Flow Cytometry Virus Quantification via Real-Time Flow Control
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Solution Overview
Problem
Current flow cytometry methods are inadequate for accurately and efficiently enumerating viruses due to limitations in size range, accuracy, and speed, particularly for nanometer-sized particles, and require labor-intensive and time-consuming traditional methods like plaque assays and transmission electron microscopy.
Innovation Solution
A system utilizing real-time measurement and control of flow rates through a feedback loop in a dual-channel flow cytometer with hydrodynamic focusing and two-color detection for improved accuracy and discrimination of virus particles, enabling real-time enumeration and reducing post-acquisition data analysis.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If traditional flow cytometry methods are used for particle analysis, then large particles (bacterium or cell size) can be analyzed, but nanometer-sized virus particles cannot be accurately enumerated
Solution Approach 1:
The patent modifies key operational parameters including flow rate (reducing to 1-100 nL/min), detection volume (minimizing to 1-10 fL), and particle concentration (increasing to 10^6-10^9 particles/mL) to enable accurate detection of nanometer-sized virus particles while maintaining flow cytometry capabilities
2Measurement precision
If plaque assay is used for virus quantification, then gold standard accuracy is achieved, but the method is labor intensive and time consuming (12 hrs to 2 weeks)
Solution Approach 1:
The patent replaces the mechanical and biological processes of plaque assay with an optical detection system that measures light scattering and fluorescence from individual virus particles in real-time, eliminating the need for cell culture and plaque formation while achieving comparable accuracy in minutes rather than weeks
3Measurement precision
If transmission electron microscopy is used for virus analysis, then high spatial resolution and morphological information are obtained, but samples must be interrogated under high vacuum conditions and the method is expensive and not widely available
Solution Approach 1:
The patent replaces the complex vacuum-based electron microscopy system with a liquid-phase optical flow cytometry system that uses lasers and detectors to achieve comparable resolution for virus particles, eliminating vacuum requirements and making the technique accessible in standard laboratory settings
4Ease of operation
If flow rate is not controlled in flow cytometry, then simple operation is maintained, but accurate quantification of particles cannot be achieved
Solution Approach 1:
The patent incorporates flow rate sensors and control systems that continuously monitor and adjust the flow rate to maintain optimal conditions for virus particle detection, automatically compensating for variations and ensuring accurate quantification without requiring manual intervention
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
This approach enhances the accuracy and speed of virus particle quantification, providing high counting precision and rapid results, overcoming the limitations of traditional methods by maintaining constant flow rates and stabilizing hydrodynamic focusing, thus improving laboratory efficiency.
Implementation Method 1
The flow rate of each fluid is measured, and also may be controlled via a feedback loop between each fluid flow sensor and a flow control device in each flow path
Implementation Method 2
Within the flow cell, the sample is commonly shaped into a narrow stream or stream of droplets by a sheath medium (either liquid or gas)
Implementation Method 3
Particle interactions with the laser(s) are monitored by one or more detectors that are used to quantify detectable properties such as forward or side light scattering or fluorescence
Implementation Method 4
Particle interactions with the laser(s) are monitored by one or more detectors that are used to quantify detectable properties such as forward or side light scattering or fluorescence
Data Source
Figure 1
Figure 2(a)~2(b)
Figure 3
AI summary
The present invention relates to methods that enable improved accuracy for quantitative particle counting in a flowing liquid stream. The methods of the present invention utilize the real-time measurement of flow rates and flow rate control through feedback mechanisms to improve quantification, and this improved quantification translates to more accurate particle counting. In certain embodiments, particles being counted are biological particles in a liquid sample, such as viruses.