Improvement of Homogeneity of Analytical Biodevices by Gene

Anal. Chem. 2004, 76, 632-638

Improvement of Homogeneity of Analytical Biodevices by Gene Manipulation Jing-Xue Shi,†,‡ Xian-En Zhang,*,† Wei-Hong Xie,† Ya-Feng Zhou,† Zhi-Ping Zhang,† Jiao-Yu Deng,† Anthony E. G. Cass,| Zhi-Ling Zhang,§ Dai-Wen Pang,§ and Cheng-Gang Zhang‡

Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan 430071, China, Shenyang Institute of Ecology, Chinese Academy of Sciences, Shenyang, China, Department of Chemistry, Wuhan University, Wuhan, China, and Department of Biochemistry, Imperial College of Science, Technology & Medicine, South Kensington, London SW7 2AY, U.K.

Homogeneity is proposed for evaluation of the quality of analytical biodevices, such as biosensors and biochips. As a demonstration, glucose oxidase (GOx) was modified at its C-terminal with a linker peptide with a cysteine residue at the end. The fusion structure (GOx-linkercysteine) enables the enzyme to immobilize on gold surfaces with a Cys-S-Au bond or to immobilize on a silanized glass surface via disulfide chemistry. With this fusion structure, the enzyme can be anchored onto the substrate with well-controlled orientation, thus forming a homogeneous biological layer on biodevices. The linker peptide between GOx and the cysteine acts as a spacer to reduce the steric hindrance caused by the bulky body of the enzyme. Biochemistry experiments showed that this genetically modified glucose oxidase (shortened to GOxm) retained most of its catalytic characteristics, with Km and Kcat similar to those of the wild-type GOx. Electrochemistry experiments showed that GOxm-modified electrode gave higher and more stable current responses than the electrode modified with GOx which has no free -SH on its surface. The coefficients of variation (used for evaluation of the interchangeability of the enzyme device from the same batch preparation) were 9.5% for the GOxm gold electrode and 20.0% for the GOx gold electrode and the GOxm oxygen electrode. The relative errors (used for evaluation of the precision of the individual enzyme device) were 2.9% for the GOxm gold electrode, 12.0% for the GOx gold electrode, and 11.2% for the GOxm oxygen electrode. Atomic force microscopy images revealed that GOxm formed a self-assembled monolayer in a hexagonal-like lattice packing arrangement on the gold surface, while GOx formed multilayer assembling or aggregated particles. The homogeneity of the protein chips, the GOxm array that was prepared through -SS- formation, and the GOx array that was prepared through nonspecific adsorption was evaluated. The coefficients of variation, calculated with the signal level of all dots, were 5.4% for the GOxm array and 81.8% for the GOx array. All experimental results pointed to the fact that 632 Analytical Chemistry, Vol. 76, No. 3, February 1, 2004

