An electrochemical biosensor for sensitive detection of live Salmonella in food via MXene amplified methylene blue signals and electrostatic immobilization of bacteriophages

An electrochemical biosensor for sensitive detection of live Salmonella in food via MXene amplified methylene blue signals and electrostatic immobilization of bacteriophages | 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 An electrochemical biosensor for sensitive detection of live Salmonella in food via MXene amplified methylene blue signals and electrostatic immobilization of bacteriophages Jingjing Zhou, Tingliu Deng, Qin Zeng, Heye Wang, Chunyan Deng This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4649888/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 21 Aug, 2024 Read the published version in Microchimica Acta → Version 1 posted 8 You are reading this latest preprint version Abstract The rapid reproduction of live foodborne pathogenic bacteria poses a significant threat to human health. In the aspect of food safety monitoring, it is crucial to develop sensitive, rapid, and specific methods for detecting foodborne pathogenic bacteria. In this study, we present a novel bacteriophage-targeted electrochemical biosensor designed for accurate and quantitative detection of live Salmonella in food samples. The biosensor is simply constructed by electrostatic immobilizing bacteriophages on the MXene-nanostructred electrodes. MXene, renowned for its high surface area, biocompatibility, and conductivity, serves as an ideal platform for bacteriophage immobilization. This allows for a high-density immobilization of bacteriophage particles, achieving approximately 71 pcs µm − 2 . Remarkably, the bacteriophages immoblized MXene nanostructured electrode still maintain their viability and functionality, ensuring their effectiveness in pathogen detection. Therefore, this proposed biosensor exhibited the enhanced sensitivity with a low limit of detection (LOD) of 5 CFU mL − 1 . Notably, the biosensor exhibits excellent specificity in the presence of other bacteria that commonly contaminate food, and can distinguish live Salmonella from a mixed population. Furthermore, it is applicable in detecting live Salmonella in food samples, which highlights its potential in food safety monitoring. This biosensor offers simplicity, convenience, and suitability for resource-limited environments, making it a promising tool for on-site monitoring of foodborne pathogenic bacteria. Phages Oriented immobilization MXene Salmonella Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 1. Introduction Food borne pathogens represent a significant and persistent danger to global public health, environment, food security as well as microeconomics stability [ 1 ]. Salmonella , a Gram-negative bacterium, has emerged as one of the leading causes of Food borne illness [ 2 ]. Most cases of salmonella are linked to the ingestion of Salmonella -contaminated food items [ 3 ]. To effectively address this pressing public health concern, the traditional microbiological and biochemical methods, including bacterial culture, ELISA, and PCR, have been widely used for the detection of bacteria in food [ 4 ]. However, they require specialized equipment, trained personnel, and expensive reagents. Moreover, they cannot distinguish live from dead cells and often take hours or days to yield results. Given these challenges, especially in resource-limited regions like Africa, it is urgent to develop a rapid, sensitive, and quantitative food testing method that can facilitate the early detection of contaminated food products, which would enable timely intervention strategies and mitigate the potential for outbreaks, thereby maintaining public health safety and security. So far, various biosensors have emerged as a promising alternative to conventional methods for bacteria detection in food and water [ 4 ]. Among them, electrochemical biosensors offer advantages such as low-cost, easy-to-use technology, high sensitivity, fast analytical speed, and the ability to be easily miniaturized [ 5 ]. These features make electrochemical sensors suitable for use as point-of-contamination monitors, and therefore it is significance to explore the agrochemical biosensor for the detection of bacteria. Bacteriophages are viruses that specifically target bacteria, naturally exhibiting remarkable specificity in recognizing and attaching to particular bacterial strains [ 6 ]. This specificity extends to their ability to sense preferred bacterial spectra. Crucially, phages can only infect and replicate within viable bacteria, thus serving as a reliable tool to discriminate between living and non-living cells. This specific recognition mechanism not only enhances the accuracy of bacterial detection but also significantly reduces the risk of false positive or negative results. Moreover, phages are ubiquitous in nature and therefore they are tolerant to extreme environmental conditions like ultrahigh temperatures, organic solvents and wide-ranging pH. This robustness ensures reliable performance even in challenging environments, making them ideal for practical applications. Also, phages are significantly less expensive to produce and can be mass-produced cost-effectively. Therefore, phages have emerged as promising biological recognition elements for bacterial detection, offering an effective alternative to traditional antibody or nucleic acid recognition methods [ 7 , 8 ]. On the other hand, when phages as recognition elements are immobilized on the biosensing interface, it is crucial to employ appropriate methods to achieve the high density of immobilized phage and maintain their ability to infect the host bacteria. Until now, the common phage immobilization strategies on solid surfaces include physic adsorption [ 9 ], covalent bonding [ 10 ] the electrostatic interaction [ 7 ] and entrapment of phages in solid matrix [ 11 ], etc. Among them, the electrostatic interaction has been proved to be excellent for immobilizing bacteriophages on electrode surfaces because it utilizes the negative charge of phage capsid proteins to form a stable bond with positively charged substrates, while the phage's tail fibers remain free to capture bacteria [ 12 ]. Moreover, this method enables the oriented immobilization of phages, a crucial factor that favors not only a high density of phage particles but also an enhanced bacteria-capture efficiency [ 8 ]. Additionally, the substrate for phage immobilization must possess high biocompatibility, which is conducive to maintaining phage viability and stability [ 7 ]. At the same time, it should offer a large surface area to accommodate a high density of immobilized phages, thereby enhancing biosensing performance [ 8 ]. MXene, a two-dimensional material comprising transition metal carbides or nitrides, has recently garnered significant attention as a potential candidate for agrochemical biosensing applications. This material boasts remarkable physicochemical properties, such as high conductivity, excellent biocompatibility, and a vast surface area. The excellent biocompatibility of MXene is conducive to preserving the viability of immobilized phages, ensuring their functionality and stability. The high conductivity of MXene enables efficient electron transfer during agrochemical reactions, thereby enhancing the agrochemical biosensor's sensitivity. Moreover, its large surface area provides ample sites for immobilizing biological recognition elements, thus improving the detection efficiency and capacity of the biosensor. Therefore, MXene nanostructured electrode substrates are highly beneficial for the immobilization of phages, enabling efficient biosensing applications. In this study, using phages as biorecognition elements, an electrochemical biosensor for the detection of live Salmonella was developed. MXene served as a signal-enhancing matrix, adsorbing methylene blue (MB) effectively. The resulting MXene@MB composite was subsequently employed as an electrode nanosubstrate and positively charged using PDDA. A“tail-up head down” orientation of phages was successfully achieved through electrostatic interaction with the positively-charged substrate. Due to the large surface area and excellent biocompatibility of MXene, a significant number of phages were immobilized while maintaining their ability to infect the host bacterium. The resulted phage/PDDA/MXene@MB modified glassy carbon (GC) electrode was used for the electrochemical detection of live Salmonella exhibited high sensitivity, excellent stability and high specificity. More significantly, this present biosensor is applicable for rapid determining the concentration of Salmonella in contaminated milk or egg samples, indicating its promising application in practical fields. 2. Experimental Section 2.1. Regents Methylene blue (MB) and Nafion (20 wt%) were bought from Macklin Biotech Co., Ltd. (Shanghai, China). SYBR Green I Nucleic Acid Gel Stain (SYBR Green I) was acquired from Solarbio Science & Technology Co., Ltd. (Beijing, China). 10 mM phosphate buffer powder (PBS, pH = 7.4) was bought from Servicebio Technology Co., Ltd. (Wuhan, China). SYTO9 Green Fluorescent Nucleic Acid Stain (SYTO9)/Propidium Iodide (PI) Live/Dead Bacterial Double Stain Kit was purchased from Shanghai Fusheng Bio-Technology Co., Ltd. (Shanghai, China). Ti 3 AlC 2 powder was purchased from Jilin One One Technology Co., Ltd. (Jilin, China). Hydrochloric acid was acquired from Chengdu Cologne Chemical Co., Ltd. (Chengdu, China). Lithium Fluoride and tryptic soy broth (TSB) were obtained from Sangon Biotech Co., Ltd. (Shanghai, China). Poly (diallyldimethylammonium chloride) (PDDA) solution, PEG 8000, potassium ferricyanide, potassium chloride, Tris, hydrofluoric acid, sodium chloride and magnesium sulfate were all purchased from Aladdin Biochemical Technology Co., Ltd. (Shanghai, China). The ITO electrodes were purchased from Luoyang Gulu Glass Co., Ltd. All phages and bacteria used in this study was kindly provided by Jiangsu Academy of Agricultural Sciences, International Phage Research Center (IPRC, Nanjing). The reagents used in the experiment were of analytical-grade purity. Deionized water (18.2 MΩ cm; Millipore M-Q System Inc., Milford, MA) was used in all experiments. 2.2. Instruments and measurements Cyclic voltammetry (CV) and differential pulse voltammetry (DPV) measurements were performed on CHI 660E electrochemical workstation (Chenhua Instruments, Shanghai, China). Electrochemical impedance spectroscopy (EIS) measurements were performed on a Gamry Reference 600 electrochemical workstation (Gamry Instruments Co., Ltd., Warminster, PA, USA). Scanning electron microscopy (SEM) morphology and energy dispersive spectra (EDS) were acquired on a scanning electron microscopy (JEOL/JSM-7610Fplus, Japan). ζ-Potential was performed on a Malvern Zetasizer Nano ZNE 3600 analyzer. Fluorescence Spectrophotometer were executed on FL-4600 Hitachi, Japan. Bacteria were quantified using an enzyme labeller (Tecan Infinite F50 analyzer, Austria). Fluorescence images were observed using a confocal laser-scanning microscope (Olympus FV3000, Tokyo, Japan) at 488 nm. 2.3. Synthesis of MXene and preparation of the MXene@MB composite Layered MXene was synthesized by etching aluminum from Ti 3 AlC 2 using a LiF/HCl solution [ 13 ]. Briefly, 1.6 g of LiF and 1.0 g of Ti 3 AlC 2 powder were combined with a 20 mL solution of 9 M HCl and stirred continuously for 48 hours at 35°C. After the etching reaction, the material was thoroughly rinsed with deionized water and centrifuged until it reached a neutral pH. The resulting precipitate was then redispersed in Milli-Q water and sonicated to produce a stock solution of MXene. The MXene@MB nanocomposite was synthesized following the procedures reported in the literature [ 14 ]. Briefly, a 1.0 mL solution of MXene (1.0 mg mL − 1 ) was prepared and mixed with 1.0 mL of MB (1.0 mM) solution. The mixture was then sonicated for 30 min to facilitate the adsorption of positively charged MB molecules onto the negatively charged MXene sheets through electrostatic interactions. To remove any excess MB, the mixture was centrifuged three times at 1000 rpm for 10 min, followed by washing. Finally, 1.0 mL of deionized water was added to the precipitate to obtain the MXene@MB solution. To improve the adhesion and durability of the nanocomposite on the electrode surface [ 15 ], 0.1 wt % Nafion was added to the resulting MXene@MB solution. 2.4. Microbial Culture Salmonella (ATCC 13076) was inoculated into TSB liquid medium and enriched by incubating it for 10 hours at 37°C. Subsequently, the bacteria were collected by centrifuging at 4000 rpm for 3 min. The resulting pellet was washed three times with SM buffer and ultimately resuspended in the same buffer. To determine the bacterial concentration, the optical density at 600 nm was measured (OD 600 ) using an enzyme labeler. The bacterial concentration was determined using the following equation: 1 OD 600 = 2.4 × 10 8 CFU mL − 1 [ 16 ]. 