Article pubs.acs.org/Langmuir
Self-Oriented Immobilization of DNA Polymerase Tagged by Titanium-Binding Peptide Motif Hirokazu Nishida,*,† Taira Kajisa,‡ Yuuya Miyazawa,‡ Yuki Tabuse,‡ Takuya Yoda,§ Haruko Takeyama,§ Hideki Kambara,† and Toshiya Sakata*,‡ †
Central Research Laboratory, Hitachi Ltd. 1-280 Higashi-Koigakubo, Kokubunji, Tokyo 185-8601, Japan Department of Materials Engineering, Graduate School of Engineering, The University of Tokyo, 7-1-3 Hongo, Bunkyo, Tokyo 113-0033, Japan § Department of Life Science and Medical Bioscience, Waseda University, 2-2 Wakamatsu-cho, Shinjuku, Tokyo 162-8480, Japan ‡
ABSTRACT: We developed a titanium-binding-peptide-1 (TBP-1)-tagged DNA polymerase, for self-oriented immobilization onto a titanium oxide (TiO2) substrate. The enzymatic function of a polymerase immobilized on a solid state device is strongly dependent on the orientation of the enzyme. The TBP-tagged DNA polymerase, which was derived from a hyperthermophilic archaeon, was designed to incorporate the RKLPDA peptide at the N-terminus, and synthesized by translation processes in Escherichia coli (E. coli). The specific binding of the TBP-tagged DNA polymerase onto a TiO2 substrate was clearly monitored by surface plasmon resonance spectroscopy (SPR) and by surface potential detection with an extended-gate field effect transistor (FET). In the SPR analyses, constant quantities of the DNA polymerase were stably immobilized on the titanium substrate under flow conditions, regardless of the concentration of the DNA polymerase, and could be completely removed by a 4 M MgCl2 wash after measurement. The FET signal showed the contribution of the molecular charge in the TBP motif to the binding with TiO2. In addition, the TBP-tagged DNA polymerase-tethered TiO2 gate electrode enabled the effective detection of the positive charges of hydrogen ions produced by the DNA extension reaction, according to the FET principle. Therefore, the self-oriented immobilization platform based on the motif-inserted enzyme is suitable for the quick and stable immobilization of functional enzymes on biosensing devices.
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INTRODUCTION Immobilization of biomolecules onto material surfaces is crucial in many biological sciences, including cell and molecular biology and analytical chemistry, as well as in practical fields such as medical diagnostics and tissue engineering.1−4 Several chemical modifications and peptide tags for immobilizing protein molecules have been developed over the past few decades. They have been utilized for purifying proteins and for monitoring enzymatic reactions on lab-on-a-chip devices. The covalent immobilization of protein molecules onto surfaces usually involves conjugation reactions between the active chemical groups on the surfaces, such as Nhydroxysuccinimide (NHS)5 or maleimide,6 and the functional © XXXX American Chemical Society
groups in the target proteins, such as amines and thiols. An amine group is present in lysine, which is commonly found on the surfaces of protein molecules. In this case, the orientation of the proteins immobilized on a surface is not uniform, because there are many possible residues participating in the conjugation reactions. On the other hand, the thiol groups of cysteines are usually oxidized to form S−S bonds and are not available for reaction unless the protein is reduced. Thus, these Received: August 7, 2014 Revised: December 12, 2014
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DOI: 10.1021/la503094k Langmuir XXXX, XXX, XXX−XXX
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binding site in the polymerase domain was distant from the metal surface, in a manner similar to an enzyme immobilized via a capture molecule, such as avidin. A few examples of TBP-1-tagged proteins have been reported. A representative example is bone morphogenetic protein, which stimulates human bone growth, fused with TBP1 to successfully induce premyoblastic cells to differentiate into an osteoblastic state on the titanium often utilized in bone implants.18 However, the application of TBP-1 for the activation of substrate surfaces in measurement systems has not been reported thus far. The application of TBP-1 reported here could be widely useful in several detection methods.
