Lysozyme Monolayers at Polymer Microparticles: Electrokinetic

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C: Surfaces, Interfaces, Porous Materials, and Catalysis

Lysozyme Monolayers at Polymer Microparticles: Electrokinetic Characteristics and Modeling Maria D#bkowska, Zbigniew Adamczyk, Micha# Cie#la, Malgorzata Iwona Adamczak, and Joanna Bober J. Phys. Chem. C, Just Accepted Manuscript • DOI: 10.1021/acs.jpcc.8b04916 • Publication Date (Web): 03 Jul 2018 Downloaded from http://pubs.acs.org on July 20, 2018

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The Journal of Physical Chemistry

Lysozyme Monolayers at Polymer Microparticles: Electrokinetic Characteristics and Modeling

Maria Dąbkowska1*, Zbigniew Adamczyk2, Michał Cieśla3, Małgorzata Adamczak4, Joanna Bober1

1

Department of Medical Chemistry, Pomeranian Medical University, Rybacka 1, 70-204 Szczecin, Poland [email protected] 2

Jerzy Haber Institute of Catalysis and Surface Chemistry Polish Academy of Sciences, Niezapominajek 8, 30-239 Krakow, Poland 3

M. Smoluchowski Institute of Physics, Jagiellonian University, Łojasiewicza 11, 30-348 Kraków, Poland 4

Department of Pharmacy, School of Pharmacy, University of Oslo, P.O. Box 1068 Blindern, 0316 Oslo, Norway

*Corresponding author,

ACS Paragon Plus Environment

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Abstract Adsorption of hen egg-white lysozyme (HEWL) molecules at negatively charged polystyrene microparticles was studied using the dynamic light scattering (DLS), electrophoresis (LDV), solution depletion techniques, enzyme-linked immunosorbent assay (ELISA) and atomic force microscopy (AFM). The measurements were carried out at pH 3.5 and 7.4 and for NaCl/PBS concentration of 10-2 and 0.15 M. Initially, the dependence of the electrophoretic mobility and zeta potential of microparticles on the bulk concentration of lysozyme was determined. These results were interpreted in terms of the general electrokinetic model. This allowed the Authors to devise a formula for precise determination of lysozyme coverage at microparticles under in situ conditions. The maximum coverage of irreversibly adsorbed lysozyme was also determined with the use of the electrokinetic concentration depletion methods and the ELISA assay. At pH 7.4 (PBS buffer) and ionic strength of 0.15 M, the maximum coverage was equal to 0.95 mg m-2 that agrees with theoretical modeling performed according to the random sequential adsorption approach. The stability of acid base properties of lysozyme monolayers was also determined via the electrophoretic mobility measurements carried out for different ionic strength and pH range 3-12. These results allowed to develop a more efficient and more sensitive method for determining lysozyme bulk concentration than ELISA assay as well as a robust procedure for preparing its stable monolayers at microparticles of well-controlled coverage and acid-base properties.


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1.

INTRODUCTION Lysozyme as one of the most abundant globular proteins, catalyzes the hydrolysis of

polysaccharides in outer cell walls of bacteria, destroys their integrity and viability. In addition to this role, lysozyme is also an important carrier of peptides and a large variety of low molecular weight drugs. Lysozyme has also been the subject of involved in various investigations of disorders induced by protein aggregation leading to the loss of their original biological activity.

For

example, the studies on the formation of amyloid-type aggregates of lysozyme may result in a better understanding of several diseases, including Alzheimer’s, Parkinson’s, type II diabetes, Creutzfeldt-Jakob’s, Huntington’s diseases, and many others 1-4. Lysozyme is secreted in milk, tears, mucus, saliva, serum, synovial fluids, linkage tissues etc. 5,6,7 as well as in egg whites. Hen egg-white lysozyme (HEWL) is a small monomeric protein (129 amino acid residues). The structure of lysozyme was determined in 1965 as the second protein structure and the first enzyme structure was solved by X-ray diffraction methods8. Its molecular weight calculated from this amino acid composition is equal to 14,325 Da (molar mass 14.325 kg mol-1). The crystalline structure of the monomer consists of the larger alpha -helical domain (1-36 and 87-129 amino acid residues) and the smaller beta-sheet domain (37-86 amino acid residues) 9,10. HEWL molecule exhibits a well-known three-dimensional structure with ca. 60% structural homology to human lysozyme10. The molecule of hen egg-white lysozyme comprises of six tryptophan (Trp) residues (Trp 28, 62, 63, 108, 111 and 123) 5,11, out of which Trp 62, 63 and 108 are present in the active site, although only Trp 62 and Trp 108 has been recognized as most dominant fluorophores in the overall geometry12. Refaee et al.13 and Fear et al.14 reported that the secondary structure of HEWL molecules in solution consists of five helices that account for 38% of the protein and one β-sheet in which 10% of the residues reside. Four disulfide bridges are responsible for the stabilization of the structure of the protein17. The shape of lysozyme molecule is approximated by a spheroid, with dimensions of 4.5 x 3 x 3 nm

15,16

with the effective cross-section area in the side-on orientation equal to 12.6 nm2.

