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Immobilization of glucose oxidase onto gold nanoparticles with enhanced thermostability
Biochemical and Biophysical Research Communications 355 (2007) 488–493 www.elsevier.com/locate/ybbrc
Immobilization of glucose oxidase onto gold nanoparticles with enhanced thermostability Dongxiang Li a, Qiang He a
a,b
, Yue Cui a, Li Duan a, Junbai Li
a,*
Beijing National Laboratory for Molecular Sciences (BNLMS), International Joint Lab, CAS Key Lab of Colloid and Interface Science, Institute of Chemistry, Chinese Academy of Sciences, Beijing 100080, PR China b Max Planck Institute of Colloids and Interfaces, 14476 Golm/Potsdam, Germany Received 27 January 2007 Available online 8 February 2007
Abstract Immobilized proteins and enzymes were widely investigated in medical field as well as in food and environmental fields. In this paper, glucose oxidase (GOD) monolayer was covalently immobilized on the surface of gold nanoparticles (AuNPs) to fabricate bioconjugate complex. The citrate-stabilized AuNPs were first functionalized by a carboxyl-terminated alkanethiol and the terminal carboxyl groups were subsequently bonded with side-chain amino groups of protein surface through EDC/NHS coupling reaction. The enzyme activity assays of the obtained bioconjugates display an enhanced thermostability and similar pH-dependence behavior in contrast with that of free enzyme. Such GOD/AuNPs bioconjugates can be considered as a catalytic nanodevice to construct nanoreactor based on glucose oxidation reaction for biotechnological purpose. 2007 Elsevier Inc. All rights reserved. Keywords: Immobilization; Glucose oxidase; Gold nanoparticles; Bioconjugate; Enzyme activity
In past decades, gold nanoparticles (AuNPs) had attracted a continuous interest due to their unusual properties in electronics, optics [1,2], especially in biotechnology and nanotechnology fields involving biosensors [3,4], DNA hybridization [5–7], and biocatalysts [8]. Many methods of AuNPs’ modification with alkanethiols provided different functional gold surfaces for facile combinations with biomolecules [9–11]. Moreover, the general covalent combination strategy between protein and solid surfaces provided a stable linkage with more feasibility as advantages [12,13]. In elctrochemistry, AuNPs attached on an electrode were covalently modified by glucose oxidase (GOD) to fabricate glucose biosensors [14]. On the other hand, GOD was also immobilized on surface of polystyrene latex or mesostructured silica to investigate the systemic stability and enzymatic properties [15–17]. McShane reported that
*
Corresponding author. Fax: +86 10 82612629. E-mail address: [email protected] (J. Li).
0006-291X/$ - see front matter 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2007.01.183
the immobilized GOD in alginate microspheres could be applied to fabricate optical glucose sensor systems for implantable biosensor purpose [18,19]. Such immobilized enzymes could be widely utilized to make chemical biosensors not only in the medical field but also in the food and environmental fields [20]. A well-known coupling reaction of N-ethyl-N 0 -(3-dimethylaminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS) was recently reported to construct protein films on the carboxyl or amino-terminated surface [21,22]. In this paper, we fabricated the covalently-linked bioconjugates of GOD/AuNPs in aqueous suspension through such coupling reaction as shown in Scheme 1. The citrate-stabilized AuNPs were first modified by 11mercaptoundecanoic acid (MUA) under protection of nonionic surfactant. Subsequently, the peripheral carboxylic groups were activated by EDC/NHS as coupling agents. The as-formed NHS-terminated AuNPs covalently combined with the side-chain amino groups on the surface of protein GOD and the target bioconjugates were
D. Li et al. / Biochemical and Biophysical Research Communications 355 (2007) 488–493
489
Scheme 1. Schematic illustration of the fabrication of the GOD/AuNPs bioconjugates.
constructed. These assembled GOD/AuNPs bioconjugates in aqueous suspension can maintain their enzyme activity with enhanced thermostability and similar pH dependence. Such bioconjugates would greatly reduce the protein leaching in further application process due to the covalent bond linkage. As an original purpose, they were designed as a fundamental catalytic nanodevice in biomimetic nanoreactor, such as phospholipids-based capsules in previous works [23,24]. Materials and methods Materials. GOD (200 U/mg) was purchased from Sigma. MUA and NHS were obtained from Aldrich. EDC, o-dianisidine, peroxidase from horseradish (HRP, 858 U/mg) were purchased from Fluka. Aurate chloride, sodium citrate, glucose, poly(oxylethene), sorbitan monolaurate (Tween-20), and other chemicals were obtained from Beijing Chemical Reagents Co., China. All chemicals were used as received. The deionized water was prepared in a three-stage Millipore Milli-Q Plus 185 purification system with a resistivity above 18.2 MX cm. All glassware were treated with potassium dichromate/sulfuric acid digestant and then cleaned with tap water and deionized water. Preparation of alkanethiol MUA-modified AuNPs. The citrate-stabilized AuNPs were freshly synthesized through the reduction of aurate chloride by sodium citrate in boiling solution as a conventional method [25]. The subsequent MUA-modified AuNPs were prepared by ligand exchange between mercapto-carboxylic acid and citrate groups under the protection of nonionic surfactant Tween-20 [26]. As a typical prescription, 2.0 mL colloidal gold (approximately 0.8 nM) was gently added 2.0 mL phosphate buffer (10 mM, pH 6.8, with 0.2 mg/mL Tween-20) and the mixture was incubated for 30 min. Then 2.0 mL MUA solution (0.5 mM in 1:3 alcohol/H2O) was added into the mixture, followed