the homogeneity of the analytical biodevices could be considerably improved by using the proposed method. Protein-based analytical biodevices, such as enzyme biosensors and protein biochips, have shown great potential for applications in the fields of clinical diagnosis,1-4 bioprocess quality control,5-7 food analysis, environmental monitoring,8,9 life science research,10-12 etc. However, the proteins are not easy to work with. They may be deposited on the substrate with various orientations, altered conformation, or different packing loads, all of which resulted in uncontrolled quality. Here, we use the word “homogeneity” to describe the quality of the biodevices. The homogeneity of a biodevice is mainly determined by the status of the plastic part (sensing layer), as the solid part is inherently stable. The biodevice on which the proteins are deposited with unique biological status can be termed “homogeneous boidevice”, as opposed to a “heterogeneous biodevice”. The homogeneous biodevices should possess good quality and uniformity, while the heterogeneous biodevices bear poor exchangeability or unsatisfactory reproducibility. Great efforts have been made to allow molecular sensing elements to be arranged in a controlled and reproducible way, to * Corresponding author. Tel.: +86 10 68512083. Fax: +86 10 68512094. E-mail: [email protected]. † Wuhan Institute of Virology. ‡ Shenyang Institute of Ecology. § Wuhan University. | Imperial College of Science, Technology & Medicine. (1) Ludi, H. Anasthesiol. Intensivmed. Notfallmed. Schmerzther. 1999, 34, 225226. (2) Rowe, C. A.; Scruggs, S. B.; Feldstein, M. J.; Golden, J. P.; Ligler, F. S. Anal. Chem. 1999, 71, 433-439. (3) Wang, P.; Tan, Y.; Xie, H.; Shen, F. Biosens. Bioelectron. 1997, 12, 10311036. (4) Ramanathan, K.; Rank, M.; Svitel, J.; Dzgoev, A.; Danielsson, B. Trends Biotechnol. 1999, 17, 499-505. (5) Scouten, W. H.; Luong, J. H. T.; Brown, R. S. Tibtech 1995, 13, 178-185. (6) Schiigerl, K. Bioprocess Monitoring; John Wilery & Sons: New York, 1997. (7) Williams, D.; Jones, K. Chem. Ind. 1995, 17, 684-688. (8) Van Emon, J. M.; Gerlach, C. L.; Bowman, K. J. Chromatogr. B, Biomed. Sci. Appl. 1998, 715, 211-228. (9) Sole, S.; Alegret, S. Environ. Sci. Pollut. Res. Int. 2001, 8, 256-264. (10) Leickt, L.; Grubb, A.; Ohlson, S. Scand. J. Clin. Lab. Invest. 2002, 62, 423429. (11) Kim, S. J.; Kim, M. Y.; Lee, J. H.; You, J. C.; Jeong, S. Biochem. Biophys. Res. Commun. 2002, 291, 925-931. (12) Laich, A.; Sim, R. B. Biochim. Biophys. Acta 2001, 1544, 96-112. 10.1021/ac020796f CCC: $27.50

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increase the quality of the biodevices and generate homogeneous biodevices. A useful method is achieved by incorporating proteins into a Langmuir-Blodgett (L-B) monolayer.13 However, films containing proteins are not stable when transferred to supports using conventional L-B methods, and further investigation is required.14 It is well known that thiol compounds can bind to gold surfaces through Au-S bonds.15 The strength of this covalent bond is 40 kcal/mol.16 On the basis of this principle, alkanethiol and alkanethiol-like molecules can form self-assembled monolayers (SAMs) on metal surfaces.17,18 It is also possible to prepare a wellorganized monolayer of proteins on the gold substrate. Unfortunately, most proteins, as far as we know, do not provide free -SH themselves for the immobilization because cysteine is a sort of low-appearance amino acid. Furthermore, the bulky body of the protein may hinder the interaction between cysteine-SH and the gold surface. Site mutagenesis could provide cysteine-SH on the protein surface;19-21 however, it does not solve the problem of space hindrance of the proteins. The application of this method is thus very limited so far. Instead of using the Au-S principle, a specific attachment through a high-affinity reaction, with a binding constant of Kd ) 37 µM,22 between streptag and strepavidin has also been reported.23 The recovery of the enzyme activity, however, is less than 40% because of steric hindrance. It is no longer an obstacle to the anchor-chain model system in our previous study, where a linker peptide was inserted between alkaline phosphatase and streptag to overcome this steric hindrance problem.24 In this study, on the basis of the anchor-chain model and the SAM principle, we used cysteine, instead of using streptag, as the “anchor”. A fusion containing glucose oxidase (GOx), a linker peptide, and a cysteine end group was constructed by gene manipulation. The new design is expected to be simpler and more efficient for the preparation of the self-assembling biodevices with good homogeneity. An enzyme electrode and an enzyme chip were constructed using the fusion method. Their homogeneity was evaluated with atomic force microscopy (AFM) and electrochemical and enzymatic indication system analysis. EXPERIMENTAL SECTION Materials. GOx (EC1.1.3.4, Aspergillus niger, type X-S), ferrocenecarboxylic acid (FCA), o-dianisidine, yeast nitrogen base (YNB), and (3-mercaptopropyl)trimethoxysilane (MPTS) were purchased from Sigma. DNA polymerase, restriction enzymes, and (13) Singhal, R.; Gambhir, A.; Pandey, M. K.; Annapoorni, S.; Malhotra, B. D. Biosens. Bioelectron. 2002, 17, 697-703. (14) Tuan, V.-D.; Cullum, B. M.; Stokes, D. L. Sens. Actuators B 2001, 74, 2-11. (15) Nuzzo, R. G.; Allara, D. L. J. Am. Chem. Soc. 1983, 105, 4481-4483. (16) Nuzzo, R. G.; Dubois, L. H.; Allara, D. L. J. Am. Chem. Soc. 1990, 112, 558-569. (17) Finklea, H. O. Encycl. Anal. Chem. 2000, 11, 10090-10115. (18) Weisshaar, D. E.; Lamp, B. D.; Porter, M. D. J. Am. Chem. Soc. 1992, 114, 5860-5862. (19) Huang, W.; Wang, J.; Bhattacharyya, D.; Bachas, L. G. Anal. Chem. 1997, 69, 4601-4607. (20) Kanno, S.; Yanagida, Y.; Haruyama, T.; Kobatake, E.; Aizawa, M. J. Biotechnol. 2000, 76, 207-214. (21) Davis, J. J.; Djuricic, D.; Lo, K. K. W.; Wallace, E. N. K.; Wong, L. L.; Hill, H. A. O. Faraday Discuss. 2000, 116, 15-22. (22) Voss, S.; Skerra, A. Protein. Eng. 1997, 10, 975-982. (23) Hengsakul, M.; Cass, A. E. G. J. Mol. Biol. 1997, 266, 621-632. (24) Shao, W.-H.; Zhang, X.-E.; Liu, H.; Zhang, Z.-P. Bioconjugate Chem. 2000, 11, 822-826.

