A multichromatic colorimetric detection method for Vibrio parahaemolyticus based on Fe3O4-Zn-Mn nanoenzyme and dual substrates

ABSTRACT A successful development of a dual-substrate colorimetric system for rapid and multicolorimetric semi-quantification of Vibrio parahaemolyticus (V. parahaemolyticus) has been achieved. The enzymatic activity of Fe3O4-Zn-MnO2-aptamer (Fe3O4-Zn-Mn-Apt) catalysts can induce the oxidation of 3,3′,5,5′-tetramethylbenzidine (TMB) to form oxidized TMB (TMB+ with a bluish green color) and ortho-phenylenediamine (OPD) to form oxidized OPD (with yellow color), resulting in a three-color comparison due to the complementary nature of bluish green and yellow. The absorbance value of TMB+ at 652 nm is used for the determination of V. parahaemolyticus. After optimizing the reaction conditions, the developed dual-substrate colorimetric method exhibits high sensitivity for V. parahaemolyticus detection (limit of detection of 1.12 cfu mL−1) and a linear range of 0–1 × 104 cfu mL−1 (R 2 = 0.9934). Additionally, as the concentration of V. parahaemolyticus increases, the reaction solution changes from bluish green to green and then to yellow, enabling semi-quantitative detection by visual observation with a visual detection limit of 10 cfu mL−1. Furthermore, the developed dual-substrate colorimetric method demonstrates excellent selectivity for V. parahaemolyticus detection and satisfactory recovery rates when applied to the determination of V. parahaemolyticus in food samples. IMPORTANCE The Fe3O4-Zn-Mn nanomimetic enzyme demonstrates significant importance in dual-substrate colorimetric detection for V. parahaemolyticus, owing to its enhanced sensitivity, selectivity, and rapid detection capabilities. Additionally, it offers cost-effectiveness, portability, and the potential for multiplex detection. This innovative approach holds promise for improving the monitoring and control of V. parahaemolyticus infections, thereby contributing to advancements in public health and food safety.

