Article
`
`pubs.acs.org/ac
`
`Method for Determination of Polyethylene Glycol Molecular Weight
`Sari Pihlasalo,* Pekka Hänninen, and Harri Härmä
`
`Department of Cell Biology and Anatomy and Medicity Research Laboratory, Institute of Biomedicine, University of Turku,
`Kiinamyllynkatu 10, 20520 Turku, Finland
`
`ABSTRACT: A method utilizing competitive adsorption
`between polyethylene glycols (PEGs) and labeled protein to
`nanoparticles was developed for the determination of PEG
`molecular weight (MW) in a microtiter plate format. Two mix-
`and-measure systems, time-resolved luminescence resonance
`energy transfer (TR-LRET) with donor europium(III)
`polystyrene nanoparticles and acceptor-labeled protein and
`quenching with quencher gold nanoparticles and fluorescently
`labeled protein were compared for their performance. MW is
`estimated from the PEG MW dependent changes in the
`competitive adsorption properties, which are presented as the
`luminescence signal vs PEG mass concentration. The curves
`obtained with the TR-LRET system overlapped for PEGs
`larger than 400 g/mol providing no information on MW. Distinctly different curves were obtained with the quenching system
`enabling the assessment of PEG MW within a broad dynamic range. The data was processed with and without prior knowledge
`of the PEG concentration to measure PEGs over a MW range from 62 to 35 000 g/mol. The demonstration of the measurement
`independent of the PEG concentration suggests that the estimation of MW is possible with quenching nanoparticle system for
`neutrally charged and relatively hydrophilic polymeric molecules widening the applicability of the simple and cost-effective
`nanoparticle-based methods.
`
`M olecular weight (MW) is a key physical property for a
`
`polymeric molecule, and thus, simple and reliable
`methods are required for its determination at wide MW
`range. Size exclusion chromatography (SEC) and light
`scattering are widely used in industry for determination of
`polymer MWs. Other commercially available methods include
`electrophoresis, NMR,
`the measurement of viscosity, mass
`spectrometry, and ultracentrifugation.1−14 However, most of
`the methods are not suitable for routine analysis needed in
`industry nor for high throughput screening. The methods can
`be time-consuming,3,4 have limitations for low or high MW
`range,15 and might require analyte concentrations as high as few
`grams per liter,12 expensive equipment, and expertise.7−9 Thus,
`there is a need to develop cost-effective and simple methods for
`fast routine analysis purposes.
`Neutral organic polymers, such as poly(vinyl alcohol),
`polyvinylpyrrolidone, polyethylene glycol (PEG), poly-
`(ethylene oxide), and dextran, are widely used and produced,
`e.g.,
`in medicine, biomedical
`laboratories, and by various
`industries such as paper, textile, pharmaceutical,
`food, and
`cosmetic industry. The determination of PEG MW is
`important, as MW affect
`its properties, such as viscosity,
`melting point, solubility, degradation, and hygroscopicity.16
`Low MW PEGs are rapidly removed from human body making
`them suitable for pharmaceutical and food products.17,18
`Instead, the increased MW causes reduced kidney excretion
`and prolonged circulation of the drug in the blood.18 PEGs
`have also different benefits to personal care and cosmetic
`
`products depending on their MW.19 PEG MW is also
`optimized for the performance in different applications. PEG
`6000 is effective in virus purification, whereas lower MW
`decreases the effectiveness in concentration of viruses.20
`Moreover, the PEG MW affects the biological or enzymatic
`activity of PEG−protein conjugates.21
`Determination of neutral or charged polymers may need
`different protocols or there might be other limitations, if for
`instance the charge of the analytes and calibrators differs.
`Electrostatic interactions affect the properties of the charged
`polymers, and thus the determination of MW depends on the
`presence of charge, especially for methods, such as measure-
`ment of viscosity and diffusion, which utilize a correlation
`between MW and size.22 In SEC, intermolecular interactions
`and adsorption to the column packing or the electrostatic
`interaction with the packing might lead to wrong estimation of
`MW.23 In vapor phase osmometry, the counterions are also
`detected, and thus MW of the charged polymers is under-
`estimated. In gel electrophoresis, the migration and the MW
`determination of neutral polymers is enabled with the binding
`of the sodium dodecyl sulfate. However, the binding to the
`neutral polymers can be relatively weak,
`identical charge-to-
`mass ratios are not achieved, and the resolution of the gel is
`
`Received: December 18, 2014
`Accepted: March 18, 2015
`Published: March 18, 2015
`
`© 2015 American Chemical Society
`
`3918
`
`DOI: 10.1021/acs.analchem.5b00736
`Anal. Chem. 2015, 87, 3918−3922
`
`See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.
