Electrochemical Nucleic Acid Sensing with Immobilization-Free CRISPR
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Solution Overview
Problem
Existing nucleic acid detection systems based on RPA-CRISPR/Cas integration primarily use fluorescence signals, which require expensive instruments and lack portability, and conventional electrochemical sensors face issues with immobilization, batch-to-batch variation, low sensitivity, and high noise due to solid-liquid reactions.
Innovation Solution
An electrochemical nucleic acid detection method using an immobilization-free reporter molecule with electrochemical active molecules and modification groups, allowing for a homogeneous phase reaction system that enhances molecular collision efficiency and reduces background noise.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If fluorescence signals are used in RPA-CRISPR/Cas detection systems, then detection sensitivity is improved, but instrument cost and device complexity increase
Solution Approach 1:
The patent replaces the optical detection system (fluorescence) with an electrochemical detection system. The CRISPR/Cas12a cleavage of the reporter molecule releases electrochemical active molecules that can be detected by simple electrochemical sensors, eliminating the need for expensive fluorescence instruments while maintaining detection sensitivity.
Solution Approach 2:
The patent changes the detection parameter from optical fluorescence signal to electrochemical signal. By using reporter molecules with electrochemical active groups instead of fluorescent groups, the detection modality is transformed, enabling the use of simple and portable electrochemical instruments.
2Measurement precision
If immobilization is used in electrochemical sensors, then signal response is improved, but batch-to-batch variation and manufacturing complexity increase
Solution Approach 1:
The patent removes the immobilization step from the electrochemical sensor system. Instead of immobilizing the reporter molecule on the electrode surface, the reporter molecule remains free in solution, and only the electrochemical active molecules are detected after CRISPR/Cas12a cleavage, eliminating batch-to-batch variation caused by immobilization inconsistencies.
Solution Approach 2:
Instead of the conventional approach where the sensing element is immobilized on the electrode, the patent inverts the approach by keeping the reporter molecule free in solution and detecting the released electrochemical active molecules, thereby avoiding immobilization-related manufacturing issues.
3Productivity
If solid-liquid reaction mode is used in electrochemical sensors, then molecular collision efficiency is improved, but background noise increases due to non-specific collisions
Solution Approach 1:
The patent introduces a specific intermediary mechanism where the CRISPR/Cas12a ternary complex acts as a selective mediator. The complex specifically recognizes and binds to the target DNA, enabling specific molecular collisions while preventing non-specific collisions between free reporter molecules and the electrode, thereby reducing background noise.
Solution Approach 2:
The patent changes the reaction mode from direct solid-liquid interaction to a solution-phase homogeneous reaction. The CRISPR/Cas12a cleavage reaction occurs in solution, releasing electrochemical active molecules that are then detected, separating the specific recognition step from the detection step and reducing non-specific background noise.
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 method achieves sensitive, rapid, accurate, and cost-effective nucleic acid detection with improved signal response and portability by utilizing a freely dispersed reporter molecule and modification groups, breaking conventional electrochemical sensor design limitations.
Implementation Method 1
The Cas protein and the crRNA are capable of forming a stable binary complex. Depending on the properties of the Cas protein (such as Cas9, Cas12, Cas13, or Cas14), crRNA can selectively bind to RNA or single-stranded DNA (ssDNA) in a sample, forming a ternary complex of Cas/crRNA/RNA or Cas/crRNA/ssDNA. The ternary complex induces conformational changes in the Cas protein that confer trans-cleavage activity toward RNA or ssDNA substrates.
Implementation Method 2
the reporter molecule includes: at least one electrochemical active molecule; and at least one nucleotide
Implementation Method 3
RPA differs from conventional quantitative fluorescence PCR in that it does not require repeated thermal cycling of denaturation-annealing-extension at 56°C, 65°C, and 95°C. Instead, as a commonly used isothermal amplification technique, RPA offers advantages of rapid reaction speed, high detection sensitivity, and low temperature requirements, enabling target nucleic acid amplification at a constant temperature of 37°C.
Data Source
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AI summary
The present application relates to the field of electrochemical analysis and detection, and in particular to an electrochemical nucleic acid detection sensing element and electrochemical nucleic acid detection method based on RPA and CRISPR/Cas. According to the present application, RPA technology, a CRISPR/Cas tool, and electrochemical detection technology are combined, and additionally, an electrochemical active molecule and a modification group are linked to a reporter molecule in a CRISPR/Cas reaction system, such that construction of an immobilization-free and homogeneous electrochemical nucleic acid detection method is realized, and detection of the target nucleic acid can be realized.