MEASUREMENT

Our project aims to develop a novel ​​four-in-one nanoflower system​​ with dual-signal amplification capabilities, engineered to achieve ​​ultrasensitive, rapid, and low-cost bacterial detection​​. This integrated platform combines ​​specific bacterial recognition​​, ​​catalytic signal generation​​, and ​​dual amplification mechanisms​​ within a single nanostructure, enabling highly efficient and accurate pathogen identification.
To ensure the ​​reliability and reproducibility​​ of the detection method, we established a standardized experimental protocol with rigorously repeatable procedures. This approach guarantees consistent performance across multiple tests and operational batches.
A comprehensive evaluation of the nanoflower system was conducted, including:
1.Synthesis of the four-in-one nanoflowers;
2.Catalytic performance assessment-incorporating temperature and reaction time optimization);
3.Bacterial detection capability validation—including ​​limit of detection (LOD)​​, ​​relative standard deviation (RSD)​​, ​​specificity​​, real-sample testing, and ​​storage stability​​.
This systematic development and validation framework ensures that the system is both experimentally robust and practically applicable for complex biological detection scenarios.

Background
The development of this ​​four-in-one nanoflower system​​ represents an advanced approach in the field of ​​bacterial detection biosensors​​. It is designed to integrate multiple functional components into a single nanostructure to achieve ​​ultrasensitive, rapid, and low-cost​​ identification of specific pathogens, such as E. coli. Traditional methods for bacterial detection often face limitations in sensitivity, speed, and complexity, especially in resource-limited settings. This system addresses these challenges by combining ​​specific biological recognition​​, ​​enzymatic catalysis​​, and ​​nanozyme-enhanced signal amplification​​ within a unified platform. The use of hybrid organic-inorganic nanoflowers, inspired by natural biomineralization processes, allows for high surface area, enhanced stability, and efficient co-immobilization of bioactive components, significantly improving detection performance [1].

Principle
The measurement principle of the four-in-one nanoflower system is based on a ​​cascade signal amplification mechanism​​ triggered by the specific recognition of target bacteria, followed by a colorimetric reaction that produces a visible output. The process can be broken down into several key steps:
1.​​Specific Bacterial Recognition​​
Phage-derived binding proteins (PBPs) can selectively bind to surface antigens of E. coli, which ensures the high specificity of the detection.
2.​​Signal Catalysis and Amplification​​
Once the target bacteria are captured, two catalytic components work synergistically to amplify the detection signal:
•​​Horseradish Peroxidase (HRP)​​: An enzyme that catalyzes the oxidation of the substrate 3,3',5,5'-Tetramethylbenzidine (TMB) in the presence of hydrogen peroxide (H₂O₂), producing a ​​blue-colored product​​.
•​​Gold-Platinum Nanozyme (AuPt)​​: This synthetic nanoparticle exhibits ​​intrinsic peroxidase-like activity​​, further enhancing the catalytic conversion of TMB. The combination of HRP and AuPt achieves ​​dual signal amplification​​, significantly boosting sensitivity and enabling detection even at low bacterial concentrations.
3.​​Signal Detection and Readout​​
The color change resulting from TMB oxidation can be quantified using simple methods:
•Visual Inspection​​: A clear color shift can be observed with the naked eye for qualitative assessment.
•RGB Analysis Based on Smartphones: To obtain quantitative results, smartphone applications can be used to analyze the RGB values of the reaction products.
4.​​Structural Support and Enhancement​​
The ​​calcium phosphate matrix (CaHPO₄)​​ forms a porous, three-dimensional nanoflower structure. This not only provides a ​​high surface area​​ for efficient immobilization of PBPs, HRP, and AuPt but also facilitates better ​​substrate diffusion​​ and ​​bacterial binding​​, enhancing overall detection efficiency.