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`Analytical Chemistry
`
`low.24 The method may fail also with low MW polymers due to
`the low binding of sodium dodecyl sulfate to small polymers.24
`Previously, we have developed the nanoparticle-based
`methods utilizing time-resolved luminescence resonance energy
`transfer (TR-LRET) and quenching for the quantification of
`proteins,25−27 eukaryotic cells,28 and detergents.29,30 Recently,
`we demonstrated the novel application of the nanoparticle-
`based method utilizing TR-LRET for the determination of MW
`for polyethylenimines and polyamino acids.31 The method
`utilized the size-dependent competitive adsorption between
`differently sized polymers and labeled protein to nanoparticles.
`These molecules have a total positive charge at the assay pH
`and polyamino acids contain hydrophobic sites giving optimal
`attractive electrostatic and hydrophobic interactions
`for
`adsorption to carboxylate-modified polystyrene particles. In
`this paper, we widened the nanoparticle-based application to
`hydrophilic PEGs having zero total charge. MWs for PEGs
`could be estimated with quenching nanoparticle system. The
`determination is based on the size-dependent adsorption to the
`nanoparticles (Figure 1). In the absence of analyte PEG, the
`
`Figure 1. Principle for determination of polymer MW. Nanoparticle-
`based TR-LRET (a) and quenching (b) systems utilize the competitive
`adsorption between labeled protein and analyte polymer molecules. At
`constant analyte mass concentration, the increase in polymer MW
`reduces the adsorption of labeled protein, which is observed as a
`change in the signal. (a) In the TR-LRET system, an acceptor-labeled
`protein is adsorbed to the donor europium(III) polystyrene
`nanoparticles. Due to the close proximity of the donor−acceptor
`pair, luminescence resonance energy is transferred. The increase in
`MW of the polymer results in the decrease in the TR-LRET signal. (b)
`In the quenching system, a labeled protein is adsorbed to the quencher
`gold nanoparticles and the luminescence is quenched. The increase in
`MW of the polymer results in the increase in the luminescence.
`
`labeled protein adsorbs to the gold nanoparticles and the
`luminescence is quenched. PEGs competed with labeled
`protein in the adsorption to the quencher gold nanoparticles
`leading to the increase in signal and the degree of
`the
`competition was related to MW. MW is assessed from the
`relationships between the luminescence signal and PEG mass
`concentration. The method was tested for PEG polymers with
`MW from 62 (monomer) to 35 000 g/mol. The assessment of
`
`Article
`
`PEG MW was enabled without prior knowledge of the PEG
`concentration, which was not achieved in our previous work.31
`
`■ EXPERIMENTAL SECTION
`Materials. γ-Globulins from bovine blood (γG), albumin
`from bovine serum (BSA), and polyethylene glycols (PEG 200,
`PEG 400, PEG 1500, PEG 3000, PEG 6000, and PEG 35000)
`were from Sigma-Aldrich Co. (St. Louis, MO). Ethylene glycol
`was from J. T. Baker (Deventer, The Netherlands). Carboxylate
`modified europium(III) polystyrene nanoparticles 92 nm in
`diameter were purchased from Seradyn Inc. (Indianapolis, IN)
`and colloidal gold particles 20 nm in diameter (having ∼100%
`monodispersity according to the manufacturer) from British
`Biocell International (Cardiff, U.K.). Alexa Fluor 680 carboxylic
`acid, succinimidyl ester was obtained from Molecular Probes
`(Eugene, OR). Dipyrrylmethene-BF2 530 (BF530) dye was
`from Arctic Diagnostics Oy (Turku, Finland). NAP-5 gel
`filtration columns were ordered from GE Healthcare (Uppsala,
`Sweden). All the reagents used to prepare buffer solutions were
`obtained from Sigma-Aldrich Co. (St. Louis, MO). High purity
`Milli-Q water was used to prepare all aqueous solutions.
`Methods. Labeling of γ-Globulin with Alexa Fluor 680
`and Bovine Serum Albumin with BF530. Alexa Fluor 680
`carboxylic acid, succinimidyl ester (Alexa) was conjugated to γ-
`globulin (γG), as
`recommended by the manufacturer.