immobilization methods are sometimes not suitable for the effective measurement of enzymatic reactions on a surface. On the other hand, noncovalent immobilization, utilizing physical adsorption or affinity immobilization, is reversible under specific conditions. For example, immobilization methods utilizing avidin−biotin or nitriloacetic acid−histidine interactions7,8 are widely used for various purposes, including protein purification. Notably, these methods permit the immobilization of proteins with a unique orientation, by using peptide tags that recognize specific chemical groups on the substrate surface. In the preceding examples, the capture molecules used for recognition, such as avidin or nitriloacetic acid, must be bound on the substrate surface before the immobilization of the enzymes. If the enzyme to be immobilized is directly bound to the substrate surface, then the modification of the surface by the capture molecule can be eliminated. However, these capture molecules sometimes exhibit inhibitory activities against effective measurements on the surface. For example, in the case of the electrode surface for a field effect transistor (FET) measurement, the length of the capture molecules may decrease the effective detectable range (the so-called “Debye length”). Hence, the direct binding of enzymes on inorganic materials, to be employed as the detector electrode, is desirable for efficient measurement systems. Numerous peptide aptamers that bind to a variety of inorganic materials have been reported (reviewed in ref 9). Notably, Sano et al. described the useful peptide aptamer sequence (RKLPDA: TBP-1 (titanium-binding peptide-1)) against titanium in an aqueous solution, in which the arginine at the first position and the aspartic acid at the fifth position are considered to interact with the -O‑ and -OH2+ groups, respectively, of the oxidized titanium.10,11 Titanium is immediately oxidized in an aqueous solution, resulting in a nonconducting film, and titanium oxide (TiO2 or Ti2O5) has been extensively utilized as a sensing membrane in an ion-sensitive field effect transistor (ISFET).12 The ISFET is suitable for detecting ionic charges released from enzymatic reactions, based on the field effect principle.13,14 We have been investigating an electrostatic detection method for biomolecular recognition events, using a biologically coupled-gate FET (bio-FET). In this method, we constructed a DNA detection system that enables the detection of charge density changes, depending on the hybridization of ss-DNA molecules immobilized on a chip, and the real-time monitoring of the pyrophosphates released during DNA strand extension reactions.15−17 A TBP-1-tagged enzyme can be directly immobilized on a bio-FET electrode and may be advantageous for monitoring the changes induced by enzymatic reactions. Here, we report the binding properties of TBP-1-tagged DNA polymerase on a titanium oxide surface, assessed by surface plasmon resonance (SPR) analysis and atomic force microscopy (AFM). We also confirmed the efficient immobilization of the enzyme, by electronic detection with a bio-FET device. Generally, the direct attachment of enzymes to metal or metal oxide surfaces is often detrimental to their structures and functions. Pyrococcus f uriosus (Pfu) DNA polymerase BI belongs to the family B DNA polymerases and comprises two independent, equally sized domains: the N-terminal exonuclease domain and the C-terminal polymerase domain. In the present structure, the TBP-1 motif was attached to the Nterminal end of the exonuclease domain so that the DNA
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MATERIALS AND METHODS
Plasmid Construction. The plasmid containing the RKLPDA (TBP-1) sequence was prepared by the inverse PCR technique, using primer-1 including a start codon and RKLP (5′- GGGGA GCTTC CTCAT GGTAT ATCTC CTTCT TA), and primer-2 comprising the DA and linker peptide GGGG sequences (5′- GACGC CGGTG GTGGT GGTGT TTTAG ATGTG GATTA CAT). The plasmid pTPOL (the structural gene for Pfu DNA polymerase BI, inserted into the pET21a vector (Novagen, Madison, WI, USA)), which was described previously,19 was directly amplified by PCR using primer-1 and primer-2. The PCR conditions involved 25 cycles of denaturation for 30 s at 95 °C, annealing for 1 min at 55 °C, and extension for 10 min at 68 °C, with PfuUltra DNA polymerase (Agilent Technologies, Santa Clara, CA, USA). The PCR solution was processed by the restriction enzyme DpnI, in order to digest the methylated template plasmid. The enzyme-processed solution was precipitated by EtOH and solubilized in TE buffer. The resultant solution was transformed into JM109 competent cells (Takara, O̅ tsu, Japan), which were cultured on Luria-Bertani (LB) agar plates, containing 100 μg/mL ampicillin, for 12 h at 37 °C. The colonies were picked from the agar plate, and each colony