Electromembrane extraction of unconjugated fluorescein

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Electromembrane extraction of unconjugated fluorescein isothiocyanate from solutions of labeled proteins prior to flow induced dispersion analysis Magnus Saed Restan, Morten Enghave Pedersen, Henrik Jensen, and Stig Pedersen-Bjergaard Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.9b00730 • Publication Date (Web): 30 Apr 2019 Downloaded from http://pubs.acs.org on April 30, 2019

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Analytical Chemistry

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Manuscript for Analytical Chemistry

Electromembrane extraction of unconjugated fluorescein isothiocyanate from solutions of labeled proteins prior to flow induced dispersion analysis

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Magnus Saed Restana, Morten E. Pedersenb , Henrik Jensenb,c, Stig Pedersen-Bjergaarda,c aDepartment

of Pharmacy, University of Oslo, P.O. Box 1068 Blindern, 0316 Oslo, Norway ApS, c/o University of Copenhagen, Universitetsparken 2 2100 Copenhagen, Denmark cDepartment of Pharmacy, Faculty of Health and Medical Sciences, University of Copenhagen, Universitetsparken 2, 2100 Copenhagen, Denmark

bFIDA-Tech

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Abstract

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In this initial research on feasibility, removal of unconjugated fluorescein isothiocyanate (FITC)

17

after fluorescent labeling of human serum albumin (HSA) with electromembrane extraction (EME)

18

was investigated for the first time. A 100 µL solution of 0.1 mg/mL HSA was fluorescently labeled

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with 0.01 mg/mL FITC in a molar ratio of 10:1 in an Eppendorf tube for 30 min under agitation

20

and absence of light. Then the labeled solution was transferred to 96-well EME with 3 µL 0.1 %

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(w/w) Aliquat 336 in 1-octanol as the supported liquid membrane (SLM) and 200 µL 10 mM NaOH

22

as waste solution. EME was performed for 10 min with a voltage of 50 V, with the anode in the

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waste solution and at 900 rpm agitation. Negatively charged and unconjugated FITC was extracted

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electro-kinetically into the SLM and to the waste solution. Analysis of purified samples, by Taylor

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dispersion analysis (TDA), showed a 92 % removal of unconjugated FITC (FITC clearance: 92 %,

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RSD: 3 %), while 79 % of the HSA/FITC complex remained in the sample (Protein retention: 79 %,

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RSD: 18 %). Conserved functionality of the HSA/FITC complex after EME was proven by a binding

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affinity study with anti-HSA using flow induced dispersion analysis (FIDA). In this real sample, the

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dissociation constant (Kd) and hydrodynamic radius of the complex were determined to be 0.8 µM

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and 5.87 nm respectively, which was in concordance with previously reported values.

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Introduction

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Electromembrane extraction (EME) is a three-phase microextraction technique developed for

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analytical applications, and was presented for the first time in 20061. The technique is a

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combination of liquid phase microextraction (LPME) and electrophoresis, and has been reported

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for extraction of chargeable analytes from aqueous samples such as biological fluids2-4,

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environmental waters5,6, and beverages7. In EME, analytes are extracted through a supported

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liquid membrane (SLM) and into aqueous acceptor phase. The SLM is an organic solvent

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immobilized inside the pores of a thin polymeric membrane. The main driving force for mass

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transfer in EME is an electrical field sustained across the SLM. Two electrodes connected to an

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external power supply are introduced into the system, one in the sample and one in the acceptor

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phase. The electrical field force charged analytes to migrate towards the electrode of opposite

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polarity. For the extraction of basic (cationic) analytes, the cathode is placed in the acceptor phase

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while the anode is placed in the sample. For acidic analytes, the direction of the electrical field is

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reversed. Since the electrical field only affects charged compounds, pH of the sample and acceptor

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phase has to be controlled to a level where the analytes of interest are fully charged (i.e two-three

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pH units from their pKa). EME can typically be completed in 5-10 minutes1,8, and the adaptation of

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EME to a 96-well format open for high-throughput operation9. In the current literature, EME has

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been used for analytical extraction of target analytes from complex samples. Target analytes have

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been transferred electro-kinetically from the original sample solution, through the SLM, and into

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pure aqueous solution containing acid or base as acceptor phase. Subsequently the analytes in the

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acceptor phase have been measured by chromatography, mass spectrometry, or related

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instrumental techniques.

