-throughput analysis.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003ch2\u003eDeclaration of Competing Interest\u003c/h2\u003e\u003cp\u003eThe authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.\u003c/p\u003e\u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e\u003cp\u003eThis work was supported by the Guangdong Basic and Applied Basic Research Foundation (2023A1515030011).\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eBao-Zhu Jia: Data curation, Formal analysis, Investigation, Methodology, Writing \u0026ndash; original draft; Wen-Feng Zhang: Data curation, Investigation, Methodology; Qing-Chun Yin: Data curation, Investigation, Methodology; Xue-Ying Rui: Data curation, Methodology, Investigation. Lin Luo: Conceptualization, Writing \u0026ndash; review \u0026amp; editing, Formal analysis, Funding acquisition; Zhen-Lin Xu: Investigation, Resources, Funding acquisition;\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eData will be made available on request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eVandervelde, E M, (2005). Enzyme‐linked immunoassay (ELISA): Its practical application to the diagnosis of hepatitis B. Journal of Medical Virology 3, 17-18. https://doi.org/10.1002/jmv.1890030105\u003c/li\u003e\n\u003cli\u003eBolarinwa, I F, Orfila, C, Morgan, M R A, (2014). Development and Application of an Enzyme-Linked Immunosorbent Assay (ELISA) for the Quantification of Amygdalin, a Cyanogenic Glycoside, in Food. Journal of Agricultural and Food Chemistry 62, 6299-6305. https://doi.org/10.1021/jf501978d\u003c/li\u003e\n\u003cli\u003eLiu, Z, Zhang, B, Sun, J, Yi, Y, Li, M, Du, D, Zhu, F, Luan, J, (2018). Highly efficient detection of salbutamol in environmental water samples by an enzyme immunoassay. Science of The Total Environment, 861-865. https://doi.org/https://doi.org/10.1016/j.scitotenv.2017.08.324\u003c/li\u003e\n\u003cli\u003eCharernchai, S, Chikae, M, Phan, T T, Wonsawat, W, Hirose, D, Takamura, Y, (2022). Automated Paper-Based Femtogram Sensing Device for Competitive Enzyme-Linked Immunosorbent Assay of Aflatoxin B1 Using Submicroliter Samples. Analytical Chemistry 94, 5099-5105. https://doi.org/10.1021/acs.analchem.1c05401\u003c/li\u003e\n\u003cli\u003eLi, Y, Wang, L, Cui, Z, Liu, S, Wang, S, Ren, J, Tian, Y, Shu, R, Luo, X, Liao, Y, Wang, J, Zhang, D, (2022). White peroxidase-mimicking nanozyme˗nanocarrier of enzyme labeled antibody to enhance catalytic performance and relieve color interference of immunoassay. Sensors and Actuators B: Chemical 364. https://doi.org/10.1016/j.snb.2022.131909\u003c/li\u003e\n\u003cli\u003eWang, Y, Bu, T, Cao, Y, Wu, H, Xi, J, Feng, Q, Xuan, C, Wang, L, (2023). A Versatile PdRu Bimetallic Nanoenzyme-Integrated Enzyme-Linked Immunosorbent Assay for Highly Sensitive Escherichia coli O157:H7 Detection. Analytical Chemistry 95, 9237-9243. https://doi.org/10.1021/acs.analchem.3c00743\u003c/li\u003e\n\u003cli\u003eYu, Z, Gong, H, Li, M, Tang, D, (2022). Hollow prussian blue nanozyme-richened liposome for artificial neural network-assisted multimodal colorimetric-photothermal immunoassay on smartphone. Biosensors and Bioelectronics 218. https://doi.org/10.1016/j.bios.2022.114751\u003c/li\u003e\n\u003cli\u003eGao, Y, Zhou, Y, Chandrawati, R, (2019). Metal and Metal Oxide Nanoparticles to Enhance the Performance of Enzyme-Linked Immunosorbent Assay (ELISA). ACS Applied Nano Materials 3, 