Diffusion-Controlled Protein Adsorption in Mesoporous Silica - The

ARTICLE pubs.acs.org/JPCB

Diffusion-Controlled Protein Adsorption in Mesoporous Silica Shan Lu, Zhihong Song, and Jing He* State Key Laboratory of Chemical Resource Engineering, Beijing University of Chemical Technology, Beijing 100029, China ABSTRACT: In this work, the pore size-dependent PPL diffusion and protein distribution in SBA-15 support have been investigated in detail by confocal laser scanning microscopy (CLSM) and X-ray diffraction (XRD) observations in combination with hindered diffusion simulation, intraparticle diffusion analysis, and apparent kinetics calculation. The CLSM observations indicate porcine pancreatic lipase (PPL) diffuses partly into the pores with a diameter of 5.6 nm and much deeper into the pores with a diameter of 8.0 or 9.7 nm. PPL distribution along the pore length has been simulated by hindered diffusion model and the result coincides well with CLSM observations. Besides pore diffusion, boundary resistance is revealed by the intraparticle diffusion analysis of adsorption data. The populations of PPL adsorbed inside the pores or on the external surface are estimated. A higher PPL uptake is found in the 8.0 nm than 9.7 nm pores, indicative of the existence of an optimal pore size to match the protein dimension for maximum adsorption capacity. The so-called “confinement” of PPL in the mesoporous supports, revealed by the XRD observation and intraparticle diffusion analysis above, is further confirmed by apparent kinetics calculation.

1. INTRODUCTION Mesoporous silicate (MPS) materials have been recognized as energetic hosts for protein adsorption due to their high surface area, tunable nanosized pores, tailorable surface property, and good mechanical stability.1 MPS with protein encapsulated has wide potential applications in biocatalysis2
4 and biosensing.5
7 In addition, MPS is a promising material to separate or purify proteins with size-exclusion and ion-exchange techniques.8 The controlled adsorption of proteins is essential in many applications, in that the adsorption rate not only is directly related to the preparation of the immobilized protein systems but also decides the separation efficiency in protein chromatography; Moreover, the distribution of proteins, i.e., being at interior or exterior surface, uniform or not, and the packing density, significantly affect enzyme intrinsic stability and activity.9
11 Protein diffusion and surface
protein binding are two steps that are of the most concern in the adsorption process. Protein bindings to modified or unmodified silica surfaces via interfacial interactions have been extensively studied using adsorption isotherm measurement12,13 as well as advanced techniques like microcalorimetry,14 mass spectrophotometry,15 and photon correlation spectroscopy.16 Protein diffusion, sensitive to spatial factor which mainly refer to the size of the mesopore, or more specifically, the relative size of mesopores and protein molecules, has also been investigated. Díaz and Balkus found that the protein amount loaded on mesoporous silica MCM-41 in a limited contact time decreased with increasing protein molecular weight.17 It is expected that the pore size of the mesochannels should be sufficiently large for “comfortable” entrapment of biomolecules. Kisler et al. demonstrated that the adsorption rate in MCM-41 material depended strongly on the protein molecular size relative to the pore size, supporting its potential for size selective separations.18 Recently, protein exclusion or r 2011 American Chemical Society

penetration has been visualized on spherical MPS with different pore sizes using confocal laser scanning microscopy (CLSM).19 But so far, most of studies rely on the uptake amount or the adsorption rate to speculate the protein diffusion. A detailed understanding of the diffusion characteristics of proteins in MPS and quantitative analysis of the resulting protein distribution still remain great challenges. Proteins diffusion in porous materials is a complex process in which the diffusion effects and protein
surface interactions are difficult to separate and several diffusion modes such as intraparticle diffusion and boundary layer diffusion might coexist. Conventional isotherm and adsorption rate studies are far insufficient to provide insight into the protein diffusion. In the pores matching biomolecules in size, hindered diffusion should be the first consideration since the diffusion coefficient of protein is much lower than in bulk solution.20 A few efforts have been devoted to predictions of hindered transport based on sizes, shapes, and electric charges of solutes and pores.20
23 An intraparticle diffusion model is useful in a wider range to analyze the adsorption data to reveal the diffusion mechanism in porous materials.24 Whether there is other diffusions besides pore diffusion and what is the rate-controlling step can be demonstrated by intraparticle diffusion analysis. Yet it would be of great help to combine the intraparticle diffusion analysis of experimental data with the hindered diffusion simulation for comprehensively understanding the protein diffusion in MPS supports. Lipase belongs to hydrolase’s family and is regarded as a versatile biocatalyst for hydrolysis, esterification, and trans-esterification Received: January 25, 2011 Revised: May 13, 2011 Published: May 19, 2011 7744