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Recently, EME was utilized for purification for the first time. In one paper, EME was used to

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remove and recycle template molecules after polymerization of molecularly imprinted polymers10.

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In the second paper, EME was used for rapid and efficient salt removal from minute volumes of

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saline samples11. From a conceptual point of view, these papers represented a new direction for

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EME and new types of applications may be developed. To explore this potential, fundamental

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research is requested, and in the present work we test for the first time EME for removal of

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unconjugated fluorescent reagent from protein samples. Fluorescence labeling is a common way

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to enhance the sensitivity and detectability of proteins and involves labeling of purified protein

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with a fluorescent reagent and subsequently detection by fluorescence spectrophotometry12. To

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ensure effective labeling of the protein, surplus fluorescent reagent is added to the protein

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solution. However, in order to suppress the background level of fluorescence, the samples need to

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be purified to remove unconjugated fluorescent reagent

13-15.


This is of general importance in

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experiments with fluorescence detection, including those conducted by flow induced dispersion

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analysis (FIDA). In FIDA, the in-capillary diffusivity of an analyte is used to calculate the analyte

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concentration16-18. FIDA can also be used in binding studies of antibodies to fluorescently labeled

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protein, and removal of unconjugated fluorescent reagent is mandatory for accurate calculation

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of binding constants through the change in diffusivity.

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The most common fluorescent reagents are derivatives of fluorescein, witch have different

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fluorescence, stability and protein binding properties. Fluorescein isothiocyanate (FITC) is one

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example, and is frequently used19. Existing techniques for removal of unconjugated FITC include

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gel filtration20, dialysis21, and purification columns for dye removal22,23. The mutual goal for these

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techniques is (1) to remove as much as possible of unconjugated fluorescent reagent (high reagent

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clearance) and (2) to avoid loss of labeled protein (high protein retention). The existing

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techniques have proven successful in the separation of labeled protein and unconjugated

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fluorescent reagent24,25 but can be time and labor intensive26, and with a loss of labeled protein26.

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The current paper is the first report on EME removal of unconjugated fluorescent reagent from

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protein solutions. The experimental work was fundamental and intended as an initial research on

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feasibility. Although real samples were included in the current experiments, it should be

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emphasized that more work is required before the concept can generally be applied as part of

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experimental work with proteins. FITC was selected as model substance for the fluorescent

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reagent. FITC is an ideal candidate for EME (Figure S1). The carboxylic group can be ionized in

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neutral and alkaline solution, which is required for EME. In addition, mass transfer is favored by

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the small size (389.4 Da) and hydrophobic character (log P 4.8) of FITC. The common proteins

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cytochrome C, human serum albumin (HSA), and myoglobin were selected as model proteins. First

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EME was optimized for removal of FITC (high FITC clearance), and for retention of protein in the

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sample (high protein retention). Second, samples of FITC labeled HSA was processed with EME

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and subjected to Taylor dispersion analysis (TDA)27-30, to further verify EME performance and to

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document the stability of the HSA/FITC complex during EME. Finally, EME was combined FIDA

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for a real sample application, where binding and affinity studies of HSA and anti-HSA antibody

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were performed.



Experimental

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Chemicals and solutions

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FITC,sodium tetraborate decahydrate, boric acid, 2-nitrophenyl octyl ether (NPOE), 1-octanol,

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methanol, acetone, horse heart myoglobin, bovine heart cytochrome C, HSA, and monoclonal

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antibody against HSA (mouse, IgG1) were purchased from Sigma Aldrich (St Louis, Missouri, USA).

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Sodium chloride and sodium hydroxide (NaOH) were purchased from Merck (Darmstadt,

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Germany). Aliquat 366 was purchased from Cognis Corporation (Cincinnati, Ohio, USA). Deionized

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water was obtained from a Milli-Q water purification system (Molsheim, France)

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A stock solution of 1 mg/mL FITC dissolved in acetone was prepared freshly each day and diluted

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with either 50 mM borate buffer pH 9.20 or the reaction buffer (50 mM borate buffer pH 9.20 with

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150 mM NaCl) to the working concentration of 0.01 mg/mL (2.57·10-5M). Solutions of each of the

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proteins were made to a concentration of 0.1 mg/mL for the optimization studies. For the FITC

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labelling reaction, 10 mg HSA was weighed and dissolved in 10 mL 50 mM borate buffer pH 9.20

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providing a molar concentration of 1.50·10-5 M. All solutions were stored in darkness at < 8°C.