2.5. Phage PA13076 propagation and purification The phage's host bacterium was Salmonella ATCC 13076. The phage, designated as vB_SenM_PA13076 (abbreviated as PA13076), was maintained at a concentration of 3.0 × 10 10 PFU mL − 1 . Routinely, it was suspended in SM buffer and preserved at 4°C. Propagation of phage PA13076 followed a previously established protocol [ 17 ]. The titer of the bacteriophage was ascertained through the utilization of a standardized plaque assay. To precipitate the phage particles, a solution comprising of 1 M NaCl and 15% (w/v) PEG 8000 was employed. Upon discarding the supernatant, the sediments were carefully resuspended in SM buffer. Subsequently, the mixture underwent chloroform extraction, preceded by centrifugation at 10000 rpm for a duration of 20 min at 4°C. The phage particles were then meticulously recovered using a pipette and preserved at 4°C. 2.6. The disc diffusion method The disc diffusion method was employed to assess the binding capacity of phage to Salmonella . Initially, 100 µL of Salmonella suspension (10 7 CFU mL − 1 ) was uniformly spread onto an agar plate. Once the bacterial solution on the agar plate had dried, 1.0 µL of phage solution was added dropwise. The agar plate was then incubated overnight at 37°C. To assess the activity of the immobilized phage, we coated ITO glass slides with PDDA/MXene@MB and immobilized phage particles onto them [ 18 ]. The agar plates were topped with electrode discs cut from the coated ITO glass slides and immobilized with phage particles. The agar plates were inspected the following day for the presence of lysis rings around the electrode disks. Additionally, a phage-free immobilized PDDA/MXene@MB-coated ITO glass slide served as a negative control. 2.7. Fluorescence imaging of bacteriophage induced bacteria lysis To investigate the binding state of bacteriophages and bacteria in solution over time, fluorescence microscopy was employed to analyze bacterial viability [ 19 ]. Salmonella with a concentration of 2.4 × 10 7 CFU mL − 1 was inoculated with phage PA13076 10 10 PFU mL − 1 and incubated at 37°C. After incubating the bacteria with the phage for a specific duration, the mixture was centrifuged at 4000 rpm for 5 min. The centrifuged bacteria were then re-suspended in SM buffer for staining. A mixture of 1.5 mL SYTO9 and 1.5 mL propidium iodide was added to 1.0 mL of the bacterial solution. The staning mixture was incubated in the dark at room temperature for 30 min to allow the dyes to bind to the cells. Subsequently, the stained samples were examined using a fluorescence microscope. Excitation was performed using a 488 nm laser, and the emitted light was observed at wavelengths of 500 nm (for SYTO9) and 635 nm (for propidium iodide, PI), respectively, using a ×100 objective lens. For each sample, three independent experiments were conducted, and three images were captured from fixed locations within the sample. This approach allowed for obtaining a statistical overview by analyzing multiple images and minimizing potential bias resulting from focusing on specific regions within the sample. 2.8. Electrode preparation GC electrode was polished using 0.05 µm alumina powder and ultrasonically cleaned before use. The MXene@MB solution containing 0.1 wt % Nafion was fixed on the surface of the GC electrode and dried at room temperature. The obtained MXene@MB/GC electrode was dried and immersed in 1 wt % PDDA solution for 20 min. Afterward, the electrode was washed three times with deionized water to remove any excess or adsorbed PDDA. The resulting PDDA/MXene@MB/GC electrode was incubated with a solution containing 3.0 × 10 10 PFU mL − 1 of phages for 60 min. After the incubation period, the electrode (phage/PDDA/MXene@MB/GC) was washed with deionized water to remove any unbound or non-specifically bound phages. Finally, the phage/PDDA/MXene@MB/GC electrodes were incubated with different concentrations of Salmonella and the electrochemical detection of bacteria was performed after culturing the bacteria on the phage/PDDA/MXene@MB/GC electrodes for 30 min. 2.9. Electrochemical characterization of the modified electrode The electrochemical experiments used a classical three-electrode system consisting of a glassy carbon electrode (2 mm) supplied by CH Instruments, a platinum wire and a saturated AgCl electrode (Ag/AgCl). The electrochemical characterisation of the tested electrodes was performed by cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) in 5 mM [Fe(CN) 6 ] 3−/4− redox pair solution (1:1 molar ratio) containing 0.1 M KCl. Cyclic voltammograms were recorded at different scan rates in the range of 100 mV s − 1 . Electrochemical impedance spectroscopy was measured in the frequency range of 0.1–10,000 Hz. Differential pulse voltammetry (DPV) was conducted in a 10 mM PBS solution at pH 7.4. The potential range for the measurement was set from − 600 mV to -200 mV, with an amplitude of 100 mV and a pulse period of 0.1 seconds. 2.10. Characterization of immobilized phages In order to study the quantities of immobilized phage particles, the immobilized phage particles on PDDA/MXene@MB/ITO electrodes were visualized using confocal microscopy. ITO used as the electrode instead of GCE is due to the difficulties in placing the rod-shaped GCE inside the confocal microscope. 100 µL of phage solution (10 10 PFU mL − 1 ) was stained with SYBR Green I, 10 µL of SYBR Green I in the dark at 4°C for 15 min. Subsequently, the mixture was precipitated with PEG/NaCl for 1 h in the dark. After centrifugation at 12,000 rpm for 20 min at 4°C, the phage-containing pellet was resuspended in SM buffer. The same experimental procedure as mentioned earlier for GCE was used to immobilize phage onto the ITO surface in the dark. The resulting ITO was used for fluorescence characterization. 2.11. The electrochemical detection In order to further determine the practical applicability of the proposed test method in actual samples, milk and eggs samples obtained from a local supermarket (Changsha, China) were subjected to analysis. For the actual sample pretreatment process, the surfaces of the eggs and milk were cleaned thoroughly with 75% ethanol to eliminate any contaminants. Subsequently, the samples were divided into separate portions and sterilized using irradiation techniques to ensure their sterility. For the spiked samples, sterile eggs and pasteurized milk samples were inoculated with varying concentrations of Salmonella , ranging from 24 CFU mL − 1 to 2.4 × 10 6 CFU mL − 1 . Once inoculated, the eggs and milk samples were analyzed using these phage/PDDA/MXene@MB/GC electrodes. 3. Results and discussion 3.1. Principal for the electrochemical biosensor As shown in Scheme 1 (A), MXene, a layered material, was initially synthesized. Subsequently, MB (methylene blue) was adsorbed onto the surface of MXene through electrostatic interactions, resulting in the formation of the MXene@MB composite. MXene as a biocompatible and highly conductive nanomaterial provides a large surface area for the immobilization of MB and phage particles, which is useful for enhancing the performance of biosensor. In Scheme 1 (B), the MXene@MB nanostructures modified electrode undergoes a crucial step of positively charging through electrostatic interaction with PDDA. This results in the formation of the PDDA/MXene@MB/GC electrode, which inherits the biocompatibility and high conductivity of MXene, while also benefiting from the large surface area provided by the MXene@MB nanostructures. This bioactive surface is not only suitable for retaining the viability and infection capabilities of the immobilized phages, but it also enhances the adsorption capacity of MB and the density of the immobilized phages. This enhancement is crucial for improving the biosensor's sensitivity and specificity. More importantly, the positively charged PDDA layer on the electrode surface interacts strongly with the negatively charged capsids (heads) of the phage particles. This interaction results in the oriented immobilization of phage particles on the electrode surface, with their tail fibers facing upwards. This "tail-up head-down" orientation maximizes the exposure of the phage's tail fibers, which are responsible for binding and infecting the host bacteria. When Salmonella bacteria are present, the tail fibers of the immobilized phage particles bind to specific receptors on the bacterial surface, triggering measurable changes in the electrochemical signals detected by the biosensor. This targeted interaction allows for the specific, accurate, and rapid detection of Salmonella in food samples. The specificity of phage to Salmonella minimizes non-specific binding or interference from other components in the sample. This ensures that the biosensor only responds to the presence of Salmonella , greatly enhancing the reliability and accuracy of the detection process. 3.2. Characterization of MXene and MXene@MB The successful synthesis of MXene was demonstrated through SEM. As exhibited in Fig. 1 (a), the SEM image clearly reveals the ultrathin nature of the produced 2D MXene nanosheets. These nanosheets possess micrometer-scale lateral dimensions, exhibiting their extensive surface area. Furthermore, the razor-sharp edges, resembling blades, are a distinctive characteristic of MXene, which aligns well with previous reports in the literature [ 20 ]. However, based on the EDS elemental mapping analysis presented in Fig. 1 (b-d), it is evident that the Al elements in the synthesized MXene have been effectively and selectively etched. The successful removal of aluminum is further confirmed by the absence of significant Al peaks in the EDS spectrum. To verify the successful loading of MB onto MXene, we conducted fluorescence spectroscopy measurements, as shown in Fig. 2 (A). The MXene solution with black color (inset: a) does not emit fluorescence (curve a), indicating the absence of inherent fluorescent properties. On the other hand, the MB solution (1 mM) displayed a darker-blue color and emitted fluorescence at an emission wavelength of 675 nm (curve b), characteristic of MB's fluorescent nature. After mixing 1.0 mL of 1.0 mg mL − 1 MXene with 1.0 mL of 10 mM MB solution, the resulted solution was centrifugated to obtain the supernatant with light-blue color, indicating there are some unadsorbed MB onto the MXene. However, a higher fluorescence intensity was observed in the supernatant compared to that of the original MB solution. In this phenomenon, the fluorescence intensity decreases as the concentration of MB increases. This can be explained by the reported concentration burst phenomenon exhibited by MB [ 21 ]. These results demonstrate that a large number of MB molecules were adsorbed onto MXene because of the electrostatic interaction and a high specific area of 2D MXene. Figure 2 (B) presents the zeta potential measurements, revealing that both MXene and MXene@MB composite possess a negative charge. However, after treatment with PDDA, the zeta potential of MXene@MB is ca. +50 mV, which indicates the electrostatic adsorption between positively-charged PDDA and negatively-charged MXene. Figure 2 (C) shows DPV responses of GC, MB/GC and MXene@MB/GC electrodes in 10 mM PBS (pH 7.0). When the GC electrode was modified by MB and MXene@MB composite, obvious oxidation peaks at ca. -0.28 V was observed, which is consistent with the redox potential of MB [ 22 ]. More significantly, comparing curve b to curve c in Fig. 2 (C), it is evident that the current of MXene@MB/GCE obviously higher than that of MB/GCE. This enhanced performance can be attributed to the unique 2D structure and high specific surface area of MXene. These properties not only facilitate the adsorption of a greater number of MB molecules but also provide a vast array of active sites for electron transfer and electrochemical reactions, thereby boosting the current response. [ 23 ]. This is useful for improving the sensing sensitivity of the MXene@MB based electrochemical biosensors. Figure 2 (D) clearly illustrates the MXene@MB/GCE's excellent operational stability across 20 consecutive CV cycles, highlighting the remarkable durability of the MXene@MB composite on the surfaces of GC electrodes. All these findings not only confirm the successful synthesis of MXene@MB but also demonstrate the ease of modifying GCE surfaces and the stability of the modified electrode. 