Sensitive Detection Of Foodborne Pathogens Research Articles

One-pot dual-CRISPR/Cas12b-mediated dUTP-LAMP for rapid and sensitive detection of foodborne pathogens without carryover contamination

Rapid and accurate detection of foodborne pathogens is of great significance for ensuring food safety. Currently, the CRISPR-Dx based on Cas12b, a new and better performing Cas enzyme, is very rare, and the vast majority require a two-step method, facing aerosol contamination. In this work, we reported a one-pot dual-CRISPR/Cas12b mediated dUTP-LAMP method for rapid and sensitive detection of foodborne pathogens without nucleic acid extraction and contamination-free. The design mechanism of sgRNAs in the one-pot CRISPR/Cas12b-mediated LAMP method was investigated in detail for the first time. Moreover, a novel PAM sequence UUN of AapCas12b was discovered, and combined with dUTP-LAMP system for one-pot dual-sgRNA detection without carryover contamination. Our developed method can achieve high specificity, nucleic acid extraction-free detection of 2.33×104 CFU/mL Salmonella within 1 h. Furthermore, accurate detection of Salmonella was successfully performed in real samples of milk, fruit and egg, demonstrating the practical significance of the developed method.

Photothermal-Based Multiplex Nested Digital PCR System for Rapid Detection of Foodborne Pathogens.

The rapid and sensitive detection of foodborne pathogens is crucial for ensuring food safety. Among virus testing methods, polymerase chain reaction (PCR) has served as the gold-standard technique in most food safety regulation organizations. However, to enhance the speed and efficiency of PCR, novel approaches are continually being explored. In this work, leveraging the photothermal effects and high thermal conductivity of gold nanoparticles, we have significantly improved the heating and cooling rates of thermal cycles, enabling ultra-fast PCR detection. Specifically, we present a pre-degassing multiplex digital PCR chip integrated with gold nanoparticles. We further developed a portable system with a light source for photothermal heating cycling, along with an optoelectronic sensor to analyze PCR amplification products after rapid thermal cycling. As proof of concept, the proposed chip and portable device was applied for the on-site detection of several types of foodborne pathogens, including Escherichia coli, Listeria monocytogenes, Staphylococcus aureus, and Salmonella. The whole system could distinguish those pathogens within 20 min, showing good potential for the rapid detection of multiple types of foodborne pathogens.

Dynamically reconfigurable complex liquid crystal emulsions

ABSTRACT Complex emulsions, containing liquid crystals (LCs) and immiscible oils such as fluorocarbon or silicone oils, present innovative functionalities in soft materials. These emulsions exhibit dynamic reconfiguration in droplet morphology and internal LC organisation, expanding applications in optics, sensing, and active matter. This review highlights the evolution and potential of LC-containing complex emulsions. Emphasis is placed on emulsions featuring dynamic and reversible morphological transformations and LC reorganisation, underpinning their suitability for tunable micro-lenses, chemical sensing, and biosensing. Nematic LCs within complex emulsions allow for topological defect-driven functionalization, attaching antibodies to induce substantial changes in the LC director field upon interfacial recognition events. This feature may facilitate the development of ultrasensitive chemical and biological sensors. Moreover, cholesteric LCs, through their selective reflection, enable the rapid and sensitive detection of foodborne pathogens. The controlled assembly of magnetic nanoparticles at interfaces illustrates advancements towards magnetic field-responsive and active colloids. Future perspectives include exploiting these properties for intelligent colloidal systems capable of autonomous behaviour, and furthering applications in dynamic photonic devices, high-security identification tags, and responsive lens systems.

Specific separation and sensitive detection of foodborne pathogens by phage-derived bacterial-binding protein-nano magnetic beads coupled with smartphone-assisted paper sensor

