Adsorption dynamics and thermodynamics of Hb on the Hb-imprinted polymer beads

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Reactive & Functional Polymers 68 (2008) 63–69

REACTIVE & FUNCTIONAL POLYMERS www.elsevier.com/locate/react

Adsorption dynamics and thermodynamics of Hb on the Hb-imprinted polymer beads Yong-qing Xia a, Tian-ying Guo a,*, Mou-dao Song a, Bang-hua Zhang a, Bao-long Zhang b a

Key Laboratory of Functional Polymer Materials (Nankai University), Ministry of Education, Institute of Polymer Chemistry, Nankai University, Tianjin 300071, China b Department of Chemistry, Nankai University, Tianjin 300071, China Received 23 October 2006; received in revised form 15 March 2007; accepted 22 October 2007 Available online 30 October 2007

Abstract The adsorption of hemoglobin (Hb) onto the Hb-imprinted polymer beads has been dynamically and thermodynamically investigated. The order of Hb adsorption rate was examined to be 0.7-order, and the rate constant k, apparent activation energy EA, and Arrhenius constant A were calculated. Equilibrium modeling of the adsorption showed that the adsorption of MIP beads to the imprinted molecule Hb was fitted well to the Freundlich equation. From the results of the thermodynamic analysis, standard free energy DG0, standard enthalpy DH0, and standard entropy DS0 were determined. Ó 2007 Elsevier Ltd. All rights reserved. Keywords: Molecularly imprinting; Protein adsorption; Dynamics; Thermodynamics; Hemoglobin

1. Introduction Molecular imprinting is a technique for creating recognition sites for a specific analyte in a synthetic polymer [1–10]. The principle of this technique involves (a) the assembly of polymerizable functional monomer around an imprinted molecule in a solution containing a ratio of cross-linker, (b) polymerization of the mixture, and (c) removal of the imprint molecule to afford the imprinted polymer.

*

Corresponding author. Tel./fax: +86 22 235 015 97. E-mail address: [email protected] (T.-y. Guo).

Chemically and mechanically stable molecularly imprinted polymers (MIPs) have been used as the stationary phase in chromatography [11], as artificial antibodies in immunoassays [12] and as recognition elements in sensors [13]. This technique is a conceptually simple and straightforward method to apply to a wide variety of target molecules. The adsorption isotherm of the MIPs was reported in literature [14–18]. Usually, the Scatchard equation was used to describe the specific binding of the MIP to the imprinted molecule because of the monolayer assumption from the Langmuir basis. Zhou and Zhu used Scatchard equation to describe the adsorption kinetics of Z-aminopyridine and

1381-5148/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.reactfunctpolym.2007.10.018

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metsulfuron by the corresponding MIP [19,20]. Tsai and Miloikovic used a Langmuir model to fit the adsorption isotherms obtained from the imprinted polymers [21,22]. The Freundlich isotherm has also been used in some reports [23,24]. For the optimal design of MIPs, an understanding of the thermodynamics and kinetics properties of the molecular recognition process of imprint molecules by the MIPs is important. Previous studies have been reported on molecularly imprinted polymers with protein with varying chemical structures or techniques, these studies have described mainly the MIPs’ adsorption capacity, their imprinting effect and the mechanism of recognition [25–29]. However, previous works did not provide information about the dynamics and thermodynamics of adsorption. In this paper, we report on the synthesis of polymeric beads which able to recognize Hb. The materials were prepared using macroporous chitosan beads as the functional supporting matrix, acrylamide and N,N0 methylenebisacrylamide as comonomers and cross-linker was polymerized to form interpenetrating polymer networks (IPNs) with chitosan beads. Herein, we focus on the adsorption dynamics and thermodynamics of Hb on the MIPs. The purpose is to increase our understanding of protein adsorption and to prepare reliably and predictably highquality MIPs. 2. Materials and methods 2.1. Materials Chitosan was purchased from Boao Bio-Technology Company, Shanghai (China); the degree of deacetylation degree was 90%. The viscosity average molecular weight of the chitosan was determined to be 503,495 Da by viscometric method. Acrylamide (Am) was purchased from Miou Chemical Factory (Tianjin, China). N,N0 -methylenebisacrylamide (MBA) was purchased from Tianjin Special Reagent Factory (China). Potassium persulfate (KPS) was obtained from Tianjin No. 3 Chemical Reagent Factory. Hemoglobin (Hb) and bovine serum albumin (BSA) were purchased from Sino-America Biotechnology Company, and the protein solutions were prepared using 0.01 M sodium dihydrogen phosphate buffer (pH 6.8). Am and KPS were recrystallized before used. Other chemicals were analytical grade and used as received.