1-21. https://doi.org/10.1021/acsanm.9b02003\u003c/li\u003e\n\u003cli\u003eSong, M, Xing, J, Cai, H, Gao, X, Li, C, Liu, C, Li, X, Fu, X, Ding, S, Cheng, W, Chen, R, (2023). Pomegranate-Bionic Encapsulating Horseradish Peroxidase Using Dopamine Flexible Scaffold-Coated Multishell Porous ZIF-8 To Enhance Immunochromatographic Diagnosis. ACS Nano 17, 10748-10759. https://doi.org/10.1021/acsnano.3c02164\u003c/li\u003e\n\u003cli\u003eChen, Y, Ma, J, Yin, X, Deng, Z, Liu, X, Yang, D, Zhao, L, Sun, J, Wang, J, Zhang, D, (2023). Joint-detection of Salmonella typhimurium and Escherichia coli O157:H7 by an immersible amplification dip-stick immunoassay. Biosensors and Bioelectronics 224, 115075. https://doi.org/https://doi.org/10.1016/j.bios.2023.115075\u003c/li\u003e\n\u003cli\u003eZhao, Y, Wang, X, Pan, S, Hong, F, Lu, P, Hu, X, Jiang, F, Wu, L, Chen, Y, (2024). Bimetallic nanozyme\u0026ndash;bioenzyme hybrid material-mediated ultrasensitive and automatic immunoassay for the detection of aflatoxin B1 in food. Biosensors and Bioelectronics 248. https://doi.org/10.1016/j.bios.2023.115992\u003c/li\u003e\n\u003cli\u003eZhang, X, Wu, D, Wu, Y, Li, G, (2021). Bioinspired nanozyme for portable immunoassay of allergenic proteins based on A smartphone. Biosensors and Bioelectronics 172. https://doi.org/10.1016/j.bios.2020.112776\u003c/li\u003e\n\u003cli\u003eDou, L, Bai, Y, Liu, M, Shao, S, Yang, H, Yu, X, Wen, K, Wang, Z, Shen, J, Yu, W, (2022). \u0026lsquo;Three-To-One\u0026rsquo; multi-functional nanocomposite-based lateral flow immunoassay for label-free and dual-readout detection of pathogenic bacteria. Biosensors and Bioelectronics 204. https://doi.org/10.1016/j.bios.2022.114093\u003c/li\u003e\n\u003cli\u003eWu, H, Bu, T, Cao, Y, Wang, Y, Xi, J, Li, M, Li, R, Jia, P, Wang, L, (2023). Double-Enzyme Active Vanadium Nanospheres-Mediated Ratiometric Multicolor Immunosensors for Sensitive Detection of the T-2 Toxin. Analytical Chemistry 95(12), 5275-5284. https://doi.org/10.1021/acs.analchem.2c05197\u003c/li\u003e\n\u003cli\u003eLiu, Z, Xianyu, Y, Zheng, W, Zhang, J, Luo, Y, Chen, Y, Dong, M, Wu, J, Jiang, X, (2018). T-1-Mediated Nanosensor for Immunoassay Based on an Activatable MnO2 Nanoassembly. Analytical chemistry 90, 2765-2771. https://doi.org/10.1021/acs.analchem.7b04817\u003c/li\u003e\n\u003cli\u003eZhu, X, Shan, J, Dai, L, Shi, F, Wang, J, Wang, H, Li, Y, Wu, D, Ma, H, Wei, Q, Ju, H, (2023). PB@PDA nanocomposites as nanolabels and signal reporters for separate-type cathodic photoelectrochemical immunosensors in the detection of carcinoembryonic antigens. Talanta 254. https://doi.org/10.1016/j.talanta.2022.124134\u003c/li\u003e\n\u003cli\u003eLiu, S, Dou, L, Yao, X, Zhang, W, Zhao, B, Wang, Z, Ji, Y, Sun, J, Xu, B, Zhang, D, Wang, J, (2020). Polydopamine nanospheres as high-affinity signal tag towards lateral flow immunoassay for sensitive furazolidone detection. Food Chemistry 315. https://doi.org/10.1016/j.foodchem.2020.126310\u003c/li\u003e\n\u003cli\u003eLai, X, Zhang, G, Zeng, L, Xiao, X, Peng, J, Guo, P, Zhang, W, Lai, W, (2020). Synthesis of PDA-Mediated Magnetic Bimetallic Nanozyme and Its Application in Immunochromatographic Assay. ACS Applied Materials \u0026amp; Interfaces 13, 1413-1423. https://doi.org/10.1021/acsami.0c17957\u003c/li\u003e\n\u003cli\u003eSu, L, Chen, Y, Wang, L, Zhang, H, Sun, J, Wang, J, Zhang, D, (2021). Dual-signal based immunoassay for colorimetric and