The isoelectric point of fatty acid free lysozyme is pH 10.6 -10.9 14-18,20. The slightly lower value of 10 reported in the literature may be characteristic of fatty acid containing samples of HEWL. This can also be due to the various buffers used in these studies. The electric charge over the HEWL molecules is not uniform but heterogeneously distributed. The largest negatively charged region is mostly due to the 2 glutamic and 7 aspartic


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acid residues, whereas the positive charge is due to 6 lysine (Lys), 11 arginine (Arg) amino acid residues, which is located at the N,C-terminal region and its opposite side. As a result, the HEWL molecule exhibits a positive net charge at pH 7 in a 0.05 M NaCl solution equal to 8 e (where e is the elementary charge) and the dipole moment of 72 D18,19. In Refs21, 22 a similar value of 7 e at pH 7.4 in 0.1 M KCl solution was recorded. The adsorption of HEWL at colloid particle has been studied mainly with the aim of determining isotherms for various physicochemical conditions. For example, Yu-Hong Cheng et al.23, determined the adsorption of HEWL molecules at negatively CeO2 and ZnO particles (of the size 4 nm and 12 nm, respectively) at pH 7.4 using UV–vis spectrophotometric measurements. The maximum adsorption amount determined with the use of the Langmuir isotherm equation was -2

equal to 2,7 and 1,8 mg m for CeO2 and ZnO, respectively. Klose, Welzel and Werner16 determined adsorption isotherms of lysozyme under flow conditions at spherical soda lime glass particles (size 74.5 nm) by frontal chromatography. The -2

maximum coverage of the protein was equal to 1.3 mg m in 0.01M PBS (pH 7.4), for its bulk concentration of 10 mg/L-1 . Bharti et al.24, determined the adsorption of lysozyme at 20 nm silica nanoparticles at pH -2

range 3-11. The maximum coverage derived via adsorption isotherm was equal to 1.1 mg m at pH 7, which corresponds to 96 protein molecules per one silica particle. Galisteo and Norde25 determined the adsorption of lysozyme on poly(styrenesulfonate) microparticles of various surface charge densities. They analyzed the role of pH (2.0 - 12) for two ionic strengths 0.005 M and 0.05 M KCl was investigated. They analysis showed that a It was observed that some fraction of the lysozyme molecules was reversibly adsorbed, whereas the -2

maximum coverage of irreversibly bound protein was equal to 2.8 mg m at pH 7 in 0.05M KCl. Analogous measurements were performed for HEWL using other carrier particles such as silicon dioxide nanoparticles 26, gold nanoparticles

27,28

hydroxyapatite,29 and magnetic iron oxide -2

nanoparticles30,31. The maximum coverage reported by these works varied between 0.1 mg m and -2

4.5 mg m . Despite a wide range the maximum coverage of lysozyme adsorption on carrier particles, two essential regularities were observed in above-mentioned works. Firstly, it was shown that there appears a fraction of irreversibly bound lysozyme whose coverage is independent of their initial concentrations in the bulk. This coverage varies between 0.25 and 1.7 mg m-2 at ionic strength of


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0.05 M depending on pH24. Secondly, there is also a considerable fraction of lysozyme whose coverage depends on bulk concentrations and varies between 0.4 and 3.1 mg m-2 as discussed in Refs.32, 33. This indicates that this fraction of lysozyme is reversibly adsorbed, which leads to the appearance of adsorption isotherms. In a few other works17, 26, 34, 35 negligible desorption of HEWL molecules from polymeric carrier particles was observed. This is probably due to the adsorption of dimers or larger aggregates because lysozyme, at larger bulk concentrations, is known to form aggregates in aqueous

solutions24, 36. Such aggregates, exhibiting higher affinity to the polymeric surfaces can

replace due to Vroman effect, single HEWL molecules, which were first adsorbed. Given the deficit of reliable experimental data, the main aim of this paper is to elucidate the mechanism of lysozyme adsorption at well-characterized polymer microparticles. The electrophoretic mobility measurements were performed to monitor the progress of protein adsorption in situ. Unlike in the previous works, the coverage of adsorbed protein was precisely determined via LDV and AFM measurements and compared with the ELISA immunoassay, thus providing reliable data for bulk lysozyme concentration as low as 0.1 mg L-1. In this way, one can unequivocally prove the irreversibility of HEWL adsorption for the low concentration range. It should be mentioned that this method had been efficiently used before to determine adsorption mechanism of human serum albumin37 and fibrinogen38 on polystyrene microparticles. The maximum coverage of irreversibly adsorbed lysozyme molecules - experimentally determined by these complementary methods at different pHs and ionic strengths - is theoretically interpreted in terms of the extended random sequential adsorption (RSA) model. On the other hand, the results of electrophoretic mobility (LDV) results are interpreted in terms of the general electrokinetic model, which allows to determine lysozyme coverage at microparticles in situ more efficiently than by using the time consuming ELISA test. The acid-base properties and a large stability of lysozyme monolayers are also determined via the electrophoretic mobility measurements carried out for different ionic strength and pH range 312.