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Electromembrane extraction (EME)

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Equipment used for EME is illustrated in Supporting Information Figure S2. A laboratory built 96-

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well stainless steel plate with 0.5 mL wells was used as compartment for the waste solutions and

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as anode (96-well waste reservoir plate). A 96-well MultiScreen-IP filter plate with polyvinylidene

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fluoride (PVDF) filter membranes with 0.45 µm pore size (Merck Millipore Ltd, Carrigtwohill,

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Ireland) was used as SLM and compartment for the samples (96-well filter plate). The volume of

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each well in the 96-well filter plate was 350 μL and this defined the maximum possible volume of

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sample. A laboratory built aluminum plate with 96 rods tailor-made for the wells of the

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MultiScreen plate, was used as cathode (96-electrode plate). A model ES 0300-0.45 (Delta

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Elektronika BV, Zierikzee, The Netherlands) was used as power supply, and a Vibramax 100

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Heidolph shaking board (Kellheim, Germany) was used to agitate the extraction system.

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EME was performed according to the following procedure for each sample: a) 200 μL of 10 mM

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NaOH was pipetted into the 96-well waste reservoir plate, b) 3 µL organic solvent was pipetted

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on to the filter membrane of the 96-well filter plate, and c) 100 µL sample was pipetted into the

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96-well filter plate. The 96-well waste reservoir plate and the 96-well filter plate were clamped,

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and the 96-electrode plate was located on top. The rod electrodes of the electrode plate was then

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in contact with the samples. The waste reservoir plate (anode) and the electrode plate (cathode)

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were connected to the power supply. The extraction time varied from 1 to 10 minutes, the voltage

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was between 1 and 100 V, and agitation was set to 900 rpm. For a couple of experiments, the

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direction of the electrical field was reversed.


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FITC clearance and protein retention

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FITC clearance is the percentage of FITC removed from the sample after EME compared to the

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non-extracted sample, as defined by equation (1). FITC clearance of 100% indicates complete

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removal of FITC from the sample after EME. Protein retention is the percentage of protein left in

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the sample after EME compared to the non-extracted sample, as defined by equation (2). Protein

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retention of 100% indicates no protein loss after EME.

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𝐹𝐼𝑇𝐶 𝑐𝑙𝑒𝑎𝑟𝑎𝑛𝑐𝑒 = 1 ― [𝐹𝐼𝑇𝐶 ]𝑠𝑎𝑚𝑝𝑙𝑒,𝑏𝑒𝑓𝑜𝑟𝑒 𝐸𝑀𝐸 ∙ 100%

135

𝑃𝑟𝑜𝑡𝑒𝑖𝑛 𝑟𝑒𝑡𝑒𝑛𝑡𝑖𝑜𝑛 =

136

FITC labeling protocol

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The fluorescent labeling was performed on HSA and the reaction was done with a molar ratio of

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10:1 FITC to HSA31. Calculation of the volume of 0.01 mg/mL FITC (VFITC(uL)) needed to obtain

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molar ratio of 10:1, was done with the general formula:

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𝑉𝐹𝐼𝑇𝐶(𝜇𝑙) = 𝑀𝑃𝑟𝑜𝑡𝑒𝑖𝑛(𝑔/𝑚𝑜𝑙) ∙ 1000 𝑚𝑔/𝑚𝑙 ∙ 𝐶𝐹𝐼𝑇𝐶 (𝑚𝑜𝑙/𝑙) ∙ 106 𝜇𝑙/𝑙

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The HSA solution was prepared to a molar concentration of 1.50·10-5 M and set to a volume of 100

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µL. The volume of 0.01 mg/mL FITC needed to obtain a molar ratio of 10:1 was 0.585 mL.