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reactions.25,26 Our previous work27
29 has demonstrated good activity and enhanced stability of porcine pancreatic lipase (PPL) immobilized on MPS. In this work, the pore sizedependent PPL diffusion and protein distribution in SBA-15 support have been investigated in detail by CLSM and X-ray diffraction (XRD) characterizations in combination with hindered diffusion simulation, intraparticle diffusion analysis, and apparent kinetics calculation. It is expected to promote the comprehension of PPL adsorption in mesoporous material. PPL has been proved more stable in conformation than other proteins.30 So the conformational changes can be negligible in protein adsorption study.

εr is defined as actual pore volume divided by starting pore volume.

2. THEORY

The initial condition is t = 0 and Cs = 0. Each pore requires two boundary conditions, one at the inlet and the other at the outlet.

εr ¼







D Aεr

DCp
D Dz




 ¼

DCp DV þ Cp V Dt Dt






ð1Þ

DCs

ADz Dt



ð2Þ where V ¼ Aεr Dz

ð3Þ

Because A and ∂z are constants, eq 4 is obtained. 
 DCp Cp Dεr DCp 1 D 1 DCs εr
D þ ¼

εr Dz εr Dt Dt εr Dt Dz

ð4Þ

The diffusivity of protein in solutions can be estimated from the Stokes
Einstein equation22 D0 ¼

RT kB T ¼ 6πμR0 AV 6πμR0

ð5Þ

ð9Þ

In order to solve eq 4, the relation of Cs and Cp can be associated with Langmuir constants derived from the adsorption isotherm.33 Cp ¼

Hindered Diffusion Stimulation. For the protein adsorption in cylindrical pores, a continuity equation can be derived as follows:31

in
out ¼ accumulation
production

πr 2 Dz r2 2 ¼
 DBJH DBJH 2 π Dz 2 2


Cs ðCT
Cs ÞKL

z ¼ 0,

at the inlet,

Cs ¼

ð10Þ

CT KC0 1 þ KC0

ð11Þ

DCp ¼0 ð12Þ Dz The boundary condition is used since each protein diffuses from the pore mouths to the middle of one channel where it is reasonable to assume that the flux is zero at z = L/2 due to the symmetry of the channel. Intraparticle Diffusion Model. Intraparticle diffusion model is based on the theory proposed by Weber and Moris.24 According to the theory, if intraparticle diffusion is the ratecontrolling factor, the uptake of adsorbate is proportional to the square root of contact time in the course of adsorption. Boundary layer diffusion involves mass diffusion to the external surface of the particle through a hypothetical boundary layer surrounding the particle. When the boundary layer diffusion gives an effect, the plot of qt versus t0.5 does not pass through the origin, and the intraparticle diffusion equation can be described as eq 13,34,35 where ki is the intraparticle diffusion rate constant. at the outlet,

z ¼ L=2,

D

qt ¼ ki t 0:5 þ C

ð13Þ

The radius R0 of protein can be inferred from the molecular long axis a and short axis b by eq 632 pffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi ða2
b2 Þ R0 ¼ ð6Þ pffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi! a þ ða2
b2 Þ ln b

An extrapolation of the linear portion of the plot back to the axis yields the intercept C which is proportional to the extent of boundary layer thickness.34 The adsorbate amount in the mesopores can be estimated from eq 14, where te is the time needed to reach the equilibrium.