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2. MATERIALS AND METHODS 2.1. Experimental Section In our studies the filtered (0.1 µm lowest binding membrane for protein solutions, PVDF Durapore, Merck Millipore) stock solutions of lysozyme (from chicken egg white, purity 98%, L4917 Sigma Aldrich) of known concentrations (typically 500 mg L-1) in the PBS buffer/ NaCl electrolyte (pH 7.4) were first prepared. To minimize errors in concentration measurements two complementary spectrophotometric techniques were used the BCA assay and UV absorbance at 280nm. Prior to each experiment, the stock solution was diluted to a desired bulk concentration typically 0.1-5 mg L-1. Ionic strength of HEWL solutions thus obtained in this way was fixed by the addition of NaCl and pH by HCl, NaOH or the PBS buffer. The real concentration of these solutions after membrane filtration was determined by commercially available enzyme-linked immunosorbent assay (ELISA kit for Lysozyme, EIAab) and by reaction of bicinchoninic acid with the cuprous cation, which is generated by reduction of cupric cation by the protein in alkaline conditions (BCA protein assay kit for low concentration, abcam). The quantitative immunotest ELISA is based on antigen–antibody reactions through the color change obtained using an enzyme- linked conjugate. Ruby muscovite mica from Continental Trade was used as a substrate for lysozyme adsorption measurements. The freshly solid pieces of mica were cleaved into thin sheets prior to every experiment. Water was purified using a Milipore Elix 5 apparatus. Chemical reagents (sodium chloride, hydrochloric acid) were commercial products of Sigma-Aldrich and were used without further purification. The temperature of experiments was kept at a constant value equal to 298 ± 0.1K. The suspension of sulfonate polystyrene microparticles was synthesized following the Goodwin39 method and was used as colloid carriers for HEWL. The stock suspension of a welldefined concentration, determined by densitometry and the dry weight method, was diluted prior to each adsorption experiment to a desired mass concentration, equal to 40 mg L-1. The diffusion coefficient of lysozyme and microparticles was determined by dynamic light scattering (DLS) ( Zetasizer Nano ZS, Malvern). The electrophoretic mobility of lysozyme molecules, bare and lysozyme, covered microparticles was measured using the Laser Doppler Velocimetry (LDV) technique. The AFM measurements of surface concentration of HEWL molecule on mica were carried out under ambient air conditions using the NanoWizard AFM (JPK Instruments AG). The samples


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were left for air-drying until the next day. AFM imaging was performed with NanoWizard® instrument (JPK Instruments, Berlin, Germany). The intermittent contact mode images were obtained in the air, using ultrasharp silicon cantilevers (NSC35/AlBS, MicroMash, Spain) and the cone angle of the tip was less than 20o. The images were recorded at the scan rate of 1 Hz for the six randomly chosen places. The images were flattened using an algorithm provided with the instrument. We captured all images in random areas within the scan size of 0.5 x 0.5 µm or 1 x 1 µm. The lysozyme surface concentration was determined using ImageJ software by gathering the number and coordinates of single protein molecules. Manual counting of HEWL molecules was based on comparing the original original image and exactly the same picture altered by digital image filters by cutting off the picture background. The adsorption of lysozyme molecules at microparticles was studied in situ using the LDV method. Initially, in these experiments, the dependence of the electrophoretic mobility of microparticles on the amount of lysozyme added to the suspension was determined. Using these data, the surface concentration of molecules was calculated using the electrokinetic model described in Refs.40,41. Since the direct LDV method seems less accurate for larger surface concentration, the maximum coverage of molecules on microparticles was obtained using different concentration depletion methods, where the unbound (residual) lysozyme concentration was determined directly by AFM imaging or by applying the two-step LDV method42,43. In the former case, the microparticle/lysozyme mixture acquired after the adsorption step was left to deposit on mica sheets placed in the diffusion cell over a controlled time. The number of lysozyme molecules was determined in randomly chosen equal-sized areas of the mica sheets using AFM imaging. Knowing this parameter, the residual bulk concentration of lysozyme and consequently their coverage on microparticles was calculated. Following the adsorption step, in the two-step LDV method, the microparticle/protein mixture was centrifuged in order to remove the microparticles and the supernatant was again contacted with bare microparticle suspension of a known concentration. Using the LDV method, the electrophoretic mobility of the complex was measured and converted into the surface concentration using calibration curves derived from the primary adsorption experiments. In parallel with the LDV measurements, the residual lysozyme concentration in the filtrate was determined by applying the ELISA immunoassay method according to the manufacturer’s protocol. Sandwich ELISA technique was employed to simultaneously monitor the maximum concentration of lysozyme in the supernatant suspensions, acquired after adsorption at microparticles.


