Rapid amperometric detection of Escherichia coli in wastewater by measuring β-D glucuronidase activity with disposable carbon sensors

An assay on the indirect amperometric quantification of the β-D-Glucuronidase (GLUase) activity was developed for the rapid and specific detection of Escherichia coli (E. coli) in complex environmental samples. The p-aminophenyl β-D-glucopyranoside (PAPG) was selected as an electrochemical substrate for GLUase measurement and the p-aminophenol (PAP) released during the enzymatic hydrolysis was monitored by cyclic voltammetry with disposable carbon screen-printed sensors. The intensity of the measured anodic peak current was proportional to the amount of GLUase, and therefore to the number of E. coli in the tested sample. Once the substrate concentration and pH values optimized, a GLUase detection limit of 10 ng mL⁻¹ was achieved. Using a procedure involving a filtration step of the bacteria followed by their incubation with the substrate solution containing both the nonionic detergent Triton X-100 as permeabilization agent and the culture media Luria broth to monitor the growth, filtered bacterial cells ranging from 5 × 10⁴ to 10⁸ UFC/membrane were detected within 3 h. The amperometric assay was applied to the determination of fecal contamination in raw and treated wastewater samples and it was successfully compared with conventional bacterial plating methods and uidA gene quantitative PCR. Owing to its ability to perform measurements in turbid media, the GLUase amperometric method is a reliable tool for the rapid and decentralized quantification of viable but also nonculturable E. coli in complex environmental samples.     

Fluorescence detection of total count of Escherichia coli and Staphylococcus aureus on water-soluble CdSe quantum dots coupled with bacteria

The aim of this paper was to demonstrate a fluorescence measurement method for rapid detection of two bacterial count by using water-soluble quantum dots (QDs) as a fluorescence marker, and spectrofluorometer acted as detection apparatus, while Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) were as detection target bacteria. Highly luminescent water-soluble CdSe QDs were first prepared by using thioglycolic acid (TGA) as a ligand, and were then covalently coupled with target bacteria. The bacterial cell images were obtained using fluorescence microscopy. Our results showed that CdSe QDs prepared in water phase were highly luminescent, stable, and successfully conjugated with E. coli and S. aureus. The fluorescence method could detect 10²-10⁷ CFU/mL total count of E. coli and S. aureus in 1-2 h and the low detection limit is 10² CFU/mL. A linear relationship of the fluorescence peak intensity and log total count of E. coli and S. aureus have been established using the equation Y = 118.68X − 141.75 (r = 0.9907).     

Development of multiplex loop mediated isothermal amplification (m-LAMP) label-based gold nanoparticles lateral flow dipstick biosensor for detection of pathogenic Leptospira

In recent years extensive numbers of molecular diagnostic methods have been developed to meet the need of point-of-care devices. Efforts have been made towards producing rapid, simple and inexpensive DNA tests, especially in the diagnostics field. We report on the development of a label-based lateral flow dipstick for the rapid and simple detection of multiplex loop-mediated isothermal amplification (m-LAMP) amplicons. A label-based m-LAMP lateral flow dipstick assay was developed for the simultaneous detection of target DNA template and a LAMP internal control. This biosensor operates through a label based system, in which probe-hybridization and the additional incubation step are eliminated. We demonstrated this m-LAMP assay by detecting pathogenic Leptospira, which causes the re-emerging disease Leptospirosis. The lateral flow dipstick was developed to detect of three targets, the LAMP target amplicon, the LAMP internal control amplicon and a chromatography control. Three lines appeared on the dipstick, indicating positive results for all representative pathogenic Leptospira species, whereas two lines appeared, indicating negative results, for other bacterial species. The specificity of this biosensor assay was 100% when it was tested with 13 representative pathogenic Leptospira species, 2 intermediate Leptospira species, 1 non-pathogenic Leptospira species and 28 other bacteria species. This study found that this DNA biosensor was able to detect DNA at concentrations as low as 3.95 × 10⁻¹ genomic equivalent ml⁻¹. An integrated m-LAMP and label-based lateral flow dipstick was successfully developed, promising simple and rapid visual detection in clinical diagnostics and serving as a point-of-care device.     

