University of Twente Student Theses


Combining CRISPR/Cas12a and gold nanoparticles to detect low sample concentrations of nucleic acids.

Vliet, Annemiek van (2023) Combining CRISPR/Cas12a and gold nanoparticles to detect low sample concentrations of nucleic acids.

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Abstract:As the pressure of healthcare in hospitals increases, a new and easy detection method for nucleic acids could bring the diagnostic part hospitals have to perform to a general practitioner or even patients' homes. This new diagnostic assay could be gold nanoparticles combined with CRISPR/Cas12a. Gold nanoparticles (AuNPs) can be functionalized with DNA to detect nucleic acids already. However, they lack sensitivity measuring low sample concentrations. CRISPR/Cas12a could be used to amplify the signal. Here, the color change of solutions with AuNPs and a linker ssDNA is analyzed. But, naked-eye detection was challenging as the changes in color were only 'visible' from absorbance values. Furthermore, the enzymatic activity of CRISPR/Cas12a is analyzed. This showed that, after target dsDNA and the CRISPR/Cas12a RNP complex were incubated for 1 hour, the cutting of CRISPR/Cas12a can be followed with a Michaelis-Menten analysis and the maximum velocity of the cutting was 2072 M/s. Also, by varying the reporter and target dsDNA concentration in the CRISPR assay and measuring the fluorescence, the relation between these was determined. A calibration curve showed the target dsDNA concentration and percentage of cut ssDNA reporters by CRISPR. From this, it can be concluded that adding a [ssDNA linker] of 444,44 nM to ssDNA functionalized AuNPs should provide a fast and sensitive colorimetric assay readable with the naked-eye. Further research could be done on optimizing the functionalizing of the AuNPs with DNA, in fact using the recommended ssDNA linker concentration and using other Cas enzymes. Also, creating a one-pot assay could provide even more advantages in detecting low sample concentrations of nucleic acids.
Item Type:Essay (Bachelor)
Faculty:TNW: Science and Technology
Subject:35 chemistry, 42 biology, 44 medicine
Programme:Biomedical Technology BSc (56226)
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