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Standard and supernatant samples containing lysozyme after centrifugation of latex suspension were added to the 96-well microplate pre-coated with capture antibody specific to HEWL; then the plate was incubated for some time and rinsed. Following this step, the detection antibody, conjugated to horseradish peroxidase (HRP) enzyme, was added and incubated. The concentration of lysozyme molecules was spectrophotometrically measured as only the wells containing HEWL molecule change color in enzyme-substrate reaction. Thus, it was possible to precisely determine to the concentration of non-adsorbed HEWL molecules at microparticle surface separated by a centrifugation process. The theoretical modeling of lysozyme adsorption on microparticles was carried out according to the random sequential adsorption (RSA) approach described in Refs.37, 38, 43-45. It is significantly more efficient than the molecular dynamics or Brownian dynamics simulations as large populations of molecules adsorbing and interfaces of spherical shape (e.g. microparticles) can be generated considering their lateral electrostatic interactions. Due to the rigidity of the lysozyme molecule, the bead model was applied where the real shape of the lysozyme molecule was approximated by a sphere of the diameter dp, that matches the hydrodynamic diameter of the molecule derived from DLS measurements. The electrostatic interactions among adsorbed molecules were calculated applying the linear superposition approach45 and the molecule interaction with microparticles, were described by the perfect sink model potential. Further details of the RSA calculation algorithm are given in the Supporting Information section.

3. RESULTS AND DISCUSSION 3.1. HEWL and Microparticle Bulk Characteristics It was determined by DLS that for the NaCl concentration of 10-2 to 0.15 M and pH 3.5, 7.4 (PBS), the diffusion coefficient of lysozyme molecules was equal to 1.1x10-6 +/- 0.1 cm2 s-1 (at T= 298 K), which agrees with the results obtained by Jachimska et al.46. Based on the known diffusion coefficient, using the Stokes–Einstein relationship, it was calculated using the Stokes–Einstein relationship that the hydrodynamic diameter dH was equal to 4.0 +/- 0.2 nm. This corresponds to the molecule cross-section area of 12. 6 nm2. The electrophoretic mobility µe of lysozyme molecules was measured for different NaCl concentrations at pH 3.5 and 7.4 (PBS) using the LDV method. The experimental data are shown in Table 1.


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Table 1. Electrophoretic mobility, zeta potentials and charge densities of microparticles and Lysozyme molecules. Microparticles (L800)

Lysozyme

NaCl/PBS µe

ζp

Qc *

Qc**

[mV]

[e ]

[e ]

2.96

56

6.2

10

-0.289

1.13

20

2.4

7.8

- 110

-0.307

0.46

8.1

1.0

1.6

- 45

-0.282

0.29

5.2

0.60

2.0

concentration

µe

ζl

σ0

[M]

[µm cm (Vs)-1]

[mV]

[e nm-2]

- 6.5

- 85

-0.166

- 3.6

- 48

- 8.7

- 3.5

[µm cm (Vs)-1]

10-2 NaCl pH 3.5

0.15 NaCl pH 3.5

10-2 PBS pH 7.4

0.15 PBS pH 7.4

Footnote:

( )

2

Qc* calculated from the formula: 3πη dH µe (Lorenz, model);

Qc*

calculated from the formula:

(Henry model).

At pH 3.5 the electrophoretic mobility of HEWL molecules was equal to 2.96 and 1.13 µm cm (Vs)-1 for NaCl concentration of 0.01 and 0.15 M, respectively that corresponds to the zeta potential of HEWL molecules equal to 56 and 20 mV, respectively, calculated using the Henry model. Using the electrophoretic mobility data, it was possible to calculate the electrokinetic charge per albumin molecule Qc, expressed in Coulombs, from the Lorenz-Stokes relationship47-49 : = 3

(1)


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where η is the dynamic viscosity of the solvent (water). Although Eq. (1) is valid for molecules of arbitrary shape, its accuracy is limited for larger ionic strengths. Therefore, in this case when the double-layer thickness attains smaller value than the protein dimension, the following equation was used in Refs.37, 43 to calculate Qc for molecules of a spherical shape and the absolute zeta potential values below 50 mV.

= 2 (1 + ) ζ = 2 p

( )

(2)

3.2 HEWL adsorption at microparticles

The lysozyme adsorption at microparticles was performed according to the following procedure: (i) the reference electrophoretic mobility of bare microparticles was measured, (ii) the lysozyme monolayers were formed mixing equal volumes of its solutions of the bulk concentration varied between 0 – 2.4 mg L-1 with the microparticle suspension of the bulk concentration 80 mg L-1. The adsorption time was 600 seconds, (iii) the electrophoretic mobility of lysozyme covered microparticles was measured and the corresponding zeta potential was calculated. This procedure enables direct in situ determination of the electrophoretic mobility and zeta potential variations as a function of the bulk concentration of lysozyme. It should also be mentioned that the relaxation time of lysozyme monolayer formation on latex particles, estimated in Ref.42 was ca. 2 seconds for the final latex concentration of 40 mg L1

. Therefore, the adsorption time of 600 seconds made it possible for all albumin molecules to

adsorb on latex particles to a full monolayer. The time scale for formation of additional layers is much longer than that of monolayer formation. In all cases, lysozyme should irreversibly adsorbed onto the surface. The experimental data obtained in the primary adsorption experiments for 0.15 M NaCl, pHs 3.5 and 7.4 (PBS) are shown in Fig. 1 as the dependence of the zeta potential of microparticles on the initial concentration of lysozyme in the suspension (after mixing) denoted by cb. It can be noted that the zeta potential abruptly increases along with the lysozyme concentration and approaches for cb. > 0.5 mg L-1 the plateau values of 10 and 1 mV, at

pH 3.5 and 7.4 (0.15 M

NaCl/PBS), respectively. The results in Fig. 1 were more conveniently analyzed by introducing the nominal coverage of HEWL