by a gentle shake for 5 h for a complete chemisorption of alkanethiol onto gold surface. The final mixture was centrifuged to remove excess alkanethiol and resuspended in phosphate buffer (10 mM, pH 6.8, with 0.2 mg/mL Tween-20). Fabrication of GOD/AuNPs bioconjugates. The combination of GOD and AuNPs was based on the EDC/NHS coupling reaction [22]. In detail, the above MUA-modified AuNPs were reacted with a mixture of freshly prepared 50 mM NHS and 200 mM EDC solution for 10 min. Then asformed NHS-terminated AuNPs were separated by centrifugation and resuspended in a phosphate buffer (10 mM, pH 6.8, with 0.2 mg/mL Tween-20) under ultrasonication for the next wash. After discarding the supernatant, the remaining NHS-terminated AuNPs were incubated with 0.8 mg/mL GOD in phosphate buffer (10 mM, pH 6.8) for more than 12 h under nitrogen atmosphere. The resultant mixture was centrifuged to discard free GOD and washed by phosphate buffer with Tween-20. The target GOD/AuNPs bioconjugates were finally dispersed under ultrasonication in phosphate buffer (0.1 M, pH 5.8) and stored at 4 C. Enzyme activity assays. The activity of the GOD/AuNPs bioconjugates and free GOD were examined spectrometrically at k = 460 nm based on the change of solution color resulting from oxidation of o-dianisidine by the reaction product hydrogen peroxide from glucose in presence of HRP, in which the chemical equations are as follows [18,27].
glucose oxidase
b-d-Glucose þ O2 þ H2 O ! d-gluconic acid þ H2 O2 peroxidase
H2 O2 þ o-dianisidine ðreducedÞ ! o-dianisidine ðoxidizedÞ þ H2 O Typically, 2.5 mL of a 0.33 mM o-dianisidine solution in 0.1 M buffer, 0.3 mL 18% glucose solution, and 0.1 mL 0.02% HRP were mixed in a quartz cell as a substrate. Then, 0.010 mL suspension of the bioconjugates or free GOD solution was added into the mixture and the light absorption of the mixture was immediately recorded for 2 min. The slope of fitted line to recorded absorbance curve was proportional to enzyme activity in the same mass of enzyme by the theoretical analysis. The equivalent volume of obtained bioconjugate suspension or free enzyme solution was employed in all assays to get comparable result. The enzyme concentration in bioconjugates suspension and free GOD solution were adjusted at an approximately same condition to reduce systemic errors. The thermostability measurements were performed after the samples were incubated at a certain temperature for 10 min in a water bath and the pH-dependence measurements were carried out in buffers with pH from 2.1 to 9.1. Characterizations and instrumentations. FT-IR spectra were recorded on a TENSOR 27 instrument (BRUCKER) by dropping the samples onto CaF2 plates followed by a vacuum drying. The UV–vis spectra of AuNPs in fabrication stages and the spectrometric absorption data in catalytic activity assays were collected on a U-3010 UV–vis spectrometer (HITACHI) using 1 cm quartz cell. Transition electron microscopy (TEM) images were obtained from a TECNAI 20 microscope (PHILIPS) operated at 120 kV. X-ray photoelectron spectroscopy (XPS) data were analyzed on an ESCALab220i-XL electron spectrometer (VG Scientific) using 300 W AlKa radiation at about 3 · 109 mbar and the binding energies were referenced to the C1s line at 284.8 eV from adventitious carbon.
Results and discussion Introduction of alkanethiol ligands with different terminal groups can functionalize the surface of AuNPs and the formed self-assembled monolayer of thiols on gold surfaces can drastically reduce nonspecific protein adsorption [28]. Herein, as shown in Scheme 1, carboxyl-terminated AuNPs were obtained by MUA ligand exchange from original citrate-stabilized AuNPs in the presence of Tween-20, which is prior physically adsorbed around gold particles as a protective nonionic surfactant [26]. Subsequently, the carboxylate groups were activated by esterification of NHS catalyzed by the water-soluble carbodiimide EDC. As the resultant NHS-terminated AuNPs were mixed with GOD solution, the side-chain amino groups on GOD surfaces displaced the terminal NHS groups of AuNPs in a phosphate buffer [21,22]. Thus a GOD monolayer was immobilized onto the surface of AuNPs through covalent bonds, that is, the GOD/AuNPs bioconjugates were obtained. Fig. 1A shows the UV–vis spectra of different AuNPs in four stages, in which the curve peak indicates the character-
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modified AuNPs, the distinct peaks appeared at 2918 and 2849 cm1 are ascribed to the vibrational stretches of –CH2 groups of long alkane chains (curve b), while the carbonyl groups of terminal carboxylic acid are shown at 1700 cm1. This indicates the successful modification of AuNPs by MUA ligands. For NHS-terminated AuNPs, it displays a different profile (curve c). A new peak at 1742 cm1 is contributed by the succinimidyl carbonyl group, and the two weak peaks at 1830 and 1782 cm1 are attributed to the band splitting of the ester carbonyl C@O stretching vibration, which is similar to the self-assembled layer of the same chemical component on planar gold surface [31]. It demonstrates the presence of NHS esterification of carboxylic groups. For the GOD/AuNPs bioconjugates, it displays the mainly characteristic peaks at 1650 and 1540 cm1 due to the secondary amide C@O stretching and at 3290 cm1 of hydroxyl groups from protein GOD (curve d) [17]. Therefore, FT-IR results provide evidential component information of different modified AuNPs. Moreover, the NHS-terminated AuNPs and the GOD/ AuNPs bioconjugates were further analyzed by XPS as shown in Fig. 2. The contributions of elements, O, N, C,
Fig. 1. UV–vis (A) and FT-IR spectra (B) of citrate-stabilized AuNPs (curve a), MUA modified AuNPs (curve b), NHS-terminated AuNPs (curve c) and the GOD/AuNPs bioconjugates (curve d).
