Electromembrane Extraction of Unconjugated Fluorescein Isothiocyanate from Solutions of Labeled Proteins Prior to Flow Induced Dispersion Analysis

• Published in: Analytical chemistry (Washington) Vol. 91; no. 10; pp. 6702 - 6708
• Main Authors: Restan, Magnus Saed, Pedersen, Morten E, Jensen, Henrik, Pedersen-Bjergaard, Stig
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 21.05.2019
• Subjects: 1-Octanol; Agitation; Aliquat; Chemistry; Dispersion; Electrokinetics; Feasibility studies; Fluorescein; Fluorescein isothiocyanate; Fluorescence; Human serum albumin; Octanol; Proteins; Serum albumin; Sodium hydroxide
• ISSN: 0003-2700; 1520-6882
• DOI: 10.1021/acs.analchem.9b00730

In this initial research on feasibility, removal of unconjugated fluorescein isothiocyanate (FITC) after fluorescent labeling of human serum albumin (HSA) with electromembrane extraction (EME) was investigated for the first time. A 100 μL solution of 0.1 mg/mL HSA was fluorescently labeled with 0.01 mg/mL FITC in a molar ratio of 10:1 in an Eppendorf tube for 30 min under agitation and absence of light. Then the labeled solution was transferred to a 96-well EME with 3 μL 0.1% (w/w) Aliquat 336 in 1-octanol as the supported liquid membrane (SLM) and 200 μL 10 mM NaOH as waste solution. EME was performed for 10 min with a voltage of 50 V, with the anode in the waste solution and at 900 rpm agitation. Negatively charged and unconjugated FITC was extracted electrokinetically into the SLM and to the waste solution. Analysis of purified samples, by Taylor dispersion analysis (TDA), showed a 92% removal of unconjugated FITC (FITC clearance: 92%, RSD: 3%), while 79% of the HSA/FITC complex remained in the sample (protein retention: 79%, RSD: 18%). Conserved functionality of the HSA/FITC complex after EME was proven by a binding affinity study with anti-HSA using flow induced dispersion analysis (FIDA). In this real sample, the dissociation constant (Kd) and hydrodynamic radius of the complex were determined to be 0.8 μM and 5.87 nm, respectively, which was in concordance with previously reported values.