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= ⁄

(3)

where vs is the volume of the suspension and Sl is the surface area of latex expressed in m2, given by Sl = 6 cl vs /dl ρl, ρl is the density of microparticles equal to 1.05x103 kg m-3. Based on the data shown in Fig. 1, it was calculated that the maximum coverage of lysozyme at microparticles is equal to ca. 0.8 and 0.9 mg m-2 at pH 3.5 and 7.4, respectively (for NaCl concentration of 0.15 M). Additionally, knowing Γ it was possible to calculate the dimensionless coverage of lysozyme Θ from the constitutive dependence

= ! "

#$

%&

' =

( #$ )* +* , %& *

(4)

where Sg is the characteristic cross-section area of the lysozyme molecule (assumed to be equal to π dH2 / 4 , i.e. 12.6 nm2), Av is the Avogadro constant and Mw is the molar mass of lysozyme. a)

Γ [mg m-2] 0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

1.8

2.0

80 HEWL bulk 0.01M 60 40 HEWL bulk 0.15M 20

ζ [mV]

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


0 HEWL bulk 0.01M -20

HEWL bulk 0.01M

-40

HEWL bulk 0.15M microparticle bulk 0.15M

-60

HEWL bulk 0.15M

-80 microparticle bulk 0.01M -100 0.0

0.2

0.4

0.6

0.8

1.0

1.2

cb [mg L-1]


11


b) Γ [mg m-2] 0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

1.8

2.0

40 20 HEWL bulk 0.15M

HEWL bulk 0.01M HEWL bulk 0.01M

0

ζ [mV]

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 12 of 35

-20 HEWL bulk 0.15M -40 microparticle bulk 0.15M -60 -80 Latex bulk 0.15M

-100

microparticle bulk 0.01M

-120 0.0

0.2

0.4

0.6

0.8

1.0

1.2

-1

cb [mg L ]

Fig. 1. The dependence of the zeta potential of microparticles ζ on the bulk concentration of lysozyme in the suspension after mixing cb (lower horizontal axis) and the nominal coverage (upper horizontal axis) Γ [mg m-2]. The points show experimental results obtained by the LDV method. Part a: pH 3.5 NaCl concentration 0.01M and 0.15 M; part b: pH 7.4 PBS concentration 0.01M and 0.15 M. The solid lines show the theoretical results calculated from Eqs.(5-6). The dashed lines show the zeta potential of bare microparticles.

It should be mentioned that the results presented in Fig. 1 show analogous trends as previously observed by Serra et al.41 for the polyclonal rabbit immunoglobulin (IgG), Bratek Skicki et al.38 for human serum fibrinogen, and rHSA, Sofińska et al.50 for recombinant serum albumin. The experimental results shown in Fig. 1 were theoretically interpreted in terms of the electrokinetic model previously applied in Refs.51,52. According to this model, the following expression for the zeta potential of interfaces covered by protein molecules can be formulated

ζ ( ) = -. ( )ζI + -/ ( )ζp

(5)


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where Fi (Θ ) , Fp (Θ ) are the dimensionless functions53 of coverage for spherically-shaped molecule hence within the limit of large ionic strength they can be approximated by the following formula: -. ( ) = 0 123 4 -/ ( ) = 1 − 0 1√728 4

(6)

where Ci , C p are constants approach 10.2 and 6.51, respectively.51, 52 It should also be mentioned that for higher coverage the Fi (Θ) function vanishes and the Fp (Θ) function attains 1 2 . As seen in Fig. 1 the theoretical results calculated from Eqs. (5-6), agree with the experimental data for both pHs. Therefore, these equations can be used in order to directly determine the lysozyme coverage at microparticles via the simple LDV measurements. Since Eqs. (5-6) can be analytically inverted, hence the formula54 is as follows: = −

9

23

ln < (ζ)

(7)

where X (ζ) is defined as the normalized zeta potential given by ζ

X (ζ ) =

ζ −ζ p / 2 ζi −ζ p / 2

=

ζ −ζ∞ ζi −ζ∞

(8)

where ζ ∞ = ζ p / 2 is the limiting zeta potential. Since X (ζ) is experimentally accessible, Eq. (8) can be used to directly determine the lysozyme coverage at microparticles via simple and reliable LDV measurements. Additionally, considering Eqs.(4), one can transform Eq.(8) can be transformed into the useful expression considering that at a fixed ionic strength and pH the zeta potential is proportional to the electrophoretic mobility , %& *

= 2

3 ( #$ )* +*

= 1 =

> 3 ln =1 =

(9)

>

Eq. (9) enables to determine the bulk concentration of lysozyme even for the range of 0.1-1 -1

mg L , i.e., for the nM concentration range, via a simple LDV measurement of the electrophoretic mobility of microparticles.