Light scattering sensing detection of pathogens based on the molecular recognition of immunoglobulin with cell wall-associated protein A

In this contribution, we report a rapid optical detection method of pathogens using Staphylococcus aureus (S. aureus) as the model analyte based on the molecular recognition of immunoglobulin with cell wall-associated Protein A (SpA). It was found that the molecular recognition of human immunoglobulin (IgG) with protein A on the cell wall of S. aureus on glass slide sensing area could result in strong surface enhanced light scattering (SELS) signals, and the SELS intensity (ΔI) increases proportionally with the concentration of S. aureus over the range of 2.5 × 10⁵-1.0 × 10⁸ CFU mL⁻¹ with right angle light scattering (RALS) signals detection mode. In order to identify the solid support based molecular recognition between IgG with SpA, we also employed water-soluble CdS quantum dots (CdS-QDs) as a fluorescent marker for IgG by immobilizing the IgG onto the surfaces of CdS-QDs through covalent binding in order to generate recognition probes for SpA on the cell wall of S. aureus. Consequently, the fluorescent method also showed that the detection for pathogens with solid supports is reliable based on the molecular recognition of IgG with SpA.     

Trace metal analysis on hafnium silicate deposited Si wafer by Total Reflection X-ray Fluorescence

Hafnium silicate is a so-called high-k material, which is a new key material in the semiconductor field. This material is difficult to analyze by a conventional W-Lβ1TXRF source due to the high background originating from Hf-Lα lines. In this paper, the capability of Ir source TXRF analysis on hafnium silicate films is investigated with intentional contamination of Ti, Cr, Fe, Ni and Cu elements. The spectral fitting is discussed where X-ray resonant Raman scattering and escape peak of Ir-Lα overlap with Ni-Kα peak. The detection limits are estimated to 0.9 × 10¹⁰ to 2 × 10¹⁰ atoms/cm² for the transition metals.     

Rapid Legionella pneumophila determination based on a disposable core–shell Fe3O4@poly(dopamine) magnetic nanoparticles immunoplatform

A novel amperometric magnetoimmunoassay, based on the use of core–shell magnetic nanoparticles and screen-printed carbon electrodes, was developed for the selective determination of Legionella pneumophila SG1. A specific capture antibody (Ab) was linked to the poly(dopamine)–modified magnetic nanoparticles (MNPs@pDA-Ab) and incubated with bacteria. The captured bacteria were sandwiched using the antibody labeled with horseradish peroxidase (Ab-HRP), and the resulting MNPs@pDA-Ab-Legionella neumophila-Ab-HRP were captured by a magnetic field on the electrode surface. The amperometric response measured at −0.15 V vs. Ag pseudo-reference electrode of the SPCE after the addition of H₂O₂ in the presence of hydroquinone (HQ) was used as transduction signal. The achieved limit of detection, without pre-concentration or pre-enrichment steps, was 10⁴ Colony Forming Units (CFUs) mL⁻¹. The method showed a good selectivity and the MNPs@pDA-Ab exhibited a good stability during 30 days. The possibility of detecting L. pneumophila at 10 CFU mL⁻¹ level in less than 3 h, after performing a membrane-based preconcentration step, was also demonstrated.     

A dual-response fluorescent probe for Zn and Al detection in aqueous media: pH-dependent selectivity and practical application

A Schiff base-type fluorescent probe (1) consist of 2-hydroxynaphthaldehyde and glutamide moieties has been designed and synthesized for detection Zn²⁺ and Al³⁺. The probe shows pH dependent dual-selectivity for Zn²⁺ and Al³⁺ in Tris-HCl buffer, viz. that can selectively recognized Zn²⁺ at pH 7.4 and Al³⁺ at pH 6.0, respectively. From Job's plots and MS data, the stoichiometric ratios of the probe with Zn²⁺ and Al³⁺ appeared to be 1:1 and 2:1, respectively. The probe can detect as low as 5.5 × 10⁻⁸ M⁻¹ Zn²⁺ and 1.27 × 10⁻⁷ M⁻¹ Al³⁺, whereas respective association constants are 4.27 × 10⁴ M⁻¹ and 3.50 × 10⁹ M⁻¹. Furthermore, it is also confirmed that the probe has good cell-permeability and could thus be used to selectively sense intracellular Zn²⁺ and Al³⁺ by bioimaging in different pH environment. Finally the probe has been used successfully for determination of the analytes in real drug samples.     