3.3. Bioactivity of Phage PA13076 and its infection to bacteria Firstly, the bioactivity of phage PA13076 utilized in this study was thoroughly examined. Figure 3 (a) clearly demonstrates that phage PA13076 forms distinct plaques on the host strain, S. Enteritidis ATCC13076. This observation indicates that PA13076 is a virulent bacteriophage capable of effectively lysing Salmonella cells and generating progeny phages. Furthermore, the activity and specificity of phage PA13076 were further proved by SEM, as displayed in Figure.3 (b). It can be seen that Salmonella cells (inset) were infected and lysed by phage PA13076, resulting in the release of offspring phages. All these results demonstrate the successful infection and lytic capacity of phage PA13076 against Salmonella bacteria but also provide insight into the interaction mechanism between phage and host cells. SYTO9, a green fluorescent dye, is known to stain live bacterial cells, providing a visible marker for intact and viable cells. PI, a red fluorescent dye, is selectively permeable to cells with compromised membranes, indicating damaged or dead cells [ 24 ]. By employing these fluorescent dyes, the dynamic changes in bacterial cell viability during phage infection can be visualized. Therefore, the intricate interaction between Salmonella and phage PA13076 in solution was further investigated by fluorescence imaging using SYTO9 and PI staining. As shown in Fig. 4 , the significant change in viable cell count at 30 min suggests that the phages attach to and invade the Salmonella cells, leading to cell death. Over time, more bacteria are infected by the phages, resulting in a gradual increase in red fluorescence. By 120 min, the cells appeared to be completely compromised, exhibiting a strong lytic effect of the phage on the bacteria. These observations provide crucial insights into the interaction between phages and Salmonella . Moreover, it can be inferred that 30 min is optimal for phage capture of whole cells for detection purposes. 3.3. Characterization of the modification of Phage/PDDA/Mxene@MB on the GC electrode EIS were employed to charactrize the modification of the electrodes. As shown in Fig. 5 (A). In comparison with that of bare GCE, the R et of MXene@MB/GCE increase. The electrostatic adsorption the PDDA layer resulted in an increasing in the R et because of the polymerisation of PDDA. Subsequently, the immobilization of phage led to a further increase in Rct as the phage layer behaved as an inert barrier, hindering electron transfer. After incubating the phage/PDDA/MXene@MB/GCE with bacteria, a significant rise in impedance values was observed, indicating successful capture of bacteria at the biointerface. The corresponding CV responses of these electrodes aligned with the EIS results, as presented in Figure S1 . On the other hand, as seen in Fig. 5 (B), the bare GCE exhibited no oxidation peak in the PBS solution. However, upon immobilizing the MXene@MB nanocomposite on the GC electrode surface, an oxidation peak appeared at approximately − 0.28 V (curve b), attributable to the oxidation of MB. Nevertheless, following treatment with PDDA, a notable reduction in the electrochemical current was observed, confirming the successful adsorption of PDDA onto the MXene@MB-modified GC electrode. After the PDDA/MXene@MB/GC electrode was incubated with phage for 90 min, a further decrease in the current response of the electrode was recorded (curve d), indicating the successful immobilization of the non-conductive phage. Upon further incubation of the sensor with bacteria, the electrochemical signal from MB diminished significantly, suggesting specific capture of bacteria. The changes in current responses measured by DPV in Fig. 5 (B) is consistent with the observations made in Fig. 5 (A). Overall, these results corroborate the successful construction of the electrochemical biosensors as intended and demonstrate the construction feasibility of the phage-based electrochemical biosensor. 3.4. The biological activity of the immobilized phages The biological activity of the immobilized phages plays a crucial role in the biosensor's performance. Initially, the disc diffusion method was employed to assess the bioactivity of the immobilized bacteriophages. Specifically, both the PDDA/MXene@MB/ITO and phage/PDDA/MXene/ITO samples were placed on agar plates inoculated with the target bacteria. The results clearly indicate that the control ITO electrode modified solely with PDDA/MXene@MB, exhibited no plaque formation. Conversely, the ITO electrode modified with phage/PDDA/MXene@MB displayed a distinct lysis ring, indicating active bacteriophage diffusion. This evidence validates that the immobilized phages maintain their lytic ability against infected Salmonella , thereby confirming their viability and functionality within the immobilized state. Furthermore, SEM images in Figure. 6(c) and 6(d) provide additional evidence for bioactivity of the immobilized phage. Figure 6 (c) confirms the successful immobilization of phages on the PDDA/MXene@MB surface, aligning with our electrochemical and disc diffusion results. Figure 6 (d) visually demonstrates that the immobilized phages not only captured bacteria but also caused partial lysis of the captured bacteria. These findings strongly further proved the bioactivity of the immobilized bacteriophages and validate the proposed phage/PDDA/MXene@MB/GCE system for the selective detection of live bacteria. 3.5. The density of the immobilized phages on the PDDA/MXene@MB/GCE SYBR Green I as known for its bright fluorescence when bound to nucleic acids, was used to stain phages in this study [ 25 ]. In our experiment, we utilized an inverted fluorescence microscope to observe the fluorescent signals of PA13076, enabling us to assess the density and distribution of bacteriophages immobilized on the PDDA/MXene@MB/GC electrode. Figure 7 (a) exhibits distinct fluorescent dots representing the stained bacteriophages in a free state, which is consistent with the phenomena described in the literature [ 26 ], indicating that bacteriophages can be labeled with SYBR Green I dye. For comparison, Fig. 7 (b) shows the fluorescent signals on the surface of PDDA/MXene@MB/ITO treated with SYBR Green I dye but without bacteriophages. The results demonstrate that the dye does not interact with the PDDA/MXene@MB/ITO surface. Figures 7 (c) and 7(d) exhibit the outcomes following the incubation of MXene@MB/ITO and PDDA/MXene@MB/ITO with stained bacteriophages, respectively. It is evident that the number of bacteriophages immobilized on the MXene@MB surface is significantly fewer than those immobilized on the PDDA/MXene@MB/ITO surface. This difference is attributed to the electrostatic interaction between the positively charged PDDA/MXene@MB/ITO surface and the negatively charged bacteriophage tails. This phenomenon suggests that positively charged surfaces are more useful for the bacteriophage immobilization. By analyzing the images in Fig. 7 (c) and (d) using ImageJ software, only 5 pcs µm -2 bacteriophage particles were detected on the MXene@MB/ITO surface. However, approximately 71 pcs µm -2 bacteriophage particles were observed on the PDDA/MXene@MB/ITO surface, which is favorable for the higher detection sensitivity. 3.6. Optimal incubation time for phage immobilizing on the PDDA/MXene@MB/GCE To optimize the performance of the phage-based biosensor, the incubation time for phage immobilization on the PDDA/MXene@MB/GCE was investigated. As shown in Fig. 8 , with incubation time of phage varying from 30 min to 90 min, the current responses of the modified electrode gradually decreased and approaching the platform after 90 min. This increase in inhibition is attributed to the nonconductive behavior of the phages. This trend indicates that the immobilization of the phage on the electrode surface reached saturation at around 90 min of incubation. Therefore, the optimal incubation time for phage immobilization on the PDDA/MXene@MB/GCE is 90 min. 3.7. Electrochemical detection of Salmonella Having proven the sensing feasibility and high stability of the phage/PDDA/MXene@MB/GCE, we employed it as the sensing element to detect salmonella at various concentrations. After incubating the phage/PDDA/MXene@MB/GCE with Salmonella suspensions of differing concentrations at 37°C for 30 min, we conducted DPV analysis across a potential range of -0.6 to 0.2 V, as illustrated in Fig. 9 (A). Notably, it was observed that as Salmonella concentration increased from 2.4 × 10 1 CFU mL − 1 to 2.4 × 10 7 CFU mL − 1 , the peak current of MB decreased. Figure 9 (B) shows a satisfactory linear range from 2.4 × 10 1 CFU mL − 1 to 2.4 × 10 7 CFU mL − 1 , with a regression coefficient ( R 2 ) of 0.9958. The limit of detection (LOD) for Salmonella was determined to be as low as 5 CFU mL − 1 . In comparison to other detection methods, our sensor system exhibits comparable sensitivity, the capability to discriminate between live and dead bacteria, a favorable LOD, and a wide linear range, as outlined in Table S1 . This exceptional analytical performance is primarily due to the high-density, oriented immobilization of phages, combined with the exceptional properties of MXene. Therefore, this proposed sensing approach holds promising potential for practical bacteria detection. 3.8. Specificity and reproducibility Since only live bacteria can cause disease, we investigated the present biosensor's ability to distinguish between live and dead bacteria. Figure 10 (A) illustrates that only live Salmonella caused the current response of the present biosensor, which is due to the specific capture of bacteria. This indicates that this present electrochemical biosensor can effectively differentiate between dead and live Salmonella . On the other hand, considering that bacteria such as Staphylococcus aureus are also significant contaminants in food and environmental pollution, it is crucial to evaluate the potential interference from these bacteria on the sensor's specificity towards Salmonella . As observed in Fig. 10 (B), there is no interference signals from other potentially coexisting bacteria. These results elucidate that this proposed biosensor exhibits remarkable specificity and selectivity, which is beneficial in reducing false positive/negative results and enhancing the detection accuracy of Salmonella . To assess the reproducibility of the proposed electrochemical biosensor, the current responses of five replicate electrodes (bacteria/phages/PDDA/MXene@MB/GCE) were investigated under the same conditions. The results from all five replicate measurements exhibited similar patterns and outcomes, thus demonstrating the excellent reproducibility of our electrochemical biosensor. 3.9. Recoveries of the propsosed biosensor for Salmonella detection in practical samples In order to validate the practical application of the proposed biosensor in the detection of foodborne pathogens, the current responses of the phage/PDDA/MXene@MB/GC electrodes were investigated after incubated with milk or egg samples spiked with different concentrations of Salmonella . The concentrations of spiked Salmonella were determined and recorded in Table 1 . The recovery rate of Salmonella detection in milk and egg samples ranged from 91.1–108%. These results demonstrate the biosensor's high accuracy and reliability when applied to real samples. Table 1 Spiking recoveries of the proposed methods for Salmonella detection in milk and eggs Samples Spiked (log 10 N CFU mL − 1 ) Detected (log 10 N CFU mL − 1 ) Recovery (%) RSD ( n = 3, %) Milk 1.38 1.36 98.6 4.6 2.38 2.34 8.3 2.7 4.38 4.39 100.2 2.4 6.38 6.40 100.3 1.5 Eggs 1.38 1.41 102.2 5.9 2.38 2.36 99.2 4.3 4.38 4.39 100.2 1.7 6.38 6.35 99.5 3.9 4. Conclusions This study presents an innovative electrochemical sensor platform that utilizes MXene@MB nanocomposite materials and bacteriophage-targeted detection for rapid, precise, and ultra-sensitive identification of live food-borne pathogenic bacteria. Taking advantages of the electrostatic properties of bacteriophages, the biosensor immobilizes their heads onto the MXene@MB modified GC electrodes, enabling their tail fibers to capture food-borne pathogens. This approach not only preserves the vitality of the bacteriophages but also enhances the sensor's sensitivity. The remarkable performance of our sensor platform is evident in its ability to detect Salmonella at concentrations as low as 5 CFU mL − 1 . Furthermore, the sensor can distinguish the live and dead bacteria, and demonstrates excellent specificity for Salmonella , even in the presence of other bacteria that commonly contaminate food, minimizing the risk of false positives. Therefore, this work provides a potential method for the detection of food-borne pathogenic bacteria, which is significant for enhancing public health protection and ensuring the safety of the food supply. Declarations Supplementary information The Supporting Information is available free of charge at… Ethical approval This research did not involve human or animal samples. Conflict of interest The authors declare no competing interests. Funding No applicable Author Contribution Jingjing Zhou: conceptualization, methodology, investigation, writing original draft. Tingliu Deng: methodology, investigation, writing original draft, writing review and editing. Qin Zeng: conceptualization, methodology, investigation. Heye Wang: conceptualization and writing review and editing. Chunyan Deng: conceptualization, supervision, writing review and editing. All authors read and approved the final manuscript. Data availability All relevant data are within the manuscript and its additional files. References Panwara S, Duggiralab KS, Yadavc P, Debnathc N, Yadavc AK, Kumar A (2023) Advanced diagnostic methods for identification of bacterial foodborne pathogens: Contemporary and upcoming challenges. 