Detection of Tetracycline with a CRISPR/Cas12a Aptasensor Using a Highly Efficient Fluorescent Polystyrene Microsphere Reporter System.
ACS Synth Biol
; 13(7): 2166-2176, 2024 Jul 19.
Article
em En
| MEDLINE
| ID: mdl-38866727
ABSTRACT
CRISPR-based diagnostics use the CRISPR-Cas system trans-cleavage activity to identify specific target sequences. When activated, this activity cleaves surrounding reporter molecules, producing a detectable signal. This technique has great specificity, sensitivity, and rapid detection, making it an important molecular diagnostic tool for medical and infectious disease applications. Despite its potential, the present CRISPR/Cas system has challenges with its single-stranded DNA reporters, characterized by low stability and limited sensitivity, restricting effective application in complex biological settings. In this work, we investigate the trans-cleavage activity of CRISPR/Cas12a on substrates utilizing fluorescent polystyrene microspheres to detect tetracycline. This innovative discovery led to the development of microsphere probes addressing the stability and sensitivity issues associated with CRISPR/Cas biosensing. By attaching the ssDNA reporter to polystyrene microspheres, we discovered that the Cas12a system exhibits robust and sensitive trans-cleavage activity. Further work revealed that the trans-cleavage activity of Cas12a on the microsphere surface is significantly dependent on the concentration of the ssDNA reporters. Building on these intriguing discoveries, we developed microsphere-based fluorescent probes for CRISPR/Cas aptasensors, which showed stability and sensitivity in tetracycline biosensing. We demonstrated a highly sensitive detection of tetracycline with a detection limit of 0.1 µM. Finally, the practical use of a microsphere-based CRISPR/Cas aptasensor in spiked food samples was proven successful. These findings highlighted the remarkable potential of microsphere-based CRISPR/Cas aptasensors for biological research and medical diagnosis.
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Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Poliestirenos
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Tetraciclina
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DNA de Cadeia Simples
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Técnicas Biossensoriais
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Sistemas CRISPR-Cas
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Microesferas
Idioma:
En
Ano de publicação:
2024
Tipo de documento:
Article