of V. parahaemolyticus infection is on the rise (6).Particularly in warm climatic conditions, bacterial proliferation accelerates, exacerbating food safety concerns.Consequently, the detection and control of VP have become increasingly crucial.
Currently, commonly employed methods for V. parahaemolyticus detection encom pass traditional culture-based techniques and molecular biology methods (7).Tradi tional culture-based methods involve inoculating samples (such as seafood and water sources) onto specific culture media and employing bacterial growth characteristics for screening and identification (8).This method necessitates a certain amount of time for bacterial cultivation, typically requiring 24-48 hours to yield results (9).Although traditional culture-based methods are widely utilized, they possess certain limitations, including the need for extended time and stringent bacterial cultivation conditions.Common molecular biology methods encompass polymerase chain reaction, real-time fluorescence polymerase chain reaction, and gene sequencing, among others (10).These techniques enable rapid and accurate detection of V. parahaemolyticus, facili tating strain typing and strain identification.Molecular biology methods and immu nological approaches exhibit high sensitivity and specificity, yet they still require expensive laboratory equipment and intricate operational procedures (11).In compar ison, rapid colorimetric detection techniques offer advantages such as simplicity of operation, cost-effectiveness, and intuitive result interpretation (12,13).V. parahaemo lyticus detection holds significant implications in terms of food safety and public health.Early detection and control of V. parahaemolyticus infection can reduce disease transmission and occurrence, safeguarding food safety and public well-being (14).Therefore, strengthening research on V. parahaemolyticus detection, improving detection methods and technologies, and enhancing detection accuracy and sensitivity were of paramount importance (15).The research background in bacterial detection focuses on the utilization of nanoprobes, which are engineered particles with high sensitivity and specificity (16).These nanoprobes have the potential to accurately identify and analyze the presence of bacteria (17), thereby enhancing the efficiency and precision of the bacterial detection method (18).Dual-substrate colorimetry is a commonly used analytical method for quantitatively measuring the concentration of specific substan ces in a solution (19,20).This method is based on the chemical reaction between a substrate and the target substance, resulting in observable color changes.The research background also involves the field of nanotechnology.In recent years, the development of nanotechnology has provided new opportunities for dual-substrate colorimetry (2).By harnessing the unique properties of nanomaterials, such as nanoenzyme activity, the sensitivity and selectivity of dual-substrate colorimetry can be enhanced (21).Therefore, researchers are exploring the application of nanomaterials in dual-substrate colorimetry to further improve its performance and expand its application range.Dual-substrate colorimetry, as a widely used analytical technique (22), holds great potential in fields such as analytical chemistry, biomedical sciences, and environmental monitoring (23,24).Researchers are continuously exploring new methods and materials to enhance the performance and application scope of dual-substrate colorimetry, providing better support for scientific research and practical applications.
In this study, a Fe 3 O 4 -Zn-Mn-Apt probe was developed through the preparation of a Fe 3 O 4 -Zn-Mn conjugated V. parahaemolyticus aptamer with oxidase activity, enabling rapid colorimetric detection of Vibrio parahaemolyticus.As depicted in Fig. 1, the pre-prepared Fe 3 O 4 -Zn-Mn-Apt was initially subjected to a rotational reaction with the V. parahaemolyticus bacterial solution for a duration of 30 min.Subsequently, magnetic separation was performed to eliminate the supernatant, followed by the addition of ortho-phenylenediamine (OPD) and 3,3′,5,5′-tetramethylbenzidine (TMB) solutions for colorimetric reaction.Based on the outcome analysis, varying concentrations of the V. parahaemolyticus bacterial solution exhibited diverse degrees of inhibition on the enzymatic activity of Fe 3 O 4 -Zn-Mn.Experimental findings revealed that the nanozyme oxidized OPD initially, and OPD oxidizes TMB.Consequently, under different concen trations of the VP bacterial solution, a multitude of colors would manifest, thereby accomplishing a multicolor spectrophotometric detection of V. parahaemolyticus.In summary, rapid colorimetric detection techniques offer advantages such as simplicity of operation, cost-effectiveness, and intuitive result interpretation.The development of a Fe 3 O 4 -Zn-Mn-Apt probe with oxidase activity and the rapid colorimetric detection of V. parahaemolyticus can be realized.This method holds promising potential for widespread application in areas such as food safety monitoring and medical diagnostics.

Assay principle of the developed VP colorimetric detection sensor
The feasibility of this approach was validated through several comparative experiments.As depicted in Fig. 2, the catalytic oxidation of OPD to oxOPD by Fe 3 O 4 -Zn-Mn-Apt exhibited a broad absorption band within the range of 350-600 nm, affirming its consistent oxidative effect on OPD.Additionally, Fe 3 O 4 -Zn-Mn-Apt also catalyzed the oxidation of TMB, as evidenced by the presence of UV-vis absorption peaks at 370 and 652 nm for TMB + .When both OPD and TMB were employed as substrates, absorption peaks for oxOPD and TMB + were observed.Furthermore, an increase in absorbance for oxOPD and a decrease in absorbance for TMB + were observed, indicating that TMB + might undergo further oxidation by OPD, generating more oxOPD, while simultaneously being reduced back to TMB.Hence, it can be inferred that this is the key mechanism underlying the generation of the multicolor chromogenic response.In the presence of V. parahaemolyticus bacterial solution, a significant decrease in the absorption peak at 650 nm was observed, accompanied by distinct color changes.Therefore, this multicolor chromogenic system holds promise for the detection of V. parahaemolyticus.