Protocols
1.AuPt Nanoparticle Solution Preparation
Materials:
Magnetic stirrer with heating capability, Analytical balance, ddH2O, Chloroplatinic acid (H₂PtCl₆), Chloroauric acid (HAuCl₄), Trisodium citrate (C6H5Na3O7), Calcium chloride (CaCl2), Potassium Carbonate (K2CO3), 10 × PBS.
Procedures:
(1)AuPt Nanoparticle Synthesis: In a 100 mL flask, combine: ddH2O: 45 mL, Chloroplatinic acid: 0.38 mL (10 mg/mL), Chloroauric acid: 0.3 mL (10 mg/mL).
(2)Heating and mixing: Stir the mixture continuously using a magnetic stirrer. Heat to boiling and maintain for 10 min.
(3)Reduction step: Add 0.2 mL of trisodium citrate solution (114.1 mg/mL) to the boiling mixture. Allow the reaction to proceed under condensation reflux for 30 min.
(4)Cooling step: Gradually cool the resulting AuPt nanoparticle solution to ambient temperature. Store at 4°C for future use.
(5)Supporting Reagent Preparation: Prepare the following reagents according to the specified concentrations and volumes.

Reagent	Concentration	Volume (mL)	Preparation Notes
K₂CO₃	0.1 M	100	Dissolve 1.38 g in ddH2O
Trisodium Citrate	114.1 mg/mL	100	Dissolve 11.41 g in ddH2O
CaCl₂	200 mM	100	Dissolve 2.94 g in ddH2O
0.5 × PBS	0.5 ×	500	Dilute 10× PBS stock 1:20 with ddH2O
0.5 × PBS (0.05% Tween-20)	contain 0.05% Tween-20	200	Add 100 μL Tween-20 to 200 mL 0.5× PBS
Chloroauric acid	10 mg/mL	3.8	Dissolve 38 mg in ddH2O
Chloroplatinic acid	10 mg/mL	3	Dissolve 30 mg in ddH2O

(6)Storage: AuPt nanoflowers store at 4°C, protected from light. Buffer solutions store at 4°C, check for precipitation before use. Metal salt solutions store at room temperature.

2.Synthesis of HRP-PBP-CaHPO₄@AuPt Nanoflowers
Materials:
2 mL microcentrifuge tubes, Centrifuge, Vortex mixer, pH meter, HRP (Horseradish Peroxidase), PBP (Phage-derived Binding Proteins, include gp17, TFP), 0.5 × PBS, CaCl₂ solution (200 mM), AuPt nanoparticle solution, K₂CO₃ solution (0.1 M), Pipettes and sterile tips
Procedure:
(1)HRP-PBP-CaHPO₄ Nanoflower Synthesis: In a 2 mL microcentrifuge tube, combine HRP solution 250 μL (2 mg/mL), PBP solution 100 μL (1 mg/mL), 0.5 × PBS 1 mL (5 mM, pH 7.4). Add 20 μL of CaCl₂ solution (200 mM) to the mixture. Mix gently by pipetting to ensure uniform distribution. Incubate at room temperature for 18 h to allow biomineralization and nanoflower formation.
(2)Purification of nanoflowers: Centrifuge the reaction mixture to pellet the nanoflowers. Wash five times with PBS to remove unreacted components. After each wash, gently vortex to resuspend the pellet. Resuspend the purified HRP-PBP-CaHPO₄ nanoflowers in 200 μL PBS.
(3)AuPt Nanoparticle Coupling: pH adjustment for electrostatic coupling, take 500 μL of AuPt nanoparticle solution (from previous synthesis). Adjust pH to 8.0 using K₂CO₃ solution to optimize electrostatic interactions. Add 200 μL of HRP-PBP-CaHPO₄ nanoflowers (1 mg/mL) to the pH-adjusted AuPt solution.
(4)Incubate at room temperature for 30 min under static conditions to allow electrostatic adsorption.
(5)Purification of hybrid nanoflowers: Centrifuge at 12,000 rpm for 2 min at 4°C to pellet the hybrid nanoflowers. Wash the pellet with PBS to remove unbound AuPt nanoparticles. Resuspend the HRP-PBP-CaHPO₄@AuPt nanoflowers in 200 μL PBS.
(6)Storage: Store the final hybrid nanoflowers at 4°C for future use. Label with preparation date and concentration.

