Cy3-Top18-biotin or Cy5-D27-biotin DNA was immobilized on a PEG-coated surface through biotin-neutravidin binding using 100 pM concentration of DNA.(14 (link)) Prior to imaging, the sample was first thoroughly rinsed with a pH 8.0 T50 buffer, containing 10 mM Tris and 50 mM NaCl, and then incubated in a pH 8.0 imaging buffer containing 25 mM Tris, 5 mM MgCl2, 0.8% (w/v) dextrose, 2 mM Trolox, 0.04 mg/mL catalase, and glucose oxidase at the concentrations specified in the text. This two-step buffer exchange procedure was carried out for each of the three glucose oxidase concentrations examined, ranging from 1.0 to 0.25 mg/mL.
Single-molecule experiments were performed with prism-type total internal reflection microscopy.(14 (link)) In brief, a 532 nm DPSS laser or 633 nm HeNe laser was used to excite the fluorophores on immobilized DNA molecules through total internal reflection. The fluorescence was collected using a 60×, 1.2 NA, water immersion objective lens from Olympus America (Center Valley, PA) and detected up to 60 s at 30 ms time resolution with an EMCCD camera from Andor (Belfast, Northern Ireland). Single molecules were identified and their intensity traces were extracted from the raw data using a custom program written in IDL. The total intensity of all the molecules identified from within the imaging area was then obtained using a program written in MATLAB. The decay in the average intensity of all molecules as a function of time was fit to a single exponential, with the decay lifetime representing the single-molecule photobleaching time, τphotobleaching. This single-molecule measurement was repeated five times in different imaging areas for both fluorophores and all glucose oxidase concentrations to provide the basis for statistical analysis.
Single-molecule experiments were performed with prism-type total internal reflection microscopy.(14 (link)) In brief, a 532 nm DPSS laser or 633 nm HeNe laser was used to excite the fluorophores on immobilized DNA molecules through total internal reflection. The fluorescence was collected using a 60×, 1.2 NA, water immersion objective lens from Olympus America (Center Valley, PA) and detected up to 60 s at 30 ms time resolution with an EMCCD camera from Andor (Belfast, Northern Ireland). Single molecules were identified and their intensity traces were extracted from the raw data using a custom program written in IDL. The total intensity of all the molecules identified from within the imaging area was then obtained using a program written in MATLAB. The decay in the average intensity of all molecules as a function of time was fit to a single exponential, with the decay lifetime representing the single-molecule photobleaching time, τphotobleaching. This single-molecule measurement was repeated five times in different imaging areas for both fluorophores and all glucose oxidase concentrations to provide the basis for statistical analysis.