istic surface plasmon resonance (SPR) of AuNPs [2]. It is obvious that the SPR of MUA-modified AuNPs (curve b) is about 525 nm with a red shift of 5 nm compared to that of original citrate-stabilized AuNPs (curve a, 520 nm). This denotes that alkanethiol molecules are adsorbed on gold surface by Au–S interaction and a soformed dielectric monolayer of alkanethiol around AuNPs results in the red shift [29]. For NHS-terminated AuNPs, the SPR peak at 535 nm (curve c) maybe implies the surface change of metal nanoparticles with a partial aggregation. This is because the NHS esterification of terminal carboxyl group can destroy carboxyl ionization and cause charge neutralization on nanoparticle surface, and result in colloidal particle aggregation. Successively, the suspension of final bioconjugates (curve d) displays a SPR peak at 539 nm. The red shift is only 14 nm compared with MUA-modified AuNPs, this denotes such bioconjugates still maintain a relatively good dispersity although a little of aggregation occurs [30]. Fig. 1B shows the FT-IR spectra of modified AuNPs at different stages. For citrate-stabilized AuNPs, absorbance at about 1690 cm1 represents the citrate component by an evidential carboxylate groups (curve a). For MUA-
Fig. 2. XPS survey spectra (A) and deconvolution of C1s spectra (B) of NHS-terminated AuNPs (curve a) and the GOD/AuNPs bioconjugates (curve b) on CaF2 plates.
D. Li et al. / Biochemical and Biophysical Research Communications 355 (2007) 488–493
S and Au atoms are displayed in Fig. 2A. The binding energy of Au 4f7/2 of both samples exhibits at 83.2 eV, which is slightly higher than that of citrate-stabilized AuNPs at 82.7 eV. It means that the chemical interaction between gold and sulfur atoms occurs. Comparing with Au, the signals of C, N, and O of the GOD/AuNPs bioconjugates are obviously stronger than those of NHS-terminated AuNPs, indicating that AuNPs in bioconjugates are densely enwrapped by protein molecules. The peak-fitting of C1s for these two samples displays three different carbon atoms with binding energy at 288.2, 286.2, and 284.8 eV, respectively, as shown in Fig. 2B. The different carbon assignment of NHS (curve a) involves the carbonyl (zone III), -SC (zone II) and -CH2 of alkane chains (zone I), while that of GOD/AuNPs (curve b) shows the individual contribution from carboxyl (zone III), peptide (zone II) to alkane chains (zone I) of protein molecules. The morphology of different modified AuNPs was characterized by TEM. Fig. 3A shows the well-dispersed cit-
491
rate-stabilized AuNPs with unique size of about 20 nm in diameter. The subsequent MUA-modified AuNPs (Fig. 3B) also exhibit a good dispersity under protection of nonionic surfactant Tween-20. Fig. 3C and D show the GOD/AuNPs bioconjugate particles before and after stained by 1% silver nitrate, respectively. It is obvious that the AuNPs cores are well dispersed after they are conjugated with GOD, but the peripheral protein layer cannot be imaged due to the low electron resistance of protein molecules in TEM examination. However, after the sample was stained by silver nitrate, the GOD layer was visualized since positive silver ions were reduced by protein GOD molecules, resulting in the formation of silver nanoparticles around protein domains as shown in Fig. 3D. It is reported that GOD immobilized in polyelectrolyte multilayer films and assembled on polystyrene particles [15] or quartz slides [32] can enhance its enzyme stability with respect to temperature and pH environments. The GOD immobilized on magnesium silicate microparticles
Fig. 3. TEM images of citrate-stabilized AuNPs (A), MUA modified AuNPs (B), and the GOD/AuNPs bioconjugates before (C) and after stained by 1% silver nitrate (D).
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exhibits a resistance to a high pH value and high buffer concentration [33]. Similarly, here we detected the enzyme thermostability and pH-dependence of such assembled bioconjugates. Fig. 4A gives the relative activity of the bioconjugates (curve a) and free enzyme (curve b) determined at different pH values. As a whole, curve a and b display a resembling profile with a maximum activity at pH 5.8 and a deactivation at pH 2.1. However, at pH 9.1 the bioconjugates still remain a tiny relative activity (5.4%). This indicates that GOD/AuNPs bioconjugates can hold the enzyme activity at different pH. Fig. 4B shows the thermostability of as-prepared bioconjugates (curve a) and free enzyme (curve b). The activity assays were examined after the samples were incubated at a certain temperature for 10 min [15]. It is seen that both immobilized GOD on bioconjugates and free GOD in solution have a maximum activity at 40 C and a sharp activity reduction occurs when the temperature is above 50 C. The relative activity of enzyme on bioconjugates remains, respectively, 57.3% and 11.1% of their activity after incubated at 60 and 70 C, which are higher than that of the free enzyme. This result shows that the GOD/AuNPs bioconjugates are more stable against high temperature in contrast with free enzyme in solution, which is consistent with the results of GOD assembled into polyelectrolyte multilayer [15]. In summary, glucose oxidase can be successfully immobilized on the surface of carboxyl-terminated alkanethiol
Fig. 4. Enzyme pH dependence (A) and thermostability (B) of the GOD/ AuNPs bioconjugates (curve a) and free GOD in solution (curve b).