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The labelling reaction was performed in a 1.5 mL low-bind Eppendorf tube (Eppendorf, Hamburg,

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Germany). 100 µL of HSA solution was mixed with 585 µL of a freshly made 0.01 mg/mL FITC

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solution (in reaction buffer) in the Eppendorf tube. The tube was wrapped in aluminium foil, for

146

light protection, and was agitated for 30 minutes at 900 rpm. After fluorescent labeling, 100 µL of

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the solution was transferred to EME.

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To avoid saturation of the fluorescence detector in the TDA and FIDA analysis, both the extracted

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and un-extracted samples were diluted with 50 mM borate buffer pH 9.20 to a theoretical

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concentration of 10 nM or 20 nM for the HSA/FITC –complex.

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Flow injection analysis with UV-detection

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In experiments reported in section 3.1 and 3.2, FITC and proteins were individually measured

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using a 3000 Ultimate HPLC-UV (Thermo Fisher, Waltham, MA, USA) for automated flow injection

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analysis with UV-detection. Thus, the HPLC column was replaced with PEEK tubing. Mobile phase

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consisted of a 50 mM borate buffer pH 9.20 and the injection volume was 20 µL. The flow of mobile

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phase was 0.1 mL/min. UV-detection was performed at 214 nm for Myoglobin and Cytochrome C,

(

[𝐹𝐼𝑇𝐶 ]𝑠𝑎𝑚𝑝𝑙𝑒, 𝑎𝑓𝑡𝑒𝑟 𝐸𝑀𝐸

(

[𝑃𝑟𝑜𝑡𝑒𝑖𝑛]𝑠𝑎𝑚𝑝𝑙𝑒,𝑎𝑓𝑡𝑒𝑟 𝐸𝑀𝐸

)

(1)

) ∙ 100%

(2)

[𝑃𝑟𝑜𝑡𝑒𝑖𝑛]𝑠𝑎𝑚𝑝𝑙𝑒,𝑏𝑒𝑓𝑜𝑟𝑒 𝐸𝑀𝐸

𝑉𝑃𝑟𝑜𝑡𝑒𝑖𝑛(𝑚𝑙) ∙ 𝐶𝑃𝑟𝑜𝑡𝑒𝑖𝑛(𝑚𝑔/𝑚𝑙)

𝑀𝑜𝑙𝑎𝑟 𝑟𝑎𝑡𝑖𝑜


(3)


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216 nm for HSA, and 495 nm for FITC. The system was washed with a pure methanol solution

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prior to each injection to avoid any carryover.

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Taylor Dispersion Analysis conditions

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The TDA experiments were conducted on a capillary electrophoresis instrument (Agilent 3D CE,

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Agilent Technologies, Waldbronn, Germany) using light emitting diode fluorescence detection

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(ZETALIF LED, Picometrics, Labège, France) with excitation wavelength 480 nm. A standard fused

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silica capillary (inner diameter: 75 µm, outer diameter: 375 µm, length total: 100 cm, length to

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detection window: 88 cm) was used. The capillary cassette was temperature-controlled to 25°C,

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and the capillary inlet, outlet and sample vials were indirectly thermostated to 24°C via the lab

166

heating, ventilation and air conditioning system.

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The capillary was rinsed and equilibrated prior to each sample analysis with 1 M NaOH at 1 bar

168

for 5 min followed by 50 mM Borate buffer pH 9.20 at 1 bar for 7 min. The HSA/FITC–complex

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sample was injected at 50 mbar for 10 s (corresponding to 1 % of the capillary volume).

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Subsequently, the injected sample was mobilized towards the detector with running buffer (50

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mM borate buffer, pH 9.20) at 50 mbar for 20 min. All samples were analyzed in triplicate.Flow-

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Induced Dispersion Analysis conditions

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The FIDA experiments were conducted on a FIDAlyzer instrument (FIDA-Tech ApS, Copenhagen,

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Denmark) using light emitting diode fluorescence detection (ZETALIF LED, Picometrics) with

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excitation wavelength 480 nm. A fused silica capillary (inner diameter: 75 µm, outer diameter:

176

375 µm, length total: 100 cm, length to detection window: 90 cm) with an inner coating of

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poly(ethylene glycol) was used. The capillary was temperature-controlled to 25°C inside the

178

FIDAlyzer instrument. Capillary inlet and samples were also temperature-controlled to 25°C.