In situ selective determination of methylmercury in river water by diffusive gradient in thin films technique (DGT) using baker's yeast (Saccharomyces cerevisiae) immobilized in agarose gel as binding phase

Saccharomyces cerevisiae immobilized in agarose gel as binding phase and polyacrylamide as diffusive layer in the diffusive gradient in thin films technique (DGT) was used for selective determination of methylmercury (MeHg). Deployment tests showed good linearity in mass uptake up to 48 h (3276 ng). When coupling the DGT technique with Cold Vapor Atomic Fluorescence Spectrometry, the method has a limit of detection of 0.44 ng L⁻¹ (pre concentration factor of 11 for 48 h deployment). Diffusion coefficient of 7.03 ± 0.77 × 10⁻⁶ cm² s⁻¹ at 23 °C in polyacrylamide gel (pH = 5.5 and ionic strength = 0.05 mol L⁻¹ NaCl) was obtained. Influence of ionic strength (from 0.0005 mol L⁻¹ to 0.1 mol L⁻¹ NaCl) and pH (from 3.5 to 8.5) on MeHg uptake were evaluated. For these range, recoveries of 84–105% and 84–98% were obtained for ionic strength and pH respectively. Potential interference due to presence of Cu, Fe, Mn, Zn was also assessed showing good recoveries (70–87%). The selectivity of the proposed approach was tested by deployments in solutions containing MeHg and Hg(II). Results obtained showed recoveries of 102–115 % for MeHg, while the uptake of Hg(II) was insignificant. The proposed approach was successfully employed for in situ measurements in the Negro River (Manaus-AM, Brazil).     

Development of an impedimetric immunosensor based on electropolymerized polytyramine films for the direct detection of Salmonella typhimurium in pure cultures of type strains and inoculated real samples

The development of a faradic impedimetric immunosensor based on electropolymerized polytyramine (Ptyr) films for the detection of S. typhimurium in milk is described for the first time. Polyclonal anti-Salmonella was cross-linked, in the presence of glutaraldehyde vapors, on Ptyr-modified gold electrodes. The dielectric behaviour of Ptyr films was evaluated with capacitance measurements, while their stability in neutral aqueous solutions was examined with impedimetric measurements. The effect of the concentration of tyramine in the forming solution on both the sensitivity and the dynamic range of the resulted immunosensors was also investigated. The alteration of the interfacial features of the electrodes due to different modification or recognition steps, was measured by faradic electrochemical impedance spectroscopy in the presence of a hexacyanoferrate(II)/(III) redox couple. At samples containing a low initial concentration of 10 cfu mL⁻¹S. typhimurium, that actually defines the LOD of the immunosensors, signal changes of 33% and 88% were achieved after 3 and 10 h incubation, respectively. To achieve the working simplicity expected by a biosensor, immunoreaction was performed directly in cultures. This resulted in the elimination of various centrifugation and washing steps, which are used for the isolation of bacteria cells from the culture, thus making the proposed immunosensors promising candidates for on-site applications. Finally, the proposed immunosensors were successfully used for the detection of S. typhimurium in experimentally inoculated milk samples.     

An ultrasensitive lysozyme chemiluminescence biosensor based on surface molecular imprinting using ionic liquid modified magnetic graphene oxide/β-cyclodextrin as supporting material

In this work, ionic liquid modified Fe₃O₄@dopamine/graphene oxide/β-cyclodextrin (ILs-Fe₃O₄@DA/GO/β-CD) was used as supporting material to synthesize surface molecularly imprinted polymer (SMIP) which then was introduced into chemiluminescence (CL) to achieve an ultrasensitive and selective biosensor for determination of lysozyme (Lys). ILs and β-CD was applied to provide multiple binding sites to prepare Lys SMIP and Fe₃O₄@DA was designed to make the product separate easily and prevent the aggregation of GO which could improve absorption capacity for its large specific surface area. The ILs-Fe₃O₄@DA/GO/β-CD-SMIP showed high adsorption capacity (Q = 101 mg/g) to Lys in the adsorption isotherm assays. The adsorption equilibrium was reached within 10 min for all the concentrations, attributing to the binding sites situated exclusively at the surface, and the adsorption model followed Langmuir isotherm. Under the suitable CL conditions, the proposed biosensor could response Lys linearly in the range of 1.0 × 10⁻⁹–8.0 × 10⁻⁸ mg/mL with a detection limit of 3.0 × 10⁻¹⁰ mg/mL. When used in practical samples in determination of Lys, the efficient biosensor exhibited excellent result with the recoveries ranging from 94% to 112%.     

