Anhydrous 1 2 dichloroethane

Poly(2,6-dimethyl-1,4-phenylene oxide) (PPO) was purchased from Asahi Kasei Corp (Tokyo, Japan). 6-bromohexanoyl chloride, aluminum chloride, 1,2-dichloroethane anhydrous, trimethyl ammonium solution (45 wt% in DI water) and N,N,N’N’-tetramethyl-1,6-hexanediamine (TMHDA) were obtained from Sigma Aldrich Korea (Yongin, Korea). Methyl alcohol (MeOH), N-methyl-2-pyrrolidone (NMP) and potassium hydroxide (KOH) were purchased from Daejung Chemical (Siheung, Korea). The polyethylene (PE) support was provided from W-SCOPE KOREA (Ochang, Korea).

One barrel of the nanopipette was filled with 5 μl of 100 μM fluorophore (ATTO 488, 41051 Sigma-Aldrich, for HeLa and ATTO 565, 75784 Sigma-Aldrich, for M059K) in 0.1 M KCl (Sigma-Aldrich). The operation was performed ensuring that dry conditions were maintained to avoid water to wet the backend of the nanopipette which represents the main cause of cross-talk between the two barrels. The second barrel was filled with 5 μl of the organic phase mixture of 10 mM THATPBCl in 1,2-dichloroethane anhydrous (Sigma-Aldrich), following the silanization of the backend of the nanopipette by exposure to trichloro(1H, 1H, 2H, 2H-perfluorooctyl)silane (Sigma-Aldrich) for 10 s. This operation makes the glass hydrophobic and reduces the cross-talk during the experiment. After filling the two barrels, the nanopipette was left for 5 min at 60°C to ensure that the back of the nanopipette was dry before the experiment. The ion current generated in the aqueous barrel was used as SICM feedback to drive the nanopipette toward and inside the cell. The electrolyte solution in the aqueous barrel has high electrical conductivity (σ_aq = 1.3 S/m) and generates an ion current with greater magnitude than the one generated in the organic barrel, where the conductivity of the organic solution is two orders of magnitude smaller (σ_org = 0.011 S/m) (30 (link)).

9-Fluorenylmethoxycarbonyl (Fmoc)-protected
amino acids were purchased from CEM (Buckingham, United Kingdom),
Fluorochem (Hadfield, United Kingdom), and Sigma-Aldrich (Gillingham,
United Kingdom), and preloaded Fmoc-Lys(Boc)-Wang resin was from Novabiochem
(Watford, United Kingdom). N,N′-Diisopropylcarbodiimide
(DIC) was from Fluorochem, piperidine, acetic anhydride, triisopropylsilane
(TIS), formic acid, anhydrous 1,2-dichloroethane (DCE), Grubbs first-generation
catalyst, and 5(6)-carboxyfluorescein (FAM) were from Sigma-Aldrich,
and ethyl 2-cyano-2-(hydroxyimino)acetate (Oxyma Pure) was obtained
from CEM. Acetonitrile (ACN), dimethylformamide (DMF), dimethyl sulfoxide
(DMSO), and diethyl ether were from VWR (Lutterworth, United Kingdom),
and dichloromethane (DCM) and trifluoroacetic acid (TFA) were obtained
from Fisher Scientific UK (Loughborough, United Kingdom).