Foodborne pathogen infection poses a significant threat to public health and is considered as one of the most serious hazards in global food safety. Herein, a sensitive and efficient method for on-site monitoring of foodborne pathogens was developed by using a smartphone-assisted paper-sensor combined with phage-derived bacterial-binding proteins-nano magnetic beads (PBPs-MBs). PBPs including tail fiber protein (TFP:gp13), cell-wall binding domain (CBD) of endolysin and tailspike protein (TSP) coated on the surface of MBs were applied for rapid separation and enrichment of targeted bacteria (Escherichia coli O157:H7, Staphylococcus aureus and Salmonella typhimurium, respectively) from food samples in 20 min before detection on paper-based sensors. The paper-based sensor was loaded with the lytic agent (polymyxin B) to induce bacterial lysis and release specific endogenous enzymes. Subsequently, three distinct chromogenic substrates were hydrolyzed by their corresponding enzymes, resulting in characteristic color changes on the paper, respectively. In addition, a smartphone APP for red-green-blue (RGB) color analysis of paper was able to directly detect three foodborne pathogens. As a result, the limit of detection (LOD) values for three foodborne pathogens were found to be 2.44 × 102, 2.68 × 104 and 4.62 × 103 CFU/mL, respectively, which were much lower than other studies (106–108 CFU/mL) based on enzymes. Moreover, the feasibility of this approach was further assessed through the successful detection of targeted bacteria in real samples with satisfactory recovery rates. In conclusion, this smartphone-assisted biosensor offers promising application potential for point-of-care testing (POCT) of foodborne pathogens in resource-scarce areas.

Enhancing Oriented Immobilization Efficiency: A One-for-Two Organism-Bispecific Nanobody Scaffold for Highly Sensitive Detection of Foodborne Pathogens.

Nanobodies have gained widespread application in immunoassays. However, their small size presents a significant challenge in achieving effective immobilization and optimal sensitivity. Here, we present a novel "one-for-two"-oriented immobilization platform based on an organism-bispecific nanobody (O-BsNb) scaffold, enabling highly sensitive detection of two bacterial pathogens. Through genetic engineering, a bispecific nanobody (BsNb) was engineered, targeting Salmonella spp. and Vibrio parahaemolyticus. The O-BsNb scaffold allowed one nanobody to bind specifically to inactivated bacteria, forming an organism-oriented immobilization platform, while the other served as the capture antibody. Consequently, the O-BsNb bioscaffold-based ELISA (O-ELISA) for individual detection of S. enteritidis and V. parahaemolyticus was established. When compared to the sandwich ELISA utilizing passive immobilization of monovalent nanobodies, the O-ELISA exhibited a remarkable 13.4- and 13.7-fold improvement in LOD for S. enteritidis and V. parahaemolyticus, respectively, highlighting the enhanced immobilization efficacy of the O-ELISA. Furthermore, the feasibility and reproducibility of the assay in practical samples were meticulously evaluated, revealing exemplary performance in terms of recovery precision and assay stability. These findings demonstrate the significant potential of the O-ELISA platform for the sensitive detection of macromolecules, opening new avenues for efficient pathogen identification in foodborne safety and clinical diagnostics.

Rapid detection of foodborne pathogens in diverse foodstuffs by universal electrochemical aptasensor based on UiO-66 and methylene blue composites.

Rapid and sensitive detection of foodborne pathogens in complex environments is essential for food protection. A universal electrochemical aptasensor was fabricated for the detection of three common foodborne pathogens, including Escherichia coli (E. coli), Staphylococcus aureus (S. aureus), and Salmonella typhimurium (S. typhimurium). The aptasensor was developed based on the homogeneous and membrane filtration strategy. Zirconium-based metal-organic framework (UiO-66)/methylene blue (MB)/aptamer composite was designed as a signal amplification and recognition probe. Bacteria were quantitatively detected by the current changes of MB. By simply changing the aptamer, different bacteria could be detected. The detection limits of E. coli, S. aureus and S. typhimurium were 5, 4 and 3CFU·mL-1, respectively. In humidity and salt environments, the stability of the aptasensor was satisfactory. The aptasensor exhibited satisfactory detection performance in different real samples. This aptasensor has excellent potential for rapid detection of foodborne pathogens in complex environments.

Preparation of a multifunctional and ultrasensitive Au@AgNPs-Van/PDMS film SERS substrate for rapid detection of foodborne pathogens in beef

We present a novel and super-sensitive vancomycin (Van) modified Au@AgNP self-assembly with polydimethylsiloxane (PDMS) (Au@AgNPs/Van-PDMS) film surface-enhanced Raman scattering (SERS) substrate for identification of foodborne pathogens in beef, without the need for in vitro bacterial culture. The results illustrated that the Au@AgNPs/Van-PDMS film exhibited high reproducibility and Raman enhancement effect (1.09 × 105). Clostridium perfringens, Bacillus subtilis and Staphylococcus aureus isolated from beef captured by Au@AgNPs/Van-PDMS film exhibited high reproducibility and significant differences. Principal component analysis score plots distinguished classes of foodborne pathogens, and the qualitative identification of linear discriminant analysis was correct at 100%. The detection limit of S. aureus in beef was as low as 3 CFU/mL. This method could provide an effective means and technical support for realizing the application of SERS technology for quick and highly sensitive detection of foodborne pathogens in complex environments.

