Automated MPN Assay Calibration for Accurate CFU Quantification
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Solution Overview
Problem
Existing MPN methods for determining colony-forming-units (CFUs) of bacteria in samples are labor-intensive and lack accuracy due to variables like lag times and growth rates, making it difficult to quantify pathogen presence and concentration efficiently.
Innovation Solution
An automated assay system using flow cytometry, PCR, and immunoassay in combination with serial dilution, along with a microfluidic cartridge and magnetophoresis, to accurately determine CFU levels by generating results from positive dilutions and applying a calibration curve for precise quantitation.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If classic MPN analysis with serial dilution and enrichment is used, then pathogen concentration can be determined, but the method is labor-intensive and lacks accuracy due to variables like lag times and growth rates
Solution Approach 1:
The patent replaces manual mechanical operations (serial dilution, enrichment, plating) with automated robotic systems and microfluidic devices. The robotic system automatically performs dilution series and transfers samples, while microfluidic cartridges handle enrichment and detection, eliminating human labor and associated variability in the MPN determination process.
Solution Approach 2:
The patent standardizes and controls critical parameters such as incubation time, temperature, and dilution factors through automated systems. By fixing these parameters and using calibrated detection methods, the system eliminates variability from manual operations while maintaining the ability to determine pathogen concentration accurately.
2Measurement precision
If multiple dilution steps are performed to determine CFU levels, then quantification accuracy can be improved, but the analysis time and processing duration increase
Solution Approach 1:
The patent performs enrichment of all dilution samples simultaneously in parallel microfluidic chambers before detection. This preliminary enrichment step allows multiple dilution levels to be processed concurrently rather than sequentially, maintaining quantification accuracy while reducing total analysis time by eliminating serial processing delays.
Solution Approach 2:
The patent transitions from sequential linear processing to parallel three-dimensional microfluidic processing. Multiple dilution samples are enriched and detected simultaneously in vertically stacked or laterally arranged microfluidic channels, enabling time compression while preserving the analytical rigor of multiple dilution steps.
3Reliability
If traditional MPN methods are used, then pathogen presence can be detected, but sensitivity and dynamic range are limited
Solution Approach 1:
The patent employs a multi-functional detection platform that combines fluorescence detection, absorbance measurement, and cell imaging capabilities within a single system. This allows the same instrument to detect pathogens across a broad dynamic range from low to high concentrations using different detection modes, enhancing both sensitivity and adaptability to various pathogen loads.
Solution Approach 2:
The patent uses adjustable detection thresholds and gain settings that can be dynamically optimized for different expected pathogen concentrations. The system adapts its detection parameters based on the dilution series results, maintaining high sensitivity for low concentrations while accurately quantifying high concentrations through automated threshold adjustment.
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
The method provides rapid and accurate quantification of CFUs with reduced dilution steps, enhancing sensitivity and dynamic range, allowing for automated determination of pathogen presence and concentration within a shorter timeframe.
Implementation Method 1
The system uses magnetophoresis to manipulate particles or cells with a ferrofluid in microfluidic channels. The ferrofluid is a colloidal suspension of polymer or surfactant stabilized superparamagnetic nanoparticles, which, under the influence of a magnetic field generated by the PCB, as well as a pumping system built into the cartridge, particles or cells and be concentrated or otherwise focused and flowed along a surface of capture zone coated with binders specific for a particular target particle or cell.
Implementation Method 2
After capture, the particles or cells can be labeled for fluorescence detection, via, for example, fluorescent DNA intercalating dyes, labeled target specific binders such as labeled antibodies, enzyme substrates specific for intracellular enzymes, and labeled nucleic acid probes that enable fluorescence in-situ hybridization detection of target specific DNA, mRNA or rRNA sequences. The optics can include a camera and microscope objective along with, for example, a specific LED and/or a filter set that enables excitation and detection of emission of the label fluorophores.
Data Source
AI summary
Embodiments of the present disclosure include systems, devices and methods for increasing the accuracy of an MPN, using assay instrumentation. For example, such embodiments can be accomplished by pre-loading the assays system with standard curves generated from measurements made with dilutions of known levels of pathogens. When such an approach is used, for each sample, the value from the last positive dilution and a calibrated assay count can both be used to provide a more accurate CFU per sample value than would be determined from just the last positive dilution alone.