photothermal detection of furazolidone. Sensors and Actuators B: Chemical 331. https://doi.org/10.1016/j.snb.2020.129431\u003c/li\u003e\n\u003cli\u003eLu, D, Jiang, H, Zhang, G, Luo, Q, Zhao, Q, Shi, X, (2021). An In Situ Generated Prussian Blue Nanoparticle-Mediated Multimode Nanozyme-Linked Immunosorbent Assay for the Detection of Aflatoxin B1. ACS Applied Materials \u0026amp; Interfaces 13, 25738-25747. https://doi.org/10.1021/acsami.1c04751\u003c/li\u003e\n\u003cli\u003eGuo, L, Zhang, Y-J, Yu, Y-L, Wang, J-H, (2020). In Situ Generation of Prussian Blue by MIL-53 (Fe) for Point-of-Care Testing of Butyrylcholinesterase Activity Using a Portable High-Throughput Photothermal Device. Analytical Chemistry 92, 14806-14813. https://doi.org/10.1021/acs.analchem.0c03575\u003c/li\u003e\n\u003cli\u003eFu, G, Sanjay, S T, Zhou, W, Brekken, R A, Kirken, R A, Li, X, (2018). Exploration of Nanoparticle-Mediated Photothermal Effect of TMB-H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e Colorimetric System and Its Application in a Visual Quantitative Photothermal Immunoassay. Analytical Chemistry 90, 5930-5937. https://doi.org/10.1021/acs.analchem.8b00842\u003c/li\u003e\n\u003cli\u003eWang, J, Xu, X, Li, Z, Li, M, Qiu, B, (2024). Sensitive electrochemical detection of benzocaine based on hollow carbon nanobowl modified electrode. Journal of Electroanalytical Chemistry 952. https://doi.org/10.1016/j.jelechem.2023.117893\u003c/li\u003e\n\u003cli\u003eUehara, S A, Andrade, D R, Takata, R, Gomes J\u0026uacute;nior, A V, Vidal, M V, (2019). The effectiveness of tricaine, benzocaine, clove oil, and menthol as anesthetics for lambari-bocarra Oligosarcus argenteus. Aquaculture 502, 326-331. https://doi.org/10.1016/j.aquaculture.2018.12.054\u003c/li\u003e\n\u003cli\u003ePysarevska, S, Dubenska, L, Plotycya, S, \u0026Scaron;vorc, Ľ, (2018). A state-of-the-art approach for facile and reliable determination of benzocaine in pharmaceuticals and biological samples based on the use of miniaturized boron-doped diamond electrochemical sensor. Sensors and Actuators B: Chemical 270, 9-17. https://doi.org/https://doi.org/10.1016/j.snb.2018.05.012\u003c/li\u003e\n\u003cli\u003eLin, S-Q, Jia, B-Z, Luo, W, Wang, H, Lei, H-T, Zhang, W-F, Xu, Z-L, Luo, L, (2023). Controllable formation of polydopamine on carbon dots for ultrasensitive detection of alkaline phosphatase and ratiometric fluorescence immunoassay of benzocaine. Food Chemistry 426. https://doi.org/10.1016/j.foodchem.2023.136582\u003c/li\u003e\n\u003cli\u003eLi, Y, Xie, Y, Wang, Z, Zang, N, Carniato, F, Huang, Y, Andolina, C M, Parent, L R, Ditri, T B, Walter, E D, Botta, M, Rinehart, J D, Gianneschi, N C, (2016). Structure and Function of Iron-Loaded Synthetic Melanin. ACS Nano 10, 10186-10194. https://doi.org/10.1021/acsnano.6b05502\u003c/li\u003e\n\u003cli\u003eChen, Y, Ren, J, Yin, X, Li, Y, Shu, R, Wang, J, Zhang, D, (2022). Vanadium Disulfide Nanosheet Boosts Optical Signal Brightness as a Superior Enzyme Label to Improve the Sensitivity of Lateral Flow Immunoassay. Analytical Chemistry 94, 8693-8703. https://doi.org/10.1021/acs.analchem.2c01008\u003c/li\u003e\n\u003cli\u003eChen, R, Chen, X, Zhou, Y, Lin, T, Leng, Y, Huang, X, Xiong, Y, (2022). \u0026quot;Three-in-One\u0026quot; Multifunctional Nanohybrids with Colorimetric Magnetic Catalytic Activities to Enhance Immunochromatographic Diagnosis. Acs Nano 16, 