`Dipyrrylmethene-BF2 530 (BF530) dye was conjugated to
`albumin from bovine serum (BSA) as described previously for
`mouse monoclonal IgG anti-hAFP.32
`Molecular Weight or Concentration Determination of
`Polymers. The MW or concentration determinations using
`nanoparticle application were performed in microtitration wells.
`In the TR-LRET system, 70 μL of the sample polyethylene
`glycol solution in 5 mM glycine buffer pH 3.0 and 10 μL of the
`europium(III) nanoparticles 92 nm in diameter in water were
`mixed. γG-Alexa was added in 10 μL of water. The final
`concentrations of europium(III) nanoparticles and γG-Alexa
`were 0.36 and 53 pM, respectively. In the quenching system, 70
`μL of the sample polyethylene glycol solution in 5 mM glycine
`buffer pH 3.0 and 20 μL of the gold nanoparticles 20 nm in
`diameter in water were mixed. BSA-BF530 was added in 20 μL
`of 1.0 mM phosphate buffer, pH 7.4, containing 0.10 mM
`potassium chloride. The final concentrations of gold nano-
`particles and BSA-BF530 were 64 and 890 pM, respectively.
`The size of europium(III) and gold nanoparticles was chosen
`according to our previous work.25,27 The utilization of small
`gold nanoparticles (20 nm) is economical, as the lower amount
`of gold (mass) is required in the assay compared to larger
`nanoparticles. Luminescence emission intensities were meas-
`ured with the Victor2 multilabel counter (Wallac, PerkinElmer
`Life and Analytical Sciences, Turku, Finland). For the TR-
`LRET system, a 340 nm excitation and a 730 nm emission
`wavelength and a 50 μs window and a 75 μs delay time were
`used. For the quenching system, the emission was measured at
`572 nm after the excitation at 530 nm.
`
`■ RESULTS AND DISCUSSION
`A simple mix-and-measure nanoparticle-based method was
`developed for the estimation of PEG MW. In this paper, the
`nanoparticle method is extended to the MW estimation of
`PEGs contrary to polyethylenimines and polyamino acids
`measurements published earlier.31 As polyethylenimines and
`polyamino acids are positively charged at the assay pH of 3 and
`
`3919
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`DOI: 10.1021/acs.analchem.5b00736
`Anal. Chem. 2015, 87, 3918−3922
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`Analytical Chemistry
`
`Article
`
`polyamino acids contain hydrophobic sites, the adsorption is
`efficient due to the attractive electrostatic and hydrophobic
`interactions to carboxylate-modified polystyrene nanoparticles.
`Here, we demonstrate that hydrophilic uncharged polymers
`such as PEGs having low affinity to the nanoparticles can also
`be measured on nanoparticles of different surface properties.
`PEGs are commercially available in a wide size range and their
`chemical structure is similar within the size series enabling the
`undistorted information on MW. We tested two nanoparticle-
`based systems utilizing TR-LRET or luminescence quenching
`to obtain the size related information for PEGs (Figure 1). The
`method exploits the adsorption competition between the
`labeled protein and the polymer
`to the europium(III)
`polystyrene nanoparticles or quencher gold nanoparticles.
`The estimation of MW is based on the size-dependent
`adsorption of the polymer, as a small polymer occupies the
`particle surface less efficiently than a large polymer. A large
`polymer may also cross-link several nanoparticles, as a single
`polymer can attach to many particles.33 The cross-linking could
`be beneficial for the method, as a large polymer would cover
`larger total nanoparticle surface area than without cross-linking
`and the cross-linking would widen the dynamic range.