2.2. Methods 2.2.1. Preparation of MIP beads Chitosan solution was prepared by dissolving 3.0 g of chitosan in 100 mL of a 2% (v/v) acetic acid. This solution was dropped through a 7-gauge needle into 2 M sodium hydroxide solutions, and the gelled spheres formed instantaneously. This process was accomplished by using a Model 100 push–pull syringe pump. The formed chitosan beads remained in the sodium hydroxide for 24 h and washed with distilled water. Then, the beads were cross-linked chemically with epichlorhydrin to glucose residue for 6 h in sodium hydroxide solution (pH 10) under 60 °C. The cross-linked chitosan beads were washed with distilled water repeatedly and used as a matrix to produce MIP beads. For the preparation of MIPs, 16.0 g wet crosslinked chitosan beads (use filter to absorb the surface water), 1.9 g Am, 0.1 g MBA, 20.4 mg KPS, 600 mg Hb, and 28 mL 0.01 M sodium dihydrogen phosphate buffer (pH 6.8) were put into a 100 mL, four-necked flask equipped with a nitrogen inlet and a mechanical stirrer. The mixture was stirred continuously under a nitrogen atmosphere for 45 min, then added 5 mL sodium dihydrogen phosphate buffer containing 0.16% (w/v) NaHSO3. The mixture was stirred under the nitrogen atmosphere for 2 h at 4 °C. The formed beads were put into a nylon stocking to press out the surrounding polyacrylamide gel, and the freed chitosan beads were washed with 10% (v/v) acetic acid containing 10% (w/v) SDS solution to desorb the hemoglobin until the color was pale (but was still somewhat colored). Then, the beads were equilibrated with 0.01 M sodium dihydrogen phosphate buffer (pH 6.8) for 24 h. 2.2.2. Adsorption experiments The adsorption capacity was measured by UV spectrophotometry (UV-9100). Different concentrations of Hb solutions were detected by UV spectrophotometer at 280 nm. The wet MIP beads (0.5 g) was placed in a 50 mL conical flask and mixed with 25 mL of a known concentration of Hb solution. The conical flask was oscillated in a constant temperature bath oscillator. The temperature of test solutions was varied in the range 289.15–308.15 K, the concentration of Hb in the solution was determined using a spectrophotometer at 280 nm. The adsorption capacity Q, was calculated based on the difference of Hb concentration before and after

Y.-q. Xia et al. / Reactive & Functional Polymers 68 (2008) 63–69

adsorption, the volume of aqueous solution and the weight of the beads as follows: Adsorption capacity Q = (C0  Ct) V/W, here C0 is the initial Hb concentration (mg/mL), Ct is the Hb concentration (mg/mL) of different time, V is the volume of Hb solution (mL), and W is the weight of the MIP beads (g).

