Multiplex Q-PCR Arrays for Simultaneous Nucleic Acid Detection
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Solution Overview
Problem
Current nucleic acid amplification and detection methods, such as PCR and microarrays, face challenges in efficiently amplifying and detecting multiple nucleic acid sequences simultaneously in a single reaction, due to limitations in sample splitting, signal interference from unbound labeled species, and washing artifacts, which hinder accurate multiplexed Q-PCR and high-throughput screenings.
Innovation Solution
A method involving nucleic acid amplification on multiple sequences in a fluid with a solid surface array of independently addressable probes, where amplicon hybridization is measured in real-time, allowing for the determination of sequence concentrations without the need for sample splitting or washing, using quenchers and fluorescent moieties to enhance signal accuracy and reduce background noise.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Quantity of substance
If conventional fluorescent-based microarrays are used to detect multiple analytes simultaneously, then the detection capacity increases, but the signal to noise ratio deteriorates due to overwhelming background signal from unbound labeled species
Solution Approach 1:
The patent extracts and removes the unbound labeled species from the detection system by using unlabeled capture probes on the array surface and labeled detection antibodies in solution. This separation eliminates the overwhelming background signal from unbound labels while maintaining high detection capacity through the specific binding between capture probes and target analytes.
Solution Approach 2:
The patent introduces an intermediary binding step where unlabeled capture probes on the array first bind to target analytes, creating a stable complex. Then labeled detection antibodies are introduced to bind to the captured targets. This intermediary capture step separates the targeting function from the signaling function, reducing background noise while maintaining detection sensitivity.
2Quantity of substance
If sample splitting is performed to enable multiplexed Q-PCR, then the number of detectable sequences increases, but the sample volume required increases and processing complexity increases
Solution Approach 1:
The patent merges multiple Q-PCR detection capabilities into a single reaction chamber by immobilizing multiple different capture probes on the array surface. Each probe targets a different nucleic acid sequence, allowing simultaneous amplification and detection of multiple sequences in one reaction without requiring sample splitting into multiple tubes.
Solution Approach 2:
The patent transitions from a one-dimensional solution-phase multiplexing approach (multiple tubes) to a two-dimensional surface-based approach (array of probes on solid surface). This dimensional change allows multiple detection channels to coexist in a single reaction volume, reducing processing complexity while maintaining high multiplexing capacity.
3Measurement precision
If washing steps are performed in conventional microarrays to remove unbound species, then the signal quality improves, but washing artifacts occur and processing time increases
Solution Approach 1:
The patent maintains continuous binding equilibrium between captured targets and detection antibodies throughout the detection process. By using an excess of labeled detection antibodies and allowing the system to reach equilibrium without washing, the method eliminates time-consuming washing steps while maintaining high signal quality through the stable captured complex.
Solution Approach 2:
The patent allows the captured target-antibody complexes to self-stabilize on the array surface through specific binding interactions. The high affinity and specificity of the biological binding interactions naturally prevent non-specific binding and background signal formation, eliminating the need for external washing interventions to remove unbound species.
4Productivity
If multiple nucleic acid sequences are amplified simultaneously in a single reaction chamber, then the productivity increases, but the measurement precision deteriorates due to signal interference between multiple amplicons
Solution Approach 1:
The patent segments the detection function across multiple spatially distinct capture probes on the array surface. Each probe is positioned at a specific location and detects a specific amplicon type. This spatial segmentation allows independent measurement of each target sequence, eliminating signal interference between multiple amplicons while maintaining high amplification throughput in a single reaction chamber.
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 the simultaneous amplification and detection of multiple nucleic acid sequences in a single reaction, improving the accuracy and efficiency of nucleic acid quantification and reducing the complexity of sample handling, thereby enhancing multiplex Q-PCR capabilities and high-throughput screenings.
Implementation Method 1
The array comprises independently addressable locations which comprise a fluorescent moiety. Hybridization of the amplicon to the probe results in quenching of the fluorescent signals from the probes.
Data Source
AI summary
This invention provides methods and systems for measuring the concentration of multiple nucleic acid sequences in a sample. The nucleic acid sequences in the sample are simultaneously amplified, for example, using polymerase chain reaction (PCR) in the presence of an array of nucleic acid probes. The amount of amplicon corresponding to the multiple nucleic acid sequences can be measured in real-time during or after each cycle using a real-time microarray. The measured amount of amplicon produced can be used to determine the original amount of the nucleic acid sequences in the sample. Also provided herein are biosensor arrays, systems and methods for affinity based assays that are able to simultaneously obtain high quality measurements of the binding characteristics of multiple analytes, and that are able to determine the amounts of those analytes in solution. The invention also provides a fully integrated bioarray for detecting real-time characteristics of affinity based assays.