Microfluidic isothermal amplification strategy coupled with improved propidium monoazide (PMAxx) for rapid detection of six viable foodborne pathogens

Based on the combination of the centrifugal microfluidic chip, programable loop-mediated isothermal amplification (pLAMP), and improved propidium monoazide (PMAxx), a new method called PMAxx-mpLAMP was developed for the quantitative determination of six viable foodborne pathogens in real food samples. Specific target genes and primers were designed and verified, and the PMAxx conditions were optimized. Treatment with PMAxx before pLAMP eliminated false-positive outcomes from dead bacteria and identified viable target bacteria with good specificity and sensitivity. The PMAxx-mpLAMP strategy could detect approximately 102 ∼ 103 CFU/mL pathogens without any pre-enrichment and 101 CFU/mL after a short period of enrichment. In addition, the PMAxx-mpLAMP strategy was performed in the presence of a high concentration of dead bacteria and non-target bacteria. Therefore, the established PMAxx-mpLAMP strategy in this study has potential value for the rapid and sensitive detection of foodborne pathogens.

Vancomycin functionalization of gold nanostars for sensitive detection of foodborne pathogens through surface‐enhanced Raman scattering

AbstractIn this paper, we report a sensitive surface‐enhanced Raman scattering (SERS) biosensor for the detection of food‐borne pathogens on the basis of recognition of bacterial cells through the specific targeting agent vancomycin. Vancomycin‐functionalized gold nanostars (GNSs–4–MBA–van) were synthesized as SERS tags for specific and sensitive quantification of food pathogens. The Raman reporter molecule 4–mercaptobenzoic acid (4–MBA) was also a linker molecule to attach vancomycin to gold nanostars. The proposed vancomycin‐based SERS technique achieved a detection limit of 5.7 and 8.2 CFU/ml for Staphylococcus aureus and Escherichia coli, respectively. The logarithm of the bacterial concentration ranged from 101 to 107 CFU/ml and demonstrated a linear relationship with the SERS intensity of the characteristic 4–MBA Raman peak at 1,081 cm−1. Principle component analysis was used to discriminate the two pathogens simultaneously by using reproducible and specific intrinsic fingerprints and intracellular components obtained through SERS. Moreover, the spiking tests revealed that GNSs–4–MBA–van is effective as a potential biosensor for detecting pathogens in the fresh meat and food‐processing industries.

Combination of filtration and immunomagnetic separation based on real-time PCR to detect foodborne pathogens in fresh-cut apple

Rapid detection methods require pre-enrichment culture in order to detect low levels of foodborne pathogens. To rapidly detect foodborne pathogens, enrichment culture processes could be replaced. Filtration and immunomagnetic separation methods have been identified to effectively concentrate and separate target pathogens from foods. In this study, a combination of filtration and immunomagnetic separation (IMS) has enabled the rapid and sensitive detection of foodborne pathogens. The pretreatment method, including separation and concentration procedures, increased sensitivity 10–100-fold. The sensitivity of a combination method using filtration and IMS to detect Escherichia coli O157:H7 and Salmonella enterica subsp. enterica serovar Typhimurium was 100–101 CFU/10 mL. In fresh-cut apples, IMS combined with filtration effectively improved the detection limit of real-time PCR to 2.70 × 101 CFU/g in E. coli O157:H7 and 1.80 × 102 CFU/g in Salmonella. The filtration simplified processing of large-volumes (250 mL) and effectively concentrated pathogens while decreasing immunomagnetic beads used in IMS. Bacterial concentration by IMS combined with filtration increased sensitivity 10–100-fold compared with control. In addition, the application of IMS effectively removed concentrated residual food material (10–15 mg/mL) after filtration, improving relative sensitivity. In conclusion, this method may detect foodborne pathogen in foods such as fresh-cut fruits in a more rapid and sensitive fashion than traditional culture-based methods.

