Functional Nucleic Acid Probes for Single-Cell Detection
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Solution Overview
Problem
Current nucleic acid probe technologies face limitations in sensitivity, specificity, and the need for cell lysis, which complicates the detection of low-copy-number proteins and nucleic acids, particularly in complex cellular environments like bacterial infections and cancer diagnosis, where drug resistance is a concern, and often rely on ensemble averaging rather than single-cell analysis.
Innovation Solution
The development of functional nucleic acid probes (FNAPs) that can catalyze reactions or bind specifically to targets, allowing for single-cell analysis without cell lysis, using allosterically and enzymatically activated deoxyribozyme cleavage cascades to generate detectable signals, which can be amplified and are effective across various cell types and pathogen species.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If conventional nucleic acid probes are used for detection, then the presence or absence of target sequences can be detected, but sensitivity is limited and cell lysis is required which complicates the analysis
Solution Approach 1:
The patent extracts the detection function from conventional probe systems by introducing functional nucleic acid probes that perform multiple functions (binding, catalysis, signal generation) without requiring cell lysis. The FNAPs are designed to remain intact within living cells while performing detection functions.
Solution Approach 2:
The functional nucleic acid probes perform multiple functions simultaneously: they bind to target sequences, catalyze reactions to amplify signals, and generate detectable outputs. This multi-functionality eliminates the need for separate reagents and procedures, simplifying the overall detection system.
2Ease of operation
If ensemble averaging methods are used, then analysis is simplified, but single-cell variability and heterogeneity information is lost
Solution Approach 1:
The patent segments the population analysis approach by enabling detection at the individual cell level. Each cell can be analyzed separately for its nucleic acid content and functional response, preserving heterogeneity information while maintaining operational simplicity through standardized single-cell protocols.
3Device complexity
If low-copy-number proteins and nucleic acids are detected without amplification, then the method is simpler, but sensitivity is insufficient below detection limits
Solution Approach 1:
The functional nucleic acid probes are self-amplifying through their catalytic activity. Once bound to the target, they catalyze reactions that produce detectable signals without requiring external amplification reagents, maintaining simplicity while achieving high sensitivity for low-copy-number targets.
4Measurement precision
If thermal cycling and high temperatures are used for nucleic acid detection, then amplification is achieved, but the workflow is more complex and time-consuming
Solution Approach 1:
The patent replaces thermal cycling mechanisms with alternative amplification strategies based on functional nucleic acid catalysis. This substitution eliminates the need for temperature-controlled cycling equipment and procedures, reducing analysis time while maintaining sensitivity through chemical catalytic amplification.
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
FNAPs enable sensitive, fast, and accurate identification and characterization of analytes at the single-cell level, providing natural amplification, cost reduction, increased stability, and simplified workflows, suitable for point-of-care devices and overcoming the limitations of existing methods by allowing analysis in confined spaces without the need for thermal cycling or high temperatures.
Implementation Method 1
Nucleic acid probes are typically short nucleic acid sequences used to detect, amplify, and quantify DNA and RNA for diagnostic and therapeutic applications. They are designed to specifically hybridize with particular complementary target nucleic acid sequences.
Implementation Method 2
functional nucleic acid probes (FNAPs) that when activated can cleave a substrate, initiating a cleavage cascade that results in a detectable signal
Data Source
AI summary
Embodiments of this invention are directed towards the sensitive, fast, and accurate identification and/or characterization of a single cell or bacterium, particularly phenotypic characterization. Certain aspects of the invention include assays that include functional nucleic acid probes (FNAPs). FNAPs can be used to generate deoxyribozyme cleavage cascades (DRCC) initiated by activation of a FNAP resulting in a detectable signal from a single cell.