Ammonium persulfate

A 1-inch electrophoretic device developed in-house was used for gel-based detection^{24 (link)}. Ten-well microcassettes with 6% T polyacrylamide gel were prepared using 40 (w/v)%-acrylamide/bis (19:1) (Nacalai Tesque, Kyoto, Japan), 5× TBE, 10% ammonium persulfate (Fujifilm Wako Pure Chemicals) and tetramethylethylenediamine (Fujifilm Wako Pure Chemicals, Osaka, Japan) and used as a 1-inch (2.5 × 2.5 cm) precast polyacrylamide gel to analyze the RNA-specific amplification or RICCA reaction products. Paper pads (1.0 × 2.0 cm) were soaked in 2 mL of 1× TBE buffer and used as a source of running buffer. One microliter of each sample was loaded in the micro gel wells with 0.5 µL of 6× gel loading dye. A 50-bp DNA ladder was also loaded in one well to confirm the size of the obtained DNA fragments and comigrated with the samples at 100 V for 5–7 min. A 300-μL volume of 10× SYBR gold nucleic acid gel stain (Thermo Fisher Scientific) was poured on top of the gel, and the bands were visualized using the blue LED flashlight installed in the palm-sized electrophoretic device. Images were acquired using a standard camera-equipped cellular phone.

An FP solution was mixed with 30% (w/v) acrylamide (Wako, 012-08023). Immediately after the addition of 10% ammonium persulfate (Wako, 202-04003) and N,N,N′,N′-tetramethylethylenediamine (Wako, 202-04003), 200 μl of the mixture was poured onto a 35-mm glass-bottom dish (Iwaki, 3911-035) and overlaid with a coverslip (No. 1, 0.13–0.17-mm thickness, Matsunami Glass). Sandwiched between the two coverslips was 1 μM FP gel embedded in 20% polyacrylamide. The protein sample was excited continuously on an inverted microscope (IX-81, Olympus) equipped with a standard 75-W xenon lamp, a ×40 objective lens (UPlanSApo ×40/0.95 NA) and a cooled CCD camera (ORCA AG, Hamamatsu Photonics). An appropriate excitation filter was used to choose the excitation wavelength. Whereas no neutral density (ND) filter was installed in the illuminator in principle, appropriate ND filters (1–12% transmittance) were used to attenuate the emitted fluorescence. Image acquisition was performed using an appropriate emission filter every 6 seconds with a short exposure time (250–360 ms). The whole system was controlled using AQUACOSMOS software (Hamamatsu Photonics).
The following experimental conditions were used for eight FP groups:

The following reagents were used for the synthesis of polyStyrene microspheres. Styrene as a monomer, methanol and ethanol as solvents, and polyvinylpyrrolidone as a dispersion stabilizer were obtained from Fujifilm Wako Pure Chemical. Azobisisobutyronitrile as a polymerization initiator was obtained from Sigma-Aldrich Japan. For the polymerization of polyaniline, aniline hydrochloride monomer and ammonium persulfate as an oxidant were obtained from Fujifilm Wako Pure Chemical. The Styrene monomer was distilled under reduced pressure before polymerization to remove the polymerization inhibitor. Other reagents were of analytical grade or better and were used as received.