A rapid method for determining Mycobacterium tuberculosis based on a bulk acoustic wave impedance biosensor

The bulk acoustic wave impedance biosensor was set up and used to monitor the growth of Mycobacterium tuberculosis (M.TB). This sensor is rapid, simple, sensitive (lower limit is 2×10³ cells ml⁻¹) and cheap (easy to generalize). The typical response curve was different from other bacteria's, such as Escherichia coli, Staphylococcus aureus, Proteus mirabilis. The frequency detection time was used to quantitatively determine M.TB. It was proportional to logarithm of the initial concentration of M.TB in the range of 2×10³-3×10⁷ cells ml⁻¹. The set up sensor was applied to the direct diagnosis of M.TB samples. The interference of other bacteria was eliminated by pretreatment. Our results confirmed that the use of the set up biosensor was reliable, sensitive. It gives out the potential use for determining M.TB.     

Dual-color upconversion fluorescence and aptamer-functionalized magnetic nanoparticles-based bioassay for the simultaneous detection of Salmonella Typhimurium and Staphylococcus aureus

A sensitive luminescent bioassay for the simultaneous detection of Salmonella Typhimurium and Staphylococcus aureus was developed using aptamer-conjugated magnetic nanoparticles (MNPs) for both recognition and concentration elements and using upconversion nanoparticles (UCNPs) as highly sensitive dual-color labels. The bioassay system was fabricated by immobilizing aptamer 1 and aptamer 2 onto the surface of MNPs, which were employed to capture and concentrate S. Typhimurium and S. aureus. NaY_0.78F₄:Yb_0.2,Tm_0.02 UCNPs modified aptamer 1 and NaY_0.28F₄:Yb_0.70,Er_0.02 UCNPs modified aptamer 2 further were bond onto the captured bacteria surface to form sandwich-type complexes. Under optimal conditions, the correlation between the concentration of S. Typhimurium and the luminescent signal was found to be linear within the range of 10¹–10⁵ cfu mL⁻¹ (R² = 0.9964), and the signal was in the range of 10¹–10⁵ cfu mL⁻¹ (R² = 0.9936) for S. aureus. The limits of detection of the developed method were found to be 5 and 8 cfu mL⁻¹ for S. Typhimurium and S. aureus, respectively. The ability of the bioassay to detect S. Typhimurium and S. aureus in real water samples was also investigated, and the results were compared to the experimental results from the plate-counting methods. Improved by the magnetic separation and concentration effect of MNPs, the high sensitivity of UCNPs, and the different emission lines of Yb/Er- and Yb/Tm-doped NaYF₄ UCNPs excited by a 980 nm laser, the present method performs with both high sensitivity and selectivity for the two different types of bacteria.     

Immunomagnetic separation and rapid detection of bacteria using bioluminescence and microfluidics

This paper describes an immunomagnetic separation of target bacterial cells from others by using magnetic bead. The surface of bead was coated with antibodies which can capture specific organism. The binding efficiency of immunomagnetic bead (IMB) capturing target bacterial cells was higher than 98% when the concentrations of target and interferent bacterial cells were at the same level. The concentration of bacteria was determined indirectly by detecting adenosine 5′-triphosphate (ATP) employing bioluminescence (BL) reaction of firefly luciferin-ATP. Benzalkonium chloride (BAC) was used as an ATP extractant from living bacterial cells. We found that BAC could enhance the light emission when the concentration of BAC was less than 5.3 × 10⁻²% (w/v) and the BL intensity reached its maximum at the concentration of BAC was 2.7 × 10⁻²%, which was 10-fold stronger than that without BAC. Based on the principle of the IMB, a microfluidic chip combined with immunofluorescence assay for separating and detecting bacteria simultaneously was also developed. The IMBs were magnetically fixed in the bead-beds of chip channels with a 3-mm diameter of NdFeB permanent magnet. The target bacterial cells can be captured magnetically and observed by a fluorescent microscope.     