3351-3361. https://doi.org/10.1021/acsnano.2c00008\u003c/li\u003e\n\u003cli\u003eMa, H, He, Y, Liu, H, Xu, L, Li, J, Huang, M, Wei, Y, (2019). Anchoring of Prussian blue nanoparticles on polydopamine nanospheres as an efficient peroxidase mimetic for colorimetric sensing. Colloids and Surfaces A: Physicochemical and Engineering Aspects 577, 622-629. https://doi.org/https://doi.org/10.1016/j.colsurfa.2019.06.035\u003c/li\u003e\n\u003cli\u003eZhou, L, Chen, J, Li, R, Wei, L, Xiong, H, Wang, C, Chai, K, Chen, M, Zhu, Z, Yao, T, Lin, Y, Dong, C, Shi, S, (2021). Metal‐Polyphenol‐Network Coated Prussian Blue Nanoparticles for Synergistic Ferroptosis and Apoptosis via Triggered GPX4 Inhibition and Concurrent In Situ Bleomycin Toxification. Small 17. https://doi.org/10.1002/smll.202103919\u003c/li\u003e\n\u003cli\u003eLiang, H, Liu, Y, Qileng, A, Shen, H, Liu, W, Xu, Z, Liu, Y, (2023). PEI-coated Prussian blue nanocubes as pH-Switchable nanozyme: Broad-pH-responsive immunoassay for illegal additive. Biosensors and Bioelectronics 219. https://doi.org/10.1016/j.bios.2022.114797\u003c/li\u003e\n\u003cli\u003eHao, W, Huang, Y, Wang, L, Liang, J, Yang, S, Su, L, Zhang, X, (2023). Smartphone-Based Photothermal Lateral Flow Immunoassay Using Rhenium Diselenide Nanosheet. ACS Applied Materials \u0026amp; Interfaces 15, 9665-9674. https://doi.org/10.1021/acsami.2c22616\u003c/li\u003e\n\u003cli\u003eZhang, G, Hu, H, Deng, S, Xiao, X, Xiong, Y, Peng, J, Lai, W, (2023). An integrated colorimetric and photothermal lateral flow immunoassay based on bimetallic Ag\u0026ndash;Au urchin-like hollow structures for the sensitive detection of E. coli O157:H7. Biosensors and Bioelectronics 225. https://doi.org/10.1016/j.bios.2023.115090\u003c/li\u003e\n\u003cli\u003eLee, H, Dellatore, S M, Miller, W M, Messersmith, P B, (2007). Mussel-Inspired Surface Chemistry for Multifunctional Coatings. Science 318, 426-430. https://doi.org/doi:10.1126/science.1147241\u003c/li\u003e\n\u003cli\u003eWang, Z, Xing, K, Ding, N, Wang, S, Zhang, G, Lai, W, (2022). Lateral flow immunoassay based on dual spectral-overlapped fluorescence quenching of polydopamine nanospheres for sensitive detection of sulfamethazine. Journal of hazardous materials 423, 127204. https://doi.org/10.1016/j.jhazmat.2021.127204\u003c/li\u003e\n\u003cli\u003eMeng, Y, Liu, P, Zhou, W, Ding, J, Liu, J, (2018). Bioorthogonal DNA Adsorption on Polydopamine Nanoparticles Mediated by Metal Coordination for Highly Robust Sensing in Serum and Living Cells. ACS nano 12, 9070-9080. https://doi.org/10.1021/acsnano.8b03019\u003c/li\u003e\n\u003cli\u003eYan, X, Song, Y, Wu, X, Zhu, C, Su, X, Du, D, Lin, Y, (2017). Oxidase-mimicking activity of ultrathin MnO\u003csub\u003e2\u003c/sub\u003e nanosheets in colorimetric assay of acetylcholinesterase activity. Nanoscale 9, 2317-2323. https://doi.org/10.1039/C6NR08473G\u003c/li\u003e\n\u003cli\u003eACD Pharmaceuticals, AS (2017) Environmental Assessment in Support of an Import Tolerance for Benzocaine in Food Derived from Atlantic Salmon and Rainbow Trout. ACD Pharmaceuticals. https://www.fda.gov/files/animal%20%26%20veterinary/published/Environmental-Assessment-for-Benzocaine-Import-Tolerance.pdf. Accessed 19 September 2025.