The pore diffusivity D is related to the bulk diffusivity D0, and normally expressed as a function of λ, the ratio between the molecule and the pore diameter. The Higdon and Muldowney equation23 is valid for 0 < λ < 0.95.

The adsorbate amount on the external surface qex, obtained by subtracting adsorbate amount in the pores from the total, just equals to the value of intercept C. The adsorption amount per unit area in internal or external surface can also be calculated. qmeso ð15Þ Qmeso ¼ Ameso

D 9 ¼ 1 þ λ ln λ
1:56034λ þ 0:528155λ2 þ 1:91521λ3 D0 8
2:81903λ4 þ 0:270788λ5 þ 1:10115λ6
0:435933λ7

qmeso ¼ ki te0:5

ð7Þ Equation 8 gives the effective pore radius r when a certain amount of proteins have been adsorbed. r ¼

DBJH 4πR0 3 1
Cs N A 2 3M

!0:5

Qex ¼

qex Aex

ð14Þ

ð16Þ

Apparent Kinetics. Consider the reversible adsorption of protein (PPL) on the contact site Ω of the support

ð8Þ

ka

PPL þ Ω T PPL
Ω kd

7745

ð17Þ

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Table 1. Pore Structure Parameters of SBA-15 Supports sample

DBJH (nm) Ameso (m2 g
1) Aex (m2 g
1) Vmeso (cm3 g
1)

SBA-15
5.6

5.6

454

29

0.61

SBA-15
8.0

8.0

422

47

0.78

SBA-15
9.7

9.7

438

87

0.97

Equation 18, derived by Poltorak et al.,36 can be used to determine the kinetic constants.36,37
 1 1
aθ 1 ln ¼ ka C0
ka k d C 0 t ð1
aÞ ð18Þ t 1
θ 2 where θ = qt/qmax and a =qmax/C0V. qmax is estimated using the kinetic data by eq 19, which is based on the hypothesis of irreversible adsorption and approximately valid when a is close to 1, in other words, when qmax is not much different from C0V.36,37 1 1 1 ¼ þ qt qmax qmax ka C0 t

ð19Þ

3. EXPERIMENTAL SECTION Materials. Porcine pancreatic lipase (PPL) from Sigma was stored at 0
4 °C and used without further purification. Fluorescein isothiocyanate (FITC) from Sigma, P123 (EO20PO70EO20, molecular weight = 5800) from Aldrich, tetraethylorthosilicate (TEOS) from Beijing Yili Chemical Reagent LTD, and other reagents of analytical purity were all used as received. Synthesis of Mesoporous Materials. SBA-15 materials were synthesized according to a published procedure38 by modifying pH and temperature during the synthetic process. Two g of P123 was dissolved in the mixture of 64 mL of deionized water and 8.2 mL of 12 M HCl solution. Thereafter, 4.43 g of TEOS was added and stirred at 45 °C for 24 h. The mixture was then transferred to a Teflon bottle and heated for 48 h. To obtain the samples with different pore sizes, the aging temperature was set at 80, 130, and 140 °C, respectively. The solid was filtered, washed with deionized water, and dried at ambient temperature. P123 was removed by calcination in air at 500 °C for 4 h. The resulting samples were designated as SBA-15-x, where x represented the pore diameter. The detailed pore parameters of the supports are listed in Table 1. Immobilization of PPL. In a typical adsorption experiment, support powder (0.5 g) was added at 25 °C with agitation to 1 mg mL
1 solution (125 mL) of PPL in pH 7.5 Tris-HCl buffer. The adsorbed amount of PPL on solid support was calculated by subtracting the PPL in supernatant from total PPL amount. The supernatants were filtered with 0.22 μm microporous membrane, and then subjected to determination of protein concentration by Bradford assay. PPL Labeling and CLSM Study. Fluorescein isothiocyanate (FITC) with excitation at 488 nm and emission at 525 nm39 was used to label PPL as follows: FITC was first dissolved in DMSO to prepare a 1.0 mg mL
1 FITC solution. For 10 mL of PPL solution (1 mg mL
1, pH 7.5), 150 μL of FITC solution was slowly added under stirring. The conjugation continued overnight in the dark at 4 °C. The unreacted FITC was removed by dialysis method using dialysis tubing (Viskase, 12000 Da cutoff). The adsorption of FITC-labeled PPL was carried out for 24 h in the batch mode following the same procedure as used for the