Detection of Aeromonas hydrophila DNA oligonucleotide sequence using a biosensor design based on Ceria nanoparticles decorated reduced graphene oxide and Fast Fourier transform square wave voltammetry

A new strategy was introduced for ssDNA immobilization on a modified glassy carbon electrode. The electrode surface was modified using polyaniline and chemically reduced graphene oxide decorated cerium oxide nanoparticles (CeO₂NPs-RGO). A single-stranded DNA (ssDNA) probe was immobilized on the modified electrode surface. Fast Fourier transform square wave voltammetry (FFT-SWV) was applied as detection technique and [Ru(bpy)₃]^2+/3+ redox signal was used as electrochemical marker. The hybridization of ssDNA with its complementary target caused a dramatic decrease in [Ru(bpy)₃]^2+/3+ FFT-SW signal. The proposed electrochemical biosensor was able to detect Aeromonas hydrophila DNA oligonucleotide sequence encoding aerolysin protein. Under optimal conditions, the biosensor showed excellent selectivity toward complementary sequence in comparison with noncomplementary and two-base mismatch sequences. The dynamic linear range of this electrochemical DNA biosensor for detecting 20-mer oligonucleotide sequence of A. hydrophila was from 1 × 10⁻¹⁵ to 1 × 10⁻⁸ mol L⁻¹. The proposed biosensor was successfully applied for the detection of DNA extracted from A. hydrophila in fish pond water up to 0.01 μg mL⁻¹ with RSD of 5%. Besides, molecular docking was applied to consider the [Ru(bpy)₃]^2+/3+ interaction with ssDNA before and after hybridization.     

Determination of puerarin and daidzein in Puerariae radix and its medicinal preparations by micellar electrokinetic capillary chromatography with electrochemical detection

The use of micellar electrokinetic capillary chromatography (MECC) with electrochemical detection is described for the determination of puerarin and daidzein in Puerariae radix and its medicinal preparations. Operated in a wall-jet configuration, a 300 μm diameter carbon-disk electrode was used as the working electrode, which exhibits good responses at +900 mV (versus SCE) for the two analytes. Under the optimum conditions, the analytes were base-line separated within 11 min in a sodium dodecyl sulphate—borax (pH 7.8) running buffer, and excellent linearity was obtained in the concentration range from 5.0×10⁻⁴ to 5.0×10⁻⁶ mol/l. The detection limit (S/N=3) was 6×10⁻⁷ and 1.1×10⁻⁶ mol/l for puerarin and daidzein, respectively. This work provides a useful method for the analysis of traditional Chinese medicines.     

Bacteria detection based on the evolution of enzyme-generated volatile organic compounds: Determination of Listeria monocytogenes in milk samples

The rapid detection of Listeria monocytogenes contamination in food is essential to prevent food-borne illness in humans. The aim of this study was to differentiate non-contaminated milk from milk contaminated with L. monocytogenes using enzyme substrates coupled with the analysis of volatile organic compounds (VOCs). The method is based on the activity of β-glucosidase and hippuricase enzymes and the detection of a specific VOC i.e. 2-nitrophenol and 3-fluoroaniline, respectively. VOCs were extracted, separated and detected by headspace-solid phase microextraction coupled to gas chromatography–mass spectrometry (HS-SPME GC–MS). This approach required the inclusion of the selective agent's cycloheximide, nalidixic acid and acriflavine HCl in the growth medium to inhibit interfering bacteria. The VOCs were liberated by L. monocytogenes provided that samples contained at least 1–1.5 × 10² CFU ml⁻¹ of milk prior to overnight incubation. This approach shows potential for future development as a rapid method for the detection of L. monocytogenes contaminated milk.     

Sensitive and selective electrochemical detection of artemisinin based on its reaction with p-aminophenylboronic acid

The electrochemical detection of artemisinin generally requires high oxidation potential or the use of complex electrode modification. We find that artemisinin can react with p-aminophenylboronic acid to produce easily electrochemically detectable aminophenol for the first time. By making use of the new reaction, we report an alternative method to detect artemisinin through the determination of p-aminophenol. The calibration curve for the determination of artemisinin is linear in the range of 2 μmol L⁻¹ to 200 μmol L⁻¹ with the detection limit of 0.8 μmol L⁻¹, which is more sensitive than other reported electrochemical methods. The relative standard deviation is 4.83% for the determination of 10 μM artemisinin. Because the oxidation potential of p-aminophenol is around 0 V, the present method is high selective. When 40 μM, 90 μM and 140 μM of artemisinin were spiked to compound naphthoquine phosphate tablet samples, the recoveries are 107.6%, 105.4% and 101.7%, respectively. This detection strategy is attractive for the detection of artemisinin and its derivatives. The finding that artemisinin can react with aromatic boronic acid has the potential to be exploited for the development of other sensors, such as fluorescence artemisinin sensors.     