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"microchimica-acta","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"miac","sideBox":"Learn more about [Microchimica Acta](https://link.springer.com/journal/604)","snPcode":"604","submissionUrl":"https://submission.springernature.com/new-submission/604/3","title":"Microchimica Acta","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Polydopamine, Prussian Blue, Nanozyme, Benzocaine, Dual-Mode Assay, Photothermal Immunoassay, Colorimetric Immunoassay","lastPublishedDoi":"10.21203/rs.3.rs-7663415/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7663415/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eA dual-mode immunosensor based on polydopamine@Prussian blue nanoparticles (PDA@PB NPs) was developed for rapid and sensitive detection of benzocaine (BZC) in aquatic food. PDA@PB NPs possess excellent peroxidase-like activity, photothermal conversion efficiency, and antibody-binding capacity, enabling simple, crosslinker-free antibody immobilization. Upon target recognition, the sensor produces a distinct blue color via TMB\u0026ndash;H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e oxidation and a robust near-infrared photothermal signal, allowing dual-mode quantitative analysis. Under optimized conditions, the sensor showed a linear range of 0.01\u0026ndash;1000 ng/mL, with detection limits of 0.33 ng/mL (colorimetric) and 0.82 ng/mL (photothermal). The integration of colorimetric and photothermal outputs improves detection accuracy, reduces matrix interference, and eliminates the need for complex instrumentation. Validation with spiked real samples confirmed its reliability and applicability. This portable, cost-effective immunosensor offers a promising tool for on-site monitoring of BZC residues in aquatic products, contributing to enhanced food safety surveillance.\u003c/p\u003e","manuscriptTitle":"Dual-Mode Photothermal and Colorimetric Immunosensor Based on Polydopamine@Prussian Blue Nanocomposite for Sensitive Detection of Benzocaine","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-11-05 09:47:26","doi":"10.21203/rs.3.rs-7663415/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-09-27T19:13:57+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-09-23T12:10:02+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-09-22T23:56:51+00:00","index":"","fulltext":""},{"type":"submitted","content":"Microchimica Acta","date":"2025-09-20T07:53:43+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"microchimica-acta","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"miac","sideBox":"Learn more about [Microchimica Acta](https://link.springer.com/journal/604)","snPcode":"604","submissionUrl":"https://submission.springernature.com/new-submission/604/3","title":"Microchimica Acta","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"1f20b449-07f3-412e-8cc1-323c826446ba","owner":[],"postedDate":"November 5th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2026-02-09T16:04:12+00:00","versionOfRecord":{"articleIdentity":"rs-7663415","link":"https://doi.org/10.1007/s00604-026-07881-3","journal":{"identity":"microchimica-acta","isVorOnly":false,"title":"Microchimica Acta"},"publishedOn":"2026-02-07 15:59:37","publishedOnDateReadable":"February 7th, 2026"},"versionCreatedAt":"2025-11-05 09:47:26","video":"","vorDoi":"10.1007/s00604-026-07881-3","vorDoiUrl":"https://doi.org/10.1007/s00604-026-07881-3","workflowStages":[]},"version":"v1","identity":"rs-7663415","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7663415","identity":"rs-7663415","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}