13


Due to the limited precision of the direct electrophoretic mobility measurements for larger lysozyme coverage, three supplementary methods were used in order to determine its maximum coverage at microparticles (i) the above mentioned two-step LDV method exploiting the calibrating measurements, (ii) the AFM imaging method and (iii) ELISA immunoassay test. The results obtained by the two-step LDV method are shown in Fig. 2 as the dependence of the zeta potential of microparticles on the initial lysozyme concentration added in the first step for two type of electrolyte at 0.15 M ionic strength. It can be noted that for low lysozyme concentration up to 0.6 mg L-1, the change in microparticle zeta potential determined in the second step is negligible, which indicates that the residual protein concentration after adsorption vanishes. Only for cb exceeding the threshold value, the microparticle zeta potential increases in an analogous manner as for the first adsorption step (shown by solid line in Fig. 2) for NaCl as well as PBS. This concentration can be precisely determined as the intersection point of this line with the horizontal line showing the zeta potential of bare microparticles. Knowing threshold value of lysozyme concentration one can calculate the maximum coverage of lysozyme from Eq.(3) was calculated giving Γmx = 0.95 mg m-2, which falls within experimental error bounds, which were previously determined by the direct micro-electrophoretic method (see Table 2). Analogously, it was possible to determine the maximum coverage of lysozyme at microparticles for ionic strength 0.01 M for both types of electrolytes: NaCl and PBS (see the Supporting Information section). Γ [mg m-2] 0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

1.8

2.0

40

1

20

ζ [mV]

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 14 of 35

0

2

-20

-40

2 -60

-80

-100 0.0

0.2

0.4

0.6

0.8

1.0

1.2

cb [mg L-1]

Fig. 2. The dependence of the zeta potential of microparticles ζ on the initial lysozyme concentration in the suspension cb (lower horizontal axis). The upper horizontal axis shows the nominal coverage of lysozyme Γ [mg m-2] corresponding to this concentration; pH 7.4, 0.15M PBS. The solid line 1 represents the theoretical results calculated


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from Eqs.(5,6) and the points show the reference experimental data obtained for the initial adsorption step; the dashed line 2 shows the fit of the experimental data obtained for the second adsorption step.

The maximum coverage of lysozyme at microparticles was also determined via the AFM imaging. This procedure consisted in determining the number concentration of lysozyme molecules adsorbed at mica (denoted by Nm ) after a fixed time (900 s). In these experiments, the native microparticle suspensions, acquired after albumin adsorption was used. Lysozyme molecules could be directly imaged, because the deposition of microparticles on mica was negligible over the time of these experiments due to their low number concentration and low diffusion coefficient compared to lysozyme molecules. Considering that in these measurements the lysozyme adsorption at mica occurred under diffusion transport conditions, its surface concentration is expressed by the formula,42 9/7

?@ = 2 (A B⁄)

(10)

where t is the adsorption time. Hence, for a fixed adsorption time (ta), the residual lysozyme concentration in the suspension *

(c ), is given by 9/7

∗ = (⁄4 A BF )

= G ?@

(11)

where Cl = (π/ 4 D ta)1/2 is a known constant. The results of such AFM measurements carried out at pH 3.5, 0.15 M NaCl and pH 7.4, 0.15 M PBS, are plotted in Fig. 3 as the dependence of c* calculated from Eq.(11) on the initial lysozyme concentration in the mixture prior to the adsorption equal to cb. One can see that c* linearly increases for cb exceeding the threshold value of the lysozyme concentration for both type of electrolytes and pH values. The threshold concentration of lysozyme can be determined as the intersection of this -1

0.53 mg L

straight line

with the horizontal axis

(see Fig. 4), which is equal to

in this case. Knowing ct,, the maximum lysozyme coverage from Eq. (3) was

calculated as equal to 0.95 mg m-2 .


15


1.2

1.0 1

2

0.8 -1 c* [mg L ]

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 16 of 35

0.6

0.4

0.2

0.0 0.0

0.3

0.6

0.9

1.2

1.5

1.8

cb [mg L-1]

Fig. 3. The dependence of residual lysozyme concentration after the adsorption step c* determined by AFM imaging at pH 3.5, 0.15 M NaCl (triangles) and pH 7.4, 0.15 M PBS (circles). The dashed lines 1 and 2 show the linear fits of experimental data for pH 7.4 and 3.5, respectively. The inset shows lysozyme monolayer at mica for the surface concentration of 500 [µm-2].

The experimental data acquired by AFM and LDV were compared with the results obtained from the ELISA immunoassay, which is sensitive, specific but also a time consuming and expensive. The dependence of the HEWL concentration in the suspension after adsorption on microparticles at its initial concentration is shown in Fig. 4 (pH 7.4, 0.01M and 0.15 M PBS). In this way we also determined using ELISA technique, lysozyme concentration in the NaCl electrolyte after adsorption at latex particle for ionic strength 0.01M as well as 0.15M for pH 3.5 (Supporting Information).


16

Page 17 of 35

1.2

1.0 2

1 0.8 -1 c* [mg L ]

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


0.6

0.4

0.2

0.0 0.0

0.3

0.6

0.9

1.2

1.5

1.8

cb [mg L-1]

Fig. 4. The dependence of residual lysozyme concentration after the adsorption step c* determined by the ELISA assay at pH 7.4 in PBS, 0.01 M (circles) and 0.15 M (triangle). The dashed lines 1, 2 show the linear fits of experimental data for 0.01 M and 0.15 M, respectively.

