Microfluidic Real-Time PCR System with Sub-Array Selection
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Solution Overview
Problem
Current methods for simultaneous and quantitative analysis of multiple analytes in polymerase chain reaction (PCR) are challenging due to limitations in detection technologies, such as intercalating dye fluorescence, which can only determine total dsDNA concentration, and require complex probe designs for multiplexing, ultimately limiting detection to about four-color analysis.
Innovation Solution
The development of a system that includes a microfluidic device with an optic system, a controller, and a sub-array selection module to selectively image and analyze subsets of microwells in a fluidic device, using magnetic particle chemistries and PCR master mixes, which allows for real-time PCR data acquisition and reduces photobleaching by optimizing dye uniformity and imaging protocols.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If conventional microtiter plate or microfluidic real-time-PCR methods are used with fluorescent probe technologies for multiplexing, then multiple analytes can be analyzed simultaneously, but the detection is ultimately limited to about four-color analysis due to instrumentation and spectral overlap between dyes
Solution Approach 1:
The invention divides the detection task into segments by using combinatorial probe sets where different probes with overlapping spectra are combined in specific patterns. Each analyte is detected by a unique combination of probes rather than requiring completely spectrally distinct dyes, enabling more than four-color multiplexing capability.
Solution Approach 2:
The invention adds a combinatorial dimension to the detection system. Instead of relying solely on spectral dimension (different wavelengths), it uses combinatorial presence/absence patterns of multiple probes to encode additional analyte information, effectively increasing the multiplexing capacity beyond the four-color limitation.
2Adaptability or versatility
If multiple reaction wells are used for multiplexing with different color probes, then concurrent analysis of multiple templates is possible, but complex probe design and optimization is required to ensure compatibility
Solution Approach 1:
The probe set is segmented into multiple individual probes that can be independently designed and optimized. Each probe targets a specific analyte or portion of an analyte, and their combinatorial use allows complex multiplexing without requiring each individual probe to be perfectly compatible with all others in the system.
Solution Approach 2:
The invention creates a universal probe platform where the same set of probes can be used across multiple analytes through combinatorial arrangements. This multi-functional approach reduces the overall design complexity compared to creating entirely separate probe systems for each analyte.
3Loss of information
If continuous imaging of microwell arrays is performed for real-time PCR data collection, then complete kinetic data is obtained, but photobleaching of fluorescent dyes occurs
Solution Approach 1:
Instead of continuous imaging, the system uses periodic imaging at specific intervals during the PCR cycles. This allows kinetic data to be captured at meaningful time points while giving the fluorescent dyes sufficient rest periods to minimize photobleaching accumulation.
Solution Approach 2:
The imaging protocol skips certain cycles or time points where imaging would provide redundant information or where photobleaching risk is highest. By strategically selecting which cycles to image, the system obtains sufficient kinetic data while minimizing total light exposure to the dyes.
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 efficient real-time PCR data collection from encoded bead microwell arrays, allowing for single-cycle resolution and reduced photobleaching, thereby overcoming the limitations of existing multiplexing methods and enhancing the analysis of multiple analytes.
Implementation Method 1
an encoded bead array, optionally while obtaining real-time PCR data
Implementation Method 2
a heat source coupled to the optic system and thermally coupled to the at least one microfluidic device held in the housing
Implementation Method 3
using magnetic particle chemistries and PCR master mixes
Data Source
AI summary
Analysis systems with a housing having a chamber sized and configured to receive at least one microfluidic device. The systems also include an optic system coupled to the housing in optical communication with the at least one microfluidic device, a controller coupled to the optic system, a heat source coupled to the optic system and thermally coupled to the at least one microfluidic device held in the housing, and a sub-array selection module in communication with the controller. The sub-array selection module is configured to select a sub-set of sets of microwells of at least one fluid channel of the microfluidic device for imaging by the optic system after a reaction step (e.g., one thermocycle) during an assay.