Figure 1. Adsorption amount of PPL on (a) SBA-15
5.6, (b) SBA15
8.0, and (c) SBA-15
9.7 as a function of time.

unlabeled PPL. The solids were recovered by centrifugation, rinsed three times with buffer, and then ultrasonically dispersed in buffer for 5 min before the CLSM analysis. Characterization. Nitrogen sorption isotherms were measured at 77 K on a Quantachrome Autosorb-1 system. The pore diameters were calculated using the BJH method based on desorption branch. The surface area and pore volume of primary mesopores and external area were calculated by Rs-plot method.40,41 Scanning electron micrographs (SEM) were taken on a Cambridge S-250MK3 microscope. Transmission electron micrographs (TEM) were taken on a FEI Tecnai 20 electron microscope operating at 200 kV. The UV absorption data were collected on a Shimadzu UV-2501 spectrometer. Powder XRD patterns were obtained on a Bruker D8 focus X-ray diffractometer with Cu KR radiation. CLSM observation was carried out on a Leica TCS SP2 confocal microscope.

4. RESULTS AND DISCUSSION PPL Adsorption. Figure 1 displays the adsorption amount of PPL on SBA-15 supports as a function of time. The pore sizes of SBA-15 supports are chosen slightly larger but still in the same order of magnitude as the dimensional sizes of PPL (1.1  2.1  4.5 nm3).42,43 The PPL adsorption on SBA-15
9.7 and SBA15
8.0 reaches a plateau amount rapidly in 4 and 5 h, whereas on SBA-15
5.6 it undergoes a rapid adsorption in the initial 2 h and then a gradual approach to the plateau in another 6 h. With the same dosage of PPL relative to SBA-15 for the immobilization in each case, the plateau amount of adsorbed PPL shows no distinct difference and yet increases slightly with elevated pore size of the support. The XRD patterns of SBA-15 materials before and after PPL adsorption are shown in Figure 2. The XRD pattern in each case exhibits well-resolved peaks indexed as (100), (110), and (200) reflections in hexagonal symmetry, revealing that each material has a well-ordered long-range mesostructure.38 SBA-15
8.0 and SBA-15
9.7 display visible reduction in the XRD reflection intensity after PPL uptake. Such a decrease in the reflection intensity can be interpreted as reduction of contrast in density between the silica walls and the pores due to the proteins inside the pores.44 The (100) intensity reduction is found more visibly for SBA-15
8.0 than SBA-15
9.7. No obvious change in the XRD intensity is observed for SBA-15
5.6 after PPL adsorption, indicative of a large amount of mesoporous spaces unoccupied. 7746

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Figure 2. XRD patterns of before (a) and after (b) PPL uptake for (A) SBA-15
5.6, (B) SBA-15
8.0, and (C) SBA-15
9.7.

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Figure 4. TEM image of SBA-15 support (SBA-15
5.6 as a demonstration).

Figure 5. Diffusion coefficient of PPL in mesopores as a function of time and pore length.

Figure 3. SEM images, CLSM images, and corresponding line-scanning light intensity profiles of SBA-15
5.6 (left column), SBA-15
8.0 (middle column), and SBA-15
9.7 (right column) with FITC-labeled PPL loaded.