It can be observed that also in this case the residual lysozyme concentration c* is a linear function of cb equal to one. The threshold concentration of lysozyme determined as the intersection of the straight line fitting the ELISA results with the horizontal axis is equal to 0.53 mg L-1, thus the maximum lysozyme coverage is equal to 0.95 mg L-1, which is close to the AFM and the LDV results. Analogous results obtained for different ionic strength and pHs are shown in Table 2. Importantly, the results obtained by various methods agree with each other within experimental error bounds. This suggests that the electrokinetic LDV method represent an attractive alternative to the time consuming and expensive ELISA assay.


17


Page 18 of 35

Table 2. Maximum coverages of lysozyme at microparticles determined by various method (pH 3.5).

Maximum coverage of lysozyme at microparticles obtained by various methods [mg m-2]

NaCl/PBS concentration [M]

Depletion, 1/(κ dH)

Electrophoretic mobility (LDV)

Depletion centrifuge

(AFM)

Theoretical ELISA

(RSA modeling)

and LDV 10-2 NaCl, 0.76

0.80+/- 0.1

0.20

0.80 +/- 0.1

0.76

0.80+/- 0.1

0.20

0.90 +/- 0.1

0.72 +/- 0.05

0.70 +/- 0.05

0.68 +/-0.05

0.60

0.80+/-0.05

0.86+/-0.05

0.85

0.85+/- 0.1

0.75+/-0.05

0.80+/-0.05

1.0

0.95+/- 0.1

0.95+/-0.05

0.96+/-0.05

1.0

pH 3.5 0.15 NaCl pH 3.5

0.84 +/- 0.05

10-2 PBS pH 7.4

0.15 PBS pH 7.4

It should be mentioned that our results agree with some data previously presented in the literature. Bharti et al.24, for instance, determined that the maximum coverage of HEWL onto 20 nm in diameter silica particles which was equal to 1.1 mg m-2. Klose et al.16 obtained the maximum coverage of HEWL at soda lime glass particles with diameter 74.5 nm which was equal to 1.3 mg/m2 for protein bulk concentration of 10 mg L -1. Similar data were also obtained for HEWL adsorption at solid substrates, e.g. silica surface22,33. However, in other works15,25,55,56,57 much larger values of the maximum coverage of HEWL were reported that can be most probably attributed to adsorption of aggregates. It is interesting to compare the experimental results obtained in this work with the theoretical predictions derived from the RSA modeling described in the Supporting Information section. In order to increase the efficiency of these calculations, the HEWL molecule was replaced


18

Page 19 of 35 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


by a sphere of the diameter dp bearing a uniform electric charge Qp, which were variable parameters. The size of the microparticle was equal to 820 nm. In Fig. 5 monolayers of lysozyme at microparticles derived from RSA modeling for NaCl concentration equal to 0.01 and 0.15 M and pH 7.4 are presented. Primary data

derived from

these calculations were the numbers of adsorbed lysozyme molecules averaged over microparticle population exceeding 103.

a)

b)

Fig. 5. Monolayers of HEWL at microparticles at the jamming (maximum coverage) limit derived from the RSA modeling for NaCl concentration of 0.15 M; part a, pH 3.5, Γ = 0.85 mg m-2, part b , pH 7.4, Γ = 1.0 mg m-2.

Knowing the average number of adsorbed molecules and the surface area of a microparticle Sg1, the coverage of model HEWL molecule was calculated based on the formula =

(* #$ %&

H ?/ I

(12)

In this way we obtained, Γmx = 0.85 and 1.0 mg m-2, for NaCl concentration equal to 0.15 M, pH 3.5 and 7.4, respectively that well agrees with the experimental data. It should also be emphasized that the theoretical value of 1.0 mg m-2 represents a limiting value pertinent to noninteracting (hard) molecules. In other words, it is expected that the monolayer coverage of single ACS Paragon Plus Environment

19


Page 20 of 35

(unaggregated) HEWL molecules should be smaller than this limiting value. Consequently, larger values reported in experiments Ref.

15, 25, 55,56,57

are likely to be attributed to multilayer adsorption

of single molecules or monolayer adsorption of aggregated molecules. The experiments demonstrated that for NaCl concentration of 0.01 M the maximum coverage equal to 0.70 mg m-2 was slightly larger than the theoretical value of 0.60 mg m-2, whereas at pH 7.4 the experimental value of 0.80 mg m-2 was slightly lower than the theoretical value of 1.0 mg m-2 (see Table 2). Therefore, considering the experimental error in these exceptionally tedious experiments, it can be assumed assume that the agreement of experimental data with theoretical predictions is satisfactory. Moreover, it can be noticed that at pH 3.5 the maximum coverage markedly increases along with ionic strength which can interpreted as being due to the decrease in the range of lateral electrostatic interactions among adsorbed HEWL molecules governed by the double layer thickness. Accordingly, for 10-2 M NaCl, the range of the interactions was equal to 3.05 nm, i.e., 0.76 of the molecule diameter whereas for 0.15 M NaCl, the range of the interactions was equal to 0.79 nm, i.e., 0.20 of the molecule diameter (see Table 2). A similar observation was reported in the case of albumin37 and fibrinogen38 adsorption at negatively charged polystyrene microparticles.