`In the TR-LRET assay, γ-globulin labeled with acceptor-dye
`Alexa Fluor 680 (γG-Alexa) adsorbed to the donor europium-
`(III) polystyrene nanoparticles and the luminescence resonance
`energy was transferred from the donor to the acceptor. The
`donor was excited at 340 nm and the sensitized emission of the
`acceptor was measured at 730 nm. The TR-LRET signal was
`decreased, as PEGs prevented the adsorption of γG-Alexa. In
`the quenching assay, albumin from bovine serum labeled with
`dipyrrylmethene-BF2 530 (BSA-BF530) adsorbed to quencher
`gold nanoparticles in the absence of
`the analyte and the
`luminescence of the BF530 dye was quenched (Figure 1b). The
`adsorption of BSA-BF530 was prevented by PEG polymers
`resulting in an increase of the luminescent intensity. The BF530
`dye was excited at 530 nm and the emission was measured at
`572 nm. The luminescence spectra of europium(III) nano-
`particles, γG-Alexa, and BSA-BF530 and the absorption
`spectrum of gold nanoparticles have been published earlier by
`us.28
`The europium(III) polystyrene nanoparticles have carboxylic
`groups giving a total negative surface charge (pKa(−SO4H) <
`−3, pKa(−SO3H) = 1.9, and pKa(COOH) = 4.7) at the assay
`pH of 3. Similarly, the total surface charge is negative for
`colloidal gold nanoparticles due to the citrate groups (pKa
`values: 3.2, 4.8, and 6.4) used in the synthesis. The negative
`total charge is also supported by ζ-potential measurements
`performed in literature studies.34−37 These ionic groups provide
`ionic and hydrophilic character for the surface. However, both
`nanoparticles contain also neutral sites,
`reduced gold at
`oxidation state 0 for gold and chains of polystyrene for
`polystyrene nanoparticles giving the hydrophobic character for
`the surface. The assay performance on these nanoparticle
`surfaces depends on the strength of the interactions of PEGs or
`labeled protein and on the degree of exchange of PEG with
`labeled protein.
`The response of both assay systems was tested for the
`competitive adsorption between labeled protein and PEG with
`different MWs and at varying PEG concentration (Figure 2).
`The curves for PEGs in Figure 2a were obtained by fitting the
`data to the modified Hill function with a constant offset.38 The
`average coefficient of variation for the replicates was 9% and 6%
`for quenching and TR-LRET assay, respectively. For the TR-
`
`Figure 2. Response curves for polyethylene glycols measured with TR-
`LRET (a) and quenching (b) systems. The initial increase of the signal
`is moved to higher concentration, as the PEG MW is decreased. The
`data measured with TR-LRET system for PEGs was fitted to the
`modified Hill function with a constant offset.38
`
`the sensitized signal decreased at PEG
`LRET system,
`concentrations above 10 g/L for all tested MWs between 400
`and 35 000 g/mol and the dose−response curves nearly
`overlapped (Figure 2a). This decrease may be related to the
`steep increase in the viscosity of PEG solutions at
`concentrations above 200 g/L and the simultaneous decrease
`in diffusion preventing the adsorption of γG-Alexa.39 As the
`adsorption of differently sized PEGs to the nanoparticle surface
`was essentially identical, the overlap of the calibration curves
`attained for PEGs between 400 and 35 000 g/mol enables the
`measurement of mass concentration independent of MW. Only
`monomeric ethylene glycol gave an opposite result, as increase
`in the signal was observed at concentrations above 3 g/L. This
`may be due to the change in the adsorbed layer of γG-Alexa in
`the presence of ethylene glycol and lower increase of viscosity
`compared to polyethylene glycols. Ethylene glycol is known to
`change the structure of γG.40−42 Thus, more γG-Alexa may be
`adsorbed or the distance of Alexa dye from the europium(III)
`polystyrene nanoparticle surface may be decreased due to the
`changes in the α-helix and β-sheet structure. PEGs with
`different MWs can not be distinguished with the TR-LRET
`system, although size-independent mass concentration can be
`measured (Figure 2a).
`As the size-related measurement failed with the TR-LRET
`system, we investigated the performance of the quencher gold
`nanoparticle system25 for the MW determination of ethylene
`glycol and six corresponding polymers. The shape of
`the
`response curves were nearly identical to different PEGs, but no
`
`3920
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`DOI: 10.1021/acs.analchem.5b00736
`Anal. Chem. 2015, 87, 3918−3922
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`Analytical Chemistry
`
`Article
`
`obvious sigmoidal curve shape was measured for the quenching
`system (Figure 2b). The luminescence signal is increased, as the
`PEG concentration is increased. However, after the initial
`adsorption, a clear plateau is found for large-MW PEGs. As the
`PEG MW was reduced, the plateau became less distinct, until
`no plateau was identified for PEG 200 and monomer ethylene
`glycol. The multiphase adsorption to silica with several plateaus
`has been reported for PEG 400 in literature and interpreted as
`conformational changes of polymeric PEG chain.43 As the PEG
`concentrations increased further, a clear increase in signal was
`observed at the PEG concentration above 100 g/L, which may
`be derived from the increase in viscosity similar
`to the
`observation in the TR-LRET system. PEGs have been shown to
`form fluorescent micelles at high concentrations with
`absorption at wavelengths below 350 nm and emission
`maximum at approximately 380 nm.44 The emission at
`wavelengths higher than 500 nm is low. Thus, we tested the
`effect of PEG micelles to the fluorescence signal of
`the
`developed assay. This was confirmed to be insignificant at high
`concentration of PEG 400 and PEG 6000 in the assay buffer in
`the absence of gold nanoparticles and BSA-BF530 (data not
`shown).
