For Achieving Direct Electron Transfer to Enzymes

The fabrication of aligned CNT electrodes used in our research first relies on the shortening of SWCNTs by modifying the procedure of Liu et al. (32) in which the tubes are oxidized in an acid mixture of concentrated sulfuric and concentrated nitric acid (3:1 ratio) while sonicating. The resultant tubes are reduced to lengths of the order of a few hundred nanometers depending on the cutting times. The ends of the shortened tubes are open and terminated with

Fig. 1. Representation of shortened SWCNTs with open end showing carboxylic acids formed as consequence of oxidative shortening process.

carboxylic acid moieties (see Fig. 1). SWCNTs shortened in this way can be self-assembled aligned normal to a surface in a number of ways including by modifying the ends with the alkanethiol cysteamine (HSCH2CH2NH2), which is then attached to a gold surface (33). Other methods include simple assembly of the carboxylic acid-terminated tubes onto a silver surface (34) or the use of a coordinating ion such as Zn2+ (35) or Fe3+ (36) to anchor the shortened SWCNTs to a surface. We have aligned the shortened SWCNT normal to a gold electrode surface modified with a SAM of cysteamine according to the procedure shown in Scheme 2. The nanotubes are covalently linked to the SAM-modified electrode by activating the carboxylic acids with dicyclo-hexylcarbodiimide (DCC). The aligned CNTs are shown in Fig. 2. Figure 2A shows what appears to be the SWCNTs standing normally from the gold surface. A cross-section of Fig. 2A is shown in Fig. 2B in which the height of the tubes attached to the surface appears to be no more than 20 nm. Furthermore, the tubes appear to be in clumps on the surface rather than as individual tubes. If the time the SAM-modified surface is incubated in the tubes is increased, then a greater density of tubes is observed on the surface. Further evidence that these images represent SWCNTs standing from the surface comes from the image in Fig. 2C, in which a feature of what appears to be a clump of tubes lying down is visible. The cross section of this image (Fig. 2D) shows a large difference in height between this feature and what we believe are the tubes standing normally.

In the activation of the shortened SWCNTs using DCC, both ends of the tubes are activated. Therefore, to the end not attached to the surface, a redox-active species such as an enzyme can be covalently attached (see Scheme 2). In our work, we have attached ferrocene and the enzymes microperoxidase

Scheme 2. Steps involved in fabricating aligned shortened SWCNT arrays for direct communication with enzymes such as microperoxidase MP-11 or glucose oxidase.

MP-11 and glucose oxidase. To explore the ability of these aligned SWCNT electrodes to allow direct electron transfer to enzymes, we first investigated the enzyme microperoxidase MP-11 (37) (Fig. 3). MP-11 is a small redox protein, 1.9 kDa, obtained by proteolytic digestion of horse heart cytochrome-c (38), in which the iron protoporphyrin IX is not shielded by a polypeptide. A number of workers (38-43) have demonstrated that electrons can be efficiently transferred between MP-11 and SAM-modified electrodes.

Attaching MP-11 to the aligned SWCNT-modified gold electrodes and subsequent electrochemical interrogation showed the characteristic peaks for the heme redox-active center of MP-11 with a formal electrode potential of -420 mV vs Ag/AgCl (Fig. 4). To verify whether the electrochemistry is owing to MP-11 attached to the ends of the SWCNTs, several control experiments were performed. If a SWCNT bed electrode was prepared by drop coating the shortened SWCNT onto a gold electrode and subsequently adsorbing MP-11 onto the nanotubes, the resultant MP-11-modified electrode showed no redox activity. This control suggests that if the MP-11 is adsorbing onto the walls of the nanotubes, as seems likely from previous work on protein adsorption onto nanotubes (15-17,22,25-27), the rate of electron transfer through the walls of the tubes is insufficient to give the discernible redox peaks observed in Fig. 4.

Fig. 2. Atomic force microscopy (AFM) images of shortened SWCNTs aligned onto gold electrode surfaces. (A) Tubes cut for 4 h followed by activation with DCC and then incubated with a cysteamine-modified gold electrode for 4 h. The typical heights of the nanotubes are 10 to 20 nm. (B) Cross-section of (A) diagonally as shown. (C) Top view AFM image showing bundle of aligned nanotubes and another bundle of tubes lying flat on surface. (D) Differences in heights between standing tubes and those lying down.