modified AuNPs through the EDC/NHS coupling reaction. The GOD/AuNPs bioconjugates have a good dispersity in aqueous suspension and have a better stability at high temperature and display similar pH dependence in contrast to the free enzyme according to enzymatic catalysis examination. Such bioconjugate GOD/AuNPs nanoparticles can be expected as catalytic nanodevice to construct nanoreactors based on glucose oxidation reaction in biological applications. Acknowledgments We acknowledge the financial supports of this research by National Nature Science Foundation of China (Nos. 20471063 and 90206035), Chinese Academy of Sciences, and the collaborated project of the German Max Planck Society. References [1] C.M. Niemeyer, Nanoparticles, proteins, and nucleic acids: biotechnology meets materials science, Angew. Chem. Int. Ed. 40 (2001) 4128–4158. [2] M.-C. Daniel, D. Astruc, Gold nanoparticles: assembly, supramolecular chemistry, quantum-size-related properties, and applications toward biology, catalysis, and nanotechnology, Chem. Rev. 104 (2004) 293–346. [3] Y. Xiao, F. Patolsky, E. Katz, J.F. Hainfeld, I. Willner, Plugging into enzymes: nanowiring of redox enzymes by a gold nanoparticle, Science 299 (2003) 1877–1881. [4] T. Liu, J. Tang, L. Jiang, The enhancement effect of gold nanoparticles as a surface modifier on DNA sensor sensitivity, Biochem. Biophys. Res. Commun. 313 (2004) 3–7. [5] Y. Cheng, C. Pun, C. Tsai, P. Chen, An array-based CMOS biochip for electrical detection of DNA with multilayer self-assembly gold nanoparticles, Sensor Actuator B 109 (2005) 249–255. [6] J.J. Storhoff, S.S. Marla, P. Bao, S. Hagenow, H. Mehta, A. Lucas, V. Garimella, T. Patno, W. Buckingham, W. Cork, U.R. Mu¨ller, Gold nanoparticle-based detection of genomic DNA targets on microarrays using a novel optical detection system, Biosens. Bioelectron. 19 (2004) 875–883. [7] S. Liu, J. Li, L. Jiang, Surface modification of platinum quartz crystal microbalance by controlled electroless deposition of gold nanoparticles and its enhancing effect on the HS-DNA immobilization, Colloid Surface A 257–258 (2005) 57–62. [8] L. Pasquato, P. Pengo, P. Scrimin, Functional gold nanoparticles for recognition and catalysis, J. Mater. Chem. 14 (2004) 3481–3487. [9] S. Park, A.A. Lazarides, C.A. Mirkin, P.W. Brazis, C.R. Kannewurf, R.L. Letsinger, The electrical properties of gold nanoparticle assemblies linked by DNA, Angew. Chem. Int. Ed. 39 (2000) 3845– 3848. [10] C. Mangeney, F. Ferrage, I. Aujard, V. Marchi-Artzner, L. Jullien, Q. Ouari, E.D. Rekai, A. Laschewsky, I. Vikholm, J.W. Sadowski, Synthesis and properties of water-soluble gold colloids covalently derivatized with neutral polymer monolayers, J. Am. Chem. Soc. 124 (2002) 5811–5821. [11] J.M. Abad, S.F.L. Mertens, M. Pita, V.M. Fernandez, D.J. Schiffrin, Functionalization of thioctic acid-capped gold nanoparticles for specific immobilization of histidine-tagged proteins, J. Am. Chem. Soc. 127 (2005) 5689–5694. [12] M.C. Parker, N. Patel, M.C. Davies, C.J. Roberts, SJB. Tendler, P.M. Willams, A novel organic solvent-based coupling method for the preparation of covalently immobilized proteins on gold, Protein Sci. 5 (1996) 2329–2332.