`The sensitivity of the quencher gold particle system for PEGs
`was improved by 5 orders of magnitude from the TR-LRET
`system. Unfortunately, the unorthodox curve shapes did not
`allow quantification of PEGs. However,
`the onset of
`the
`luminescence signal increase at varying specific concentration of
`differently sized PEGs indicates that MW of an unknown PEG
`sample could be estimated. We correlated the concentration at
`the luminescence signal
`level of 700 cts to the PEG MW
`(Figure 3a). This calibration requires a prior knowledge of
`analyte mass concentration and allowed the determination of
`the largest polymer PEG 35000 clearly below 1 mg/L and the
`smallest PEG 200 below the 100 g/L concentration. The entire
`MW range of PEGs from ethylene glycol to 35 000 g/mol could
`be measured. We also processed the data by presenting the
`range of plateau as a function of PEG MW (Figure 3b). The
`range of plateau was assessed as
`the ratio of PEG
`concentrations at the onset and end of plateaus. The end
`value was determined at the signal value two times the plateau
`signal value and the onset signal value was 700 cts. The plateau
`signal value was calculated as an average of values showing
`maximally 15% deviation from the plateau. For ethylene glycol
`and PEG 200 with no identified plateau, the concentration was
`assessed at signal value of 2500. The PEG MW could be
`assessed equally well from both calibrations. However, the data
`processing from the range of plateau requires no prior
`knowledge of PEG concentration. The curves in Figure 3
`were obtained by fitting the data (PEG concentration vs MW
`or range of plateau vs MW) to the modified Hill function with a
`constant offset.38
`The response of the quenching system was observed at
`different concentrations
`for PEGs with different MWs.
`Conversely, no size-dependent response was observed for
`TR-LRET system. The difference between the systems may be
`related to the different surface properties of the nanoparticle
`materials. Both surfaces contain hydrophilic and negatively
`charged groups and less hydrophilic neutral parts. However,
`gold is regarded as clearly more hydrophilic than polystyrene,45
`which potentially affects to the adsorption properties of PEGs
`with both hydrophilic and hydrophobic characteristics. The
`method utilizes the competitive adsorption and thus,
`the
`labeled protein may partly replace the adsorbed PEG
`
`Figure 3. Polyethylene or ethylene glycol concentration at the signal
`level of 700 (a) or the range of plateau (b) as a function of MW for
`quenching system. The data was fitted to the modified Hill function
`with a constant offset.38
`
`depending on its size. However, the exact mechanisms of the
`competitive adsorption and the differences between the systems
`remain unclear.
`
`■ CONCLUSIONS
`the quenching
`We have demonstrated the applicability of
`nanoparticle system for the determination of PEG MW in a
`high throughput
`format. The PEG MW was successfully
`measured over a range from 62 to 35 000 g/mol. The method is
`fast to perform in the microtiter plate format with standard
`luminescence plate readers, which is in contrast to the existing
`methods, such as mass spectrometry, analytical ultracentrifuga-
`tion, and chromatography, requiring expensive instrumentation
`and expertise. Furthermore, we showed that the determination
`of PEG MW can be performed without prior knowledge of
`analyte concentration. This study with PEG suggests that the
`method is suitable also for neutral polymers, and thus the
`applicability shown by us earlier for charged compounds,
`polyethylenimines and polyamino acids, is widened. Thus, this
`paper indicates that the method is of potential value in research
`and industry for a wide range of polymers with different
`physical properties.
`
`■ AUTHOR INFORMATION
`Corresponding Author
`*S. Pihlasalo. E-mail: sari.pihlasalo@utu.fi. Tel.: +358 2 333
`7692.
`
`3921
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`DOI: 10.1021/acs.analchem.5b00736
`Anal. Chem. 2015, 87, 3918−3922
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`Analytical Chemistry
`
`Notes
`The authors declare no competing financial interest.
`
`■ ACKNOWLEDGMENTS
`This work was supported by the funding from the Academy of
`Finland (grant 258617) and the Graduate School of Chemical
`Sensors and Microanalytical Systems.
`
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`DOI: 10.1021/acs.analchem.5b00736
`Anal. Chem. 2015, 87, 3918−3922
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