was cultured in 10 mL of LB medium containing 100 μg/mL ampicillin. The plasmids were extracted and purified from the culture medium, using a Wizard Plus SV Miniprep kit (Promega, Madison, WI, USA). After sequencing, the plasmid that contained the gene with the expected insertion was selected. Protein Expression and Purification. The purification of TBP-1tagged Pfu DNA polymerase BI (TBP-Pol) was performed, as described previously with slight modifications.20 The plasmids were transformed into Escherichia coli (E. coli) BL21-CodonPlus(DE3)-RIL cells (Agilent). The cells were grown at 37 °C in LB medium, supplemented with ampicillin (100 μg/mL) and chloramphenicol (20 μg/mL). When the culture reached an A600 = 0.6, protein expression was induced by an incubation in the presence of 1 mM isopropyl-β-Dthiogalactopyranoside (IPTG). The harvested cells were disrupted by sonication in buffer A, containing 50 mM Tris-HCl, pH 8, 1 mM ethylenediaminetetraacetic acid (EDTA), 50 mM NaCl, and 10% (w/ v) glycerol. The supernatant was incubated at 80 °C for 15 min, to denature and precipitate most of the E. coli proteins. The heat-treated supernatant was subjected to anion-exchange chromatography (HiTrap Q; GE Healthcare, Pittsburgh, PA, USA). The fractions that eluted at about 100 mM NaCl, containing the TBP-Pol protein, were subjected to cation-exchange chromatography (HiTrap SP, GE Healthcare). TBP-Pol was eluted with a linear gradient of 150−200 mM NaCl. The eluted fractions were dialyzed against buffer A. The equilibrated sample was concentrated and loaded onto a gel-filtration column (Superdex 200 HiLoad 26/60, GE Healthcare). The fractions that eluted in a single peak corresponding to TBP-Pol were collected and used for the following experiments. Activities of the Purified DNA Polymerases. The TBP-Pol activity was confirmed by a polymerase chain reaction (PCR) experiment, and compared to the wild type Pfu DNA polymerase BI (WT-Pol). The PCR conditions involved 30 cycles of denaturation for 30 s at 95 °C, annealing for 1 min at 55 °C, and extension for 1 min at 68 °C, with WT-Pol and TBP-Pol for amplifications of 1 and 2 kb fragments. The plasmid pTPOL was prepared as the template B
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substrate, to form a 400 μL volume well on the extended-gate electrode. The principle of bio-FET used in this study is based on the detection of a change in the charge density on the gate surface, which is induced by ionic molecules.21 The probes for detecting biomolecular recognition events are immobilized on the surface of the gate insulator. The FET is immersed in a measurement solution, together with an Ag/AgCl reference electrode with a saturated KCl solution. The potential of the measurement solution is controlled and fixed by the gate voltage through the reference electrode. The threshold voltage (VT) shift was measured using the semiconductor parameter analyzer B1500A (Agilent), and the surface potential change on the gate was monitored in a real-time manner with a custom-made potentiometric analyzer. The drain current and voltage were fixed to 0.7 mA and 1.0 V, respectively, throughout the experiment. The well was initially filled with 300 μL of phosphate buffered saline (PBS), containing 8.1 mM Na2HPO4, 1.47 mM KH2PO4 (pH 7.4), 137 mM NaCl, and 2.7 mM KCl. Next, 20 μL of a 13.3 μM protein solution in PBS was gently applied onto the PBS within the well. The final concentrations of the enzymes in the well were 832 nM for both WT-Pol and TBP-Pol. When the voltage shifts by the addition of the protein became constant, we washed the electrode with 100 μL of PBS, by directly flushing the surface of the substrate. FET Measurement of Surface Potential Change Based on the DNA Extension Reaction by the TBP-Pol-Immobilized TiO2 Surface. To verify that the TiO2-immobilized DNA polymerase is actually active in bio-FET measurements, we applied the solutions of TBP-Pol with/without the primed-DNA substrate into the well fabricated on the titanium-coated extended bio-FET gate. The devices and the system for the bio-FET experiment were the same as those described previously. The well was initially filled with 400 μL of HEPES buffer, containing 10 mM HEPES, and 100 mM NaCl. Next, 20 μL of a 13.3 μM TBP-Pol solution with/without an equimolar amount of the primed-DNA substrate, in which a 25mer ssDNA (5′-AGGCA GTAGT CAGAT CGTTG GTGGA-3′) was hybridized with a 70mer ssDNA (5′-TGCCG CCTCC AATTC TAATA CGACT CACTA TAGGG AGAAG GAAAC TCCAC CAACG ATCTG ACTAC TGCCT-3′), in HEPES buffer was gently applied onto the well at 25 °C. TBP-Pol with the primed-DNA substrate was preincubated at 35 °C for 5 min. When the gate voltage shifts became constant after several dozen minutes, 10 μL of 40 mM MgCl2 and 10 mM dNTP substrate in dH2O were gently applied onto the PBS within the well to start the DNA synthesis reaction.