D. Li et al. / Biochemical and Biophysical Research Communications 355 (2007) 488–493 [13] B.L. Frey, R.M. Corn, Covalent attachment and derivatization of poly(L-lysine) monolayers on gold surfaces as characterized by polarization-modulation FT-IR spectroscopy, Anal. Chem. 68 (1996) 3187–3193. [14] S. Zhang, N. Wang, H. Yu, Y. Niu, C. Sun, Covalent attachment of glucose oxidase to an Au electrode modified with gold nanoparticles for use as glucose biosensor, Bioelectrochemistry 67 (2005) 15–22. [15] F. Caruso, C. Schu¨ler, Enzyme multilayers on colloid particles: assembly, stability, and enzymatic activity, Langmuir 16 (2000) 9595– 9603. [16] M. Fang, P.S. Grant, M.J. McShane, G.B. Sukhorukov, V.O. Golub, Y.M. Lvov, Magnetic bio/nanoreactor with multilayer shells of glucose oxidase and inorganic nanoparticles, Langmuir 18 (2002) 6338–6344. [17] J.L. Blin, C. Ge´rardin, C. Carteret, L. Rodehu¨ser, C. Selve, M.J. Ste´be´, Direct one-step immobilization of glucose oxidase in wellordered mesostructured silica using a nonionic fluorinated surfactant, Chem. Mater. 17 (2005) 1479–1486. [18] H. Zhu, R. Srivastava, J.Q. Brown, M.J. Mcshane, Combined physical and chemical immobilization of glucose oxidase in alginate microspheres improves stability of encapsulation and activity, Bioconjugate Chem. 16 (2005) 1451–1458. [19] J.Q. Brown, R. Srivastava, H.G. Zhu, M.J. Mcshane, Enzymatic fluorescent microsphere glucose sensors: evaluation of response under dynamic conditions, Diabetes Technol. Therap. 8 (2006) 288–295. [20] J.F. Liang, Y.T. Li, V.C. Yang, Biomedical application of immobilized enzymes, J. Pharm. Sci. 89 (2000) 979–990. [21] H. Wang, D.G. Castner, B.D. Ratner, S. Jiang, Probing the orientation of surface-immobilized immunoglobulin G by time-of-flight secondary ion mass spectrometry, Langmuir 20 (2004) 1877–1887. [22] N. Patel, M.C. Davies, M. Hartshorne, R.J. Heaton, C.J. Roberts, S.J.B. Tendler, P.M. Williams, Immobilization of protein molecules onto homogeneous and mixed carboxylate-terminated self-assembled monolayers, Langmuir 13 (1997) 6485–6490.
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Immobilization of glucose oxidase onto gold nanoparticles with enhanced thermostability Dongxiang Li a, Qiang He a
a,b
, Yue Cui a, Li Duan a, Junbai Li
a,*
Beijing National Laboratory for Molecular Sciences (BNLMS), International Joint Lab, CAS Key Lab of Colloid and Interface Science, Institute of Chemistry, Chinese Academy of Sciences, Beijing 100080, PR China b Max Planck Institute of Colloids and Interfaces, 14476 Golm/Potsdam, Germany Received 27 January 2007 Available online 8 February 2007
Abstract Immobilized proteins and enzymes were widely investigated in medical field as well as in food and environmental fields. In this paper, glucose oxidase (GOD) monolayer was covalently immobilized on the surface of gold nanoparticles (AuNPs) to fabricate bioconjugate complex. The citrate-stabilized AuNPs were first functionalized by a carboxyl-terminated alkanethiol and the terminal carboxyl groups were subsequently bonded with side-chain amino groups of protein surface through EDC/NHS coupling reaction. The enzyme activity assays of the obtained bioconjugates display an enhanced thermostability and similar pH-dependence behavior in contrast with that of free enzyme. Such GOD/AuNPs bioconjugates can be considered as a catalytic nanodevice to construct nanoreactor based on glucose oxidation reaction for biotechnological purpose. 2007 Elsevier Inc. All rights reserved. Keywords: Immobilization; Glucose oxidase; Gold nanoparticles; Bioconjugate; Enzyme activity
In past decades, gold nanoparticles (AuNPs) had attracted a continuous interest due to their unusual properties in electronics, optics [1,2], especially in biotechnology and nanotechnology fields involving biosensors [3,4], DNA hybridization [5–7], and biocatalysts [8]. Many methods of AuNPs’ modification with alkanethiols provided different functional gold surfaces for facile combinations with biomolecules [9–11]. Moreover, the general covalent combination strategy between protein and solid surfaces provided a stable linkage with more feasibility as advantages [12,13]. In elctrochemistry, AuNPs attached on an electrode were covalently modified by glucose oxidase (GOD) to fabricate glucose biosensors [14]. On the other hand, GOD was also immobilized on surface of polystyrene latex or mesostructured silica to investigate the systemic stability and enzymatic properties [15–17]. McShane reported that
*
Corresponding author. Fax: +86 10 82612629. E-mail address: [email protected] (J. Li).
0006-291X/$ - see front matter 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2007.01.183
the immobilized GOD in alginate microspheres could be applied to fabricate optical glucose sensor systems for implantable biosensor purpose [18,19]. Such immobilized enzymes could be widely utilized to make chemical biosensors not only in the medical field but also in the food and environmental fields [20]. A well-known coupling reaction of N-ethyl-N 0 -(3-dimethylaminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS) was recently reported to construct protein films on the carboxyl or amino-terminated surface [21,22]. In this paper, we fabricated the covalently-linked bioconjugates of GOD/AuNPs in aqueous suspension through such coupling reaction as shown in Scheme 1. The citrate-stabilized AuNPs were first modified by 11mercaptoundecanoic acid (MUA) under protection of nonionic surfactant. Subsequently, the peripheral carboxylic groups were activated by EDC/NHS as coupling agents. The as-formed NHS-terminated AuNPs covalently combined with the side-chain amino groups on the surface of protein GOD and the target bioconjugates were
D. Li et al. / Biochemical and Biophysical Research Communications 355 (2007) 488–493
489
Scheme 1. Schematic illustration of the fabrication of the GOD/AuNPs bioconjugates.