T4 DNA ligase were obtained from Takara. DNA purification columns and Q Sepharose fast-flow ion-exchange chromatography systems were obtained from Qiagen and Pharmacia, respectively. The oligonucleotides were synthesized from Sangon (Shanghai, China). 6-Mercapto-1-hexanol (MCH) was obtained from Fluka. The yeast Pichia pastoris GS115 and its expression vector, pPIC9, were kindly donated by Professor Heping Dai and Professor Shengli Yang, respectively. GLT (GOx-linker-(lys)10) and plasmid pGEM-TGOL, which contains the gene of GOx with a peptide (Ser-Gly)5 at its C-terminus, were constructed in our previous studies.25,26 Escherichia coli DH5R was used for cloning. All other reagents used were of analytical reagent grade. Milli-Q water (18 MΩ‚cm) was used throughout to prepare all solutions. Construction of Yeast Expression Vector. The synthesized oligonucleotides encoding (Lys)10-cysteine were hybridized to form a double-stranded DNA fragment that had endorestriction enzyme HindIII and NotI recognition sites at its terminus for cloning and linking:

The double-stranded fragment and GOx-(Ser-Gly)5, which had SnaBI and HindIII sites, were inserted into the SnaBI/NotI site of pPIC9 by ligase, yielding GOx-linker-cysteine (shortened as GOxm), fusing the enzyme expression vector pPIC-GOxm. Transformation of P. pastoris. pPIC-GOxm was digested with StuI. The linearized plasmid fragment containing the GOxm coding sequence was transformed into GS115 as described by Cregg et al.27 Integration of linearized GOxm coding sequence was confirmed by both PCR, using yeast genomic DNA as template, and detection of glucose oxidase activity of GOxm expressed. Expression, Purification, and Kinetic Analysis of the Recombinant Enzyme. GOxm was expressed in P. pastoris under the control of the AOXI promoter. Yeast cells were cultured at 30 °C in a 400-mL BMGY medium (containing (per L) 3.4 g yeast nitrogen base, 10 g yeast extract, 20 g tryptone, 400 µg biotin, 10 g glycerol and 0.01 M pH 6.0 potassium) for 12 h. The cells were harvested by centrifugation when the culture reached an optical density (OD600) of 1.2-1.5. The cells were then resuspended in 250 mL of BMMY medium (containing (per L) 3.4 g yeast nitrogen base, 10 g yeast extract, 20 g tryptone, 400 µg biotin, 0.5% methnaol, and 0.01 M pH 6.0 potassium) to induce the production of the recombinant enzyme. After 5 days of induction, 500 mL of the culture supernatant was concentrated to 50-60 mL using the Filtron stirred cell system and dialyzed against 1000 mL of potassium phosphate buffer (0.01 M, pH 6.0) overnight. A Q Sepharose fast-flow ion-exchange chromatography column was used to purify the recombinant enzyme. The column was equilibrated with potassium phosphate (0.01 M, pH 6.0). The enzyme was eluted from the column using a linear gradient of 0-0.15 M KCl in 0.01 M phosphate buffer (pH 6.0). Fractions (50-60 mL) (25) Chen, L.-Q.; Zhang, X.-E.; Xie, W.-H.; Zhou, Y.-F.; Zhang, Z.-P.; Cass, A. E. G. Biosens. Bioelectron. 2002, 17, 851-857. (26) Zhou, Y.-F.; Zhang, X.-E.; Liu, H.; Zhang, Z.-P.; Zhang, C.-G.; Cass, A. E. G. Bioconjugate Chem. 2001, 12, 924-931. (27) Cregg, J. M.; Barringer, K. J.; Hessler, A. Y.; Madden, K. R. Mol. Cell. Biol. 1985, 5, 3376-3385.

