Abi3400 dna rna synthesizer

The aptamer selected for the CCRF-CEM cells, sgc8c, (5′-FTIC-ATC TAA CTG CTG CGC CGC CGG GAA AAT ACT GTA CGG TTA GA-NH2-3) and library DNA containing a randomized sequence of 41 nucleotides were synthesized at a μmol scale using an ABI3400 DNA/RNA synthesizer (Applied Biosystems, Foster City, CA). Both sgc8c and DNA library were coupled with fluorescein isothiocyanate dye (FTIC) modifier at the 5’ end and amine (-NH₂) modifier at the 3’ prime end during synthesis. After synthesis, they were deprotected in AMA (ammonium hydroxide /40% aqueous methylamine, 1:1) at 65°C for 30 min. After deprotection, all sequences were purified by reversed-phase HPLC (ProStar-Varian, Walnut Creek, CA) with a C18 column (Econosil, 5μm, 250mm length, 4.6mm diameter) from Alltech (Deerfield, IL) using a mobile phase containing 100 mM triethylamine acetic acid buffer (TEAA, pH 7.5) and acetonitrile (0-30min, 10-00%). All purified DNA solutions were dried in acid-resistant centriVap centrifugal vacuum concentrators (Labconco, Kansas City, MO). The dried DNA at the bottom of 2 mL tubes was dissolved in 50-100μL of DNA grade water. The concentrations of all DNA were determined by measuring the absorbance values at 260 nm.

The sgc8 aptamer and control DNA with biotin group or desired dye modifications were synthesized on an ABI 3400 DNA/RNA synthesizer (Applied Biosystems, Foster City, CA, USA). The aptamer with FITC labeling was used primarily for flow cytometry, while the aptamer with TAMRA labeling was used for confocal fluorescence microscopy. The aptamer without dye labeling was used for the cytotoxicity assay. Detailed sequence information is provided in the Supporting Information (Table S1). The aptamers with and without FITC labeling were deprotected in 3 mL of AMA solution (ammonium hydroxide: 40% aqueous methylamine = 1:1) at 65°C for 25 min. The TAMRA-labeled aptamers were deprotected in 3 mL of TAMRA deprotection solution (methanol:tert-butylamine:water = 1: 1: 2) at 65°C for 4 h. All deprotected sequences were precipitated by adding 250 µL of 3 M NaCl and 6 mL of cold ethanol. Then the precipitated aptamers were collected by centrifugation and dissolved in 400 µL of triethylammonium acetate (TEAA) for further purification by reversed-phase high-pressure liquid chromatography (ProStar, Varian, Walnut Creek, CA, USA) using a C18 column and acetonitrile–TEAA solvent. Finally, these aptamers were quantified by measuring their absorbance at 260 nm.

All oligonucleotides were synthesized on an ABI3400 DNA/RNA synthesizer
(Applied Biosystems, Foster City, CA, USA) with/without thiol labeling. The
sequences were then purified by reversed-phase HPLC (ProStar, Varian Walnut
Creek, CA, USA). TCEP and MCH were purchased from Sigma-Aldrich (St. Louis, MO,
USA). FITC-conjugated anti-human EpCAM antibody and isotype antibody were
purchased from BD Biosciences (Sparks, MD, USA). Streptavidin-gold nanoparticles
were purchased from Electron Microscopy Sciences (Hatfield, PA, USA). The
electrochemical detection was performed on disposable screen-printed electrodes
with 4 mm diameter working area (DropSens, LIanera, Spain) connected with a
portable potentiostat (Digi-Ivy, Austin, TX, USA), with the input parameters
optimized in detail. Binding buffer for cells and exosomes was prepared with
Dulbecco’s phosphate saline using mM/L MgCl₂, 2 g/mL bovine
sesrum albumin (BSA, Fisher Scientific), and 100 mg/L yeast tRNA
(Sigma-Aldrich). Washing buffer contained 5 mM/L MgCl₂ and 2 g/mL
BSA. All chemicals were of analytical grade, and all solutions were prepared
with ultrapure water obtained from an ELGA water purification system (Evoqua
Water Tech., Lowell, MA, USA).