Evaluation of the effect of spacers with the HTDQ probe. Gradient particles were synthesized using HTDQ, HTDQ with a 6-thymine spacer at the 5′ end (HTDQ-6T), and HTDQ with a 12-thymine spacer at the 5′ end (HTDQ-12T). (A) Static image of a gradient particle incubated with 100 pM thrombin and 10 nM PAb-T. Longer thymine spacers lead to higher signal. (B) Bar graph showing the fold increase in signal when HTDQ-12T (orange) and HTDQ-6T (green) regions are compared to the HTDQ region, over a range of thrombin concentrations (10 pM, 50 pM, 100 pM, 500 pM). All particles were incubated with 10 nM PAb-T.