Tween 20 and Conjugates. When it comes to stability, DHFR is stable in 50 mM sodium phosphate buffer, however, these conditions are not ideal for AuNPs. AuNP are stable in citrate buffer and salt-less solutions, and they will start to aggregate once placed in high salt containing buffer. To combat these non-ideal conditions, DHFR-AuNP conjugates were initially stored in 10 mM sodium phosphate buffer at 4°C. However, the activity of the conjugated DHFR would decrease rapidly. It was hypothesized that the reason this was happening was due to aggregation of the DHFR that were caused by empty regions of the AuNPs binding together. Previous studies have used Tween 20, thiol groups and other heterocyclic compounds to cap these empty regions.33-35 For the purpose of these studies Tween 20 was used because Tween 20 has been used as a surfactant with DHFR in previous studies.36,37 FG Loop Mutant-15 nm AuNP and Alpha Helix Mutant-15 nm AuNP conjugates were used to test the effect of 0.005% Tween 20. Activity assays with these conjugates were run to determine the turnovers over multiple days to determine conjugate stability, and it was determined that the conjugates were active in Tween 20 for approximately 10 days when stored at 4C. The activity of the covalently bound conjugates increases when the samples are stored in 0.005% Tween 20, which is indicative of stability (Table 3.2). The pipette tips used were all lo-bind; however, conjugates would stick to them. This problem would have consequences by altering the concentration, causing errors when determining concentration and activity. However, Tween 20 also solved this problem (Figure 3.3). Tween 20 prevented the loss of sample on lo-bind pipette tips, most likely due to the capping of empty spaces on the AuNPs with Tween 20 to prevent exposed AuNPs from attaching to pipette tips. Sample Type Day 1 Turnover (s-1) Without Tween 20 Day 1 Turnover (s-1) With Tween 20 Day 7 Turnover (s-1) With Tween 20 FG Loop Mutant- 15 nm AuNP 0.3 ± 0.2 0.83 ± 0.07 0.71± 0.08 Alpha Helix Mutant- 15 nm AuNP 0.2 ± 0.1 0.71 ± 0.08 0.72± 0.04 Table 3.2: Enzyme turnovers for the DHFR-15 nm AuNP conjugates day one and seven days after conjugate preparation. (n=3) Figure 3.3 Pipette tips after transfer of DHFR-15 nm AuNP without Tween 20 (left) and with Tween 20 (right). The pink stain on the pipette tips is indicative of the conjugates sticking to the lo-bind pipette tips that were used throughout the experiments. Part IV: DHFR-AuNP Conjugate Characterization