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Biosensors 11(9):346. https://doi.org/10.3390/bios11090346 Kuss S, Couto RA, Evans RM, Lavender H, Tang CC, Compton RG (2019) Versatile electrochemical sensing platform for bacteria. Anal Chem 91(7):4317–4322. https://doi.org/10.1021/acs.analchem.9b00326 Xu J, Chau Y, Lee Yk (2019) Phage-based electrochemical sensors: A review. Micromachines 10(12):855. https://doi.org/10.3390/mi10120855 Farooq U, Ullah MW, Yang Q, Aziz A, Xu J, Zhou L, Wang S (2020) High-density phage particles immobilization in surface-modified bacterial cellulose for ultra-sensitive and selective electrochemical detection of Staphylococcus aureus . Biosens Bioelectron 157:112163. https://doi.org/10.1016/j.bios.2020.112163 Zhou Y, Marar A, Kner P, Ramasamy RP (2017) Charge-directed immobilization of bacteriophage on nanostructured electrode for whole-cell electrochemical biosensors. Anal Chem 89(11):5734–5741. https://doi.org/10.1021/acs.analchem.6b03751 Mejri M, Baccar H, Baldrich E, Del Campo F, Helali S, Ktari T, Simonian A, Aouni M, Abdelghani A (2010) Impedance biosensing using phages for bacteria detection: Generation of dual signals as the clue for in-chip assay confirmation. Biosens Bioelectron 26(4):1261–1267. https://doi.org/10.1016/j.bios.2010.06.054 Shabani A, Zourob M, Allain B, Marquette CA, Lawrence MF, Mandeville R (2008) Bacteriophage-modified microarrays for the direct impedimetric detection of bacteria. Anal Chem 80(24):9475–9482. https://doi.org/10.1021/ac801607w Arter JA, Taggart DK, McIntire TM, Penner RM, Weiss GA (2010) Virus-PEDOT nanowires for biosensing. Nano Lett 10(12):4858–4862. https://doi.org/10.1021/nl1025826 Nanduri V, Sorokulova IB, Samoylov AM, Simonian AL, Petrenko VA, Vodyanoy V (2007) Phage as a molecular recognition element in biosensors immobilized by physical adsorption. 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J Microbiol Methods 98:94–98 Bao H, Zhou Y, Shahin K, Zhang H, Cao F, Pang M, Zhang X, Zhu S, Olaniran A, Schmidt S, Wang R (2020) The complete genome of lytic Salmonella phage vB_SenM-PA13076 and therapeutic potency in the treatment of lethal Salmonella Enteritidis infections in mice, Microbiological Research. 237:126471. https://doi.org/10.1016/j.micres.2020.126471 Yang Q, Deng S, Xu J, Farooq U, Yang T, Chen W, Zhou L, Gao M, Wang S (2021) Poly (indole-5-carboxylic acid)/reduced graphene oxide/gold nanoparticles/phage-based electrochemical biosensor for highly specific detection of Yersinia pseudotuberculosis. Microchim Acta 188:1–13. https://doi.org/10.1007/s00604-020-04676-y Low HZ, Böhnlein C, Sprotte S, Wagner N, Fiedler G, Kabisch J, Franz CM (2020) Front Microbiol 11:602444. https://doi.org/10.3389/fmicb.2020.602444 . Fast and easy phage-tagging and live/dead analysis for the rapid monitoring of bacteriophage infection Hu Y, Zeng Q, Hu Y, He J, Wang H, Deng C, Li D (2024) MXene/zinc ion embedded agar/sodium alginate hydrogel for rapid and efficient sterilization with photothermal and chemical synergetic therapy. Talanta 266:125101. https://doi.org/10.1016/j.talanta.2023.125101 Gui YX, Chen KY, Sun Y, Tan YH, Luo WS, Zhu DX, Xiong Y, Yan DY, Wang D (2023) Tang, Strategies for Improving the Brightness of Aggregation-Induced Emission Materials at Aggregate Level, Chinese of journal Chemistry. 41:1249–1259. https://doi.org/10.1002/cjoc.202200660 Zhang J, Yang L, Pei J, Tian Y, Liu J (2022) A reagentless electrochemical immunosensor for sensitive detection of carcinoembryonic antigen based on the interface with redox probe-modified electron transfer wires and effectively immobilized antibody. Front Chem 10:939736. https://doi.org/10.3389/fchem.2022.939736 Liu X, Wang M, Qin B, Zhang Y, Liu Z, Fan H (2022) 2D-2D MXene/ReS 2 hybrid from Ti 3 C 2 T x MXene conductive layers supporting ultrathin ReS 2 nanosheets for superior sodium storage. Chem Eng J 431:133796. https://doi.org/10.1016/j.cej.2021.133796 Honney C (2019) Novel detection system for microbial contamination on meat: the use of a near real time all-fibre fluorometer to detect fluorescently stained bacterial samples. ResearchSpace@ Auckl Allers E, Moraru C, Duhaime MB, Beneze E, Solonenko N, Barrero-Canosa J, Amann R, Sullivan MB (2013) Single‐cell and population level viral infection dynamics revealed by phage FISH, a method to visualize intracellular and free viruses. Environ Microbiol 15(8):2306–2318. https://doi.org/10.1111/1462-2920.12100 Han JH, Wang MS, Das J, Sudheendra L, Vonasek E, Nitin N, Kennedy IM (2014) Capture and detection of T7 bacteriophages on a nanostructured interface. ACS Appl Mater Interfaces 6(7):4758–4765. https://doi.org/10.1021/am500655r Scheme 1 Scheme 1 is available in the Supplementary Files section. Additional Declarations No competing interests reported. Supplementary Files Scheme1.png Scheme 1. Schematic diagram for the electrochemical detection of Salmonella based on the MXene nanostructed electrodes. SupplementaryinformationMA.docx Cite Share Download PDF Status: Published Journal Publication published 21 Aug, 2024 Read the published version in Microchimica Acta → Version 1 posted Reviews received at journal 14 Jul, 2024 Reviews received at journal 07 Jul, 2024 Reviewers agreed at journal 07 Jul, 2024 Reviewers agreed at journal 04 Jul, 2024 Reviewers invited by journal 02 Jul, 2024 Editor assigned by journal 01 Jul, 2024 Submission checks completed at journal 01 Jul, 2024 First submitted to journal 27 Jun, 2024 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-4649888","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":326947645,"identity":"7bf523c6-a4d2-4684-950f-d0ffd689dfaa","order_by":0,"name":"Jingjing Zhou","email":"","orcid":"","institution":"Capital Medical University","correspondingAuthor":false,"prefix":"","firstName":"Jingjing","middleName":"","lastName":"Zhou","suffix":""},{"id":326947648,"identity":"82a56a68-9c87-415b-92c7-f6d16f7d133c","order_by":1,"name":"Tingliu Deng","email":"","orcid":"","institution":"Central South University","correspondingAuthor":false,"prefix":"","firstName":"Tingliu","middleName":"","lastName":"Deng","suffix":""},{"id":326947652,"identity":"8d172a33-0a69-463b-9cbc-d274c13edadd","order_by":2,"name":"Qin Zeng","email":"","orcid":"","institution":"Central South University","correspondingAuthor":false,"prefix":"","firstName":"Qin","middleName":"","lastName":"Zeng","suffix":""},{"id":326947654,"identity":"63206a92-237d-43dd-baf7-2163da45935a","order_by":3,"name":"Heye Wang","email":"","orcid":"","institution":"Jiangsu Academy of Agricultural Sciences","correspondingAuthor":false,"prefix":"","firstName":"Heye","middleName":"","lastName":"Wang","suffix":""},{"id":326947656,"identity":"40fcb465-c30d-4672-b6f3-9cdd5916cc17","order_by":4,"name":"Chunyan Deng","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA7klEQVRIiWNgGAWjYDCCA0DM2ABmMj6ACCUQr4XZgGQtbBJEaeE73nv4xc8dh/MMjp89VvHjz2EGfvYcA4afO3BrkTxzLs2y98zhYoMzeWk3e9sOM0j2vDFg7D2DW4vBjRwzY8a2w4kbDuSY3WZsOAwSMWBmbMOj5f4bqJbzb8yKGYAOsyeo5QaP8WOwFqB1zAxsQFskCGiRPJNjxtjblp4488YbY0kgg0fizLOCg714tPAdP2P84WebdWLf+RzDDz/+WMvxtydvfPATjxYGWHQoHIDweEDEAbwagJH+AUTKNxBQNgpGwSgYBSMXAAAL8Fr2HsLG6QAAAABJRU5ErkJggg==","orcid":"","institution":"Central South University","correspondingAuthor":true,"prefix":"","firstName":"Chunyan","middleName":"","lastName":"Deng","suffix":""}],"badges":[],"createdAt":"2024-06-27 16:15:36","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4649888/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4649888/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s00604-024-06610-y","type":"published","date":"2024-08-21T15:57:06+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":60869443,"identity":"13466a95-02ba-44b6-bc9a-35e6f6e1b20d","added_by":"auto","created_at":"2024-07-23 04:24:04","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":966437,"visible":true,"origin":"","legend":"\u003cp\u003e(a) SEM image of MXene. (b, c, d) the corresponding EDS elemental-mapping images (Al, Ti and C elements) of MXene. (e) EDS spectra of MXene.\u003c/p\u003e","description":"","filename":"Fig1.png","url":"https://assets-eu.researchsquare.com/files/rs-4649888/v1/2959d30ad13072dfc307b15b.png"},{"id":60869812,"identity":"16af4596-a898-4028-914d-d5e27a96d252","added_by":"auto","created_at":"2024-07-23 04:32:04","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":258911,"visible":true,"origin":"","legend":"\u003cp\u003e(A) Zeta potentials of Mxene, Mxene@MB and PDDA/Mxene@MB composites. (B) Fluorescence spectra of 1.0 mg mL\u003csup\u003e-1\u003c/sup\u003e MXene (a), 1.0 mg mL\u003csup\u003e-1\u003c/sup\u003e MB (b), MXene@MB solution (c), centrifuged supernatant following MXene@MB sltrasound (d). (C) DPV of the bare GC (a), MB/GC (b), and MXene@MB/GC electrodes (c) in 10 mM PBS (pH 7.0); (D) The opertional stability of MXene@MB modified GC electrode after 20 cycles of continuous CV measurement.\u003c/p\u003e","description":"","filename":"Fig2.png","url":"https://assets-eu.researchsquare.com/files/rs-4649888/v1/e2953a46e4577d79a82c3359.png"},{"id":60869446,"identity":"90644390-c58d-4084-8e8d-0ece87463a4d","added_by":"auto","created_at":"2024-07-23 04:24:04","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1265683,"visible":true,"origin":"","legend":"\u003cp\u003e(a) the plaques of the phage PA13076; (b) SEM imges of \u003cem\u003eSalmonella\u003c/em\u003e infected by PA13076. Inset: the morphology of \u003cem\u003eSalmonella\u003c/em\u003e.\u003c/p\u003e","description":"","filename":"Fig3.png","url":"https://assets-eu.researchsquare.com/files/rs-4649888/v1/1f5b4aefc34294fa3b561930.png"},{"id":60869451,"identity":"03b3e4f9-0ebd-4789-9fbf-e07de5229d08","added_by":"auto","created_at":"2024-07-23 04:24:04","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1052081,"visible":true,"origin":"","legend":"\u003cp\u003eSYTO9/PI staining analyzed with confocal microscopy. Fluorescence images of bacterial cell lysis monitored at 500 nm (green) for SYTO9 signal, 635 nm (red) for PI signal and merged images were shown.\u003c/p\u003e","description":"","filename":"Fig4.png","url":"https://assets-eu.researchsquare.com/files/rs-4649888/v1/a30aa6d545ae2eb83697e43b.png"},{"id":60870402,"identity":"5b91be2f-3cab-44fa-95fd-b74dad3223c9","added_by":"auto","created_at":"2024-07-23 04:40:04","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":95511,"visible":true,"origin":"","legend":"\u003cp\u003e(A) EIS spectra, (B) DPV responses of the bare GC (a), MXene@MB/GCE (b), PDDA/MXene@MB/GCE (c), Phage/PDDA/MXene@MB/GCE (d), bacteria/Phage/PDDA/MXene@MB/GCE (e).\u003c/p\u003e","description":"","filename":"Fig5.png","url":"https://assets-eu.researchsquare.com/files/rs-4649888/v1/ccfe53d6df527eaca2164b55.png"},{"id":60869816,"identity":"d2d17b16-74d3-452e-8e70-c12ae4ab4f3e","added_by":"auto","created_at":"2024-07-23 04:32:04","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1179085,"visible":true,"origin":"","legend":"\u003cp\u003eThe bioactivity of immobilized phages. (a) the ITO electrode modified only with PDDA/MXene@MB, (b) ITO electrode modified with Phage/PDDA/MXene@MB. SEM images of (c) the immobilized phages on the PDDA/MXene@MB and (d) following bacterial capture.\u003c/p\u003e","description":"","filename":"Fig6.png","url":"https://assets-eu.researchsquare.com/files/rs-4649888/v1/5f2e0375f240b69642625d99.png"},{"id":60869449,"identity":"db03ae9b-3710-462d-ac80-5ba3a5b9f083","added_by":"auto","created_at":"2024-07-23 04:24:04","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":186059,"visible":true,"origin":"","legend":"\u003cp\u003eThe confocal microscopy images of (a) free phages stained with SYBR Green I, (b) stained PDDA/MXene@MB/ITO, (c) MXene@MB/ITO incubated with stained phages particles, (d) PDDA/MXene@MB/ITO treated with stained phage particles.\u003c/p\u003e","description":"","filename":"Fig7.png","url":"https://assets-eu.researchsquare.com/files/rs-4649888/v1/a43c5eb7cc4aa2c527cc93a7.png"},{"id":60869815,"identity":"a2a6ac05-1556-41fc-a126-a1d825d32265","added_by":"auto","created_at":"2024-07-23 04:32:04","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":155960,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of incubation time of phage on the performance of PDDA/MXene@MB/GCE. (A) DPV measurements of the modified electrode after incubated in PBS (10 mM, pH=7.4) containing 3 × 10\u003csup\u003e10\u003c/sup\u003e PFU mL\u003csup\u003e-1 \u003c/sup\u003efor different times. (B) The corresponding relationship between phage incubation time and current responses of the modified electrode. \u003cem\u003eI\u003c/em\u003e\u003csub\u003e0 \u003c/sub\u003eand \u003cem\u003eI \u003c/em\u003eare the peak currents of the modified electrodes before and after phage incubation, respectively.\u003c/p\u003e","description":"","filename":"Fig8.png","url":"https://assets-eu.researchsquare.com/files/rs-4649888/v1/8e90c01abd23dea0db957f23.png"},{"id":63300089,"identity":"091496f6-558c-4dd2-ab53-271ffe53c331","added_by":"auto","created_at":"2024-08-26 16:10:58","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":6834115,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4649888/v1/56e0e59c-e7cb-4d3a-952f-3b614af9f9b2.pdf"},{"id":60869811,"identity":"3546bba4-a91f-4a10-a0b4-ac5ea1140710","added_by":"auto","created_at":"2024-07-23 04:32:04","extension":"png","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":713969,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eScheme 1. \u003c/strong\u003eSchematic diagram for the electrochemical detection of \u003cem\u003eSalmonella\u003c/em\u003ebased on the MXene nanostructed electrodes.