Electrogenerated chemiluminescence biosensing for the detection of prostate PC-3 cancer cells incorporating antibody as capture probe and ruthenium complex-labelled wheat germ agglutinin as signal probe

A highly selective and sensitive electrogenerated chemiluminescence (ECL) biosensor for the detection of prostate PC-3 cancer cells was designed using a prostate specific antibody as a capture probe and ruthenium complex-labelled wheat germ agglutinin as a signal probe. The ECL biosensor was fabricated by covalently immobilising the capture probe on a graphene oxide-coated glassy carbon electrode. Target PC-3 cells were selectively captured on the surface of the biosensor, and then, the signal probe was bound with the captured PC-3 cells to form a sandwich. In the presence of tripropylamine, the ECL intensity of the sandwich biosensor was logarithmically directly proportion to the concentration of PC-3 cells over a range from 7.0 × 10² to 3.0 × 10⁴ cells mL⁻¹, with a detection limit of 2.6 × 10² cells mL⁻¹. The ECL biosensor was also applied to detect prostate specific antigen with a detection limit of 0.1 ng mL⁻¹. The high selectivity of the biosensor was demonstrated in comparison with that of a lectin-based biosensor. The strategy developed in this study may be a promising approach and could be extended to the design of ECL biosensors for highly sensitive and selective detection of other cancer-related cells or cancer biomarkers using different probes.     

N-Methylimidazolium modified magnetic particles-assisted highly sensitive Escherichia coli detection based on polymerase chain reaction and capillary electrophoresis

Effective bacteria detection and quantification are essential prerequisite for the prevention and treatment of infectious diseases. Herein, we report a method for the detection and quantification of Escherichia coli (E. coli).N-Methylimidazolium modified magnetic particles (MIm-MPs) are synthesized successfully and used as an efficient magnetic material for the isolation and concentration of E. coli. The factors including pH of binding buffer, concentration of elution buffer and elution time which may affect the capture and elution efficiencies are optimized. The linear correlation between bacteria concentration and peak area of polymerase chain reaction (PCR) product analyzed by capillary electrophoresis (CE) is determined. Rapid preconcentration of trace amount of E. coli (10¹ cfu mL⁻¹) in large volume of aqueous sample (500 mL) is achieved, and the capture efficiency can reach 99%. The quantification of bacteria in large volume of spiked tap water and mineral water samples is realized. The recoveries for different concentrations of E. coli in tap and mineral water samples are in the range between 83% and 93%. The results demonstrate that this MIm-MPs-PCR-CE method can be applied to detect and quantify bacteria in real samples.     

A novel fluorescence immunoassay for the sensitive detection of Escherichia coli O157:H7 in milk based on catalase-mediated fluorescence quenching of CdTe quantum dots

Immunoassay is a powerful tool for rapid detection of food borne pathogens in food safety monitoring. However, conventional immunoassay always suffers from low sensitivity when it employs enzyme-catalyzing chromogenic substrates to generate colored molecules as signal outputs. In the present study, we report a novel fluorescence immunoassay for the sensitive detection of E. coli O157:H7 through combination of the ultrahigh bioactivity of catalase to hydrogen peroxide (H₂O₂) and H₂O₂-sensitive mercaptopropionic acid modified CdTe QDs (MPA-QDs) as a signal transduction. Various parameters, including the concentrations of anti-E. coli O157:H7 polyclonal antibody and biotinylated monoclonal antibody, the amounts of H₂O₂ and streptavidin labeled catalase (CAT), the hydrolysis temperature and time of CAT to H₂O₂, as well as the incubation time between H₂O₂ and MPA-QDs, were systematically investigated and optimized. With optimal conditions, the catalase-mediated fluorescence quenching immunoassay exhibits an excellent sensitivity for E. coli O157:H7 with a detection limit of 5 × 10² CFU/mL, which was approximately 140 times lower than that of horseradish peroxidase-based colorimetric immunoassay. The reliability of the proposed method was further evaluated using E. coli O157:H7 spiked milk samples. The average recoveries of E. coli O157:H7 concentrations from 1.18 × 10³ CFU/mL to 1.18 × 10⁶ CFU/mL were in the range of 65.88%–105.6%. In brief, the proposed immunoassay offers a great potential for rapid and sensitive detection of other pathogens in food quality control.