Multicolor and Ultrasensitive Enzyme-Linked Immunosorbent Assay Based on the Fluorescence Hybrid Chain Reaction for Simultaneous Detection of Pathogens

Abstract

Various pathogens may coexist in one sample; however, detection methods that rely on traditional selective culture media or immune agents designed specifically for a certain target are unsuitable for multiple targets. It is important to develop a simultaneous and sensitive detection method for multiple pathogens. Here, a multicolor and ultrasensitive enzymelinked immunosorbent assay (ELISA) platform based on the fluorescence hybridization chain reaction (HCR) was developed. In the assay, multicolor fluorescence concatemers formed as signal amplifiers and signal reporters in the presence of target pathogens. When HCR occurred, Escherichia coli O157:H7, Salmonella serotype Choleraesuis, and Listeria monocytogenes were detected simultaneously with three different fluorescences. Additionally, the limits of detection for E. coli O157:H7, Salmonella Choleraesuis, and L. monocytogenes were 3.4 × 10 1 , 6.4 × 10 0 , and 7.0 × 10 1 CFU/mL, respectively. The assay achieved ultrasensitive, specific, and simultaneous detection of three pathogens and can be applied to the detection of pathogens in milk samples. Therefore, this multicolor and ultrasensitive ELISA platform has great potential in the application of simultaneous detection of pathogens.

Multiplex Recombinase Polymerase Amplification Assay for the Simultaneous Detection of Three Foodborne Pathogens in Seafood

“…Therefore, the rapid, sensitive, and reliable detection of Staphylococcus aureus, Vibrio parahaemolyticus, and Salmonella Enteritidis is essential in reducing the risk factor caused by the three pathogens. Numerous methods have been established to detect pathogenic bacteria in food, such as real-time PCR [27], enzyme-linked immunosorbent assays [28], and electrochemical biosensors [29]. However, these approaches are not suitable for wide application in the field due to the need for specific equipment and the requirement of time-consuming procedures.…”

Metal Nanoparticles‐Enhanced Biosensors: Synthesis, Design and Applications in Fluorescence Enhancement and Surface‐enhanced Raman Scattering

“…However, simultaneous analysis of multiple pathogens is difficult due to the weak single colour generated by ELISA method. To address this issue, Lv et al [121] have developed an ELISA base on fluorescence hybrid chain reaction, which enables simultaneous analysis of multiple pathogens using dye-labelled antibodies of different colours. In Figure 7.…”

“…The confocal fluorescence images shows the detection of megalin in the tubules was coloured by the Alexa Fluor 488 (green) while the podocin in the glomeruli was coloured by the Alexa Fluor 647 (red), [127] g) Schematic illustration of ultrasensitive ELISA based MEF biosensor for simultaneous multicolour detection of pathogens. [121] this approach, different types of metal NPs such as gold nanorods (AuNR) or nanosphere (AuNS) are gathered together to form the plasmonic hotspots. Figure 9d illustrates the sensing principle of this MEF biosensors for DNA detection through aggregation-induced plasmonic hotspots.…”

“…(C) Detection of point mutation based on change in the fluorescence of MEF sensor in the presence of PPi or Cu 2+ , e) Schematic illustration of the “turn‐on” MEF probe for detecting γ‐glutamyl transpeptidase, f) Antibody‐functionalized AuNPs as MEF probe for fluorescence imaging. The confocal fluorescence images shows the detection of megalin in the tubules was coloured by the Alexa Fluor 488 (green) while the podocin in the glomeruli was coloured by the Alexa Fluor 647 (red), g) Schematic illustration of ultrasensitive ELISA based MEF biosensor for simultaneous multicolour detection of pathogens …”

Application of Biosensors for Detection of Pathogenic Food Bacteria: A Review

“…The word biosensor refers to an effective and creative analytical device that has a biological sensing function with a broad variety of applications such as food safety, environmental monitoring, biomedicine, and drug discovery [91]. More specifically, biosensors are widely used in the identification and detection of bacteria and have attracted great interest as one of the most efficient and accurate methods of food analysis and food safety monitoring [92][93][94]. In addition, biosensors typically deliver fast, on-site tracking and thus provide real-time details throughout the production process [95,96].…”