D-Amino Acid Bacterial Labeling for Rapid Live Cell Quantification
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Solution Overview
Problem
Current methods for quantifying total live bacteria are time-consuming, relying on colony formation and serial dilutions, taking up to 48 hours, and lack a wide dynamic range for quantitation, necessitating a more efficient and rapid alternative.
Innovation Solution
A method involving D-amino acid probes that covalently incorporate into bacterial cell walls, allowing for automated, single-cell counting and detection of live bacteria within 3 hours, using fluorescence detection and bioorthogonal chemistry to label and quantify bacteria without forming colonies.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If traditional plate count methods are used, then accurate quantification of live bacteria is achieved, but the process takes up to 48 hours due to colony formation requirements
Solution Approach 1:
The patent replaces the mechanical/biological process of colony formation with a chemical labeling approach. D-amino acid probes with fluorophores are incorporated into bacterial cell walls through enzymatic activity, allowing direct detection of individual cells via fluorescence microscopy or flow cytometry without requiring colony development. This substitution reduces quantification time from 48 hours to approximately 3 hours while maintaining accuracy.
Solution Approach 2:
The patent utilizes fluorophore-based color/fluorescence changes to detect and quantify bacteria. The D-amino acid probes contain fluorophores that emit light when incorporated into bacterial cell walls, enabling rapid identification and counting of live bacteria through fluorescence detection. This optical detection method eliminates the need for visual colony counting and significantly accelerates the quantification process.
2Measurement precision
If serial dilutions are performed for quantitation, then a wide dynamic range is achieved, but the process complexity and time requirements increase
Solution Approach 1:
The patent replaces the mechanical process of serial dilution with a direct fluorescent labeling and counting approach. By labeling all live bacteria in the sample with D-amino acid probes and detecting them individually through fluorescence, the method achieves a wide dynamic range (from 10^2 to 10^9 CFU/mL) without requiring sample dilution. This eliminates multiple dilution steps, reduces procedural complexity, and decreases hands-on time.
Solution Approach 2:
The D-amino acid probe system serves multiple functions simultaneously: it labels all live bacteria across a wide concentration range, provides specific fluorescence signals for detection, and enables direct counting without additional sample preparation steps. This multi-functionality eliminates the need for separate dilution and counting procedures, simplifying the overall process while maintaining quantitation accuracy.
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
Enables rapid quantification of live bacteria in less than 3 hours with a wide dynamic range, eliminating the need for serial dilutions and providing a user-friendly, automated process.
Implementation Method 1
The bacteria covalently incorporates the at least one D-amino acid probe
Implementation Method 2
the amino acid probe includes a covalently attached fluorophore... detecting the labeled bacteria
Data Source
AI summary
The present disclosure provides methods, systems, and devices for the in situ labeling of complex environmental samples, specifically mixed populations of total live bacteria, using D-amino acid analogs, such as fluorescent D-amino acids or D-amino acids with a biorthogonal tag to enable chemical conjugation of a fluorophore (e.g. by click chemistry). The disclosure further provides methods for quantifying the TVB by cell manipulation using ferrofluid within a microfluidic device.