Characteristics
We performed a comprehensive characterization of the synthesized magnetic nano zyme Fe 3 O 4 -Zn-Mn, encompassing a series of systematic investigations.As depicted in Fig. 3A, the synthesized Fe 3 O 4 nanoparticles exhibited a uniform and well-dispersed spherical porous structure, with an average diameter of 155 nm, consistent with previous reports.Prior to the synthesis of Fe 3 O 4 -Zn-Mn, a series of characterizations were performed.Figure 3B reveals a similar morphology for the MnO 2 nanoparticles, with numerous Zn nanoparticles uniformly distributed on the surface of the membrane.Furthermore, elemental mapping and analysis using transmission electron microscopy (TEM)-energy-dispersive X-ray spectroscopy (EDS) confirmed strong signals of Zn and Mn (Fig. S1).It is evident that the Zn signal originates from the Zn nanoparticles, while the Mn signal arises from the MnO 2 nanoparticles.Upon coating the Zn-MnO 2 onto the surface of Fe 3 O 4 (Fig. 3C), the average size increased to 195 nm.EDS analysis was employed to determine the elemental composition of the prepared Fe 3 O 4 -Zn-Mn composite material (Fig. 4).The TEM elemental mapping of Fe 3 O 4 -Zn-Mn demonstrated the uniform distribution of Fe, Zn, and Mn throughout the spherical structure, indicating the presence of Zn-Mn on the encapsulated Fe 3 O 4 surface.Additionally, the elemental analysis results (Fig. S2) revealed simultaneous peaks for Fe, Zn, and Mn elements.These findings confirm the successful integration of Zn-MnO 2 with Fe 3 O 4 .Subsequently, TEM imaging of the Fe 3 O 4 -Zn-Mn-Apt probe for V. parahaemolyticus captured (Fig. 3D) clearly demonstrates the successful capture of V. parahaemolyticus by the Fe 3 O 4 -Zn-Mn-Apt probe.The dual-substrate chromogenic reaction was further analyzed using Fourier-transform infrared (FTIR) spectroscopy (Fig. S3).The obtained spectrum revealed the presence of an -NH 2 functional group at 3,454 cm −1 , attributed to the stretching vibration of O-H.Additionally, peaks at 2,358 cm −1 indicated the presence of -C≡N, while the bending vibrations of C-O were observed at 1,484 cm −1 .The presence of -C=Cbonds was confirmed by the peak at 1,634 cm −1 , and the -C-C-bonds were identified by the peak at 1,083 cm −1 (19,20).These spectral features are likely responsible for the multicolor reaction observed in the dual-substrate system.

Optimizing conditions for detecting V. parahaemolyticus
In order to optimize the performance of the constructed V. parahaemolyticus detec tion system, several factors that could potentially affect the experimental results were investigated.Firstly, the optimal capture performance of the Fe 3 O 4 -Zn-Mn-Apt probe toward V. parahaemolyticus was optimized, as shown in Fig. S4.With an increase in the concentration of Fe 3 O 4 -Zn-Mn-Apt, the quantity of V. parahaemolyticus in the super natant gradually decreased, while the capture efficiency sharply increased.When the concentration of Fe 3 O 4 -Zn-Mn-Apt reached 0.5 mg mL −1 , the capture efficiency reached a remarkable 94.85%, indicating that the majority of V. parahaemolyticus in the system could be captured.Therefore, a concentration of 0.5 mg mL −1 of Fe 3 O 4 -Zn-Mn-Apt was chosen for subsequent experiments.
For the Fe 3 O 4 -Zn-Mn-Apt probe concentration of 0.5 mg mL −1 , the influence of TMB concentration on the catalytic effect of Fe 3 O 4 -Zn-Mn was studied, as depicted in Fig. S5A.The absorbance at 652 nm gradually increased with an increase in TMB concentration, but the increasing trend became less pronounced after reaching 8 mM.Subsequently, to construct a multicolor chromogenic system, the impact of OPD on the detection performance was investigated.The detection of V. parahaemolyticus was found to be sensitive while ensuring multicolor changes in Fe 3 O 4 -Zn-Mn solutions of different concentrations.As shown in Fig. S5B, the solution stabilized at approximately 0.15 mM OPD.Additionally, Fig. S6 displayed the color changes in the solution under different OPD concentrations (0.05, 0, 0.10, 0.15, 0.20, and 0.25 mM) and different Fe 3 O 4 -Zn-Mn concentrations (0.1, 0.2, 0.3, 0.4, and 0.5 mg).It can be observed that when the OPD concentration was 0.05 mM, the solution only exhibited a bluish green change.As the OPD concentration increased, the proportion of yellow in the solution gradually increased.When the OPD concentration reached 0.10 mM, the solution displayed a single yellow change at different depths.Based on the color contrast chart, under the conditions of 0.10, 0.15, and 0.20 mM OPD, the solution was likely to exhibit multicolor changes.Therefore, considering the results in this figure, an OPD concentration of 0.15 mM was selected to meet the sensitivity requirements for V. parahaemolyticus detection.
To achieve the maximum benefit of the color reaction, the same amount of TMB was subjected to different reaction times with Fe 3 O 4 -Zn-Mn catalysis.It was found that the absorbance increased continuously with time and reached a plateau at 5 min, as shown in Fig. S5C.Finally, Fe 3 O 4 -Zn-Mn-Apt was incubated with the same V. parahaemolyticus solution for different durations, followed by the addition of the same amount of TMB for color development.The optimal incubation time was determined to be 30 min, as depicted in Fig. S5D.In summary, based on a comprehensive analysis of the experimental results, subsequent experiments were conducted using Fe 3 O 4 -Zn-Mn-Apt (0.5 mg), TMB (8 mM), OPD (0.15 mM), a color development time of 5 min, and an incubation time of 30 min.

