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High-Throughput Flow-Through Direct Immunoassays for Targeted Bacteria Detection.

Parthena Chorti1, Abbas Parvez Kazi1, Michael Wiederoder2

  • 1Department of Chemistry, University of Massachusetts Lowell, Lowell, Massachusetts 01854, United States.

Analytical Chemistry
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A new flow-through direct immunoassay (FTDI) method enables bacteria detection in large liquid volumes. This technique offers rapid and ultrasensitive options for analyzing samples like water and juice, improving upon existing methods.

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Area of Science:

  • Microbiology
  • Analytical Chemistry
  • Biotechnology

Background:

  • Regulatory bodies mandate bacterial detection methods for large sample volumes (e.g., 100 mL).
  • Existing methods like Membrane Filtration and Most Probable Number analyze large volumes, while others (ELISA, LFA) analyze significantly smaller volumes.
  • There is a need for new methods capable of analyzing large volumes with improved efficiency and sensitivity.

Purpose of the Study:

  • To introduce and describe a novel methodology, flow-through direct immunoassays (FTDI), for targeted bacterial detection in liquid samples of variable volumes.
  • To develop and validate two versions of FTDI assays for detecting *E. coli* in 10 mL samples.
  • To assess the performance of FTDI assays in terms of speed, sensitivity, robustness, precision, and accuracy across different sample matrices.

Main Methods:

  • Developed FTDI assays utilizing fluid-permeable microwells with membranes for bacterial trapping.
  • Implemented direct immunoassay detection of trapped bacteria within the microwells.
  • Created a rapid FTDI assay (<2.5 h) and an ultrasensitive FTDI assay with an integrated culturing step (<5.5 h).
  • Validated assays in tap water, river water, and apple juice using 96-well filter plates, vacuum manifolds, and multichannel peristaltic pumps for high-throughput analysis.

Main Results:

  • Two FTDI assays for *E. coli* detection in 10 mL samples were successfully developed.
  • The rapid FTDI assay achieved detection limits of 17 CFU/mL.
  • The ultrasensitive FTDI assay, incorporating a culturing step, reached detection levels below 1 CFU/mL.
  • Assays demonstrated robustness, precision, and accuracy in diverse sample types.
  • The methodology supports high-throughput analysis through parallel processing in 96-well plates.

Conclusions:

  • Flow-through direct immunoassays (FTDI) provide a versatile and effective platform for bacterial detection in large liquid volumes.
  • FTDI assays offer both rapid and ultrasensitive detection options, adaptable to regulatory requirements and research needs.
  • The integrated, in-well assay process minimizes contamination risk and enhances user safety and ease of use.
  • FTDI technology shows significant potential for improving bacterial analysis in various applications, including environmental monitoring and food safety.