To confirm the PPL uptake and investigate the protein distribution, PPL has been labeled with FITC and then subjected to adsorption on SBA-15 supports. Figure 3 displays the SEM and CLSM images of the SBA-15 materials with FITC-labeled PPL adsorbed. The corresponding light intensity profiles scanned along a line on single particles are also illustrated in Figure 3. As can be seen from the SEM images, the SBA-15 materials consist of rope-like aggregates. The rope length ranges from several to tens of micrometers. In each case, PPL loading is clearly visualized in the CLSM images. However, from the linescanning light intensity, it can be resolved that, for SBA-15
5.6, PPL is more concentrated at the edges than at the center of the particle. However, for SBA-15
8.0 and SBA-15
9.7, PPL population is found uniform in the whole particle. Hindered Diffusion Stimulation. The distribution of the proteins loaded on SBA-15 is further comprehended in this

work by simulating the protein diffusion. As shown in Figure 4, the SBA-15 supports applied here have well-ordered onedimensional cylindrical pores. Since the pore sizes (5.6, 8.0, and 9.7 nm) approach the diameter of the PPL molecule, PPL diffusion in the mesopores is typically hindered or restricted. The simulation is thus implemented with the hindered diffusion model. The model was described by second-order partial differential equations and numerically solved using MATLAB. The viscosity μ of the PPL solution is 0.0129 g cm
1 s
1, measured by viscometer, and the PPL radius R0 is 2.12 nm according to eq 6. The D0 of PPL in the solution is calculated to be 7.99  10
11 m2 s
1 by eq 5. The λ values between PPL and the pore size of 5.6, 8.0, and 9.7 nm are 0.76, 0.53, and 0.44. Equation 7 is therefore befitting to calculate D. The diffusion coefficient (D) of PPL as a function of time and pore length is shown in Figure 5. When there is no protein adsorbed, as the pore size increases from 5.6 to 8.0 nm, the pore diffusivity increases more than 20 times, from 1.42  10
13 to 2.89  10
12 m2 s
1. D in SBA-15
9.7 is estimated as 6.22  10
12 m2 s
1, about 2.2 times as large as in SBA-15
8.0 and 42 times higher than that in SBA-15
5.6. For the three mesopores, since the effective pore radius decreases with time due to the binding of proteins on the surface, D gradually decreases. D is minimized at the pore opening due to the densest proteins, as low as 1.78  10
14 m2 s
1 in the case of SBA-15
5.6, indicating considerable hindrance of diffusion in narrow pores. Figure 6 shows the simulation results of the distribution of PPL adsorbed in the mesopores. PPLs are allowed to gradually 7747

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Figure 7. Intraparticle diffusion plots for PPL adsorption on (a) SBA15
5.6, (b) SBA-15
8.0, and (c) SBA-15
9.7.

Figure 6. Normalized amount of PPL (Cs/CT) adsorbed in the pores of (A) SBA-15
5.6, (B) SBA-15
8.0, and (C) SBA-15
9.7 as a function of time and pore length.

diffuse into the pore with time increment. PPL diffusion gets faster and more PPLs enter deep into the pores with increasing support pore size. After 24 h, proteins have diffused into more than 25 μm in both SBA-15
8.0 and SBA-15
9.7 and display uniform distribution along the pore length. For SBA-15
5.6, the adsorption amount sharply reduces along the pore length and PPL can reach the pore as deep as 5 μm. The simulation illustrates that the hindered diffusion for PPL is much more significant in the pore of 5.6 nm than in larger pores, in accordance with the CLSM observations. Intraparticle Diffusion Analysis. The PPL into the pore of 5.6 nm is much fewer than into the larger pores as revealed by the