65

-4.4 R2=0.985

-4.5

log -dC0/dt

-4.6 -4.7 -4.8

3. Results and discussion -4.9

3.1. Adsorption dynamics When the adsorbent MIP beads is constant, the main factors that influence the rate constant of the adsorption process are temperature and Hb concentration, so the order of adsorption of Hb on the MIP is using the following rate equation dcA ¼ kcnA dt   dcA log  ¼ log k þ n log cA dt 

ð1Þ ð2Þ

The order of reaction of Hb adsorption n can be determined from the linear plot of log {dcA/dt} versus log cA. To avoid other interference, we use initial concentration to determine the order. Five groups of different Hb initial concentrations are 0.1, 0.2, 0.3, 0.4 and 0.5 mg/mL, respectively. Then the according five groups of the relationship of concentrations and time were determined, as shown in Fig. 1. From the values of the slope of initial concentration, we can get Fig. 2 of log {dc0/

B--C0=0.1 C--C0=0.2

0.50

D--C0=0.3

Concentration (mg/mL)

0.45

E--C0=0.4

0.40

F--C0=0.5

0.35 0.30 0.25 0.20 0.15 0.10 0.05 0

2000

4000

6000

8000

10000

12000

Time (s)

Fig. 1. Relationship between the concentration and time. V = 25 mL; C0 = 0.1, 0.2, 0.3, 0.4, 0.5 mg/mL; the sample quantity (MIP): 0.5 g; T = 25 °C. Error bars represent the standard deviation of data.

-5.0 -1.1

-1.0

-0.9

-0.8

-0.7

-0.6

-0.5

-0.4

-0.3

-0.2

logC0

Fig. 2. Determination of the reaction order. Error bars represent the standard deviation of data.

dt} versus log c0 We can see that the relationship of log {dcA/dt} versus log c0 is linear, its slope is 0.702, so the adsorption reaction order of the MIP beads to Hb is about 0.7. From the adsorption reaction equation: 

dcA ¼ kc0:7 A dt

ð3Þ

Integrating the above equation, we get c0:3 ¼ 0:3kt þ c0:3 0

ð4Þ

The relationship of c0.3 versus t is linear, the slope of the line is 0.3k. For getting the rate constant k, 0.5 g of MIP beads were used and the temperature of the test solutions were 289.3, 293.1, 297.6, 303.4 and 307.6 K, respectively. The initial concentration of Hb is 0.2 mg/mL. From the relationship of the concentration c and time t, we can get the figure of c0.3 versus t at different temperature (Fig. 3), and c0.3 versus t are linear at different temperatures Then, the rate constant k of different temperature is shown in Table 1. Thus, the adsorption rate of MIP beads increased slightly with increasing temperature. A plot of the natural logarithm of the rate constant vs. the reciprocal of absolute temperature is shown in Fig. 4. According to the Arrhenius equation k ¼ AeEa =RT , we know ln k = Ea/RT + ln A, the value of apparent activation energy EA calculated from the slope of the line was 24.6 kJ/mol, the Arrhenius constant A was 0.58, so the adsorption rate constant k = 0.58 exp (24.58  103 J/mol/ RT) s1. Compared with small molecule, the determined rate constant k for the adsorption of Hb to Hb-imprinted polymer in our study, is larger than

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Y.-q. Xia et al. / Reactive & Functional Polymers 68 (2008) 63–69 289.3K 293.1K 297.6K 303.4K 307.6K

0.62 0.60

0.56

0.52 0.50 0.48 0

2000

4000

6000

8000

10000

12000

Time (s)

Fig. 3. Relationship between c0.3 and t at different temperature to get the rate constant k. V = 25 mL; C0 = 0.2 mg/mL; the sample quantity (MIP): 0.5 g; temperature: 289.3, 293.1, 297.6, 303.4 and 307.6 K.

