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Inverted Tetrahedral DNA Reporters Enable Label-Free Ratiometric CRISPR Electrochemical Aptasensing of Kanamycin
1Shanghai Engineering Research Center of Food Rapid Detection, School of Health Science and Engineering, University of Shanghai for Science and Technology, Shanghai 200093, China.
Journal of Agricultural and Food Chemistry
|October 21, 2025
Summary
This study introduces an inverted tetrahedral DNA reporter for enhanced electrochemical CRISPR aptasensing, improving accuracy and reliability in point-of-care diagnostics for kanamycin detection.
Area of Science:
- Biotechnology
- Nanotechnology
- Biosensing
Background:
- CRISPR technology combined with electrochemical sensing offers potential for point-of-care testing.
- Challenges remain in reporter immobilization, trans-cleavage efficiency, and overall reliability of electrochemical sensing.
Purpose of the Study:
- To develop an improved reporter system for electrochemical CRISPR aptasensing.
- To enhance the accuracy and reliability of CRISPR-based electrochemical detection methods.
- To create a label-free, ratiometric electrochemical aptasensing method for kanamycin detection.
Main Methods:
- Development of an inverted tetrahedral DNA reporter (TDN) with self-assembled, thiol-modified oligonucleotides.
- Utilizing CRISPR/Cas12a trans-cleavage activity to detach TDNs from the electrode surface.
- Employing a ratiometric signal output based on changes in electron transfer and adsorbed methylene blue signals.
Main Results:
- Successfully demonstrated a label-free, ratiometric electrochemical aptasensing method for kanamycin.
- Achieved sensitive detection of kanamycin down to 0.35 pM within 50 minutes.
- Validated the method's practical applicability by detecting kanamycin in spiked milk samples.
Conclusions:
- The inverted tetrahedral DNA reporter offers a novel approach for electrochemical CRISPR sensing.
- This method enhances sensitivity, accuracy, and reliability for point-of-care applications.
- The developed aptasensing platform shows promise for detecting various analytes in complex matrices.