Controlled PAH-mediated method with enhanced optical properties for simple, stable immunochromatographic assays

Despite their potential for signal amplification in immunochromatographic assays (ICAs) with Au nanoparticles (AuNPs) as probes, metal growth methods are of limited practical applicability given their complex non-specificity and lack of robust growth schemes. Here, we propose a novel method of polyallylamine hydrochloride (PAH)-mediated metal growth for the detection of Escherichia coli O157:H7 by AuNP-ICA. The developed method relies on the highly controlled growth of Cu shells on the AuNP core and allows one to achieve highly enhanced colorimetric signals by controlling PAH as the growth framework. The introduction of PAH eliminates the non-specific adsorption of Cu ions on the nitrocellulose membrane and thus provides maximized and effective signal-to-noise ratios to achieve a detection limit of 9.8 CFU/mL for E. coli O157:H7. Moreover, the newly developed detection method exhibits good reproducibility (coefficient of variation <13%), remarkable stability, and practical applicability. The PAH-mediated signal enhancement system paves the way to the realization of stable metal growth methods based on Au, Ag, and other metals and is well suited for the rapid, stable, and sensitive detection of food-borne pathogens using the AuNP-ICA platform.

“Lighting-up” methylene blue-embedded zirconium based organic framework triggered by Al3+ for advancing the sensitivity of E. coli O157:H7 analysis in dual-signal lateral flow immunochromatographic assay

The sensitive detection of foodborne pathogens is of great significance for ensuring food safety and quality. Herein, on the basis of methylene blue-embedded zirconium based organic framework (UIO@MB) as the remarkable capture carrier and signal indicator, with the Al3+-assisted the fluorescent signal response, we developed a label-free and dual-signal lateral flow immunochromatographic assay (LDLFIA) for sensitive detection of Escherichia coli (E. coli) O157:H7. The UIO@MB sensing carrier without monoclonal antibodies (mAbs) was manufactured, which adhered to bacteria to form the UIO@MB-E. coli O157:H7 conjugate, resulting in visible blue band. Then the fluorescent response of the OH-rich UIO@MB was excited by introducing Al3+, arising from capturing of Al3+ by -OH through coordination and electrostatic affinity, thus generating a green fluorescent band. Impressively, a smartphone-based portable reading system was developed that can reflect the test results of UIO@MB-LDLFIA immediately. Under optimum conditions, UIO@MB-LDLFIA can complete colorimetric and fluorescent mode detection within 90 min, with a detection sensitivity of 103 CFU/mL, which were 100 times lower than traditional gold nanoparticles-based LFIA and polymerase chain reaction (PCR). Moreover, the feasibility of the method was further evaluated by the determination of E. coli O157: H7 in drinking water and cabbage with average recoveries of 85.1-123.0%.

Rapid detection of Salmonella in milk by a nuclear magnetic resonance biosensor based on the streptavidin-biotin system and O-carboxymethyl chitosan target gadolinium probe

Rapid and sensitive detection of foodborne pathogens is of great importance for food safety. Here, a set of nuclear magnetic resonance (NMR) biosensors based on a O-carboxymethyl chitosan target gadolinium (Gd) probe was developed to quickly detect Salmonella in milk by combining NMR technology and bioimmunotechnology with membrane filtration technology. First, O-carboxymethyl chitosan (O-CMC) was biotinylated to prepare biotinylated O-carboxymethyl chitosan (biotin-O-CMC) through amide reaction, and biotinylated magnetic complexes (biotin-O-CMC-Gd) were obtained by using O-CMC, which has strong chelating adsorption on Gd. The target probe was obtained by combining biotin-O-CMC-Gd with the biotinylated antibody (biotin-antibody) via streptavidin (SA) by introducing the SA-biotin system. Then, Salmonella was captured by the target probe through antigen-antibody interaction. Finally, NMR was used to measure the longitudinal relaxation time (T1) of the filtrate collected by membrane filtration. This NMR biosensor with good specificity and high efficiency can detect Salmonella with the sensitivity of 1.8 × 103 cfu/mL within 2 h; in addition, it can realize the detection of complex samples because of its strong anti-interference capability and may open up a new method for rapid detection of Salmonella, which has a great application potential.

Biosensors Coupled with Signal Amplification Technology for the Detection of Pathogenic Bacteria: A Review.

Foodborne disease caused by foodborne pathogens is a very important issue in food safety. Therefore, the rapid screening and sensitive detection of foodborne pathogens is of great significance for ensuring food safety. At present, many research works have reported the application of biosensors and signal amplification technologies to achieve the rapid and sensitive detection of pathogenic bacteria. Thus, this review summarized the use of biosensors coupled with signal amplification technology for the detection of pathogenic bacteria, including (1) the development, concept, and principle of biosensors; (2) types of biosensors, such as electrochemical biosensors, optical biosensors, microfluidic biosensors, and so on; and (3) different kinds of signal amplification technologies applied in biosensors, such as enzyme catalysis, nucleic acid chain reaction, biotin-streptavidin, click chemistry, cascade reaction, nanomaterials, and so on. In addition, the challenges and future trends for pathogenic bacteria based on biosensor and signal amplification technology were also discussed and summarized.