Table 1 Adsorption rate constants for the adsorption of Hb onto the MIP beads and correlation coefficient for the fit lines at different temperature Temperature (K)

289.3

293.1

297.6

303.4

307.6

k (mg/mL)0.3 s1 105 (s1) R2

1.89 0.982

2.65 0.991

3.06 0.984

3.27 0.981

3.76 0.976

R2=-0.973 -4.60

log(k, (mg/ml)0.3s-1)

3.2.1. Langmuir isotherm The equilibrium data were analyzed using the following linearized equation for Langmuir adsorption isotherm, Ce Ce 1 ¼ þ Qe Qmax bQmax

0.54

0.3

Ce (mg/mL)

0.3

0.58

3.2. Adsorption isotherms

-4.64

-4.68

-4.72

-4.76

3.25

3.30

3.35

3.40

ð5Þ

Here Ce is the equilibrium or final concentration of Hb (mg/mL), Qe is the adsorption capacity of Hb adsorbed per unit weight of MIP at equilibrium concentration (mg/g), Qmax and b are the Langmuir adsorption equilibrium constants related to the capacity (mg/g) and energy/intensity (g/mL) of adsorption respectively. A good fit of this equation reflects monolayer adsorption. 3.2.2. Freundlich isotherm The well known Freundlich isotherm used for isothermal adsorption is a special case for heterogeneous surface energy, in which the energy term in the Langmuir equation varies as a function of surface coverage strictly due to variation of the sorption. The use of the Freundlich isotherm to characterize MIP has certain advantages. First, the Freundlich isotherm is based on a heterogeneous exponentially decaying distribution, which fits well to the tailing portion of the heterogeneous distribution of MIPs. Secondly, the logarithmic form of the Freundlich isotherm is easily applied as it can be transformed into a linear function (Eq. (6)). Isotherms that are well fit by the Freundlich isotherm will fall on as straight line when plotted in ln–ln format (ln Qe versus ln Ce). This analysis is a useful diagnostic for identifying sources of error, as deviations from linearity are visually evident. Finally, the slope of the straight line fit yields n, which is a measure of the heterogeneity of a system. A more homogeneous system will have an n value approaching

3.45

1000/T (1/K)

Fig. 4. Relationship between log k and 1000/T to determination activation energy Ea. Error bars represent the standard deviation of data.

the data reported for another molecularly imprinted system [20], in which Lehmann found a k = 5.60 mL lmol1 min1 for the affinity of L-Bocphenylalanine anilide (BFA) to L-BFA-imprinted polymers at room temperature.

Table 2 Langmuir and Freundlich constants for the adsorption of Hb onto the MIP beads Temperature (K)

293.1 297.6 303.4 307.6

Langmuir

Freundlich

Qmax (mg/g)

b (g/ mL)

R2L

Qf (mg/g)

n

R2F

25.46 28.24 36.43 32.86

5.40 4.73 4.76 7.21

0.979 0.984 0.959 0.984

35.41 39.56 55.71 58.90

0.63 0.67 0.70 0.65

0.999 0.999 0.998 0.998

Y.-q. Xia et al. / Reactive & Functional Polymers 68 (2008) 63–69

C =0.1mg/mL 0 C0=0.2mg/mL C0=0.3mg/mL C0=0.4mg/mL C0=0.5mg/mL

5.4

ln(Kc, mg/g)

5.2

C0=0.6mg/mL

5.0 4.8 4.6 4.4 4.2 4.0 3.24 3.26 3.28 3.30 3.32 3.34 3.36 3.38 3.40 3.42

1000/T (1/K)

Fig. 5. Relationship between ln Kc and 1000/T to determination of DH0 at different concentration.

unity and a more heterogeneous system will have an n value approaching zero [30–33]. The Freundlich isotherm is able to produce a direct measurement of the binding properties. Through the calculation of the fitting coefficients, Nt, N, and Qf, the total

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number of binding sites can be calculated to be 0.85 ± 0.03 mg g1 (Eq. (7)) [33]. Qe ¼ Qf C ne