\u003c/p\u003e","description":"","filename":"Scheme1.png","url":"https://assets-eu.researchsquare.com/files/rs-4649888/v1/af5781dbc4c1b07becf32daf.png"},{"id":60869813,"identity":"41bfbc54-526d-4ef1-a6f2-fc842abe38a8","added_by":"auto","created_at":"2024-07-23 04:32:04","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":133776,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryinformationMA.docx","url":"https://assets-eu.researchsquare.com/files/rs-4649888/v1/7cfc7a3a9e0524e45d124be8.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"An electrochemical biosensor for sensitive detection of live Salmonella in food via MXene amplified methylene blue signals and electrostatic immobilization of bacteriophages","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eFood borne pathogens represent a significant and persistent danger to global public health, environment, food security as well as microeconomics stability [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. \u003cem\u003eSalmonella\u003c/em\u003e, a Gram-negative bacterium, has emerged as one of the leading causes of Food borne illness [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Most cases of salmonella are linked to the ingestion of \u003cem\u003eSalmonella\u003c/em\u003e-contaminated food items [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. To effectively address this pressing public health concern, the traditional microbiological and biochemical methods, including bacterial culture, ELISA, and PCR, have been widely used for the detection of bacteria in food [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. However, they require specialized equipment, trained personnel, and expensive reagents. Moreover, they cannot distinguish live from dead cells and often take hours or days to yield results. Given these challenges, especially in resource-limited regions like Africa, it is urgent to develop a rapid, sensitive, and quantitative food testing method that can facilitate the early detection of contaminated food products, which would enable timely intervention strategies and mitigate the potential for outbreaks, thereby maintaining public health safety and security. So far, various biosensors have emerged as a promising alternative to conventional methods for bacteria detection in food and water [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Among them, electrochemical biosensors offer advantages such as low-cost, easy-to-use technology, high sensitivity, fast analytical speed, and the ability to be easily miniaturized [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. These features make electrochemical sensors suitable for use as point-of-contamination monitors, and therefore it is significance to explore the agrochemical biosensor for the detection of bacteria.\u003c/p\u003e \u003cp\u003eBacteriophages are viruses that specifically target bacteria, naturally exhibiting remarkable specificity in recognizing and attaching to particular bacterial strains [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. This specificity extends to their ability to sense preferred bacterial spectra. Crucially, phages can only infect and replicate within viable bacteria, thus serving as a reliable tool to discriminate between living and non-living cells. This specific recognition mechanism not only enhances the accuracy of bacterial detection but also significantly reduces the risk of false positive or negative results. Moreover, phages are ubiquitous in nature and therefore they are tolerant to extreme environmental conditions like ultrahigh temperatures, organic solvents and wide-ranging pH. This robustness ensures reliable performance even in challenging environments, making them ideal for practical applications. Also, phages are significantly less expensive to produce and can be mass-produced cost-effectively. Therefore, phages have emerged as promising biological recognition elements for bacterial detection, offering an effective alternative to traditional antibody or nucleic acid recognition methods [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eOn the other hand, when phages as recognition elements are immobilized on the biosensing interface, it is crucial to employ appropriate methods to achieve the high density of immobilized phage and maintain their ability to infect the host bacteria. Until now, the common phage immobilization strategies on solid surfaces include physic adsorption [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e], covalent bonding [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e] the electrostatic interaction [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e] and entrapment of phages in solid matrix [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e], etc. Among them, the electrostatic interaction has been proved to be excellent for immobilizing bacteriophages on electrode surfaces because it utilizes the negative charge of phage capsid proteins to form a stable bond with positively charged substrates, while the phage's tail fibers remain free to capture bacteria [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Moreover, this method enables the oriented immobilization of phages, a crucial factor that favors not only a high density of phage particles but also an enhanced bacteria-capture efficiency [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Additionally, the substrate for phage immobilization must possess high biocompatibility, which is conducive to maintaining phage viability and stability [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. At the same time, it should offer a large surface area to accommodate a high density of immobilized phages, thereby enhancing biosensing performance [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. MXene, a two-dimensional material comprising transition metal carbides or nitrides, has recently garnered significant attention as a potential candidate for agrochemical biosensing applications. This material boasts remarkable physicochemical properties, such as high conductivity, excellent biocompatibility, and a vast surface area. The excellent biocompatibility of MXene is conducive to preserving the viability of immobilized phages, ensuring their functionality and stability. The high conductivity of MXene enables efficient electron transfer during agrochemical reactions, thereby enhancing the agrochemical biosensor's sensitivity. Moreover, its large surface area provides ample sites for immobilizing biological recognition elements, thus improving the detection efficiency and capacity of the biosensor. Therefore, MXene nanostructured electrode substrates are highly beneficial for the immobilization of phages, enabling efficient biosensing applications.\u003c/p\u003e \u003cp\u003eIn this study, using phages as biorecognition elements, an electrochemical biosensor for the detection of live \u003cem\u003eSalmonella\u003c/em\u003e was developed. MXene served as a signal-enhancing matrix, adsorbing methylene blue (MB) effectively. The resulting MXene@MB composite was subsequently employed as an electrode nanosubstrate and positively charged using PDDA. A\u0026ldquo;tail-up head down\u0026rdquo; orientation of phages was successfully achieved through electrostatic interaction with the positively-charged substrate. Due to the large surface area and excellent biocompatibility of MXene, a significant number of phages were immobilized while maintaining their ability to infect the host bacterium. The resulted phage/PDDA/MXene@MB modified glassy carbon (GC) electrode was used for the electrochemical detection of live \u003cem\u003eSalmonella\u003c/em\u003e exhibited high sensitivity, excellent stability and high specificity. More significantly, this present biosensor is applicable for rapid determining the concentration of \u003cem\u003eSalmonella\u003c/em\u003e in contaminated milk or egg samples, indicating its promising application in practical fields.\u003c/p\u003e"},{"header":"2. Experimental Section","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Regents\u003c/h2\u003e \u003cp\u003eMethylene blue (MB) and Nafion (20 wt%) were bought from Macklin Biotech Co., Ltd. (Shanghai, China). SYBR Green I Nucleic Acid Gel Stain (SYBR Green I) was acquired from Solarbio Science \u0026amp; Technology Co., Ltd. (Beijing, China). 10 mM phosphate buffer powder (PBS, pH\u0026thinsp;=\u0026thinsp;7.4) was bought from Servicebio Technology Co., Ltd. (Wuhan, China). SYTO9 Green Fluorescent Nucleic Acid Stain (SYTO9)/Propidium Iodide (PI) Live/Dead Bacterial Double Stain Kit was purchased from Shanghai Fusheng Bio-Technology Co., Ltd. (Shanghai, China). Ti\u003csub\u003e3\u003c/sub\u003eAlC\u003csub\u003e2\u003c/sub\u003e powder was purchased from Jilin One One Technology Co., Ltd. (Jilin, China). Hydrochloric acid was acquired from Chengdu Cologne Chemical Co., Ltd. (Chengdu, China). Lithium Fluoride and tryptic soy broth (TSB) were obtained from Sangon Biotech Co., Ltd. (Shanghai, China). Poly (diallyldimethylammonium chloride) (PDDA) solution, PEG 8000, potassium ferricyanide, potassium chloride, Tris, hydrofluoric acid, sodium chloride and magnesium sulfate were all purchased from Aladdin Biochemical Technology Co., Ltd. (Shanghai, China). The ITO electrodes were purchased from Luoyang Gulu Glass Co., Ltd. All phages and bacteria used in this study was kindly provided by Jiangsu Academy of Agricultural Sciences, International Phage Research Center (IPRC, Nanjing). The reagents used in the experiment were of analytical-grade purity. Deionized water (18.2 MΩ cm; Millipore M-Q System Inc., Milford, MA) was used in all experiments.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2. Instruments and measurements\u003c/h2\u003e \u003cp\u003eCyclic voltammetry (CV) and differential pulse voltammetry (DPV) measurements were performed on CHI 660E electrochemical workstation (Chenhua Instruments, Shanghai, China). Electrochemical impedance spectroscopy (EIS) measurements were performed on a Gamry Reference 600 electrochemical workstation (Gamry Instruments Co., Ltd., Warminster, PA, USA).\u003c/p\u003e \u003cp\u003eScanning electron microscopy (SEM) morphology and energy dispersive spectra (EDS) were acquired on a scanning electron microscopy (JEOL/JSM-7610Fplus, Japan). ζ-Potential was performed on a Malvern Zetasizer Nano ZNE 3600 analyzer. Fluorescence Spectrophotometer were executed on FL-4600 Hitachi, Japan. Bacteria were quantified using an enzyme labeller (Tecan Infinite F50 analyzer, Austria). Fluorescence images were observed using a confocal laser-scanning microscope (Olympus FV3000, Tokyo, Japan) at 488 nm.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3. Synthesis of MXene and preparation of the MXene@MB composite\u003c/h2\u003e \u003cp\u003eLayered MXene was synthesized by etching aluminum from Ti\u003csub\u003e3\u003c/sub\u003eAlC\u003csub\u003e2\u003c/sub\u003e using a LiF/HCl solution [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Briefly, 1.6 g of LiF and 1.0 g of Ti\u003csub\u003e3\u003c/sub\u003eAlC\u003csub\u003e2\u003c/sub\u003e powder were combined with a 20 mL solution of 9 M HCl and stirred continuously for 48 hours at 35\u0026deg;C. After the etching reaction, the material was thoroughly rinsed with deionized water and centrifuged until it reached a neutral pH. The resulting precipitate was then redispersed in Milli-Q water and sonicated to produce a stock solution of MXene. The MXene@MB nanocomposite was synthesized following the procedures reported in the literature [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Briefly, a 1.0 mL solution of MXene (1.0 mg mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) was prepared and mixed with 1.0 mL of MB (1.0 mM) solution. The mixture was then sonicated for 30 min to facilitate the adsorption of positively charged MB molecules onto the negatively charged MXene sheets through electrostatic interactions. To remove any excess MB, the mixture was centrifuged three times at 1000 rpm for 10 min, followed by washing. Finally, 1.0 mL of deionized water was added to the precipitate to obtain the MXene@MB solution. To improve the adhesion and durability of the nanocomposite on the electrode surface [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e], 0.1 wt % Nafion was added to the resulting MXene@MB solution.