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containing higher GOx activity were collected and concentrated using the Filtron stirred cell system (MW cutoff 10 000). The enzyme purity was verified by sodium dodecyl sulfide-polyacrylamide gel electrophoresis (SDS-PAGE, 12%), and the concentration was determined by the Bio-Rad method28 using bovine serum albumin as standard. GOx activity was determined according to the method described by Frederick et al.,29 using fresh wild-type GOx as reference. Electrochemical Measurement. Four gold electrodes (2 mm in diameter, CH Instruments, Inc.) were used throughout the experiment. The electrodes were polished to a mirror surface with an alumina slurry (particle size in sequence of 1.0, 0.3, and 0.05 µm), ultrasonicated in Milli-Q water for 10 min to remove residues from the polishing materials, and finally cleaned by cycling between the potentials of -0.3 and +1.5 V vs Ag/AgCl in 0.5 M H2SO4 solution, until stable scans were obtained. The cleaned electrodes were immediately inserted into the degassed enzyme solutions (2 U/µL) and stored at 4 °C for 4 days to achieve the highest enzyme loadings. As an alternative, the enzyme-modified electrodes were incubated with MCH (10 mM) overnight in order to minimize the physical adsorption of the enzymes or contaminated proteins. The GOx enzyme electrodes, using oxygen electrodes as transducers, were prepared according to Qu et al.30 Cyclic voltammetry and chronoamperometric experiments were performed on an EG&G potentiostat/galvanostat (model 273A, Princeton Applied Research, Princeton, NJ) connected to a Gateway 2000 computer (North Sioux City, SD). A three-electrode configuration of a platinum auxiliary electrode and a Ag/AgCl/ KCl reference electrode was used. Chronoamperometry was recorded at a potential of 450 mV when the current reached a platform at the 30th second. Cyclic voltammogram was performed at a potential range of 0.2-0.6 V, with a scan rate of 20 mV/s. The glucose stock solutions were prepared in phosphate buffer (0.1 M, pH 5.6) and allowed to mutarotate overnight before use. Unless otherwise stated, electrochemical measurements were carried out in the phosphate buffer solution containing 10 mM glucose and 0.03 mM FCA (pH 5.6). AFM Experiment. The atomic force microscopy (AFM) images were obtained by using a Picoscan (Molecular Imaging Co., Tempe, AZ) operating under a magnetic AC (MAC) mode with a Maclever Type I. The AFM tip had a force constant of 0.6 N/m and resonant frequency of 75 kHz in air. Tapping working mode was employed. Gold (Au 111) substrates (Molecular Imaging Co.) were used for enzyme immobilization in AFM experiments. The size of Au (111) on the mica substrate is 2.4 cm × 1.60 cm, with a thickness of 1500 Å. Before being used, the substrate was annealed in a hydrogen flame and then cooled at room temperature. Ten microliters of the enzymes was then dropped onto the gold slides and incubated in a humid chamber for 2 days. Before scanning, the slides were rinsed with Milli-Q water. (28) Bradford, M. M. Anal. Biochem. 1976, 72, 248-254. (29) Frederick, K. R.; Tung, J.; Emerick, R. S.; Masiarz, F. R.; Chamberlain, S. H.; Vasavada, A.; Rosenberg, S.; Chakraborty, S.; Schopter, L. M.; Massey, V. J. Biol. Chem. 1990, 265, 3793-3802. (30) Qu, H.-B.; Zhang, X.-E.; Zhang, S.-Z. Anal. Food Nutr. Clin. Methods Sect. 1995, 52, 187-192.

