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

Abstract

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 (K d ) 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.

Liquid-Phase Microextraction or Electromembrane Extraction?

“…On the other hand, EME was superior in terms of equilibrium time and flux. Recently, highly concentrated samples have been purified for inorganic salts, polymerization chemicals, and fluorescent probes by membrane-based microextraction techniques. ,, We expect development of numerous similar purification protocols in the near future, and for this research, we believe that the current fundamental study will help to understand the mass transfer, and be of great importance for how to rationally select an efficient and suitable membrane-based microextraction technique according to the target concentration.…”

“…Recently, EME was used to remove interfering substances, such as phospholipids from human plasma samples and high level salts from saline samples . Most recently, EME was used to recover templates after molecular imprinting at a high level and to remove the excess fluorescent tag from protein solutions …”

Electromembrane extraction and mass spectrometry for liver organoid drug metabolism studies

“…Prior to the extraction, 50 μL of the heroin-exposed liver organoid samples (containing 0.11 M FA) was added to 40 μL water and 10 μL of the 1.5 μM or 3 μM internal standard solution. The samples were then loaded into the wells of an in-house built 96-well stainless-steel plate ( Figure 2A ), previously described by Restan et al 28 . A volume of 3 μL DEHP/NPOE (10/90, w/w ) was immobilized into the membrane pores (0.45 μm pore size) of a 96-well MultiScreen-IP polyvinylidene fluoride (PVDF) filter plate from Merck Millipore ( Figure 2B ).…”

“…Such clean-up is highly important prior to liquid chromatography-mass spectrometry to avoid ion suppression or enhancement. EME has recently advanced to the 96-well plate format 26–28 (Parallel-EME), and chip systems 29,30 . Considering its documented traits regarding simple sample clean-up, we focus on using EME for organoids, which can be costly and limited in availability.…”

Size-Based Characterization of Adalimumab and TNF-α Interactions Using Flow Induced Dispersion Analysis: Assessment of Avidity-Stabilized Multiple Bound Species

“…Characterization of biomolecular interactions using FIDA is often based on end-point measurements at equilibrium 14,15,29 , where binding kinetics are not probed. The experimental time frame can be precisely controlled in FIDA assays using an approach where the binding partners are mixed directly in the capillary 30 (Fig.…”