Fluoroskan ascent fl 2

Intracellular ROS level was determined using 2′, 7′-dichlorodihydrofluorescein diacetate (DCFH-DA) that can diffuse into the cell. DCFH-DA is deacetylated by cellular esterase and converted to non-fluorescent 2′, 7′-dichlorodihydrofluorescein. It is then oxidized to fluorescent 2′, 7′-dichlorodihydrofluorescein (DCF) by intracellular ROS. The measured fluorescence intensity is proportional to the level of ROS in the cell [47 (link)]. After standard trypsinization, the cells were seeded at 8 × 10³ cells/well in 96-well black plates for 24 h. The cells were washed with PBS thrice after 24 h of culture in a normal growth medium. After the wash, cells were incubated at 50 µM DCFH-DA with a serum-free medium for 4 h. Excess DCFH-DA was removed by washing three times with PBS, and DCFH-DA-loaded cells were obtained. The cells were exposed to different concentrations of Fe-Ni ANPs (0.1; 0.5; 1; 2; 4; 8; 16; 32; 64; 128 µg/mL) for 24 h. Fluorescence values were determined kinetically at 37 °C with a fluorometric plate reader (Fluoroskan Ascent FL 2.6, Thermo Fisher Scientific, Waltham, MA, USA) at 480 nm/530 nm. The experiment was performed in triplicate. The results are given as the mean relative fluorescence unit (RFU) ± SD 6, 12, 18, and 24 h (after subtracting background values caused by Fe-Ni ANPs in the absence of dye).

A fluorescence instrument consisting of a Fluoroskan Ascent FL 2.6 instrument (Thermo Fisher Scientific, Espoo, Finland) equipped with Ascent software version 2.6. All the chemicals were weighed using a PB1501 scale (Metter Toledo, Columbus, OH, USA) with a precision of ±0.1 g. The ultraviolet (UV) emitter to 360–370 nm from NICHIA Corporation was supplied by Power Light Systems (Berlin, Germany). Electronic components were supplied by Farnell Element 14 (Chicago, IL, USA) and Mouser Electronics (Mansfield, TX, USA). Arduino^® UNO board, a serial port color camera module Link Sprit^® (LS-Y201-Infrared, Longmont, CO, USA) with a CMOS sensor and transistor-transistor logic (TTL) interface and the software employed was MATLAB R2011a^®.

All fluorescence measurements were performed in the standard 96-well opaque microplates (Thermo Fisher Scientific, Denmark). Fluoroskan Ascent FL 2.6 equipped with an Ascent software version 2.6 (Thermo Scientific, Finland) was used to carry out the FAM’s fluorescence single-point measurements with excitation and emission wavelengths of 485 and 538 nm, respectively. The final volume per well was fixed at 200 μL.
Before each experiment, the aptamer solution was placed in a thermocycler (Mastercycler personal Eppendorf VWR, Leuven, Belgium) with the following temperature profile: heating at 90 °C for 8 min to the initial denaturation step, followed by a structure maintain step at 4 °C for 5 min, then a stabilization step at room temperature for 15 min.
A UV-visible spectrophotometer (UV-1800, Shimadzu, Japan) equipped with the TCC controller (TCC 240A) was used to measure the absorption characteristics of FAM-APT, DNA duplex, and target-induced aptamer folding. For this purpose, a Nanodrop cuvette (Traycell, Hellma) was used to record UV spectra with minimal samples volume fixed at 3 µL and a dilution factor cap of 10.
Fluorescence spectroscopy measurements for concept validation were carried out using an FP-8300 Spectrofluorometer from JASCO International Co.(Tokyo, Japan).