Sensitive detection of foodborne pathogens based on CRISPR-Cas13a.

Salmonella, being one of the most widespread foodborne pathogens, is a compulsory test item required by national food safety standard of China and many other countries. More sensitive and specific Salmonella detection method is still needed since traditional methods are time consuming and highly dependent on enormous manpower and material resources. In this research, a bacteria detection method based on CRISPR-Cas13a system (where CRISPR is Clustered Regularly Interspaced Short Palindromic Repeats) was proposed. The target DNA was amplified by PCR and transcribed into RNA by T7 transcriptase, which can activate the RNase activity of the Cas13a protein. The self-folding quenched fluorescent probe can be cleaved by the activated Cas13a protein to generate fluorescent signal. We named this method as PCF detection (PCR-CRISPR-Fluorescence based nucleic acid detection). In this study, PCF detection showed excellent sensitivity, which can detect Salmonella genomic DNA with a minimum of 101 aM or 10° CFU/ml Salmonella bacteria in 2hr. It also showed good specificity with no cross-reaction with other common foodborne bacteria. PRACTICAL APPLICATION: The PCF detection method proposed in this article can detect Salmonella sensitively and specifically, providing a novel strategy for the detection of foodborne pathogens in food and has great application potential in other microbial detection fields.

Biotin exposure–based immunomagnetic separation coupled with sodium dodecyl sulfate, propidium monoazide, and multiplex real-time PCR for rapid detection of viable Salmonella Typhimurium, Staphylococcus aureus, and Listeria monocytogenes in milk

In this study, we established a rapid and sensitive method for the detection of viable Salmonella Typhimurium, Staphylococcus aureus, and Listeria monocytogenes in milk using biotin-exposure-based immunomagnetic separation (IMS) combined with sodium dodecyl sulfate (SDS), propidium monoazide (PMA), and multiplex real-time PCR (mRT-PCR). We used IMS to lessen the assay time for isolation of target bacteria. We then optimized the coupling conditions and immunomagnetic capture process. The immunoreaction and incubation times for 5 μg of mAb coupled with 500 μg of streptavidin-functionalized magnetic beads using a streptavidin-biotin system were 90 and 30 min, respectively. Treatment with SDS-PMA before mRT-PCR amplification eliminated false-positive outcomes from dead bacteria and identified viable target bacteria with good sensitivity and specificity. The limit of detection of IMS combined with the SDS-PMA-mRT-PCR assay for the detection of viable Salmonella Typhimurium, Staph. aureus, and L. monocytogenes in spiked milk matrix samples was 10 cfu/mL and remained significant even in the appearance of 106 cfu/mL of nontarget bacteria. The entire detection process was able to identify viable bacteria within 9 h. The combination of biotin-exposure-mediated IMS and SDS-PMA-mRT-PCR has potential value for the rapid and sensitive detection of foodborne pathogens.

Rapid and highly sensitive detection of Escherichia coli O157:H7 in food with loop-mediated isothermal amplification coupled to a new bioluminescent assay.

Testing for bioluminescent pyrophosphate is a convenient method of DNA detection without complex equipments, but it is insufficiently sensitive and offers no particular time advantage over other rapid detection methods. The shortcomings of the traditional bioluminescent pyrophosphate method have been addressed by using 2-deoxyadenosine-5-(α-thio)-triphosphate (dATPαS) instead of dATP for LAMP, thus reducing the high background signal and generating a constant background value. In this study, LAMP coupled to a novel bioluminescent pyrophosphate assay was developed to detect E. coli O157:H7. The new method has a limit of detection of <10 copies/μL or 5CFU/mL; its sensitivity is higher than that of the conventional LAMP assay. Moreover, a food-borne pathogen can be detected when a single DNA template is included in the LAMP assay, making it 100 times more sensitive than the traditional LAMP method. Three hundred food samples were tested with this assay and the accuracy of detection was verified with a culture method and MALDI Biotyper. The assay only took 90-120 min and detected <10 copies of the pathogen. This method had the advantages of rapidity, sensitivity, and simplicity, so it is very competitive for the rapid and highly sensitive detection of food-borne pathogens.