constructed. These assembled GOD/AuNPs bioconjugates in aqueous suspension can maintain their enzyme activity with enhanced thermostability and similar pH dependence. Such bioconjugates would greatly reduce the protein leaching in further application process due to the covalent bond linkage. As an original purpose, they were designed as a fundamental catalytic nanodevice in biomimetic nanoreactor, such as phospholipids-based capsules in previous works [23,24]. Materials and methods Materials. GOD (200 U/mg) was purchased from Sigma. MUA and NHS were obtained from Aldrich. EDC, o-dianisidine, peroxidase from horseradish (HRP, 858 U/mg) were purchased from Fluka. Aurate chloride, sodium citrate, glucose, poly(oxylethene), sorbitan monolaurate (Tween-20), and other chemicals were obtained from Beijing Chemical Reagents Co., China. All chemicals were used as received. The deionized water was prepared in a three-stage Millipore Milli-Q Plus 185 purification system with a resistivity above 18.2 MX cm. All glassware were treated with potassium dichromate/sulfuric acid digestant and then cleaned with tap water and deionized water. Preparation of alkanethiol MUA-modified AuNPs. The citrate-stabilized AuNPs were freshly synthesized through the reduction of aurate chloride by sodium citrate in boiling solution as a conventional method [25]. The subsequent MUA-modified AuNPs were prepared by ligand exchange between mercapto-carboxylic acid and citrate groups under the protection of nonionic surfactant Tween-20 [26]. As a typical prescription, 2.0 mL colloidal gold (approximately 0.8 nM) was gently added 2.0 mL phosphate buffer (10 mM, pH 6.8, with 0.2 mg/mL Tween-20) and the mixture was incubated for 30 min. Then 2.0 mL MUA solution (0.5 mM in 1:3 alcohol/H2O) was added into the mixture, followed by a gentle shake for 5 h for a complete chemisorption of alkanethiol onto gold surface. The final mixture was centrifuged to remove excess alkanethiol and resuspended in phosphate buffer (10 mM, pH 6.8, with 0.2 mg/mL Tween-20). Fabrication of GOD/AuNPs bioconjugates. The combination of GOD and AuNPs was based on the EDC/NHS coupling reaction [22]. In detail, the above MUA-modified AuNPs were reacted with a mixture of freshly prepared 50 mM NHS and 200 mM EDC solution for 10 min. Then asformed NHS-terminated AuNPs were separated by centrifugation and resuspended in a phosphate buffer (10 mM, pH 6.8, with 0.2 mg/mL Tween-20) under ultrasonication for the next wash. After discarding the supernatant, the remaining NHS-terminated AuNPs were incubated with 0.8 mg/mL GOD in phosphate buffer (10 mM, pH 6.8) for more than 12 h under nitrogen atmosphere. The resultant mixture was centrifuged to discard free GOD and washed by phosphate buffer with Tween-20. The target GOD/AuNPs bioconjugates were finally dispersed under ultrasonication in phosphate buffer (0.1 M, pH 5.8) and stored at 4 C. Enzyme activity assays. The activity of the GOD/AuNPs bioconjugates and free GOD were examined spectrometrically at k = 460 nm based on the change of solution color resulting from oxidation of o-dianisidine by the reaction product hydrogen peroxide from glucose in presence of HRP, in which the chemical equations are as follows [18,27].
glucose oxidase
b-d-Glucose þ O2 þ H2 O ! d-gluconic acid þ H2 O2 peroxidase
H2 O2 þ o-dianisidine ðreducedÞ ! o-dianisidine ðoxidizedÞ þ H2 O Typically, 2.5 mL of a 0.33 mM o-dianisidine solution in 0.1 M buffer, 0.3 mL 18% glucose solution, and 0.1 mL 0.02% HRP were mixed in a quartz cell as a substrate. Then, 0.010 mL suspension of the bioconjugates or free GOD solution was added into the mixture and the light absorption of the mixture was immediately recorded for 2 min. The slope of fitted line to recorded absorbance curve was proportional to enzyme activity in the same mass of enzyme by the theoretical analysis. The equivalent volume of obtained bioconjugate suspension or free enzyme solution was employed in all assays to get comparable result. The enzyme concentration in bioconjugates suspension and free GOD solution were adjusted at an approximately same condition to reduce systemic errors. The thermostability measurements were performed after the samples were incubated at a certain temperature for 10 min in a water bath and the pH-dependence measurements were carried out in buffers with pH from 2.1 to 9.1. Characterizations and instrumentations. FT-IR spectra were recorded on a TENSOR 27 instrument (BRUCKER) by dropping the samples onto CaF2 plates followed by a vacuum drying. The UV–vis spectra of AuNPs in fabrication stages and the spectrometric absorption data in catalytic activity assays were collected on a U-3010 UV–vis spectrometer (HITACHI) using 1 cm quartz cell. Transition electron microscopy (TEM) images were obtained from a TECNAI 20 microscope (PHILIPS) operated at 120 kV. X-ray photoelectron spectroscopy (XPS) data were analyzed on an ESCALab220i-XL electron spectrometer (VG Scientific) using 300 W AlKa radiation at about 3 · 109 mbar and the binding energies were referenced to the C1s line at 284.8 eV from adventitious carbon.