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4. Microbial Culture\u003c/h2\u003e \u003cp\u003e \u003cem\u003eSalmonella\u003c/em\u003e (ATCC 13076) was inoculated into TSB liquid medium and enriched by incubating it for 10 hours at 37\u0026deg;C. Subsequently, the bacteria were collected by centrifuging at 4000 rpm for 3 min. The resulting pellet was washed three times with SM buffer and ultimately resuspended in the same buffer. To determine the bacterial concentration, the optical density at 600 nm was measured (OD\u003csub\u003e600\u003c/sub\u003e) using an enzyme labeler. The bacterial concentration was determined using the following equation: 1 OD\u003csub\u003e600\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;2.4 \u0026times; 10\u003csup\u003e8\u003c/sup\u003e CFU mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5. Phage PA13076 propagation and purification\u003c/h2\u003e \u003cp\u003eThe phage's host bacterium was \u003cem\u003eSalmonella\u003c/em\u003e ATCC 13076. The phage, designated as vB_SenM_PA13076 (abbreviated as PA13076), was maintained at a concentration of 3.0 \u0026times; 10\u003csup\u003e10\u003c/sup\u003e PFU mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. Routinely, it was suspended in SM buffer and preserved at 4\u0026deg;C. Propagation of phage PA13076 followed a previously established protocol [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. The titer of the bacteriophage was ascertained through the utilization of a standardized plaque assay. To precipitate the phage particles, a solution comprising of 1 M NaCl and 15% (w/v) PEG 8000 was employed. Upon discarding the supernatant, the sediments were carefully resuspended in SM buffer. Subsequently, the mixture underwent chloroform extraction, preceded by centrifugation at 10000 rpm for a duration of 20 min at 4\u0026deg;C. The phage particles were then meticulously recovered using a pipette and preserved at 4\u0026deg;C.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6. The disc diffusion method\u003c/h2\u003e \u003cp\u003eThe disc diffusion method was employed to assess the binding capacity of phage to \u003cem\u003eSalmonella\u003c/em\u003e. Initially, 100 \u0026micro;L of \u003cem\u003eSalmonella\u003c/em\u003e suspension (10\u003csup\u003e7\u003c/sup\u003e CFU mL \u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) was uniformly spread onto an agar plate. Once the bacterial solution on the agar plate had dried, 1.0 \u0026micro;L of phage solution was added dropwise. The agar plate was then incubated overnight at 37\u0026deg;C. To assess the activity of the immobilized phage, we coated ITO glass slides with PDDA/MXene@MB and immobilized phage particles onto them [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. The agar plates were topped with electrode discs cut from the coated ITO glass slides and immobilized with phage particles. The agar plates were inspected the following day for the presence of lysis rings around the electrode disks. Additionally, a phage-free immobilized PDDA/MXene@MB-coated ITO glass slide served as a negative control.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7. Fluorescence imaging of bacteriophage induced bacteria lysis\u003c/h2\u003e \u003cp\u003eTo investigate the binding state of bacteriophages and bacteria in solution over time, fluorescence microscopy was employed to analyze bacterial viability [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. \u003cem\u003eSalmonella\u003c/em\u003e with a concentration of 2.4 \u0026times; 10\u003csup\u003e7\u003c/sup\u003e CFU mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e was inoculated with phage PA13076 10\u003csup\u003e10\u003c/sup\u003e PFU mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and incubated at 37\u0026deg;C. After incubating the bacteria with the phage for a specific duration, the mixture was centrifuged at 4000 rpm for 5 min. The centrifuged bacteria were then re-suspended in SM buffer for staining. A mixture of 1.5 mL SYTO9 and 1.5 mL propidium iodide was added to 1.0 mL of the bacterial solution. The staning mixture was incubated in the dark at room temperature for 30 min to allow the dyes to bind to the cells. Subsequently, the stained samples were examined using a fluorescence microscope. Excitation was performed using a 488 nm laser, and the emitted light was observed at wavelengths of 500 nm (for SYTO9) and 635 nm (for propidium iodide, PI), respectively, using a \u0026times;100 objective lens. For each sample, three independent experiments were conducted, and three images were captured from fixed locations within the sample. This approach allowed for obtaining a statistical overview by analyzing multiple images and minimizing potential bias resulting from focusing on specific regions within the sample.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.8. Electrode preparation\u003c/h2\u003e \u003cp\u003eGC electrode was polished using 0.05 \u0026micro;m alumina powder and ultrasonically cleaned before use. The MXene@MB solution containing 0.1 wt % Nafion was fixed on the surface of the GC electrode and dried at room temperature. The obtained MXene@MB/GC electrode was dried and immersed in 1 wt % PDDA solution for 20 min. Afterward, the electrode was washed three times with deionized water to remove any excess or adsorbed PDDA. The resulting PDDA/MXene@MB/GC electrode was incubated with a solution containing 3.0 \u0026times; 10\u003csup\u003e10\u003c/sup\u003e PFU mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e of phages for 60 min. After the incubation period, the electrode (phage/PDDA/MXene@MB/GC) was washed with deionized water to remove any unbound or non-specifically bound phages. Finally, the phage/PDDA/MXene@MB/GC electrodes were incubated with different concentrations of \u003cem\u003eSalmonella\u003c/em\u003e and the electrochemical detection of bacteria was performed after culturing the bacteria on the phage/PDDA/MXene@MB/GC electrodes for 30 min.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e2.9. Electrochemical characterization of the modified electrode\u003c/h2\u003e \u003cp\u003eThe electrochemical experiments used a classical three-electrode system consisting of a glassy carbon electrode (2 mm) supplied by CH Instruments, a platinum wire and a saturated AgCl electrode (Ag/AgCl). The electrochemical characterisation of the tested electrodes was performed by cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) in 5 mM [Fe(CN)\u003csub\u003e6\u003c/sub\u003e]\u003csup\u003e3\u0026minus;/4\u0026minus;\u003c/sup\u003e redox pair solution (1:1 molar ratio) containing 0.1 M KCl. Cyclic voltammograms were recorded at different scan rates in the range of 100 mV s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. Electrochemical impedance spectroscopy was measured in the frequency range of 0.1\u0026ndash;10,000 Hz. Differential pulse voltammetry (DPV) was conducted in a 10 mM PBS solution at pH 7.4. The potential range for the measurement was set from \u0026minus;\u0026thinsp;600 mV to -200 mV, with an amplitude of 100 mV and a pulse period of 0.1 seconds.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e2.10. Characterization of immobilized phages\u003c/h2\u003e \u003cp\u003eIn order to study the quantities of immobilized phage particles, the immobilized phage particles on PDDA/MXene@MB/ITO electrodes were visualized using confocal microscopy. ITO used as the electrode instead of GCE is due to the difficulties in placing the rod-shaped GCE inside the confocal microscope. 100 \u0026micro;L of phage solution (10\u003csup\u003e10\u003c/sup\u003e PFU mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) was stained with SYBR Green I, 10 \u0026micro;L of SYBR Green I in the dark at 4\u0026deg;C for 15 min. Subsequently, the mixture was precipitated with PEG/NaCl for 1 h in the dark. After centrifugation at 12,000 rpm for 20 min at 4\u0026deg;C, the phage-containing pellet was resuspended in SM buffer. The same experimental procedure as mentioned earlier for GCE was used to immobilize phage onto the ITO surface in the dark. The resulting ITO was used for fluorescence characterization.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e2.11. The electrochemical detection\u003c/h2\u003e \u003cp\u003eIn order to further determine the practical applicability of the proposed test method in actual samples, milk and eggs samples obtained from a local supermarket (Changsha, China) were subjected to analysis. For the actual sample pretreatment process, the surfaces of the eggs and milk were cleaned thoroughly with 75% ethanol to eliminate any contaminants. Subsequently, the samples were divided into separate portions and sterilized using irradiation techniques to ensure their sterility. For the spiked samples, sterile eggs and pasteurized milk samples were inoculated with varying concentrations of \u003cem\u003eSalmonella\u003c/em\u003e, ranging from 24 CFU mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e to 2.4 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e CFU mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. Once inoculated, the eggs and milk samples were analyzed using these phage/PDDA/MXene@MB/GC electrodes.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results and discussion","content":"\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e3.1. Principal for the electrochemical biosensor\u003c/h2\u003e \u003cp\u003eAs shown in Scheme \u003cspan refid=\"Sch1\" class=\"InternalRef\"\u003e1\u003c/span\u003e(A), MXene, a layered material, was initially synthesized. Subsequently, MB (methylene blue) was adsorbed onto the surface of MXene through electrostatic interactions, resulting in the formation of the MXene@MB composite. MXene as a biocompatible and highly conductive nanomaterial provides a large surface area for the immobilization of MB and phage particles, which is useful for enhancing the performance of biosensor. In Scheme \u003cspan refid=\"Sch1\" class=\"InternalRef\"\u003e1\u003c/span\u003e(B), the MXene@MB nanostructures modified electrode undergoes a crucial step of positively charging through electrostatic interaction with PDDA. This results in the formation of the PDDA/MXene@MB/GC electrode, which inherits the biocompatibility and high conductivity of MXene, while also benefiting from the large surface area provided by the MXene@MB nanostructures.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThis bioactive surface is not only suitable for retaining the viability and infection capabilities of the immobilized phages, but it also enhances the adsorption capacity of MB and the density of the immobilized phages. This enhancement is crucial for improving the biosensor's sensitivity and specificity. More importantly, the positively charged PDDA layer on the electrode surface interacts strongly with the negatively charged capsids (heads) of the phage particles. This interaction results in the oriented immobilization of phage particles on the electrode surface, with their tail fibers facing upwards. This \"tail-up head-down\" orientation maximizes the exposure of the phage's tail fibers, which are responsible for binding and infecting the host bacteria. When \u003cem\u003eSalmonella\u003c/em\u003e bacteria are present, the tail fibers of the immobilized phage particles bind to specific receptors on the bacterial surface, triggering measurable changes in the electrochemical signals detected by the biosensor. This targeted interaction allows for the specific, accurate, and rapid detection of \u003cem\u003eSalmonella\u003c/em\u003e in food samples. The specificity of phage to \u003cem\u003eSalmonella\u003c/em\u003e minimizes non-specific binding or interference from other components in the sample. This ensures that the biosensor only responds to the presence of \u003cem\u003eSalmonella\u003c/em\u003e, greatly enhancing the reliability and accuracy of the detection process.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e3.2. Characterization of MXene and MXene@MB\u003c/h2\u003e \u003cp\u003eThe successful synthesis of MXene was demonstrated through SEM. As exhibited in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e(a), the SEM image clearly reveals the ultrathin nature of the produced 2D MXene nanosheets. These nanosheets possess micrometer-scale lateral dimensions, exhibiting their extensive surface area. Furthermore, the razor-sharp edges, resembling blades, are a distinctive characteristic of MXene, which aligns well with previous reports in the literature [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. However, based on the EDS elemental mapping analysis presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e(b-d), it is evident that the Al elements in the synthesized MXene have been effectively and selectively etched. The successful removal of aluminum is further confirmed by the absence of significant Al peaks in the EDS spectrum.