A smart microfluidic platform for rapid multiplexed detection of foodborne pathogens

Rapid and sensitive detection of foodborne pathogens in food industry is of high importance in day-to-day practice to ensure safe food. To address this issue, multiple foodborne pathogens are targeted for rapid identification based in DNA amplification. A 3D PDMS sponge was fabricated using salt crystals as scarifying mold and functionalized with a ligand, apolipoprotein-H (ApoH), to test bacterial capturing for both Gram positive (L. monocytogenes) and negative bacteria (Salmonella spp.), in a microfluidic device. Pure culture of both pathogens in a range of ~10–105 CFU/mL were tested and the application of the developed automated pre-concentration protocol in real samples was verified using spiked surface samples after swab sampling. Bacterial DNA was extracted directly from the sponge and used for Real Time quantitative Polymerase Chain Reaction (qPCR) detection. The sponges did not show any significant resistance to sample flow and could easily be incorporated in a microfluidic device. A capture efficiency above 70% was observed for both targeted (Gram positive and Gram negative) pathogens and a Limit of Detection (LoD) in the range of 103 and 104 CFU/mL was obtained for Salmonella spp. and L. monocytogenes, respectively. Using this approached, we are able to perform multiplexed (Gram positive and Gram negative) capturing and reduce the enrichment time compared to the gold standard plate culture (over 1-day) method. The use of a 3D sponge for direct capturing of multiplexed pathogen on microfluidic device, followed by qPCR detection is an efficient and versatile method to stratify the presence of bacteria. This approach and methodology has potential to be integrated in full automatized device and used as point of need (PoN) system for foodborne pathogen stratification in food packaging/production industries.

Microfluidic-Based Approaches for Foodborne Pathogen Detection.

Food safety is of obvious importance, but there are frequent problems caused by foodborne pathogens that threaten the safety and health of human beings worldwide. Although the most classic method for detecting bacteria is the plate counting method, it takes almost three to seven days to get the bacterial results for the detection. Additionally, there are many existing technologies for accurate determination of pathogens, such as polymerase chain reaction (PCR), enzyme linked immunosorbent assay (ELISA), or loop-mediated isothermal amplification (LAMP), but they are not suitable for timely and rapid on-site detection due to time-consuming pretreatment, complex operations and false positive results. Therefore, an urgent goal remains to determine how to quickly and effectively prevent and control the occurrence of foodborne diseases that are harmful to humans. As an alternative, microfluidic devices with miniaturization, portability and low cost have been introduced for pathogen detection. In particular, the use of microfluidic technologies is a promising direction of research for this purpose. Herein, this article systematically reviews the use of microfluidic technology for the rapid and sensitive detection of foodborne pathogens. First, microfluidic technology is introduced, including the basic concepts, background, and the pros and cons of different starting materials for specific applications. Next, the applications and problems of microfluidics for the detection of pathogens are discussed. The current status and different applications of microfluidic-based technologies to distinguish and identify foodborne pathogens are described in detail. Finally, future trends of microfluidics in food safety are discussed to provide the necessary foundation for future research efforts.

Fluorescence immunoassay through histone-ds-poly(AT)-templated copper nanoparticles as signal transductors for the sensitive detection of Salmonella choleraesuis in milk

The rapid and sensitive detection of foodborne pathogens is one of the most important issues in food safety control. In this work, we developed a novel fluorescence immunoassay method for the sensitive detection of Salmonella choleraesuis. The method uses the fluorescent signals of histone-ds-poly(AT)-templated copper nanoparticles (His-pAT CuNP) as signal transducers and glucose oxidase as an alternative for horseradishperoxidase for the generation of hydrogen peroxide (H2O2) through the catalysis of glucose. The H2O2 is then further converted into hydroxyl radical (·OH) by Fenton reagents. Owing to the ultrahigh sensitivity of His-pAT CuNP synthesis toward ·OH, the proposed fluorescence immunoassay method exhibited excellent sensitivity for S. choleraesuis, with a limit of detection of 8.04 × 101 cfu/mL, which is 3 orders of magnitude lower than that of the tetramethylbenzidine-based traditional immunoassay. The reliability of the proposed method was evaluated by using spiked milk samples with S. choleraesuis concentration ranging from 8.8 × 101 to 8.8 × 104 cfu/mL. The average recoveries for the intra- and inter-assay ranged from 73.52 to 96.59% and from 66.99 to 98.24% with a coefficient of variation from 6.85 to 31.26% and 5.46 to 17.99%, respectively. These results indicated that the proposed fluorescence immunoassay possesses a great potential for ultra-sensitive detection of foodborne pathogens in food safety control.