Polycolorimetry for the detection of V. parahaemolyticus
Based on the dual-substrate colorimetric method, a variety of colors are generated in the presence of Fe 3 O 4 -Zn-Mn, catalyzed to produce multicolor.Exploiting this phenom enon, a convenient multicolor comparison method has been established for quantitative and semi-quantitative detection of V. parahaemolyticus.When V. parahaemolyticus is present, certain sites on the surface of Fe 3 O 4 -Zn-Mn-aptamer are occupied, resulting in a gradual decrease in the production of TMB 2+ with increasing V. parahaemolyticus concentrations.Consequently, the solution exhibits a multicolor optical signal.These colors correspond to different concentrations of V. parahaemolyticus and can be visually distinguished.As depicted in Fig. 5A, in the absence of V. parahaemolyticus, all the Fe 3 O 4 -Zn-Mn-aptamer surfaces catalyze TMB to generate TMB + , displaying a bluish green color.As the concentration of V. parahaemolyticus increases, the absorbance at 652 nm gradually decreases.Additionally, the color of the corresponding solution transitions from bluish green to green and then to yellow, enabling semi-quantitative assessment by the naked eye.The visual detection limit for V. parahaemolyticus is 10 cfu mL −1 , indicating a distinct color difference compared to the control sample.In Fig. 5B, the linear regression equation is Y = −0.2215X+ 0.9405, with R 2 = 0.9934, where Y represents absorbance, X is LogC V. parahaemolyticus , and C denotes the concentration of V. parahaemo lyticus (cfu mL −1 ).The limit of detection (LOD) for this method is 1.12 cfu mL −1 (LOD = 3σ, where σ is the standard deviation of the blank sample).The multicolor comparison method proposed in this study exhibits outstanding performance in terms of detection time, steps, and limits compared to other reported methods (Table S2).
Therefore, these results demonstrate the feasibility of utilizing the dual-substrate multicolor system for V. parahaemolyticus analysis.The method developed in this study possesses a wider linear range and lower LOD detection advantage compared to other methods.Furthermore, the establishment of the dual-substrate colorimetric system endows it with the capability of multicolor comparison.In summary, the aforementioned experimental results highlight the exceptional performance of the developed method in terms of detection time, steps, and limits compared to other reported methods.

Selective research
The selective and interference studies were conducted to develop a detection method for V. parahaemolyticus.Four common pathogens, including Staphylococcus aureus, Listeria monocytogenes, Salmonella typhimurium, and Escherichia coli O157:H7 (each at a concentration of 10 3 mg mL −1 ), were chosen as potential coexisting bacteria.Phosphate buffer solution (PBS) samples were used as reagent blanks.Under optimal conditions, the interference bacteria and V. parahaemolyticus were separately added to the detection system, and the detection was performed using a UV-vis spectrophotometer.The blank group, the four common bacteria wells, and the mixed bacteria well all appeared bluish green, indicating that the surface sites of Fe 3 O 4 -Zn-Mn-aptamer were not captured.In the presence of V. parahaemolyticus alone or in combination with other bacteria, the color of the test tube turned light green, and significant changes in absorbance were observed in samples containing interference bacteria (Fig. 6).Conversely, when V. parahaemolyticus was present, the absorbance change in the sample tube was minimal, indicating that the presence of these interference bacteria had little impact on the detection results.These results demonstrate that the method exhibits high specificity for the test tube and can differentiate it from other bacteria.In summary, the above experimental results highlight the high specificity of the developed method for the test tube and its ability to distinguish it from other bacteria.