ð6Þ 2

N t ¼ 2:303Qf nð1  n Þe

2:303n log K

ð7Þ

From linear plots of (Ce/Qe) versus Ce and ln Q versus ln Ce, the parameters in the Langmuir and Freundlich equations can be determined. The Langmuir and Freundlich constants with the correlation coefficients are given in Table 2. It can be concluded that the linear fit with the Freundlich equation were comparably better (R2 > 0.998), which means that the adsorption of Hb onto the MIP beads is a multiple layer adsorption. Therefore, although the adsorption processes were complicated, there was a tendency for a physical adsorption to exist at the surface between the Hb protein and the Hbimprinted polymer beads. 3.3. Thermodynamic studies In order to explain the effect of temperature on the adsorption thermodynamic parameters, standard free energy DG0, standard enthalpy DH0, and

Table 3 The thermodynamic parameters for the adsorption of Hb onto the MIP beads Temperature (K)

Adsorption (%)

Kc

DG0 (kJ/mol)

DH0 (kJ/mol)

DS0 (J/mol)

Concentration (mg/mL)

293.1 297.6 303.4 307.6

73.2 72 78.2 81.9

137.08 128.71 179.31 226.13

11.99 11.10 13.07 13.84

29.78

142.54 140.64 141.41 142.06

0.1

293.1 297.6 303.4 307.6

66.1 66.8 71.6 78.3

98.22 100.56 126.02 180.92

11.17 11.39 12.18 13.27

32.77

149.98 148.67 148.37 149.96

0.2

293.1 297.6 303.4 307.6

60.5 61.4 70.8 74.7

76.95 79.53 122.44 147.63

10.58 10.81 12.11 12.75

38.84

168.67 167.16 168.15 168.04

0.3

293.1 297.6 303.4 307.6

59.1 60.8 66.3 71.5

72.01 77.39 98.26 125.44

10.42 10.74 11.56 12.33

30.28

138.92 138.12 138.09 138.82

0.4

293.1 297.6 303.4 307.6

54.8 56.4 65.6 67.7

60.66 64.75 95.53 104.98

10.00 10.30 11.49 11.88

32.98

146.70 145.72 146.76 146.12

0.5

293.1 297.6 303.4 307.6

53.3 54.6 64.2 67.6

57.14 60.07 89.67 104.24

9.86 10.11 11.33 11.86

35.47

154.70 153.48 154.45 154.17

0.6

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standard entropy DS0 were determined. To calculate the value of the parameters, the following equations were used: 0

DG ¼ RT ln K c ln K c ¼

DH 0 þc RT

ð8Þ ð9Þ

DH 0  DG0 ð10Þ T Here R is the gas constant and Kc is equilibrium constant. The equilibrium constant was calculated from DS ¼

Kc ¼

Cp Cs

ð11Þ

Here Cp and Cs are the equilibrium concentrations of Hb on the MIP beads and solution, respectively. A linear plot of ln Kc against 1000/T with a slope of DH0/(R  10,000) at different initial Hb concentrations are presented in Fig. 5. The values of DH0 at different initial concentrations obtained for the adsorption of Hb onto the MIP beads were from 29.8 to 38.8 kJ/mol, which directly indicates that the adsorption was endothermic. Higher temperature was favored for the adsorption. The calculated thermodynamic parameters for the adsorption of Hb onto the MIP beads at different temperature and different initial concentrations are given in Table 3. A similar protein adsorption study were designed to experimentally measure thermodynamic parameters for the adsorption of mid-chain lysine residues on a hydrophilic silica glass surface in phosphate buffered saline [34]. This work resulted in values of DH0 = 0.97 ± 0.55 kJ/mol, DG0 = 0.30 ± 0.17 kJ/mol, and TDS0 = 0.71 ± 0.55 kJ/mol. That means the response is unfavorable to adsorption, that behavior can be explained by the effect of water on each of the functional groups during adsorption. The negative values of standard free energy DG0 in our molecularly imprinting methods are indicative of the spontaneous nature of the adsorption process. Compared with the different temperature, the higher temperature has lower standard free energy; compared with different initial concentrations, lower initial concentration has lower standard free energy. Therefore, low initial concentration or higher temperature favored the Hb protein to be adsorbed onto the MIP beads in our study system.

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