Integrated Micropillar Polydimethylsiloxane Accurate CRISPR Detection (IMPACT) System for Rapid Viral DNA Sensing

Kenneth N. Hass; Mengdi Bao; Qian He; Myeongkee Park; Peiwu Qin; Ke Du

With an optimal washing condition in hand, we then studied the effect of incubation time for biotin labeled DNA binding on streptavidin coated PDMS surface. After channel surface modification and streptavidin treatment, fluorescent reporter probe was introduced to bind on the solid surface. Then, the washing protocol was performed, followed by UV light exposure to retrieve the reporter probe. As shown in Fig. 3 , for streptavidin coated surface, the number of DNA immobilized on the surface does not show significant change with an incubation time between 10 to 60 min as the integrated fluorescence intensity of the retrieved DNA ranges between 50,000 to 60,000 counts. However, the binding capacity increases ~25% with 3 hrs incubation when compared to 10 min incubation. Although the binding capacity further increases with 24 hrs incubation , it requires refrigeration as the streptavidin protein could denature at room temperature. On the other hand, the negative sample without streptavidin coating does not show a correlation to the incubation time, indicating less specific binding. Moreover, the negative sample always shows much lower fluorescence intensity than the positive sample. For example, with 3 hrs incubation, the retrieved DNA from positive sample is ~2.6 times higher than the negative sample. It further indicates that surface modification can enhance the probe binding capacity. Therefore, we used 3 hrs incubation to prepare the IMPACT chip for solid phase CRISPR detection.