fragment in the PCR amplification. The forward primer PriF (5′GCTAT TCAAA AAAGA GAACG GAAAA TTTAA) and the reverse primer PriR1 (5′- CCTGT GCTTG ACCTT GAAAC ATCCC ATAAC) were used for the 1 kb fragment amplification, and PriF and another reverse primer, PriR2 (5′- CAACA GCTAC GTGAG GACCT ATCGC CTTAT), were used for the 2 kb fragment amplification. The primers were purchased from Sigma Genosys, Ltd. (Hokkaido, Japan). The reaction products were analyzed by electrophoresis on an agarose gel (1%) in TAE (40 mM Tris/Tris-acetate; 1 mM EDTA). After staining with SYBR Gold (Life Technologies, Grand Island, NY, USA), the gel was analyzed with a FluoroImager 595 (GE Healthcare). SPR Experiments. Titanium-coated plates were prepared by the gas flow sputtering method, based on the SIA kit Au (GE Healthcare), in which one side of a glass plate was covered by a thin gold film, as a substrate. By use of the sputtering method, the formation of a thin film of TiO2 was performed at the rate of 7.5 nm/min and at room temperature by an Ar gas flow, under vacuum conditions. A 15 nm thick titanium layer was built on the gold film by the sputtering process, and the chip was then soaked in distilled water for 12 h, in order to fully oxidize the titanium surface. The SPR experiment was performed with a Biacore X100 system (GE Healthcare). All measurements were performed at a continuous flow rate of 30 μL/min at 25 °C. WT-Pol and TBP-Pol were diluted (1, 2, 4, and 8 μM) in HBS-P buffer, containing 10 mM HEPES (pH 7.4), 150 mM NaCl, and 0.005% surfactant P20. HBS-P buffer was used for running buffer. The purified proteins were concentrated to 100 mg/mL (1.1 mM), and then diluted 138−1100 times by HBS-P buffer in order to avoid a buffer effect. The injection time for all samples was 60 s, and the chip was monitored 60 s postinjection before washing. At the end of each cycle, the bound protein was removed, by washing the chip with 4 M MgCl2 for 120 s. The evaluations of the inhibitory effect of the TBP-1 molecule, diluted (1, 2, 4, 8, and 16 μM) in the same buffer as the proteins, on the interactions of WT-Pol and TBP-Pol with the titanium substrate were performed, according to the same procedure described earlier. The concentrations of the proteins were fixed at 4 μM in this experiment. When the interaction of the TBP-1 molecule with the titanium substrate was monitored, the concentrations of the peptide were adjusted to 32 or 16 μM. We also performed the measurement of the interaction between the titanium-surface-immobilized TBP-Pol and the primed-DNA substrate (p20/t50), in which a 20mer ssDNA (5′-GAAAA AAAAA GAAAA AAAAA-3′) was hybridized with a 50mer ssDNA (5′-GCAGC AGCAG CAGCA GCAGC AGCAG CAGCA TTTTT TTTTC TTTTT TTTTC-3′). After the injection of 2μM TBP-Pol for 60 s and a 90 s interval, the primed-DNA substrate (2μM) was injected for 60 s. And we assessed the direct interaction of the primed-DNA (2 μM, injected for 60 s) with the bare TiO2 surface. All SPR data were evaluated by the BIAevaluation software (GE Healthcare). Values are the means of two independent experiments. AFM Experiments. AFM imaging was performed using an Agilent Technologies 5500 scanning probe microscope at room temperature (25 °C). An 10 μL aliquot of TBP-Pol (4 μM in HBS-P buffer) was pipetted on the 75 nm Ti-sputtered Au plane forcibly oxidized by plasma treatment, which was then rinsed with distilled water. Images were obtained in the AC mode (AC-AFM) with a fabricated silicon cantilever (PPP-NCHR-10, NanoWorld, Neuchâtel, Switzerland). The nominal spring constant was 40 N/m, and the tip had a 128 μm length and a 30 μm width. The resonance frequency was around 323 kHz, and the scan speed was 0.7 lines/s. The PicoView software, version 1.6, was used for image analysis. Measurement of Surface Potential Change Based on Immobilization of Peptide-Tagged DNA Polymerase Using an FET Sensor. To confirm that the titanium-coated extended gate is actually useful for activation by enzyme molecules in bio-FET measurements, we applied the solutions of WT-Pol and TBP-Pol into the well fabricated on the titanium-coated extended gate of bioFET. The plastic cylinder was attached to the titanium-coated