3.Transmission Electron Microscopy
Apparatus and Materials:
Nanoflower sample, Deionized (DI) water, TEM Grids, Transmission Electron Microscope: Talos F200x TEM (Thermo Fisher Scientific, USA) or an equivalent analytical TEM, Sonicator, Fine-tipped tweezers for handling TEM grids.
Procedure:
•Sample preparation
(1)Sample Dilution: Dilute the raw nanoflower solution with deionized (DI) water at a 1:2 ratio.
(2)Dispersion: Briefly sonicate the diluted sample for 30-60 seconds in a bath sonicator to ensure a homogeneous suspension and break up large aggregates before deposition.
(3)Sample Deposition: Using fine tweezers, hold a TEM grid (carbon film facing up). Pipette 5 µL of the diluted and sonicated nanoflower solution and carefully drop it onto the center of the carbon film.
(4)Drying: Place the grid inside a covered petri dish to protect it from dust and allow it to dry completely at room temperature for at least 1-2 hours or until all solvent has evaporated.
(5)TEM Imaging: Carefully load the prepared TEM grid into a suitable TEM holder according to the instrument’s specific operating manual.

Results
The synthesized ​​four-in-one nanoflowers​​ were subjected to detailed microstructural and compositional characterization using ​​transmission electron microscopy (TEM)​​. TEM analysis confirmed the successful formation and uniform dispersion of ​​gold-platinum (AuPt) nanozyme particles across the nanoflower scaffold. These metallic nanoparticles were consistently anchored across the calcium phosphate-based matrix via ​​electrostatic adsorption and surface complexation. This homogeneous distribution is critical for maximizing catalytic accessibility and enhancing signal amplification efficiency in subsequent biosensing applications.
Discussion
As the carrier for bacterial detection, the uniformity of nanoflowers is of vital importance. Their size and concentration will affect the detection outcome. Therefore, we need to pay attention to the control of reaction conditions during the preparation of nanoflowers, including the regulation of parameters such as temperature, pH value, ionic concentration, and reaction time. This will improve the reproducibility between batches.

Background
Traditional detection methods often struggle to strike a balance among sensitivity, speed, cost-effectiveness, and ease of operation, especially in field testing or in resource-poor environments. Catalytic systems based on nanoenzymes have emerged as a promising solution to this problem, as they can achieve signal amplification effects beyond those of natural enzymes, and have advantages in stability and tunability.
It is crucial to conduct the assessment under different time and temperature conditions. This assessment is based on the understanding that the biosensors used in practical applications will be exposed to various environments. Confirming that the nanoflowers maintain high activity under various environmental temperatures and can complete the detection within clinically relevant timeframes is a key step in proving their practical application value in rapid bacterial detection.

Principle
The measurement principle revolves on quantifying the ​​peroxidase-like catalytic activity​​ of the nanoflowers by employing a classic colorimetric reaction. The oxidation of the colorless substrate ​​3,3',5,5'-tetramethylbenzidine (TMB)​​ in the presence of ​​hydrogen peroxide (H₂O₂)​​ generates a blue-colored product (oxidized TMB), the intensity of which is directly proportional to the catalytic activity of the nanoflowers.
This reaction can be schematically summarized as:
  ​​Nanoflower (Catalyst) + H₂O₂ (Oxidant) + TMB (Chromogen) → Oxidized TMB (Blue) + H₂O​

Protocols
•Materials:
Microplate reader, 96-well microplates, Incubators, Timer, AuPt nanoparticles, HRP-PBP-CaHPO₄ nanoflowers, HRP-PBP-CaHPO₄@AuPt nanoflowers, TMB solution, H₂O₂ solution, pipettes and tips, Microcentrifuge tubes.

•Procedure:
(1)Comparative Catalytic Activity Assessment
Replicates: All experiments performed in triplicate
Test samples: Three different catalyst systems
  a.AuPt nanoparticles alone
  b.HRP-PBP-CaHPO₄ nanoflowers
  c.HRP-PBP-CaHPO₄@AuPt hybrid nanoflowers
Catalytic Activity Assay: Reaction mixture preparation: For each well, add the following components in order 10 μL of respective nanoflower suspension. 50 μL TMB solution. 150 μL H₂O₂ solution.
Reaction conditions: Room temperature (25°C). Reaction 30 min. Gently mix by pipetting or plate shaking.
Detection and documentation: Measure absorbance at 650 nm using a microplate reader. Take photos with smartphone to document color development. Record all measurements in triplicate.
(2)Reaction Condition Optimization
  a.Temperature Optimization: Test catalytic activity at 4°C, 25°C, 30°C, 37°C, 50°C. Reaction time: 30 min at each temperature. Place reaction plates in appropriate temperature-controlled environments.
  b.Time Course Optimization: Monitor catalytic activity at 5, 10, 15, 20, 25, 30 min.
  Use the same reaction system with optimal temperature from previous experiment. Take absorbance readings at each time point. Document color development progression.