Results and discussion Introduction of alkanethiol ligands with different terminal groups can functionalize the surface of AuNPs and the formed self-assembled monolayer of thiols on gold surfaces can drastically reduce nonspecific protein adsorption [28]. Herein, as shown in Scheme 1, carboxyl-terminated AuNPs were obtained by MUA ligand exchange from original citrate-stabilized AuNPs in the presence of Tween-20, which is prior physically adsorbed around gold particles as a protective nonionic surfactant [26]. Subsequently, the carboxylate groups were activated by esterification of NHS catalyzed by the water-soluble carbodiimide EDC. As the resultant NHS-terminated AuNPs were mixed with GOD solution, the side-chain amino groups on GOD surfaces displaced the terminal NHS groups of AuNPs in a phosphate buffer [21,22]. Thus a GOD monolayer was immobilized onto the surface of AuNPs through covalent bonds, that is, the GOD/AuNPs bioconjugates were obtained. Fig. 1A shows the UV–vis spectra of different AuNPs in four stages, in which the curve peak indicates the character-
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modified AuNPs, the distinct peaks appeared at 2918 and 2849 cm1 are ascribed to the vibrational stretches of –CH2 groups of long alkane chains (curve b), while the carbonyl groups of terminal carboxylic acid are shown at 1700 cm1. This indicates the successful modification of AuNPs by MUA ligands. For NHS-terminated AuNPs, it displays a different profile (curve c). A new peak at 1742 cm1 is contributed by the succinimidyl carbonyl group, and the two weak peaks at 1830 and 1782 cm1 are attributed to the band splitting of the ester carbonyl C@O stretching vibration, which is similar to the self-assembled layer of the same chemical component on planar gold surface [31]. It demonstrates the presence of NHS esterification of carboxylic groups. For the GOD/AuNPs bioconjugates, it displays the mainly characteristic peaks at 1650 and 1540 cm1 due to the secondary amide C@O stretching and at 3290 cm1 of hydroxyl groups from protein GOD (curve d) [17]. Therefore, FT-IR results provide evidential component information of different modified AuNPs. Moreover, the NHS-terminated AuNPs and the GOD/ AuNPs bioconjugates were further analyzed by XPS as shown in Fig. 2. The contributions of elements, O, N, C,
Fig. 1. UV–vis (A) and FT-IR spectra (B) of citrate-stabilized AuNPs (curve a), MUA modified AuNPs (curve b), NHS-terminated AuNPs (curve c) and the GOD/AuNPs bioconjugates (curve d).
istic surface plasmon resonance (SPR) of AuNPs [2]. It is obvious that the SPR of MUA-modified AuNPs (curve b) is about 525 nm with a red shift of 5 nm compared to that of original citrate-stabilized AuNPs (curve a, 520 nm). This denotes that alkanethiol molecules are adsorbed on gold surface by Au–S interaction and a soformed dielectric monolayer of alkanethiol around AuNPs results in the red shift [29]. For NHS-terminated AuNPs, the SPR peak at 535 nm (curve c) maybe implies the surface change of metal nanoparticles with a partial aggregation. This is because the NHS esterification of terminal carboxyl group can destroy carboxyl ionization and cause charge neutralization on nanoparticle surface, and result in colloidal particle aggregation. Successively, the suspension of final bioconjugates (curve d) displays a SPR peak at 539 nm. The red shift is only 14 nm compared with MUA-modified AuNPs, this denotes such bioconjugates still maintain a relatively good dispersity although a little of aggregation occurs [30]. Fig. 1B shows the FT-IR spectra of modified AuNPs at different stages. For citrate-stabilized AuNPs, absorbance at about 1690 cm1 represents the citrate component by an evidential carboxylate groups (curve a). For MUA-
Fig. 2. XPS survey spectra (A) and deconvolution of C1s spectra (B) of NHS-terminated AuNPs (curve a) and the GOD/AuNPs bioconjugates (curve b) on CaF2 plates.
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S and Au atoms are displayed in Fig. 2A. The binding energy of Au 4f7/2 of both samples exhibits at 83.2 eV, which is slightly higher than that of citrate-stabilized AuNPs at 82.7 eV. It means that the chemical interaction between gold and sulfur atoms occurs. Comparing with Au, the signals of C, N, and O of the GOD/AuNPs bioconjugates are obviously stronger than those of NHS-terminated AuNPs, indicating that AuNPs in bioconjugates are densely enwrapped by protein molecules. The peak-fitting of C1s for these two samples displays three different carbon atoms with binding energy at 288.2, 286.2, and 284.8 eV, respectively, as shown in Fig. 2B. The different carbon assignment of NHS (curve a) involves the carbonyl (zone III), -SC (zone II) and -CH2 of alkane chains (zone I), while that of GOD/AuNPs (curve b) shows the individual contribution from carboxyl (zone III), peptide (zone II) to alkane chains (zone I) of protein molecules. The morphology of different modified AuNPs was characterized by TEM. Fig. 3A shows the well-dispersed cit-
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rate-stabilized AuNPs with unique size of about 20 nm in diameter. The subsequent MUA-modified AuNPs (Fig. 3B) also exhibit a good dispersity under protection of nonionic surfactant Tween-20. Fig. 3C and D show the GOD/AuNPs bioconjugate particles before and after stained by 1% silver nitrate, respectively. It is obvious that the AuNPs cores are well dispersed after they are conjugated with GOD, but the peripheral protein layer cannot be imaged due to the low electron resistance of protein molecules in TEM examination. However, after the sample was stained by silver nitrate, the GOD layer was visualized since positive silver ions were reduced by protein GOD molecules, resulting in the formation of silver nanoparticles around protein domains as shown in Fig. 3D. It is reported that GOD immobilized in polyelectrolyte multilayer films and assembled on polystyrene particles [15] or quartz slides [32] can enhance its enzyme stability with respect to temperature and pH environments. The GOD immobilized on magnesium silicate microparticles
Fig. 3. TEM images of citrate-stabilized AuNPs (A), MUA modified AuNPs (B), and the GOD/AuNPs bioconjugates before (C) and after stained by 1% silver nitrate (D).
