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Prof. Tina Perica

Why did we contact them?

We contacted Prof. Tina Perica, Professor of Biochemistry and Systems Biology, to gain insights into possible detection outputs for our diagnostic test and to discuss enzyme stability after freeze-drying. We wanted to learn more about options for colorimetric vs. fluorescent readouts, the use of CRISPR-Cas in combination with RPA, and whether her department has a lyophilization machine that could be useful for our project.

Discussion

Detection output options

  • Prof. Tina Perica suggested several possible alternatives for achieving a visible signal beyond fluorescence. Maybe biotin on Cas enzyme instead of fluorescence/quencher.
  • Cas could also be linked to nanogold particles to accumulate and create a visible band, though this might be difficult to engineer and pathogen-specificity could be unclear.
  • She suggested Alkaline Phosphatase (AP) as a promising option for a colorimetric readout. Unlike horseradish peroxidase, AP does not require hydrogen peroxide. AP can even be lyophilized, though the enzyme activity must be carefully matched to flow speed to avoid false positives.

DNA amount and enzyme concentrations

  • The visibility of results depends more on the concentration of the dye than on the amount of DNA or Cas enzymes.
  • With lower Cas concentrations, reactions may simply take more time.

CRISPR-Cas practical considerations

  • On spatially separating RPA and CRISPR-Cas: she was not sure and suggested further expert input.
  • Immobilizing Cas enzymes was also uncertain; she recommended contacting Prof. Alexandria Deliz-Liang (Chemistry) for advice on paper type and immobilization chemistry.

Freeze-drying and enzyme stability

  • She does not have a lyophilization machine herself but confirmed that there should be one available in the department. She offered to follow up.

Main Takeaways

  • Colorimetric results might be achievable with Alkaline Phosphatase.
  • Enzyme activity must be balanced with flow rate to avoid false positives.
  • There is likely a lyophilization machine in the department.
  • Dye concentration is more critical than DNA or Cas concentration.
  • Potential new contact: Prof. Alexandria Deliz-Liang for immobilization questions.

Integration

We implemented Prof. Tina Perica’s advice by

  • Exploring Alkaline Phosphatase as a colorimetric detection option.
  • Planning to contact Prof. Alexandria Deliz-Liang for immobilization guidance.
  • Following up with the department about access to a lyophilization machine.
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