RESULTS

Our team designed an ​​integrated four-in-one nanoflower system​​ for ultra-sensitive, rapid, and low-cost bacterial detection. This platform combines ​​specific recognition, catalytic signal generation, and dual signal amplification​​ within a single nanostructure, enabling highly efficient pathogen identification. Our experimental part can mainly be divided into two parts:
•The first part is the preparation of Phage-derived Binding Proteins (PBP) through molecular biology.
•The second part is the preparation and performance testing of the four-in-one nano flowers.

PBP preparation
We obtained the amino acid sequences of TFP, gp17 and BSCBS from GenBank, detailed information is shown in Table 1.

Name	Source	Genbank
TFP	tail fiber protein [Escherichia phage T7]	NP_042005.1
gp17	tail fiber protein [Enterobacteria phage T3]	NP_523342.1
BSCBD	Bacillus phage B103	NP_690649.1

After sending the genes to GenScript Company for synthesis, we obtained a strain containing the target gene. In order to obtain a large amount of target proteins, we cloned the target gene into the plasmid vector of pET28a, which is a prokaryotic expression vector featuring a T7 promoter (for IPTG-inducible strong expression) and a C-terminal His-tag (for affinity purification). Thus, we constructed three recombinant plasmids (Figure 1).



Plasmid Construction
The insert fragments (TFP, gp17 and BSCBD) were amplified by PCR from their respective plasmids using gene-specific primers. All samples were separated on a 1% agarose gel stained with YeaGreen nucleic acid dye. Electrophoresis was performed at 180 V for 15 minutes. The gel confirms the successful amplification of target genes. The PCR products for TFP, gp17 and BSCBD are single, sharp bands at the correct sizes, indicating high purity and specificity of amplification (Figure 2).



Using Gibson Assembly, the purified target genes and linearized vector were ligated with 2× CloneExpress Mix in a stoichiometrically optimized system to facilitate homologous recombination. Subsequent colony PCR tests were conducted on the transformed organisms, as shown in Figure 3, which revealed the presence of expected amplification products (TFP: 1995 bp; gp17: 2010 bp; BSCBD: 1125 bp). Finally, Sanger sequencing demonstrated that the cloned fragments were 100% consistent with the target sequence, thereby confirming the accuracy of the plasmid assembly (Figure 3C).



Protein expression and purification
The sequence-verified recombinant plasmids pET28a-BSCBD was then transformed into chemically competent E. coli BL21(DE3) cells via heat shock method. After colony PCR verification, positive transformants were cultured for protein expression. The protein expression was induced with 0.1 mM IPTG at 25°C overnight. Cells were harvested by centrifugation, lysed by sonication, and the soluble and insoluble fractions were separated. His-tagged proteins were purified using Ni-NTA affinity chromatography. Samples were analyzed by 12% SDS-PAGE followed by Coomassie Blue staining. The results demonstrate successful expression of TFP (Theoretical molecular weight: 65.4 kDa), and gp17 (Theoretical molecular weight: 65.8 kDa). The presence of strong induced bands confirms IPTG-induced expression in the precipitation (Figure 4). The purified recombinant proteins (TFP/gp17) were then subjected to dialysis-assisted refolding to obtain soluble, functional proteins. However, the SDS-PAGE result showed that no target band of BSCBD was observed (Theoretical molecular weight: 33.1 kDa).

The failure of BSCBD expression in E. coli is likely attributable to lysin-induced host cell toxicity and insufficient control of premature expression during the pre-induction phase. Lysins, such as BSCBD, are designed to hydrolyze bacterial cell walls. When produced intracellularly in E. coli, even low levels of leakage expression can compromise cell wall integrity, leading to reduced viability, poor growth, and ultimately failed protein production [1].

Nanoflowers preparation
After successfully obtaining the TFP and gp17 proteins targeting E. coli, we will prepare four-in-one nano-flowers for subsequent bacterial detection. This four-in-one nano flower is mainly composed of the following components:
Phage-derived Binding Proteins (PBPs): Engineered TFP and gp17 proteins provide species-specific bacterial recognition.
Horseradish Peroxidase (HRP): Catalyzes 3,3',5,5'-Tetramethylbenzidine (TMB) oxidation for visible signal generation.
Gold-Platinum Nanozyme (AuPt): Enhances catalytic activity with peroxidase-like properties.
Calcium Phosphate Matrix (CaHPO4): Forms 3D nanoflowers structure providing high surface area.
The preparation of the four-in-one nanoflower mainly consists of three steps. The first step is to synthesize AuPt nanoparticles. The second step is to prepare a calcium-based nano-flower scaffold (i.e., a three-in-one nanoflower). The third step is to adsorb AuPt nanoparticles onto the nanoized scaffold through electrostatic adsorption, thereby obtaining a four-in-one nano-flower (Figure 5). For the detailed preparation method, please refer to the Experiment page.



Nanoflowers Characterization
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.



Catalytic Performance Testing
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 7). 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.



Optimization of reaction conditions
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 8). These optimized conditions will be applied in subsequent bacterial detection assays to ensure maximum sensitivity and reliability.



Bacterial detection performance test
Subsequently, serial dilutions of E. coli (ranging from 10¹ to 104CFU/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 9). Compared with the four-component nano-flower of gp17, TFP has a higher detection signal value. We have verified this on the Model page. The results show that TFP has a higher affinity than gp17, enabling it to bind to more E. coli during the detection process, thereby achieving a better detection outcome.



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 2).

	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 = 3~4) 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 12).



Storage stability test
Finally, we conducted an investigation into the storage stability of the four-in-one nano flowers in room temperature (25 ℃）). 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 13). 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.