Multiplex Nucleic Acid Assay Using Segmented CRISPR Fluorescence
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Solution Overview
Problem
Existing multiplex assays face challenges in achieving rapid, accurate detection of multiple analytes without requiring immediate preparation and large equipment, particularly for on-site applications, and lack the ability to detect nucleic acids without amplification.
Innovation Solution
A multiplex assay utilizing a substrate with physically separated assay locations, each containing a nucleotide-targeting enzyme, guide RNA, quencher-labeled oligonucleotide, and fluorophore-labeled oligonucleotide, which allows for the detection of target nucleic acids through fluorescence without amplification, using CRISPR-associated proteins like Cas12 or Cas13, and enabling detection with a hand-held light device.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If multiplex assays are performed simultaneously for multiple analytes, then detection efficiency and throughput are improved, but assay complexity and difficulty of interpretation increase
Solution Approach 1:
The assay is divided into multiple spatially separated assay locations on a single substrate, with each location dedicated to detecting a specific analyte. This segmentation allows simultaneous detection of multiple analytes while keeping each individual assay location simple and easy to interpret, resolving the contradiction between productivity and complexity.
2Loss of time
If nucleic acid detection is performed without amplification, then detection speed and simplicity are improved, but detection sensitivity and accuracy deteriorate
Solution Approach 1:
The assay employs engineered parameters including optimized guide RNA sequences, tailored enzyme concentrations, and adjusted buffer compositions to enable accurate nucleic acid detection without amplification. These parameter optimizations maintain detection precision while eliminating time-consuming amplification steps.
3Quantity of substance
If multiple reagents are combined in a single multiplex assay, then reagent consumption and cost are reduced, but reagent stability and assay reliability worsen
Solution Approach 1:
Multiple reagent sets are spatially segmented into separate assay locations on the same substrate rather than being mixed together. This physical separation maintains the stability and reliability of each reagent while still achieving multiplex detection and reducing overall reagent consumption through shared substrate and readout systems.
4Measurement precision
If assay preparation is performed immediately before use, then detection accuracy is improved, but preparation time and operational complexity increase
Solution Approach 1:
The assay substrate is pre-prepared with multiple assay locations containing immobilized reagents, capture probes, and detection components before use. This preliminary preparation eliminates time-consuming setup steps while maintaining detection accuracy, as the pre-positioned reagents are stable and ready for immediate use.
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, accurate detection and quantification of multiple analytes in a single assay, simplifying sample preparation and eliminating the need for large equipment, allowing on-site analysis.
Implementation Method 1
a quencher configured to absorb energy emitted by an adjacent fluorophore
Implementation Method 2
a fluorophore configured to emit light upon excitation in the absence of the quencher
Implementation Method 3
The gRNA forms a complex with the nucleotide-targeting enzyme. A sequence of the gRNA may be complementary to a sequence of a target nucleic acid
Implementation Method 4
activating the nucleotide-targeting enzyme to cleave nucleic acids, including the quencher-labeled oligonucleotide
Data Source
AI summary
A multiplex assay for nucleic acid detection includes a substrate, a sample, and a fluorophore-labeled oligonucleotide. The substrate has a plurality of physically separated assay locations, each of which includes a nucleotide-targeting enzyme configured to cleave nucleic acids, a guide ribonucleic acid (gRNA), and a quencher-labeled oligonucleotide. A portion of the sample is distributed to each assay location. The gRNA recognizes target nucleic acid in the sample, thereby activating the nucleotide-targeting enzyme to cleave nucleic acids, including the quencher-labeled oligonucleotide. The fluorophore-labeled oligonucleotide is subsequently added to each assay location, which facilitates identification of a presence of the target nucleic acid in the sample via detection of unquenched light emitted by the fluorophore in one or more of the plurality of assay locations.