A Combined Aqueous Two-Phase System and Spot-Test Platform for the Rapid Detection of Escherichia coli O157:H7 in Milk

Abstract

Foodborne illnesses are a public health concern in the United States and worldwide. Recent outbreaks of Escherichia coli O157:H7 have brought to light the need for improved ways to detect foodborne pathogens and minimize serious outbreaks. Unfortunately, current methods for the detection of foodborne pathogens are time intensive and complex. In this study, we designed a spot immunoassay that uses a UCON-potassium phosphate salt aqueous two-phase system (ATPS) for the preconcentration of O157:H7. This platform was tested with samples of O157:H7 spiked in phosphate-buffered saline and milk. The ATPS was found to improve the detection limit of the spot test, yielding detection at 10 cfu/mL within 30 min. This is the first known application of ATPSs to spot immunoassays. Moreover, detection was successfully achieved without upstream processing or dilution of the sample prior to testing, thereby further simplifying the detection process. This technology's ease of use, sensitivity, and short time to result highlight its potential to advance the spot test as a viable diagnostic tool for foodborne pathogens.

Aqueous two-phase systems: Towards novel and more disruptive applications

“…DNA sample preparation) and enhanced isothermal DNA amplification (by using the thermophilic helicase-dependent amplification method) [37]. A similar approach was carried by the same research group, by applying ATPS to spot immunoassays and detect foodborne illnesses [98]. In this work, an UCON/phosphate salt ATPS was used for the preconcentration of E. coli 0157:H7 cells, improving the detection limit of the spot test within 30 min and allowing the detection without upstream processing or dilution procedures.…”

Point-of-Need Diagnostics for Foodborne Pathogen Screening

“…31 Our group has previously shown that a UCON–potassium phosphate salt ATPS concentrates E. coli cells in a milk sample by more than 10 times, improving the LOD on a spot test immunoassay. 32 Spot test assays utilize the sandwich LFA format, but the antibodies are immobilized on a spot on the membrane as opposed to a line. Additionally, modifications to the GN reporter probes have been designed to increase binding of the target pathogens in the detection zone.…”

Ionic Liquid Aqueous Two-Phase Systems for the Enhanced Paper-Based Detection of Transferrin and Escherichia coli

“…Through the use of conventional polymer-salt and micellar systems, ATPSs combined with existing detection technologies have been shown to enhance sensitivity in detecting various biomarkers, including 10-fold improvements in LFA detection of parasitic biomarkers (Pereira et al, 2015 ) and viruses (Mashayekhi et al, 2010 ). In a similar manner, the ATPS was also shown to improve sensitivity and decrease time-to-detection of a paper-based spot immunoassay (Cheung et al, 2017 ). However, despite the efficacy of these systems, the limited polarity range of these ATPSs can restrict their use (Freire et al, 2012 ), particularly regarding the partitioning of small hydrophilic proteins.…”