CRISPR Protein Single-Molecule Nucleic Acid Detection
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Solution Overview
Problem
Current devices and systems for detecting target DNA are insufficient for single molecule detection due to analog detection methods and the need for calibration, which limits their ability to accurately quantify target DNA.
Innovation Solution
A bioelectronic device comprising a first electrode, a second electrode, and a CRISPR-associated protein, where the protein is chemically modified to form a chemical bond with the electrodes, allowing an electrical current to pass through and detecting shifts in current upon binding to a target nucleic acid.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If analog detection method is used with multiple RNPs bound to graphene channel, then detection sensitivity is improved, but measurement precision deteriorates due to continuous current change requiring calibration
Solution Approach 1:
The patent transitions from analog detection with multiple RNPs to single-molecule detection by segmenting the detection process. Each RNP is individually attached to the graphene channel at specific sites, enabling discrete, calibration-free detection of individual target molecules through distinct current blockage events.
Solution Approach 2:
The patent creates multiple identical detection sites on the graphene channel by attaching multiple copies of the same RNP at defined locations. This allows replicate measurements of single molecules, improving statistical reliability without requiring calibration, as each RNP-target interaction produces the same characteristic signal.
2Ease of operation
If CRISPR-associated protein is chemically modified to bond with electrodes, then electrical current can pass through the protein, but device complexity increases due to modification requirements
Solution Approach 1:
The patent uses a mediator molecule (such as a redox-active tag or conductive linker) to bridge the CRISPR-associated protein and the electrode. This intermediary enables electrical current passage and signal detection without requiring direct chemical modification of the protein's active sites, preserving its function while enabling electronic readout.
Solution Approach 2:
The patent replaces traditional biochemical detection methods with an electrical measurement system. By attaching the CRISPR protein to electrodes and measuring current changes upon target binding, the system substitutes mechanical/biochemical assays with electronic detection, simplifying the overall measurement process.
3Reliability
If nuclease activity is inhibited to trap target DNA, then target binding is improved, but detection capability is reduced without enzymatic signal amplification
Solution Approach 1:
The patent extracts the detection function from the enzymatic activity of the CRISPR system. Instead of relying on nuclease-mediated signal amplification, the detection is achieved by directly measuring the physical presence and binding of the target molecule through its effect on electrical current, eliminating the need for catalytic amplification.
Solution Approach 2:
The target nucleic acid molecule itself serves as the signal source. Its binding to the CRISPR protein complex directly modulates the electrical current through the protein, providing a self-sufficient detection mechanism that does not require external signal amplification enzymes or additional reagents.
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
The device enables sequence-specific detection of nucleic acid targets using current fluctuations as a readout, allowing for precise detection and identification of nucleic acid targets at the single molecule level.
Implementation Method 1
the binding of the CRISPR-associated protein to a target nucleic acid causes a shift in the current
Data Source
AI summary
The present disclosure provides devices, systems, and methods related to single molecule detection. In particular, the present disclosure provides devices and methods for sequence-specific detection of a nucleic acid target using current fluctuations as a readout for protein binding to the nucleic acid target. As described herein, certain aspects of the bioelectronic devices and method can be used to detect and identify any nucleic acid target for the purpose of diagnosis and/or treatment.