Results
After successfully obtaining the four-in-one nanoflowers, we measured and compared their catalytic performance. The experimental results indicated that, compared to individual AuPt nanoparticles and the three-in-one nanoflowers (HRP-PBP-CaHPO₄), the four-in-one nanoflowers (HRP-PBP-CaHPO₄@AuPt) exhibited the highest catalytic performance (Figure 2). This is attributed to the surface of the four-in-one nanoflowers being able to adsorb a large number of AuPt nanoparticles, endowing them with a dual signal amplification function. This also suggests that we can potentially use trace amounts of the four-in-one nanoflowers to achieve significant detection results.

The catalytic activity of the synthesized TFP-nanoflowers and gp17-nanoflowers was then assessed under varying conditions to determine optimal performance. For time optimization, reactions were conducted at fixed temperature (25°C) with TMB/H₂O₂ substrate, and absorbance at 650 nm was measured at different time points (5, 10, 15, 20, 25, 30 min). For temperature optimization, reactions were carried out for a fixed duration (30min) at different temperatures (4°C, 25°C, 30°C, 37°C, 50°C). All measurements were performed in triplicate. The results demonstrate that both nanoflower constructs maintain optimal catalytic activity at physiological temperature (37°C) with reaction completion within 20-30 minutes (Figure 3). These optimized conditions will be applied in subsequent bacterial detection assays to ensure maximum sensitivity and reliability.

Discussion
The experimental results show that the four-component nano-flower (HRP-PBP-CaHPO₄@AuPt) is superior to the AuPt nanoparticles and the three-component nano-flower (HRP-PBP-CaHPO₄).
This can be attributed to two main factors:
a)The synergistic effect between HRP enzyme and AuPt nanozyme
b)The three-dimensional nano-flower-like structure has an extremely high surface area, providing reaction sites
The optimization experiments demonstrated that the nanoflowers exhibited the best activity at physiological temperature (37°C) and reached the maximum signal intensity within 20 to 30 minutes. This characteristic is highly suitable for practical biosensing applications as it conforms to the conditions of many biological samples and meets the requirements for rapid diagnosis. Moreover, their activity remains stable across different temperature ranges, which indicates a certain degree of stability and is beneficial for practical applications.

Background
During the development and validation of diagnostic testing methods, especially for bacterial detection, it is crucial to establish reliable and stable performance characteristics, which can ensure the practicality of the method in clinical applications and the feasibility of its translational applications. This process requires a comprehensive assessment of the key analytical parameters that define the sensitivity, precision, specificity, and actual robustness of the testing method, and these parameters need to be evaluated under actual conditions.

Principle
The measurement principle is centered on a ​​colorimetric assay​​ that quantifies bacterial concentration by measuring the catalytic oxidation of a chromogenic substrate. The process leverages the ​​synergistic catalytic activity​​ of the nanoflowers' components.
The detection limit (LOD) is a fundamental measurement indicator, which defines the lowest concentration of an analyte that can be effectively distinguished from the blank sample. It is a crucial metric for evaluating the analytical sensitivity of a detection method.
The relative standard deviation (RSD) is used to measure the reproducibility and repeatability of the detection method. A lower relative standard deviation value (< 5%) indicates that the measurement has high accuracy.
Specificity refers to the property of the detection method that it can accurately identify the target analyte without being affected by cross-reactions with interfering substances present in non-target organisms or sample matrices, thus avoiding the occurrence of false positive results.
Using real samples for testing is of crucial importance. Because the good performance demonstrated in a well-controlled buffer system does not guarantee success in complex clinical, environmental or food matrices.
Finally, the stability storage test will evaluate the performance changes of the nanoflowers over time under specific storage conditions.

