Electrochemical Biosensor Using AC Field for Immobilization-Free DNA Detection
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Solution Overview
Problem
Existing nucleic acid detection methods, such as PCR and CRISPR-based biosensors, require complex equipment, skilled personnel, and lengthy procedures, limiting their accessibility and efficiency for DNA detection.
Innovation Solution
An immobilization-free, electrochemical method using a detection mixture comprising a nucleic acid probe, a Class 2 Cas protein with trans-cleavage activity, and a guide RNA, which is applied with an alternating current (AC) electric field and a direct current (DC) offset to concentrate nucleic acids and release an electroactive probe for detection.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If electrochemical probes are immobilized on the electrode surface, then the biosensor can detect nucleic acids, but the immobilization process is complicated and time-consuming
Solution Approach 1:
The patent extracts the probe from the electrode surface and places it in free solution, eliminating the immobilization step entirely. The detection mixture containing the probe is simply applied to the electrode, and the probe remains in solution during the CRISPR reaction, thereby removing the complicated and time-consuming immobilization process while maintaining detection capability.
Solution Approach 2:
The patent introduces an intermediary signal amplification mechanism where the CRISPR-Cas12a system generates numerous cleaved probe fragments from a single target DNA molecule. This intermediary amplification step compensates for the lack of surface immobilization and enables sensitive detection without requiring complex probe attachment procedures.
2Reliability
If electrochemical probes are immobilized on the electrode surface, then the biosensor can function, but cleavage efficiency and selectivity are reduced due to steric hindrance
Solution Approach 1:
The probe is extracted from the electrode surface and placed in free solution, eliminating steric hindrance caused by surface immobilization. This allows the CRISPR-Cas12a system to access and cleave the probe with full efficiency and selectivity, as the probe can freely diffuse and interact with the Cas complex without spatial constraints.
3Measurement precision
If fluorescence detection is used for CRISPR-based nucleic acid detection, then high sensitivity is achieved, but expensive and complicated fluorescence detectors are required
Solution Approach 1:
The patent substitutes the optical fluorescence detection system with an electrochemical detection system. Instead of using fluorophores and expensive fluorescence detectors, the invention uses electroactive probes that generate electrical signals through redox reactions at the electrode surface, thereby achieving comparable sensitivity with simpler and more cost-effective equipment.
Solution Approach 2:
The patent changes the detection parameter from optical fluorescence intensity to electrochemical current signal. By using electroactive probes that undergo oxidation or reduction reactions, the system translates the CRISPR cleavage event into an electrical signal that can be measured with simple potentiostats or voltmeters, replacing complex optical instrumentation.
4Measurement precision
If PCR methods are used for nucleic acid detection, then high sensitivity and specificity are achieved, but bulky equipment, well-trained personnel, and long turnover times are required
Solution Approach 1:
The patent replaces the thermal cycling equipment and complex PCR instrumentation with a simple electrochemical detection system. The CRISPR-based isothermal amplification method eliminates the need for thermal cyclers, and the electrochemical readout replaces complex optical detection systems, thereby reducing equipment bulk and personnel training requirements while maintaining detection precision.
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
This method enables rapid, sensitive, and versatile DNA detection without the need for immobilization processes, improving cleavage efficiency and selectivity, and allowing for point-of-care applications.
Implementation Method 1
Class 2 Cas protein trans-cleaved electroactive probe or electrochemical hybridization indicator is released from the nucleic acid probe when the target DNA sequence is present
Implementation Method 2
applying an electric field comprising an alternating current (AC) electric field and a direct current (DC) offset to the detection mixture to concentrate nucleic acids in the sample and a nucleic acid probe on a positively charged working electrode
Implementation Method 3
measuring, after applying the electric field, the current of the detection mixture as potential is applied (for example, measuring the current of the detection mixture as the potential is varied and/or examining the electrochemical activity of the detection mixture, after applying the electric field, via voltammetry and/or differential pulse voltammetry (DPV))
Data Source
AI summary
Described herein is an immobilization-free, electrochemical method of detecting a target DNA sequence in a sample. The method includes: incubating the sample with a detection mixture, applying an electric field including an alternating current electric field and a direct current offset to the detection mixture to concentrate nucleic acids in the sample and the nucleic acid probe on a positively charged working electrode, wherein a Class 2 Cas protein trans-cleaved electroactive probe is released from the nucleic acid probe when the target DNA is present in the sample and diffuses toward a negatively charged electrode; and measuring the current as potential is applied, wherein detection of a current in the detection mixture indicates the presence of the target DNA sequence in the sample.