Electrochemical biosensor and uses thereof
Inventors
Assignees
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Publication Number
US-12297485-B2
Publication Date
2025-05-13
Expiration Date
Abstract
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.
Core Innovation
The invention relates to an immobilization-free, electrochemical method for detecting a target DNA sequence in a sample using an electric-field-enhanced electrochemical CRISPR biosensor. A detection mixture is incubated with a nucleic acid probe that includes an ssDNA sequence having a negative charge, where the ssDNA sequence hybridizes with a nucleic acid sequence of a target DNA sequence that is covalently linked to an electroactive probe having a neutral or positive charge. The detection mixture also includes a Class 2 CRISPR-associated Cas protein or enzyme having trans-cleavage activity for ssDNA and a guide ribonucleic acid (gRNA) with a scaffold sequence interacting with the Class 2 Cas protein and a nucleic acid sequence hybridizing with a sequence of the target DNA sequence.
After incubation, an electric field comprising an AC electric field and a DC offset is applied to concentrate nucleic acids and the nucleic acid probe on a positively charged working electrode. The electroactive probe is released from the nucleic acid probe by the Class 2 Cas protein when the target DNA sequence is present in the detection mixture. Current is then measured after applying the electric field as potential is applied, where detection of current indicates the presence of the target DNA sequence in the sample.
The disclosed approach combines the Class 2 CRISPR trans-cleavage of an ssDNA linked electrochemical hybridization indicator with electric-field concentration and charge-based electrode attraction. An AC electric field together with a DC offset is used to concentrate the relevant nucleic-acid components toward a positively charged working electrode, while the negatively charged probe components are described as being concentrated in the presence of the electric field. The method is described in a point-of-care diagnostic context and supports target DNA detection without immobilizing the probe on an electrode.
Claims Coverage
The disclosed claim set includes one independent claim directed to an immobilization-free electrochemical detection method using a negatively charged ssDNA probe, a neutral or positive electroactive probe, Class 2 Cas trans-cleavage, and an AC electric field with a DC offset, followed by current measurement. The independent claim contains four main inventive features.
Immobilization-free electrochemical detection mixture with charged ssDNA probe and electroactive probe
Incubating a detection mixture comprising a nucleic acid probe with an ssDNA sequence that hybridizes with a target DNA sequence covalently linked to an electroactive probe, wherein the nucleic acid probe has a negative charge and the electroactive probe has a neutral or positive charge; a Class 2 Cas protein or enzyme having trans-cleavage activity for ssDNA; and a gRNA including a scaffold sequence interacting with the Class 2 Cas protein and a nucleic acid sequence hybridizing with a sequence of the target DNA sequence.
Electric-field concentration using AC electric field and DC offset toward a positively charged electrode
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 and the nucleic acid probe on a positively charged working electrode.
Cas-mediated release of the electroactive probe upon target presence
Releasing the electroactive probe from the nucleic acid probe by the Class 2 Cas protein when the target DNA sequence is present in the detection mixture.
Current measurement after field application as indicator of target DNA presence
Measuring, after applying the electric field, a current of the detection mixture as potential is applied, where detection of a current indicates the presence of the target DNA sequence in the sample.
The claims cover an immobilization-free electrochemical DNA detection workflow that integrates charged ssDNA/electroactive-probe hybridization, Class 2 Cas trans-cleavage triggered by target DNA, electric-field concentration using AC with DC offset toward a positively charged working electrode, and current measurement after field application to indicate target DNA presence.
Stated Advantages
Immobilization-free electrochemical detection.
Electric-field concentration of nucleic acids and the nucleic acid probe on a positively charged working electrode.
Documented Applications
Detection of a target DNA sequence in a sample in a point-of-care diagnostics context.
Detection of HPV-16 L1 gene using unamplified and amplified (RPA) sample types, including clinical swab samples.
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