Protocols
•Materials:
96-well microplates, Microplate reader, Incubator, PBP solution (10 μg/mL in PBS), HRP-PBP-CaHPO₄@AuPt nanoflowers (1 mg/mL), 0.5 × PBS (0.05% Tween-20), Blocking solution (5% BSA in PBS), TMB solution, H₂O₂ solution, Bacterial samples
•Procedure:
(1)PBP Immobilization in 96-Well Plates
a.Protein coating: Add 100 μL of PBP solution (10 μg/mL in PBS) to each well of the 96-well plate.
b.Incubation: Cover the plate and incubate overnight at 4°C to allow protein adsorption to the plate surface.
c.Washing: Remove unbound PBP by washing with 200 μL washing solution. Perform 3-5 wash cycles. After each wash, gently tap the plate and invert on absorbent paper to remove residual liquid.
d.Blocking: Add 200 μL blocking solution (5% BSA in PBS) to each well. Incubate at room temperature for 1 h to ensure effective blocking. Store at 4°C if not used immediately (avoid freeze-thaw cycles).
(2)Bacterial Detection Assay
a.Sample Preparation: Prepare bacterial samples at concentrations of 10¹-10⁷ CFU/mL in PBS. Use PBS as negative control. Perform 5 independent experiments, each with triplicate wells.
b.Detection Protocol: Add 200 μL of diluted bacterial sample to each test well. Add 10 μL of HRP-PBP-CaHPO₄@AuPt nanoflowers (1 mg/mL),
c.Incubation: Place in 37°C incubator for 20 min to allow bacterial binding and nanoflower attachment. d.Washing and Signal Development: Remove liquid and tap dry on absorbent paper. Add 200 μL washing solution to each well. Let stand for 1 minute, then discard washing solution. Tap dry on absorbent paper. Repeat washing 5 times,
e.Colorimetric detection: Add 50 μL TMB substrate and 50 μL H₂O₂ solution to each well. For enhanced color reaction, use H₂O₂ at 0.01%-1% concentration. Incubate at 37°C in the dark for 10 min.
f.Measurement and documentation: Measure optical density at 650 nm using microplate reader. Photograph results with smartphone for visual documentation.
(3)Specificity Test
Test nanoflowers functionalized with TFP/gp17 against:
•E. coli
•B. subtilis
•Mixed culture of E. coli and B. subtilis
- Compare RGB values with controls.
(4)Storage Stability
Store HRP, HRP-TFP-CaHPO₄, and HRP-TFP-CaHPO₄-AuPt NPs at room temperature (25°C) for 4 weeks, measuring their catalytic activity weekly using the same detection method as in point 21.
(5)Real Sample Detection
- Dilute milk/tea with PBS (1:10, w/v); mix thoroughly.
- Spike with bacteria (10⁰, 10², 10⁴ CFU/mL);
- Detect as above.
(6)Data Analysis
a.RGB Analysis Protocol: Use an online tool Image Color Picker to analyze smartphone photographs. Extract Red (R), Green (G), and Blue (B) values from each well. Calculate B/R ratios for enhanced discrimination.
b.Statistical Analysis: Plot bacterial concentration vs. RGB values and B/R ratios. Establish linear relationship equations.
Calculate Limit of Detection (LOD) using the formula:
                                                    LOD = 3 × (SD/K)
Calculate Relative Standard Deviation (RSD) using the formula
                                                 RSD = (SD/mean) × 100%
(Where LOD = Limit of Detection, the lowest concentration or amount that the method can reliably detect. 3 = Confidence level constant representing 99% confidence. SD = standard deviation of blank response. K = slope of calibration curve. Mean = Arithmetic mean value of the dataset).

Results
Serial dilutions of E. coli (ranging from 10¹ to 10⁴ CFU/mL) were prepared and detected using four different detection systems for each protein construct (TFP and gp17). The detection results showed that the four-component nanoflower systems (HRP-PBP-CaHPO₄-AuPt NPs) demonstrate significantly enhanced detection sensitivity compared to three-component and control systems. The incorporation of AuPt nanoparticles provides synergistic catalytic amplification, greatly improving the detection limit and dynamic range. The performance trends of TFP and gp17 are similar, indicating that this nano-flower-like structure has extremely high sensitivity in bacterial detection (Figure 4). Compared with the four-component nano-flower of gp17, TFP has a higher detection signal value.

We performed linear regression analysis on bacterial concentration versus B/R ratio and found a good linear relationship for both quadruple-combination nanoflowers within the detection range of 10¹–10⁴ CFU/mL (R² values of 0.9434 and 0.9286, respectively), as shown in Figure 10. These standard curves can serve as a reference for us in the subsequent detection of unknown concentrations of bacteria.
Additionally, we calculated the LOD and RSD values, with results shown in the table below. The LOD values for both quadruple-combination nanoflowers ranged from 10.1 to 19.4 CFU/mL. Within the detection range of 10¹ to 10⁴ CFU/mL, the RSD values were 3.07–5.23% and 3.54–6.51%, respectively, indicating satisfactory reproducibility and repeatability (Table 1).

