Fluorescent Dye Calibration via Checkerboard Plate and Polynomial Modeling
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Solution Overview
Problem
The existing dye calibration processes for biological analysis systems, such as qPCR instruments, are time-consuming and complex, especially when calibrating multiple fluorescent dyes, and require significant operator input, leading to potential errors and inefficiencies.
Innovation Solution
A checkerboard pattern calibration plate design and a computer-implemented system that uses a 2D quadratic polynomial function for global modeling to calibrate multiple dyes simultaneously, reducing the number of plates and process steps required, and employing a computer system to process and analyze fluorescence data for accurate dye calibration.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If traditional dye calibration processes are used for multiple fluorescent dyes, then calibration accuracy can be maintained, but the process becomes time-consuming and complex requiring multiple plates and significant operator input
Solution Approach 1:
The patent combines multiple dye calibration processes into a single integrated workflow using a multi-well plate format. Multiple dyes are calibrated simultaneously in different wells of the same plate, eliminating the need for separate calibration plates for each dye and reducing the overall calibration time while maintaining accuracy through consistent imaging conditions across all dyes.
Solution Approach 2:
The calibration plate is designed with multi-functionality to accommodate multiple fluorescent dyes with different spectral properties. The plate and imaging system are configured to handle various dye types (e.g., SYBR Green, EvaGreen, RoxPlex) in a unified calibration process, allowing a single plate to serve multiple calibration purposes that traditionally required separate specialized plates.
2Measurement precision
If traditional dye calibration processes are used for multiple fluorescent dyes, then calibration accuracy can be maintained, but the process requires significant operator input leading to potential errors
Solution Approach 1:
The system implements automated data processing and analysis capabilities that reduce reliance on operator intervention. The imaging system automatically captures fluorescence data from multiple wells, and software algorithms automatically analyze the data to determine calibration factors and assess dye performance, minimizing manual operations and reducing the potential for operator errors while maintaining calibration accuracy.
Solution Approach 2:
The calibration process incorporates automated feedback mechanisms where the system evaluates the quality of calibration data in real-time and can identify problematic wells or dyes. The software provides feedback on calibration success criteria and can prompt for corrective actions only when necessary, reducing routine operator input while ensuring accuracy through systematic quality control.
3Manufacturing precision
If multiple dyes are calibrated on separate plates, then each dye can be optimized individually, but the overall calibration process becomes complex and time-consuming
Solution Approach 1:
The calibration plate is segmented into multiple wells, each dedicated to a specific dye type. This segmentation allows individual optimization of calibration conditions for each dye while maintaining a unified plate structure. Each well or region can be tailored for specific dye characteristics (e.g., different buffer compositions, DNA concentrations) without affecting other dyes, yet all are processed simultaneously in a single calibration run.
Solution Approach 2:
The patent transitions from a sequential, single-dye-per-plate approach to a parallel, multi-dye-per-plate approach by utilizing the spatial dimension of the multi-well plate format. Multiple dyes are arranged in different wells across the plate's surface, allowing simultaneous calibration in a single two-dimensional space rather than requiring multiple separate plates stacked in time, thereby reducing process complexity while maintaining dye-specific optimization.
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 simplifies the dye calibration process, reduces time, and maintains accuracy by allowing multiple dyes to be calibrated on a single plate, decreasing operator error and increasing efficiency through automated data processing and modeling.
Implementation Method 1
systems and methods for calibrating emission data or other information signals collected during a polymerase chain reaction (PCR), amplification reaction, assay, process, or other reaction
Data Source
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AI summary
A computer-implemented method (500) for calibrating a fluorescent dye is described. The method (500) comprises imaging a sample holder, loaded into an instrument, at more than one channel. The sample holder comprises a plurality of reaction sites and more than one dye type, with each dye occupying more than one reaction site. The method further comprises identifying a peak channel for each dye on the sample holder (508), normalizing each channel to the peak channel for each dye (510), and producing a dye matrix (518) that comprises a set of dye reference values.