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exhibits a resistance to a high pH value and high buffer concentration [33]. Similarly, here we detected the enzyme thermostability and pH-dependence of such assembled bioconjugates. Fig. 4A gives the relative activity of the bioconjugates (curve a) and free enzyme (curve b) determined at different pH values. As a whole, curve a and b display a resembling profile with a maximum activity at pH 5.8 and a deactivation at pH 2.1. However, at pH 9.1 the bioconjugates still remain a tiny relative activity (5.4%). This indicates that GOD/AuNPs bioconjugates can hold the enzyme activity at different pH. Fig. 4B shows the thermostability of as-prepared bioconjugates (curve a) and free enzyme (curve b). The activity assays were examined after the samples were incubated at a certain temperature for 10 min [15]. It is seen that both immobilized GOD on bioconjugates and free GOD in solution have a maximum activity at 40 C and a sharp activity reduction occurs when the temperature is above 50 C. The relative activity of enzyme on bioconjugates remains, respectively, 57.3% and 11.1% of their activity after incubated at 60 and 70 C, which are higher than that of the free enzyme. This result shows that the GOD/AuNPs bioconjugates are more stable against high temperature in contrast with free enzyme in solution, which is consistent with the results of GOD assembled into polyelectrolyte multilayer [15]. In summary, glucose oxidase can be successfully immobilized on the surface of carboxyl-terminated alkanethiol
Fig. 4. Enzyme pH dependence (A) and thermostability (B) of the GOD/ AuNPs bioconjugates (curve a) and free GOD in solution (curve b).
modified AuNPs through the EDC/NHS coupling reaction. The GOD/AuNPs bioconjugates have a good dispersity in aqueous suspension and have a better stability at high temperature and display similar pH dependence in contrast to the free enzyme according to enzymatic catalysis examination. Such bioconjugate GOD/AuNPs nanoparticles can be expected as catalytic nanodevice to construct nanoreactors based on glucose oxidation reaction in biological applications. Acknowledgments We acknowledge the financial supports of this research by National Nature Science Foundation of China (Nos. 20471063 and 90206035), Chinese Academy of Sciences, and the collaborated project of the German Max Planck Society. References [1] C.M. Niemeyer, Nanoparticles, proteins, and nucleic acids: biotechnology meets materials science, Angew. Chem. Int. Ed. 40 (2001) 4128–4158. [2] M.-C. Daniel, D. Astruc, Gold nanoparticles: assembly, supramolecular chemistry, quantum-size-related properties, and applications toward biology, catalysis, and nanotechnology, Chem. Rev. 104 (2004) 293–346. [3] Y. Xiao, F. Patolsky, E. Katz, J.F. Hainfeld, I. Willner, Plugging into enzymes: nanowiring of redox enzymes by a gold nanoparticle, Science 299 (2003) 1877–1881. [4] T. Liu, J. Tang, L. Jiang, The enhancement effect of gold nanoparticles as a surface modifier on DNA sensor sensitivity, Biochem. Biophys. Res. Commun. 313 (2004) 3–7. [5] Y. Cheng, C. Pun, C. Tsai, P. Chen, An array-based CMOS biochip for electrical detection of DNA with multilayer self-assembly gold nanoparticles, Sensor Actuator B 109 (2005) 249–255. [6] J.J. Storhoff, S.S. Marla, P. Bao, S. Hagenow, H. Mehta, A. Lucas, V. Garimella, T. Patno, W. Buckingham, W. Cork, U.R. Mu¨ller, Gold nanoparticle-based detection of genomic DNA targets on microarrays using a novel optical detection system, Biosens. Bioelectron. 19 (2004) 875–883. [7] S. Liu, J. Li, L. Jiang, Surface modification of platinum quartz crystal microbalance by controlled electroless deposition of gold nanoparticles and its enhancing effect on the HS-DNA immobilization, Colloid Surface A 257–258 (2005) 57–62. [8] L. Pasquato, P. Pengo, P. Scrimin, Functional gold nanoparticles for recognition and catalysis, J. Mater. Chem. 14 (2004) 3481–3487. [9] S. Park, A.A. Lazarides, C.A. Mirkin, P.W. Brazis, C.R. Kannewurf, R.L. Letsinger, The electrical properties of gold nanoparticle assemblies linked by DNA, Angew. Chem. Int. Ed. 39 (2000) 3845– 3848. [10] C. Mangeney, F. Ferrage, I. Aujard, V. Marchi-Artzner, L. Jullien, Q. Ouari, E.D. Rekai, A. Laschewsky, I. Vikholm, J.W. Sadowski, Synthesis and properties of water-soluble gold colloids covalently derivatized with neutral polymer monolayers, J. Am. Chem. Soc. 124 (2002) 5811–5821. [11] J.M. Abad, S.F.L. Mertens, M. Pita, V.M. Fernandez, D.J. Schiffrin, Functionalization of thioctic acid-capped gold nanoparticles for specific immobilization of histidine-tagged proteins, J. Am. Chem. Soc. 127 (2005) 5689–5694. [12] M.C. Parker, N. Patel, M.C. Davies, C.J. Roberts, SJB. Tendler, P.M. Willams, A novel organic solvent-based coupling method for the preparation of covalently immobilized proteins on gold, Protein Sci. 5 (1996) 2329–2332.
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