	LOD (CFU/mL)	RSD (%)
HRP-TFP-CaHPO₄@AuPt	19.4	3.07-5.23
HRP-gp17-CaHPO₄@AuPt	10.1	3.54-6.51

Specificity Test
To test the specificity of the four-in-one nano-flower, we introduced different types of strains (B. subtilis and E. coli). Test results indicated that both types of quadruple-combination nanoflowers produce only weak signal values (B/R < 1) for B. subtilis, while exhibiting high signal values (B/R = 6~8 for HRP-TFP-CaHPO4@AuPt, B/R = 3~4 for HRP-gp17-CaHPO4@AuPt) for E. coli (Figure 11). Furthermore, in a mixed culture of B. subtilis and E. coli, the nanoflowers maintain high signal values, demonstrating that their detection performance remains unaffected in the presence of different bacterial species.

Detection of real samples
In order to test whether the four-in-one nano flowers can perform detection in complex environments, we conducted tests on real samples. We employed HRP-TFP-CaHPO₄@AuPt to detect E. coli in real samples. The results demonstrated successful detection of bacteria in milk (milky white) and tea (light brown). Signal intensity increased proportionally with bacterial concentration, indicating that our detection method reliably avoids interference from sample coloration and exhibits both reliability and universality (Figure 7).
Storage stability test
Finally, we conducted an investigation into the storage stability of the four-in-one nano flowers. The findings indicated that the reduction in enzyme activity of the HRP-PBP-CaHPO4@AuPt nanoflowers was significantly less in comparison to that of free HRP and HRP-PBP-CaHPO4 nanoflowers (Figure 8). These results suggested that the prepared HRP-PBP-CaHPO4@AuPt nanoflowers exhibited great resistance to high temperature and storage ability at room temperature because of the exceptional stability of AuPt.

Discussion
The experimental results show that in terms of detection sensitivity, the lower limit of detection (LOD) value achieved was 10.1 - 19.4 CFU/mL, which is an extremely low figure. This remarkable sensitivity is directly attributed to the synergistic catalytic effect between HRP enzyme and AuPt nanozymes.
This system demonstrates an excellent linear dynamic range (10¹ - 10⁴ CFU/mL), with a correlation coefficient (R² > 0.92) that is also relatively high, enabling reliable quantification of bacterial loads. The relatively low relative standard deviation (RSD) values (3.07 - 6.51%) indicate that this detection method has high repeatability and reproducibility.
Specificity tests have confirmed the accuracy of this platform. These nanoflowers only produce strong signals for the target E. coli and have minimal cross-reactions with Bacillus subtilis, even in mixed cultures. This high specificity is crucial for avoiding false positive results in complex samples.
Perhaps the most impressive aspect is that the system has demonstrated its practical application value in actual sample testing. It successfully achieved detection in complex matrices such as milk and tea (these matrices themselves have colors and may interfere with colorimetric readings), which indicates the robustness of the method and its resistance to matrix interference.
The storage stability test further highlights the advantages of the nano-flower design. Compared with the individual HRP or three-component system, the stability of the four-in-one nano-flower has significantly improved, mainly due to the protective effect of the inorganic CaHPO₄ matrix and the inherent stability of the gold-platinum nanozyme. This ensures the persistence of the reagents.
In conclusion, this four-in-one nano-flower system successfully integrates multiple functions into a powerful single platform. The innovative measure of using gold-platinum nanoenzymes for dual signal amplification is the key, which significantly enhances the detection sensitivity. Its high specificity, stability in actual samples, and excellent storage stability make it an excellent choice for the next generation of rapid, reliable, and applicable candidate technologies for bacterial detection in food safety, environmental monitoring, and clinical diagnosis.

Reference
[1] Hong, B., Qin, T., Wang, W., Luo, L., Li, Y., Ma, Y., & Wang, J. (2025). Rapid and ultrasensitive detection of Salmonella typhimurium based on dual signal amplification of four-in-one AuPt nanozyme coated enzyme-antibiotic-inorganic nanoflowers. Sensors and Actuators B: Chemical, 426, 137097.