3.3 Electrokinetic characteristics of lysozyme monolayers at microparticles

Another series of experiments determined the acid-base properties and the stability of HEWL monolayers at microparticles. Various cycles were performed where pH ranged between 3.5 and 12 keeping the ionic strength changes negligible (typically less than 5%) at constant protein coverage at microparticles. The results of these measurements, performed for the initial coverage of lysozyme equal to 0.9 mg m-2 and 0.15 M NaCl concentration are presented in Fig. 6. One can observe that the differences between the first and the third pH cycle are insignificant, which confirms the lack of monolayer stability. It is interesting to mention that analogous stability of lysozyme monolayers at mica at pH ranging from 3 to 10 was confirmed by Jachimska et al.46 via the contact angle measurements. The large stability of lysozyme monolayers at microparticles compared to their bulk solutions has practical significance because it makes it feasible through investigations of their interactions with various ligands, e.g. immunoglobulins. At pH above 8, the zeta potential of the lysozyme monolayer becomes negative. This limiting pH value is slightly smaller than the bulk isoelectric point of lysozyme equal to 9 as reported in Ref 47. The relatively large degree of uncertainty of this value is caused by a relatively


20

Page 21 of 35

low zeta potential of lysozyme molecules in the bulk, which become smaller than 8 mV for pH > 6 (see curve 1 in Fig. 6). As a result, the influence of the zeta potential of microparticles is largely negative at this pH range (see curve 3 in Fig. 6) becomes significant that can be predicted from Eqs. (5,6). Indeed at pH 7.4 considering that ζi = -110 mV one can predict that the correction factor Fi (Θ )ζ i / F p (Θ ) is ca. -8 mV that is comparable with the lysozyme molecule zeta potential.

60 adsorption

40

ζ [mV]

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


20 1 2 0

-20 cycle reversal 3

-40

-60 2

4

6

8

10

12

pH

Fig. 6. The dependence of the zeta potential of the HEWL monolayer (adsorbed at pH 7.4, 0.15 M NaCl, coverage Γ= 0.90 mg m-2) on pH cycling starting from 3.5 to 12 and back to 3.5 (three cycles for each curve were made). The points denote experimental results obtained from the LDV measurements for NaCl and PBS concentration of 0.15 M (experimental data interpolated by curve 2) The upper line (1) denotes the lysozyme zeta potential in the bulk and the lower line (3) shows the interpolated results for microparticles.

It should be mentioned that analogous shift in the isoelectric point as that shown in Fig. 6 was previously observed for fibrinogen monolayers at microparticles

38,42

and human serum albumin

37

dimer .

4. Conclusions Electrophoretic mobility measurements supplemented by the AFM and ELISA concentration depletion methods were the basis for performing a quantitative analysis of the adsorption of HEWL molecules at polymer microparticles for the nM concentration range. These results are interpreted in terms of the general electrokinetic model, which allowed to specify a functional dependence given by Eq.(7) enabling to determine in situ the lysozyme coverage at microparticles. This method is more robust than the time consuming ELISA test.


21


Page 22 of 35

The maximum coverage of irreversibly adsorbed lysozyme molecules was also determined using four various concentration depletion methods including the ELISA immunoassay. The results obtained by these techniques agreed with each other and with theoretical modeling performed according to the extended RSA approach. The experimental maximum coverage of HEWL at pH 7.4 was equal to 0.80 and 0.96 mg m-2 for ionic strength of 10-2 and 0.15 M, respectively compared with the theoretical value of 1.0 mg m-2. The increase in the maximum coverage with ionic strength was interpreted as due to the decrease in the range of lateral electrostatic interactions among adsorbed HEWL molecules. This effect was even more pronounced at pH 3.5, where the protein molecule charge is significantly larger. Therefore, it may be concluded that these electrostatic interactions play a significant role in the HEWL adsorption at oppositely charged polymer microparticles. The acid-base properties of lysozyme monolayers adsorbed at microparticles were also determined via the pH cycling experiments, which confirmed their large stability. These monolayers of well controlled coverage and acid-base properties can be used as reference substrates for studying interactions and binding of various ligands.

Supporting information Short Description 1.

Theoretical modeling of lysozyme adsorption on microparticles was carried out

according to the random sequential adsorption (RSA) approach described extensively in Supporting Information File. 2.

Determining the maximum coverage of HEWL molecules on microparticle via the

LDV and ELISA methods for various electrolyte conditions: pH 7.4, 0.01 M PBS (Fig. S1), pH 3.5, 0.01 M NaCl (Fig. S2), pH 3.5, 0.01/0.15 M NaCl (Fig. S3).

ACKNOWLEDGMENTS This work was partially supported by the National Research Center Research project: UMO2015/19/B/ST5/00847. Maria Dąbkowska acknowledges a kind gift of microparticle suspension (latex) received from Jerzy Haber Institute of Catalysis and Surface Chemistry Polish Academy of Sciences (Research Project POIG 01.01.02.-12-028/09 FUNANO).


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Lysozyme Monolayers at Polymer Microparticles: Electrokinetic

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