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo verify the successful loading of MB onto MXene, we conducted fluorescence spectroscopy measurements, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e(A). The MXene solution with black color (inset: a) does not emit fluorescence (curve a), indicating the absence of inherent fluorescent properties. On the other hand, the MB solution (1 mM) displayed a darker-blue color and emitted fluorescence at an emission wavelength of 675 nm (curve b), characteristic of MB's fluorescent nature. After mixing 1.0 mL of 1.0 mg mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e MXene with 1.0 mL of 10 mM MB solution, the resulted solution was centrifugated to obtain the supernatant with light-blue color, indicating there are some unadsorbed MB onto the MXene. However, a higher fluorescence intensity was observed in the supernatant compared to that of the original MB solution. In this phenomenon, the fluorescence intensity decreases as the concentration of MB increases. This can be explained by the reported concentration burst phenomenon exhibited by MB [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. These results demonstrate that a large number of MB molecules were adsorbed onto MXene because of the electrostatic interaction and a high specific area of 2D MXene. Figure\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e(B) presents the zeta potential measurements, revealing that both MXene and MXene@MB composite possess a negative charge. However, after treatment with PDDA, the zeta potential of MXene@MB is ca. +50 mV, which indicates the electrostatic adsorption between positively-charged PDDA and negatively-charged MXene. Figure\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e(C) shows DPV responses of GC, MB/GC and MXene@MB/GC electrodes in 10 mM PBS (pH 7.0). When the GC electrode was modified by MB and MXene@MB composite, obvious oxidation peaks at ca. -0.28 V was observed, which is consistent with the redox potential of MB [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. More significantly, comparing curve b to curve c in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e(C), it is evident that the current of MXene@MB/GCE obviously higher than that of MB/GCE. This enhanced performance can be attributed to the unique 2D structure and high specific surface area of MXene. These properties not only facilitate the adsorption of a greater number of MB molecules but also provide a vast array of active sites for electron transfer and electrochemical reactions, thereby boosting the current response. [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. This is useful for improving the sensing sensitivity of the MXene@MB based electrochemical biosensors. Figure\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e(D) clearly illustrates the MXene@MB/GCE's excellent operational stability across 20 consecutive CV cycles, highlighting the remarkable durability of the MXene@MB composite on the surfaces of GC electrodes. All these findings not only confirm the successful synthesis of MXene@MB but also demonstrate the ease of modifying GCE surfaces and the stability of the modified electrode.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e3.3. Bioactivity of Phage PA13076 and its infection to bacteria\u003c/h2\u003e \u003cp\u003eFirstly, the bioactivity of phage PA13076 utilized in this study was thoroughly examined. Figure\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e(a) clearly demonstrates that phage PA13076 forms distinct plaques on the host strain, S. Enteritidis ATCC13076. This observation indicates that PA13076 is a virulent bacteriophage capable of effectively lysing \u003cem\u003eSalmonella\u003c/em\u003e cells and generating progeny phages. Furthermore, the activity and specificity of phage PA13076 were further proved by SEM, as displayed in Figure.3 (b). It can be seen that \u003cem\u003eSalmonella\u003c/em\u003e cells (inset) were infected and lysed by phage PA13076, resulting in the release of offspring phages. All these results demonstrate the successful infection and lytic capacity of phage PA13076 against \u003cem\u003eSalmonella\u003c/em\u003e bacteria but also provide insight into the interaction mechanism between phage and host cells.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eSYTO9, a green fluorescent dye, is known to stain live bacterial cells, providing a visible marker for intact and viable cells. PI, a red fluorescent dye, is selectively permeable to cells with compromised membranes, indicating damaged or dead cells [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. By employing these fluorescent dyes, the dynamic changes in bacterial cell viability during phage infection can be visualized. Therefore, the intricate interaction between \u003cem\u003eSalmonella\u003c/em\u003e and phage PA13076 in solution was further investigated by fluorescence imaging using SYTO9 and PI staining. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e, the significant change in viable cell count at 30 min suggests that the phages attach to and invade the \u003cem\u003eSalmonella\u003c/em\u003e cells, leading to cell death. Over time, more bacteria are infected by the phages, resulting in a gradual increase in red fluorescence. By 120 min, the cells appeared to be completely compromised, exhibiting a strong lytic effect of the phage on the bacteria. These observations provide crucial insights into the interaction between phages and \u003cem\u003eSalmonella\u003c/em\u003e. Moreover, it can be inferred that 30 min is optimal for phage capture of whole cells for detection purposes.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e3.3. Characterization of the modification of Phage/PDDA/Mxene@MB on the GC electrode\u003c/h2\u003e \u003cp\u003eEIS were employed to charactrize the modification of the electrodes. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e(A). In comparison with that of bare GCE, the \u003cem\u003eR\u003c/em\u003e\u003csub\u003eet\u003c/sub\u003e of MXene@MB/GCE increase. The electrostatic adsorption the PDDA layer resulted in an increasing in the \u003cem\u003eR\u003c/em\u003e\u003csub\u003eet\u003c/sub\u003e because of the polymerisation of PDDA. Subsequently, the immobilization of phage led to a further increase in Rct as the phage layer behaved as an inert barrier, hindering electron transfer. After incubating the phage/PDDA/MXene@MB/GCE with bacteria, a significant rise in impedance values was observed, indicating successful capture of bacteria at the biointerface. The corresponding CV responses of these electrodes aligned with the EIS results, as presented in Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e. On the other hand, as seen in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e(B), the bare GCE exhibited no oxidation peak in the PBS solution. However, upon immobilizing the MXene@MB nanocomposite on the GC electrode surface, an oxidation peak appeared at approximately \u0026minus;\u0026thinsp;0.28 V (curve b), attributable to the oxidation of MB. Nevertheless, following treatment with PDDA, a notable reduction in the electrochemical current was observed, confirming the successful adsorption of PDDA onto the MXene@MB-modified GC electrode. After the PDDA/MXene@MB/GC electrode was incubated with phage for 90 min, a further decrease in the current response of the electrode was recorded (curve d), indicating the successful immobilization of the non-conductive phage. Upon further incubation of the sensor with bacteria, the electrochemical signal from MB diminished significantly, suggesting specific capture of bacteria. The changes in current responses measured by DPV in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e(B) is consistent with the observations made in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e(A). Overall, these results corroborate the successful construction of the electrochemical biosensors as intended and demonstrate the construction feasibility of the phage-based electrochemical biosensor.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003e3.4. The biological activity of the immobilized phages\u003c/h2\u003e \u003cp\u003eThe biological activity of the immobilized phages plays a crucial role in the biosensor's performance. Initially, the disc diffusion method was employed to assess the bioactivity of the immobilized bacteriophages. Specifically, both the PDDA/MXene@MB/ITO and phage/PDDA/MXene/ITO samples were placed on agar plates inoculated with the target bacteria. The results clearly indicate that the control ITO electrode modified solely with PDDA/MXene@MB, exhibited no plaque formation. Conversely, the ITO electrode modified with phage/PDDA/MXene@MB displayed a distinct lysis ring, indicating active bacteriophage diffusion. This evidence validates that the immobilized phages maintain their lytic ability against infected \u003cem\u003eSalmonella\u003c/em\u003e, thereby confirming their viability and functionality within the immobilized state. Furthermore, SEM images in Figure. 6(c) and 6(d) provide additional evidence for bioactivity of the immobilized phage. Figure\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e(c) confirms the successful immobilization of phages on the PDDA/MXene@MB surface, aligning with our electrochemical and disc diffusion results. Figure\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e(d) visually demonstrates that the immobilized phages not only captured bacteria but also caused partial lysis of the captured bacteria. These findings strongly further proved the bioactivity of the immobilized bacteriophages and validate the proposed phage/PDDA/MXene@MB/GCE system for the selective detection of live bacteria.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003e3.5. The density of the immobilized phages on the PDDA/MXene@MB/GCE\u003c/h2\u003e \u003cp\u003eSYBR Green I as known for its bright fluorescence when bound to nucleic acids, was used to stain phages in this study [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. In our experiment, we utilized an inverted fluorescence microscope to observe the fluorescent signals of PA13076, enabling us to assess the density and distribution of bacteriophages immobilized on the PDDA/MXene@MB/GC electrode. Figure\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e(a) exhibits distinct fluorescent dots representing the stained bacteriophages in a free state, which is consistent with the phenomena described in the literature [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e], indicating that bacteriophages can be labeled with SYBR Green I dye. For comparison, Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e(b) shows the fluorescent signals on the surface of PDDA/MXene@MB/ITO treated with SYBR Green I dye but without bacteriophages. The results demonstrate that the dye does not interact with the PDDA/MXene@MB/ITO surface. Figures\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e(c) and 7(d) exhibit the outcomes following the incubation of MXene@MB/ITO and PDDA/MXene@MB/ITO with stained bacteriophages, respectively. It is evident that the number of bacteriophages immobilized on the MXene@MB surface is significantly fewer than those immobilized on the PDDA/MXene@MB/ITO surface. This difference is attributed to the electrostatic interaction between the positively charged PDDA/MXene@MB/ITO surface and the negatively charged bacteriophage tails. This phenomenon suggests that positively charged surfaces are more useful for the bacteriophage immobilization. By analyzing the images in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e(c) and (d) using ImageJ software, only 5 pcs \u0026micro;m\u003csup\u003e-2\u003c/sup\u003e bacteriophage particles were detected on the MXene@MB/ITO surface. However, approximately 71 pcs \u0026micro;m\u003csup\u003e-2\u003c/sup\u003e bacteriophage particles were observed on the PDDA/MXene@MB/ITO surface, which is favorable for the higher detection sensitivity.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003e3.6. Optimal incubation time for phage immobilizing on the PDDA/MXene@MB/GCE\u003c/h2\u003e \u003cp\u003eTo optimize the performance of the phage-based biosensor, the incubation time for phage immobilization on the PDDA/MXene@MB/GCE was investigated. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e, with incubation time of phage varying from 30 min to 90 min, the current responses of the modified electrode gradually decreased and approaching the platform after 90 min. This increase in inhibition is attributed to the nonconductive behavior of the phages. This trend indicates that the immobilization of the phage on the electrode surface reached saturation at around 90 min of incubation. Therefore, the optimal incubation time for phage immobilization on the PDDA/MXene@MB/GCE is 90 min.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003e3.7. Electrochemical detection of \u003cem\u003eSalmonella\u003c/em\u003e\u003c/h2\u003e \u003cp\u003eHaving proven the sensing feasibility and high stability of the phage/PDDA/MXene@MB/GCE, we employed it as the sensing element to detect \u003cem\u003esalmonella\u003c/em\u003e at various concentrations. After incubating the phage/PDDA/MXene@MB/GCE with \u003cem\u003eSalmonella\u003c/em\u003e suspensions of differing concentrations at 37\u0026deg;C for 30 min, we conducted DPV analysis across a potential range of -0.6 to 0.2 V, as illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e(A). Notably, it was observed that as \u003cem\u003eSalmonella\u003c/em\u003e concentration increased from 2.4 \u0026times; 10\u003csup\u003e1\u003c/sup\u003e CFU mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e to 2.4 \u0026times; 10\u003csup\u003e7\u003c/sup\u003e CFU mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, the peak current of MB decreased. Figure\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e(B) shows a satisfactory linear range from 2.4 \u0026times; 10\u003csup\u003e1\u003c/sup\u003e CFU mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e to 2.4 \u0026times; 10\u003csup\u003e7\u003c/sup\u003e CFU mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, with a regression coefficient (\u003cem\u003eR\u003c/em\u003e\u003csup\u003e2\u003c/sup\u003e) of 0.9958. The limit of detection (LOD) for \u003cem\u003eSalmonella\u003c/em\u003e was determined to be as low as 5 CFU mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. In comparison to other detection methods, our sensor system exhibits comparable sensitivity, the capability to discriminate between live and dead bacteria, a favorable LOD, and a wide linear range, as outlined in Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e. This exceptional analytical performance is primarily due to the high-density, oriented immobilization of phages, combined with the exceptional properties of MXene. Therefore, this proposed sensing approach holds promising potential for practical bacteria detection.