hindered diffusion predication and XRD results. However, the actual PPL adsorption amount was observed to display no distinct discrepancy (Figure 1). As reported previously,45 the conventional rope-like SBA-15 had a much larger external area than spherical SBA-15 (e5 m2 g
1). It ranges from 29 to 87 m2 g
1 (Table 1) for the SBA-15 prepared in this work. Therefore a part of PPL might be adsorbed on the external surface. The PPL adsorption is thus to be analyzed using an intraparticle diffusion model, to make a distinction between the PPL adsorbed inside and outside of mesopores. Furthermore, the hindered diffusion in the mesopores is not enough to represent the whole diffusion of PPL; other steps such as boundary layer diffusion should be taken into considerations. The intraparticle diffusion plots for PPL adsorption on the SBA-15 supports are shown in Figure 7. These plots have the same general features, consisting of initial curved and followed linear portions, and finally a plateau. The initial curved portion indicates that boundary layer resistance is involved at the early stage of PPL adsorption and the final plateau represents a state of equilibrium. From the linear portion, the intraparticle diffusion rate can be compared. It is found that ki of SBA-15
8.0 and SBA15
9.7 are similar and much higer than that of SBA-15
5.6 (Table 2). The large intercept suggests that the process is largely of external surface adsorption.35 According to eqs 14
16, about 65% and 54% of adsorbed PPL are distributed inside the pores for SBA-15
8.0 and SBA-15
9.7. For SBA-15
5.6, the percentage decreases to 35%, and a large proportion of PPL stays on the external surface. Apparent Kinetics. Although the amount of PPL in the mesopores with varied pore sizes has been estimated by the intraparticle diffusion analysis, an inconsistency between the hindered diffusion and intraparticle diffusion results emerges. As the pore size increases from 8.0 to 9.7 nm, the PPL amount within the pore (Qmeso) is calculated to decrease (Table 2). In contrast, more PPL has been predicted to diffuse into the pores with 9.7 nm by the hindered diffusion stimulation. The equilibrium adsorption capacity is supposed to be correlative with the intrinsic adsorption rate constant and diffusion effect. To make this point clear, the diffusion-controlled reversible adsorption kinetics is investigated. The apparent adsorption rate constant ka and desorption rate constant kd, depicting the diffusion-adsorption behavior inside the mesopores, can be determined from experimental adsorption data according to eq 18. The corresponding kinetic plots based 7748

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Table 2. Intraparticle Diffusion Parameters for PPL on SBA15 Supports ki sample

C

Qex

Qmeso

(mg g
1 min
0.5) (mg g
1) (mg m
2) (mg m
2) qex:qmeso

SBA-15
5.6 SBA-15
8.0

2.72 7.28

121 66

4.19 1.41

0.14 0.30

65:35 35:65

SBA-15
9.7

7.18

100

1.15

0.26

46:54

indicating easier diffusion and more uniform distribution of proteins in the mesopores sized 8.0 and 9.7 nm than 5.6 nm. Besides pore diffusion, boundary resistance is revealed by the intraparticle diffusion model. The pore with a size of 5.6 nm shows much lower pore diffusion rate and larger boundary thickness than the pores with larger sizes in the PPL adsorption. Approximately 65% of adsorbed PPL are located at the external surface of SBA-15 with a pore size of 5.6 nm. Both of the highest PPL uptake in the mesopore of 8.0 nm and correspondingly the most reduction in XRD intensity suggest the existence of an optimal pore size for “confinement” of PPL. The confinement of PPL has been confirmed by apparent kinetics calculation. These fundamental insights obtained here into the protein adsorption are expected to ultimately contribute to the development of biocatalysts, biosensors, and other biodevices.

’ AUTHOR INFORMATION Corresponding Author

*E-mail: [email protected]. Tel: 86-10-64425280. Fax: 86-1064425385.

Figure 8. Kinetic plots of PPL adsorption on (a) SBA-15
5.6, (b) SBA-15
8.0, and (c) SBA-15
9.7.

Table 3. Kinetic Parameters for PPL Adsorption on SBA-15 Supports sample

ka (mL mg
1 h
1)

kd (h
1)

ka/kd (mL mg
1)

SBA-15
5.6 SBA-15
8.0

0.541 0.770

0.064 0.076

8.41 10.07

SBA-15
9.7

1.181

0.122

9.68

on the adsorption data when the intraparticle diffusion is predominant are shown in Figure 8. Good linear correlation is obtained in each case. From the slope and intercept, ka and kd can be calculated. Both ka and kd decrease as pore size is reduced (Table 3). This attests that small pores not only restrict protein adsorption but also impede the process of protein desorption. The ka/kd quotient is actually a hint of the final adsorption amount. The highest value of ka/kd is found for SBA-15
8.0, indicating that a pore size of about 8.0 nm is the best to hold the PPL inside the pores. It may be the as-called “confinement”. The result is consistent well with the highest uptake of PPL (Table 2) and the most marked decrease of XRD intensity (Figure 2). Smaller pore (