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Figure 1. Constructed vector for expression of GOxm.

Immobilization of Enzymes on Glass Slides by S-S Bond. Silanization of the glass slides was in accordance with the method described by Rogers et al.31 The glass slides were first pretreated with 25% ammonium hydroxide solution overnight, rinsed with running Milli-Q water for 10 min, and finally rinsed briefly with anhydrous EtOH. The -SH group was introduced by immersing the slides in a mixture of 1% MPTS, 95% EtOH, and 16 mM acetic acid (pH 4.5) for 30 min at room temperature, followed by rinsing with 95% EtOH/16 mM acetic acid (pH 4.5) and curing under dry nitrogen overnight. Three microliters of each the enzymes (GOxm and GOx) was respectively dropped onto the silanized glass slide to form a 4 × 4 array protein chip. The chips were incubated in a humid chamber overnight at room temperature and then blocked with 10 mM MCH again overnight. The colors that developed from the enzymatic reaction on the chips were quantitatively analyzed using WASABI software from Hamamatsu Photonics Deutschland GmbH. RESULT AND DISCUSSION Expression and Kinetic Analysis of the Recombinant Protein. The constructed expression vector is shown in Figure 1. The enzymatic assay showed that the plasmid had directed the synthesis and secretion of the active protein. The fused peptide chain was introduced at the C-terminus of the enzyme, based on the fact that the N-terminus of GOx is buried inside the enzyme and the C-terminus is exposed to the surface. Our previous work25,26 has shown that the linker peptide, a rigid R-helix, could reduce the geometric influence of the added chain on the protein folding, so the genetically modified enzymes remained their bioactivity. Although one GOx subunit contains three cysteines, two of them forming a disulfide bridge and the other buried in (31) Rogers, Y. H.; Jiang-Baucom, P.; Huang, Z.-J.; Bogdanov, V.; Anderson, S.; Boyce-Jacino, M. T. Anal. Biochem. 1999, 266, 23-30.

Figure 3. Cyclic voltammograms of the enzymes electrodes: (a) GOxm, (b) GLT, and (c) GOx. The curves were obtained in a phosphate buffer containing 10 mM glucose and 0.03 mM FCA, pH 5.6, and scan rate ) 20 mV/s.

Figure 2. Three-dimensional structure of the subunit of the GOxm fusion enzyme. The structure was modulated using Swiss-Pdb Viewer (version 3.5b1) and displayed as the Rasmol version (version 2.5).

the enzyme, the introduced cysteine would not interact with GOx’s cysteine because the protein folding starts from its N-terminal. Figure 2 shows the three-dimensional structure of the subunit of GOxm simulated using the software Swiss-Pdb Viewer (version 3.5b1). The kinetic constants of the GOxm and GOx were investigated. The Km and Kcat values of GOxm were 40.2 ( 1.5 mM and 2744.0 ( 4.5 S-1, and the Km and Kcat of GOx were 33.2 ( 1.2 mM and 2015.2 ( 4.0 S-1, respectively. The evidence shows that this genetic modification has no influence on GOx’s catalytic specificity. Electrochemical Experiments. With an aim to evaluate the efficiency of the enzyme modification for its immobilization on the electrode surface, electrochemical experiments were carried out with GOxm, GOx, and GLT. The structure of GLT is similar to the GOxm, except for the absence of a cysteine residue at the end of the linker peptide chain. Ferrocenecarboxylic acid (FCA) was used to mediate the electrochemical catalytic reaction of GOx and the electrode.32,33 Figure 3 shows the voltammgrams recorded at the enzyme-modified electrodes in the presence of glucose and FCA. The anodic current for the GOxm-modified electrode was 9.59 µA, while it was 8.15 µA at the GLT electrode and 7.79 µA at the GOx electrode. Subtracting the background difference, the anodic current of the GOxm electrode was 0.64 µA, and thus at least 1.0 µA bigger than those of the GLT and the GOx electrodes. It can also be seen that the voltammgram of the GOxm-modified electrode is less symmetric than those of the GLT and GOx electrodes. The cyclic voltammgrams indicated that the catalytic reaction occurred more completely on the GOxm electrode than on the GLT and GOx electrodes, which implied that the GOxmmodified electrode retained more enzyme activity. In fact, at the lower concentration range of glucose (