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The HSA/FITC–complex, purified via EME was diluted with 67 mM phosphate buffer pH 7.4 to a

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fixed indicator concentration of 20 nM in varying analyte concentrations of anti-HSA antibody (0

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– 5 µM). All samples were pre-incubated to reach equilibrium prior to analysis.

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The capillary was flushed and equilibrated prior to each sample analysis with 67 mM phosphate

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buffer at 1500 mbar for 5 min. The analyte sample (anti-HSA at varying concentrations) was

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injected at 1500 mbar for 45 s, subsequently the indicator sample (HSA/FITC–complex, mixed

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with anti-HSA at varying concentrations) was injected at 50 mbar for 10 s (39 nL, corresponding

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to 1 % of the capillary volume). The injected indicator sample was then mobilized towards the

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detection point with the analyte sample at 400 mbar for 180 s. All samples were analyzed in

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triplicate.

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TDA and FIDA data analysis

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The Taylorgrams were processed using FIDA Data Analysis Software, version 01 (FIDA-Tech ApS,

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Copenhagen, Denmark) in order to calculate the hydrodynamic radii of two species in one signal

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with identical residence time, i.e. unconjugated FITC and HSA/FITC complex. Subsequently the

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software was used for plotting the binding curve. The software utilized equations (S1), (S2), (S3)

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and (S4) (Supporting Information)18.



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Results and discussion

197 198

Concept and principle

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The purpose of this fundamental research was to investigate the potential of EME for removal of

200

unconjugated fluorescent reagent after protein labeling. The workflow included (1) labeling of

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protein with fluorescent reagent, (2) EME and (3) final measurement by TDA or flow-induced

202

dispersion analysis (FIDA). The EME device is presented in Figure S2, and consisted of (a) a 96-

203

well waste reservoir plate, (b) a 96-well filter plate, (c) a 96-electrode plate, (d) an agitator, and (e)

204

an external power supply. The three 96-plates (a,b,c) were sandwiched, and provided 96

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individual extraction channels. This enabled simultaneous processing of up to 96 samples with

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labeled protein (one sample per channel).

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EME involved the following four steps: (1) loading the supported liquid membrane (SLM), (2)

208

sample loading, (3) waste solution loading, and (4) extraction. During SLM loading (1), 3 μL organic

209

solvent was pipetted on to a filter on the 96-well filter plate (b). This procedure was repeated for

210

the number of samples with labeled protein to be processed. The organic solvent rapidly diffused

211

into the pore volume of the filter and was immobilized by capillary forces, creating the SLM.

212

During sample loading (2), samples were pipetted above the loaded SLMs in the 96-well filter

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plate (b). The samples contained labelled protein and fluorescent reagent. During waste solution

214

loading (3), a waste solution was pipetted into the 96-well waste reservoir (a) for each sample,

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and the three plates (a,b,c) were clamped together and placed in the agitator (d). Finally, during

216

EME (4) the electrical field was applied by connecting the 96-well waste reservoir plate and the

217

96-electrode plate to the external power supply (5). The anode (positive potential) was located

218

in the waste solution while the cathode was in the samples.

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FITC was selected as model fluorescent reagent. Three model proteins were selected to cover

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different molecular sizes and isoelectric point, namely HSA (MW = 66.5 kDa, pI = 5.8)32, equine

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heart myoglobin (MW = 17 kDa, pI = 6.7 (1) and 7.2(2))33 and bovine heart cytochrome C (MW =

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12kDa, pI = 10.5)34.

223 224

Operational parameters for efficient FITC clearance and protein retention

225

Major operational EME parameters were considered to be (1) the chemical composition of the

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SLM, (2) voltage, and (3) time. 1-octanol and NPOE are both commonly used as SLM solvents in

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EME, mainly for the extraction of hydrophobic (log P > 1.5) acidic and basic drug compounds,


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respectively8,35. In initial EME experiments, FITC clearance was investigated with 1-octanol and

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NPOE. However, both 1-octanol and NPOE proved to be ineffective for extraction, and FITC

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clearance was limited to 5-10 % and

Electromembrane extraction of unconjugated fluorescein

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