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This study introduces a novel biosensor combining terminal deoxynucleotidyl transferase (TdT) and CRISPR-Cas12a for ultra-sensitive detection. The biosensor accurately quantifies alkaline phosphatase activity in cancer cells, enabling early diagnosis and monitoring.

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Area of Science:

  • Biotechnology
  • Molecular Biology
  • Biosensor Technology

Background:

  • Sensitive detection of biomarkers is crucial for early cancer diagnosis.
  • Existing biosensors face limitations in sensitivity and specificity for complex biological samples.

Purpose of the Study:

  • To develop an ultra-sensitive biosensor integrating TdT and CRISPR-Cas12a.
  • To validate the biosensor's performance in detecting alkaline phosphatase (ALP) activity in cancer cells.

Main Methods:

  • Integration of terminal deoxynucleotidyl transferase (TdT) catalytic activity with CRISPR-Cas12a trans-cleavage.
  • Characterization of biosensor performance, including linear detection range and limit of detection.
  • Quantification of ALP activity in cervical cancer cells and HeLa cell lysates.

Main Results:

  • Achieved ultra-sensitive biomolecular detection with a low limit of detection (1.7 × 10^-3 U L^-1).
  • Demonstrated a broad linear detection range (0–0.2 U L^-1) and high specificity.
  • Successfully quantified ALP activity in cancer cells at single-cell resolution, even at high dilutions (10^6-fold).

Conclusions:

  • The TdT-CRISPR-Cas12a biosensor offers a robust, sensitive, and cost-effective platform for cancer diagnosis.
  • This technology shows significant potential for serological tumor screening, treatment monitoring, and enzyme inhibitor screening.
  • The biosensor facilitates advancements in translational medicine for enhanced cancer management and early disease detection.