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RESULTS AND DISCUSSION Expression and Purification of TBP-Pol. The procedures for the expression and purification of the TBP-Pol protein were exactly the same as those used for WT-Pol, and both the expression levels in the cell culture and the quantities obtained after the purification process were equal. The addition of the TBP-1 sequence to WT-Pol did not seem to affect the folding process of TBP-Pol, since no inclusion bodies were formed throughout the expression and purification steps. Activities of Purified DNA Polymerases. The molecular structure of TBP-Pol, estimated from the crystal structure of WT-Pol (PDB code: 3A2F) and the reported structure of TBP1, is shown in Figure 1.10,22 The arginine and aspartic acid residues in the RKLPDA peptide region are expected to interact with the -O− and -OH2+ groups, respectively, of the oxidized titanium (close-up view in Figure 1). The N-terminus of WTPol is on the opposite side from the active site for the DNA synthetic reaction, and thus we did not expect the TBP insertion to have any effects on its DNA synthetic activity. The activity of TBP-Pol was evaluated by PCR and was found to be the same as that of WT-Pol (Figure 2). As expected, the incorporation of the RKLPDA sequence (via the four glycine C
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excess TBP-Pol was washed away in the dissociation phase (Figure 3A, right). The TBP-Pol immobilized on the substrate was completely removed by the 4 M MgCl2 washing solution. To clarify the reason why TBP-Pol is immobilized on the TiO2 surface, we assessed the inhibitory effect of the addition of TBP-1 on the interactions between the proteins and the substrate. The protein concentrations were 4 μM in these experiments. The sensorgrams of WT-Pol with/without the same concentration of TBP-1 (4 μM) were equivalent to each other (Figure 3B, left). Therefore, the interaction of WT-Pol was not affected by the addition of the peptide. On the other hand, the sensorgram of TBP-Pol with 1 μM of TBP-1 (Figure 3B, right, red line) was considerably lower than that without TBP-1 (blue line). Judging from the sensorgrams of TBP-Pol obtained with various TBP-1 concentrations (0, 1, 2, 4, 8, and 16 μM), the binding of TBP-Pol to a TiO2 surface was competitively inhibited or replaced by TBP-1, suggesting that the immobilization of TBP-Pol on the surface was largely attributable to the TBP-1 moiety of the protein molecule. When a 4-fold larger amount of the TBP-1 peptide (16 μM) was mixed with TBP-Pol (Figure 3B, right, orange line), a substantial amount (200 resonance units (RU)) of interaction was observed, which might be attributed to the TBP-1 interaction in place of TBP-Pol, judging from the case when TBP-1 (16 μM) was solely added to the surface (Figure 3C). When TBP-1 was solely added to the surface, it was slowly adsorbed as compared to the absorption of WT-Pol, but was stably immobilized on the surface (Figure 3C). TBP-Pol binding may occur by the fast binding mode observed with WT-Pol (Figure 3A), which is not stably bound to the surface, and the slow binding mode of the TBP-1 moiety, which might adjust the orientation relative to the TiO2 surface, resulting in stable immobilization. To demonstrate the substrate binding by TBP-Pol, the primed-DNA substrate (p20/t50) was added after the immobilization of TBP-Pol on the surface (Figure 3D, left). The primed-DNA seemed to be stably captured. The immobilized TBP-Pol generated about 500 RU, while the captured DNA substrate produced about 100 RU, implying that most of the added DNA substrate (Mr = 21,500) was bound to the immobilized TBP-Pol (Mr = 91,000). On the other hand, when the DNA substrate was added on the bare TiO2 surface, the substrate interacted with the surface to some extent (about 100 RU), but it was completely washed out by the subsequent buffer flow (Figure 3D, right). Observation of the Surface Morphology Using AFM. Surface characterization using AFM imaging is difficult to perform on TiO2 surfaces in the presence of adsorbed proteins, because of the softness of biological materials and the mobility of the adsorbed biomolecules at the solid/liquid interface. However, thanks to the development of the AC mode analysis method, we can now easily measure a soft material on a hard substrate. Morphological changes were observed only when the droplets of 4 μM TBP-Pol were applied onto the TiO2 surface and subsequently rinsed with distilled water, as expected from the SPR experimental results described earlier. While the roughness of the bare TiO2 surface was lower than 3 nm (Figure 4A), the addition of TBP-Pol and the following wash with distilled water led to the appearance of particles distributed on the surface, with an appreciable increase of the surface roughness up to 10 nm (Figure 4B). The sizes of the small particles observed most frequently on the surface, except