Stability research
A stability analysis was conducted to assess the absorbance and color stability of the test samples over a period of 2 hours.The samples were monitored at regular intervals to evaluate any changes in absorbance and color.As Fig. S7 shows, during the stability analysis, it was observed that the absorbance values remained consistent and did not show significant fluctuations over the 2-hour period.This indicates that the test system maintained its optical properties and provided reliable measurements throughout the duration of the experiment.Furthermore, the color of the samples remained stable and did not exhibit any noticeable changes during the stability analysis.This suggests that the dual-substrate to detect V. parahaemolyticus retained its ability to generate the desired color response, ensuring the accuracy and reliability of the detection method.Overall, the stability analysis demonstrated that the developed method exhibits excellent stability in terms of absorbance and color over a 2-hour period.This stability is crucial for obtaining reliable and consistent results in practical applications.

Real sample analysis
To further investigate the potential application of the developed multicolor method in V. parahaemolyticus food analysis, common food samples (mackerel and seaweed) contaminated with V. parahaemolyticus were tested using the colorimetric assay to determine the practicality of the method.The food samples were processed according to the literature and tested following the same procedure.Additionally, V. parahaemolyticus at 7 and 70 cfu mL −1 levels were measured in triplicate in the samples.The summarized results are presented in Table 1, with recovery rates within the range 89.14%-115.86%and RSD within the range 3.4%-13.89%(n = 3).These results indicate the potential application of the method for V. parahaemolyticus detection in real food samples.In conclusion, the multicolor method shows promise for the analysis of V. parahaemolyticus.By successfully detecting and quantifying V. parahaemolyticus in common food samples, the method demonstrates its practicality and potential for real-world applications.

Conclusions
This study presents a sensitive, multicolor, and selective spectrophotometric method for the detection of V. parahaemolyticus based on a dual-substrate colorimetric system.The method enables high sensitivity and selective detection of V. parahaemolyticus, exhibiting three-color changes in the presence of different concentrations of the bacteria.This allows for both quantitative detection using UV-vis spectrophotometry and semi-quantitative detection by visual observation.The experimental results demonstrate that this sensing method offers a wide linear range, low detection limit, high selectivity, and excellent stability for V. parahaemolyticus detection.Notably, the sensor shows great potential for detecting V. parahaemolyticus in food samples.This research provides a novel reference for multicolor detection of V. parahaemolyticus and opens up possibili ties for future exploration of simultaneous multitarget detection in clinical diagnostics, environmental chemistry, and food safety.
All the UV-vis spectra were obtained using the SHIMADZU UV-2600i UV-vis spectro photometer (SHIMADZU, Japan).Fourier-transform infrared spectroscopy from 4,000 to 500 cm −1 was recorded using a Nicolet 6700 FTIR spectrometer (Thermo Inc., USA).All the solutions were prepared through sonication in the ultrasonic cleaner (Kunshan Ultrasonic Instrument Co., Ltd., Kunshan, China).

FIG 1
FIG 1 Schematic diagram of the proposed colorimetric assay for the detection of V. parahaemolyticus.(A) The incubation process; (B) the procedures of colorimetry method to detect V. parahaemolyticus.

FIG 2
FIG 2 Feasibility of the developed method.

FIG 5 (
FIG 5 (A) The UV-vis spectrum corresponding to the colorimetric detection of Vibrio parahaemolyticus is presented, accompanied by an illustrative photograph showcasing the corresponding array of colors.(B) Construction of a standard curve using absorbance values at 652 nm and logarithm of TMB concentration (LogC V. parahaemolyticus ).

FIG 6
FIG6 Selectivity of V. parahaemolyticus detection system.

TABLE 1
The recoveries and RSD values of detecting bacteria in spiked samples (x̄ ± s, n = 3)