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec23\" class=\"Section2\"\u003e \u003ch2\u003e3.8. Specificity and reproducibility\u003c/h2\u003e \u003cp\u003eSince only live bacteria can cause disease, we investigated the present biosensor's ability to distinguish between live and dead bacteria. Figure\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003e (A) illustrates that only live \u003cem\u003eSalmonella\u003c/em\u003e caused the current response of the present biosensor, which is due to the specific capture of bacteria. This indicates that this present electrochemical biosensor can effectively differentiate between dead and live \u003cem\u003eSalmonella\u003c/em\u003e. On the other hand, considering that bacteria such as \u003cem\u003eStaphylococcus aureus\u003c/em\u003e are also significant contaminants in food and environmental pollution, it is crucial to evaluate the potential interference from these bacteria on the sensor's specificity towards \u003cem\u003eSalmonella\u003c/em\u003e. As observed in Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003e(B), there is no interference signals from other potentially coexisting bacteria. These results elucidate that this proposed biosensor exhibits remarkable specificity and selectivity, which is beneficial in reducing false positive/negative results and enhancing the detection accuracy of \u003cem\u003eSalmonella\u003c/em\u003e. To assess the reproducibility of the proposed electrochemical biosensor, the current responses of five replicate electrodes (bacteria/phages/PDDA/MXene@MB/GCE) were investigated under the same conditions. The results from all five replicate measurements exhibited similar patterns and outcomes, thus demonstrating the excellent reproducibility of our electrochemical biosensor.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section2\"\u003e \u003ch2\u003e3.9. Recoveries of the propsosed biosensor for \u003cem\u003eSalmonella\u003c/em\u003e detection in practical samples\u003c/h2\u003e \u003cp\u003eIn order to validate the practical application of the proposed biosensor in the detection of foodborne pathogens, the current responses of the phage/PDDA/MXene@MB/GC electrodes were investigated after incubated with milk or egg samples spiked with different concentrations of \u003cem\u003eSalmonella\u003c/em\u003e. The concentrations of spiked \u003cem\u003eSalmonella\u003c/em\u003e were determined and recorded in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. The recovery rate of \u003cem\u003eSalmonella\u003c/em\u003e detection in milk and egg samples ranged from 91.1\u0026ndash;108%. These results demonstrate the biosensor's high accuracy and reliability when applied to real samples.\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\u003eSpiking recoveries of the proposed methods for \u003cem\u003eSalmonella\u003c/em\u003e detection in milk and eggs\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSamples\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSpiked\u003c/p\u003e \u003cp\u003e(log \u003csub\u003e10\u003c/sub\u003e N CFU mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eDetected\u003c/p\u003e \u003cp\u003e(log \u003csub\u003e10\u003c/sub\u003e N CFU mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eRecovery\u003c/p\u003e \u003cp\u003e(%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eRSD\u003c/p\u003e \u003cp\u003e(\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;3, %)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"3\" rowspan=\"4\"\u003e \u003cp\u003eMilk\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1.38\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1.36\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e98.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e4.6\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e2.38\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2.34\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e8.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e2.7\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e4.38\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e4.39\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e100.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e2.4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e6.38\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e6.40\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e100.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1.5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"3\" rowspan=\"4\"\u003e \u003cp\u003eEggs\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1.38\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1.41\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e102.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e5.9\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e2.38\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2.36\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e99.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e4.3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e4.38\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e4.39\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e100.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1.7\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e6.38\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e6.35\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e99.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e3.9\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"4. Conclusions","content":"\u003cp\u003eThis study presents an innovative electrochemical sensor platform that utilizes MXene@MB nanocomposite materials and bacteriophage-targeted detection for rapid, precise, and ultra-sensitive identification of live food-borne pathogenic bacteria. Taking advantages of the electrostatic properties of bacteriophages, the biosensor immobilizes their heads onto the MXene@MB modified GC electrodes, enabling their tail fibers to capture food-borne pathogens. This approach not only preserves the vitality of the bacteriophages but also enhances the sensor's sensitivity. The remarkable performance of our sensor platform is evident in its ability to detect \u003cem\u003eSalmonella\u003c/em\u003e at concentrations as low as 5 CFU mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. Furthermore, the sensor can distinguish the live and dead bacteria, and demonstrates excellent specificity for \u003cem\u003eSalmonella\u003c/em\u003e, even in the presence of other bacteria that commonly contaminate food, minimizing the risk of false positives. Therefore, this work provides a potential method for the detection of food-borne pathogenic bacteria, which is significant for enhancing public health protection and ensuring the safety of the food supply.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eSupplementary information\u003c/h2\u003e \u003cp\u003eThe Supporting Information is available free of charge at\u0026hellip;\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eEthical approval\u003c/strong\u003e \u003cp\u003eThis research did not involve human or animal samples.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eConflict of interest\u003c/strong\u003e \u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eNo applicable\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eJingjing Zhou: conceptualization, methodology, investigation, writing original draft. Tingliu Deng: methodology, investigation, writing original draft, writing review and editing. Qin Zeng: conceptualization, methodology, investigation. Heye Wang: conceptualization and writing review and editing. Chunyan Deng: conceptualization, supervision, writing review and editing. All authors read and approved the final manuscript.\u003c/p\u003e\u003ch2\u003eData availability\u003c/h2\u003e \u003cp\u003eAll relevant data are within the manuscript and its additional files.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003ePanwara S, Duggiralab KS, Yadavc P, Debnathc N, Yadavc AK, Kumar A (2023) Advanced diagnostic methods for identification of bacterial foodborne pathogens: Contemporary and upcoming challenges. 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ACS Appl Mater Interfaces 6(7):4758\u0026ndash;4765. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1021/am500655r\u003c/span\u003e\u003cspan address=\"10.1021/am500655r\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Scheme 1","content":"\u003cp\u003eScheme 1 is available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"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":"Phages, Oriented immobilization, MXene, Salmonella","lastPublishedDoi":"10.21203/rs.3.rs-4649888/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4649888/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe rapid reproduction of live foodborne pathogenic bacteria poses a significant threat to human health. In the aspect of food safety monitoring, it is crucial to develop sensitive, rapid, and specific methods for detecting foodborne pathogenic bacteria. In this study, we present a novel bacteriophage-targeted electrochemical biosensor designed for accurate and quantitative detection of live \u003cem\u003eSalmonella\u003c/em\u003e in food samples. The biosensor is simply constructed by electrostatic immobilizing bacteriophages on the MXene-nanostructred electrodes. MXene, renowned for its high surface area, biocompatibility, and conductivity, serves as an ideal platform for bacteriophage immobilization. This allows for a high-density immobilization of bacteriophage particles, achieving approximately 71 pcs \u0026micro;m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e. Remarkably, the bacteriophages immoblized MXene nanostructured electrode still maintain their viability and functionality, ensuring their effectiveness in pathogen detection. Therefore, this proposed biosensor exhibited the enhanced sensitivity with a low limit of detection (LOD) of 5 CFU mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. Notably, the biosensor exhibits excellent specificity in the presence of other bacteria that commonly contaminate food, and can distinguish live \u003cem\u003eSalmonella\u003c/em\u003e from a mixed population. Furthermore, it is applicable in detecting live \u003cem\u003eSalmonella\u003c/em\u003e in food samples, which highlights its potential in food safety monitoring. This biosensor offers simplicity, convenience, and suitability for resource-limited environments, making it a promising tool for on-site monitoring of foodborne pathogenic bacteria.\u003c/p\u003e","manuscriptTitle":"An electrochemical biosensor for sensitive detection of live Salmonella in food via MXene amplified methylene blue signals and electrostatic immobilization of bacteriophages","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-07-23 04:23:59","doi":"10.21203/rs.3.rs-4649888/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"editorInvitedReview","content":"","date":"2024-07-14T19:17:03+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-07-07T06:46:38+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"245616412873842056921592677482367583089","date":"2024-07-07T06:37:22+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"82012967745543007169870248873568715070","date":"2024-07-04T13:13:29+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-07-02T12:08:40+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-07-01T08:40:33+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-07-01T08:39:50+00:00","index":"","fulltext":""},{"type":"submitted","content":"Microchimica Acta","date":"2024-06-27T06:09:51+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":"fb4c70b1-a6e5-47b5-90b3-fce9a4db1b45","owner":[],"postedDate":"July 23rd, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-08-26T15:59:51+00:00","versionOfRecord":{"articleIdentity":"rs-4649888","link":"https://doi.org/10.1007/s00604-024-06610-y","journal":{"identity":"microchimica-acta","isVorOnly":false,"title":"Microchimica Acta"},"publishedOn":"2024-08-21 15:57:06","publishedOnDateReadable":"August 21st, 2024"},"versionCreatedAt":"2024-07-23 04:23:59","video":"","vorDoi":"10.1007/s00604-024-06610-y","vorDoiUrl":"https://doi.org/10.1007/s00604-024-06610-y","workflowStages":[]},"version":"v1","identity":"rs-4649888","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4649888","identity":"rs-4649888","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}