Figure 1. Schematic diagram of TBP-Pol. TBP-1 (RKLPDA) peptide is attached to the N-terminus of Pfu DNA polymerase, with elongating DNA in synthesis.23 The DNA polymerase molecule is depicted by a ribbon diagram, in which the polymerase domain and the exonuclease domain are colored light purple and magenta, respectively. The TBP-1 moiety is shown by sphere models, in which R1 and D5 in TBP-1 are colored light blue and pink, respectively (oxygen, red; nitrogen, blue). These two residues are considered to interact with the titanium oxide surface. R1 and D5 are simultaneously directed to the same surface by the peptide kink at the cis-peptide bond of P4, resulting in R1 and D5 electrostatically interacting with -Ti−O‑ and -Ti−OH2+, respectively (right box; adapted with permission from ref 10. Copyright 2003 American Chemical Society). Bound DNA in synthesis is flanked by TBP-Pol (orange). The DNA is extending upward in the figure (orange arrow), in the opposite direction from the immobilized protein on the TiO2 surface at the bottom (calculated from protein coordination data in ref 24).
Figure 2. Comparison of the DNA synthetic activities of WT-Pol and TBP-Pol, assessed by PCR. Agarose gel shows the PCR products for the amplifications of 1 and 2 kb fragments for each DNA polymerase, indicating the similar activities of WT-Pol and TBP-Pol.
spacer) at the N-terminus of WT-Pol did not inhibit its DNA polymerization activity. The direction of DNA elongation during the synthesis is also opposite from the N-terminus of the enzyme, suggesting that the N-terminal immobilization of TBPPol on a TiO2 surface would not affect the DNA polymerase activity. SPR Experiment. The sensorgrams showed that WT-Pol has weak affinity to a TiO2 surface (Figure 3A, left). Most of the adsorbed WT-Pol was removed by washing with the HBS-P buffer, but some residual nonspecific adsorption of WT-Pol was observed. On the other hand, a substantial amount of the injected TBP-Pol was retained on the surface after the HBS-P buffer wash, and the amount of immobilized TBP-Pol was almost independent of its concentration (1, 2, 4, or 8 μM). The D
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Figure 3. continued
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Figure 3. Series of sensorgrams obtained from SPR experiments using titanium-coated sensor chips. (A) Overlaid sensorgrams representing the interactions of WT-Pol (left) and TBP-Pol (right) with an oxidized titanium-coated Au chip. The protein concentrations were 1, 2, 4, and 8 μM. The injection period for each sample was 60 s. (B) Inhibitory effect of the TBP-1 (RKLPDA) peptide on the interactions of WT-Pol (left) and TBP-Pol (right) with the oxidized titanium-coated Au chip. In the case of WT-Pol, the sensorgram of WT-Pol (4 μM) alone, as compared with that of WTPol (4 μM) with TBP-1 peptide (4 μM), revealed that the interaction of WT-Pol was not affected by the addition of the peptide. On the other hand, the binding of TBP-Pol was competitively inhibited by the peptide. (C) Overlaid sensorgrams representing the interactions of the TBP-1 peptide with an oxidized titanium-coated Au chip. The peptide concentrations were 16 and 32 μM. (D) Interaction of TBP-Pol and a 20mer/50mer primedDNA substrate on the TiO2 surface (left) and interaction of the DNA substrate on the bare TiO2 surface (right).
Figure 4. AFM height images of the Ti-coated chips. AFM height images of (A) the bare Ti substrate without the enzyme treatment, (B, C) after the enzyme adsorptions and the subsequent rinsing by distilled water for TBP-Pol (B), and WT-Pol (C). Cross sections were taken at the locations indicated by the dashed lines. The positions possibly corresponding to single enzyme molecules are marked by asterisks on the magnified figure of the cross section (B, bottom).
On the other hand, when the WT-Pol was added on the surface with subsequent washing by distilled water, only a few nonspecifically bound particles were detected (Figure 4C). The lower roughness of WT-Pol (