Fig. 2. Atomic force microscopy (AFM) images of shortened SWCNTs aligned onto gold electrode surfaces. (A) Tubes cut for 4 h followed by activation with DCC and then incubated with a cysteamine-modified gold electrode for 4 h. The typical heights of the nanotubes are 10 to 20 nm. (B) Cross-section of (A) diagonally as shown. (C) Top view AFM image showing bundle of aligned nanotubes and another bundle of tubes lying flat on surface. (D) Differences in heights between standing tubes and those lying down.

To verify that the electrochemistry was not because of the MP-11 adsorbing onto any cysteamine SAM still accessible between the aligned nanotubes, a cysteamine-modified gold electrode was exposed to MP-11 in an analogous manner to the SWCNT-modified electrodes. In this case, very small MP-11 peaks were observed (Fig. 4A), but they were significantly smaller than those observed in Fig. 4B. Therefore, we can conclude that the electrochemistry observed in Fig. 4B is owing to MP-11 attached to the ends of the SWCNTs with the nanotubes allowing effective communication between the electrode and an enzyme located many nanometers away.



Fig. 3. Microperoxidase MP-11 showing redox-active site and short undecapeptide backbone.



Fig. 3. Microperoxidase MP-11 showing redox-active site and short undecapeptide backbone.

Fig. 4. Cyclic voltammograms of (A) Au/cysteamine after immersion into DMF and MP-11 solution and (B) Au/cysteamine/SWCNTs/MP-11 in 0.05 M phosphate buffer solution (pH 7.0) containing 0.05 KCl under argon gas at scan rate of 100 mV s-1 vs Ag/AgCl.
Fig. 5. Cyclic voltammogram of glucose oxidase attached to aligned CNT-modi-fied gold electrode measured in 0.05 M phosphate buffer solution (pH 7.0) containing 0.5 M KCl under argon gas at scan rate of x10 mV s-1.

The variation in cyclic voltammograms with scan rate can be used to calculate the rate of electron transfer between the enzyme and the electrode according to the diffusionless model of Laviron (44). The rate of electron transfer for MP-11 attached to an SWCNT-modified electrode in which the tubes were shortened for 4 h was 2.8 ± 0.9 s-1. This value is only slightly lower than MP-11 attached directly to cysteamine-modified gold electrode (6.1 ± 0.9 s-1) and a 3-mercapto-propionic acid-modified gold electrode (9.4 ± 0.6 s-1). The very similar rate of electron transfer for the MP-11 attached to the nanotubes compared with the equivalent electrode with the nanotubes removed (the cysteamine-modi-fied electrode) demonstrates the efficiency of the nanotubes as molecular wires.

The virtue of SWCNTs in allowing direct electron transfer to redox proteins is reliant on being able to communicate with enzymes in which the redox-active center is imbedded deep within the glycoprotein. The classic example of such a redox protein, as discussed in Subheading 1.1., is glucose oxidase. Figure 5 shows the electrochemical response of SWCNT-modified electrodes in which glucose oxidase is attached to the ends of the SWCNTs. The small redox peaks, at a formal potential of about -430 mV vs Ag/AgCl, are attributed to the FAD redox center of glucose oxidase. These preliminary experiments indicate that direct electron transfer to glucose oxidase can be achieved. The remainder of this chapter outlines in detail the experimental protocols used to achieve the results presented in this section.

2. Materials

1. Gold working electrode. We used polycrystalline bulk gold electrodes prepared from 1-mm-diameter gold wire (Aldrich, Sydney, Australia) sealed in glass using epoxy (see Note 1) as described previously (45). Alternatively, particularly for imaging surfaces, molecularly smooth gold surfaces were prepared by evaporation onto hot mica as described by Mazurkiewicz et al. (46).

2. Ag/AgCl reference electrode (BAS, Lafayette, IN).

3. Platinum flag counterelectrode (homemade) or, alternatively, from BAS.

4. Electrochemical cell for a three-electrode setup (BAS). Alternatively, we use 20-mL sample tubes with the appropriate number of holes cut into the plastic lid.

5. Polishing cloth and 1-, 0.3-, and 0.05-| alumina polishing powder (Buehler, Lake Bluff, IL).

6. Ultrasonic cleaner (Unisonics, Sydney, Australia).

7. Potentiostat (BAS 100B).

8. UV-VIS Cary 20 dual beam spectrometer.

9. Scanning Probe Microscopy/Atomic Force Microscopy Digital Instruments Multimode System with a Nanoscope 4 Controller.

10. Phillips CM 200 transmission electron microscope.

11. Carbon nanotubes (Carbon Nanotechnologies, Houston, TX).

12. Solution of concentrated sulfuric acid (98%) and concentrated nitric acid (70%) (3:1 [v/v]).

14. Dimethylformamide (DMF) (Prolabo, Manchester UK).

16. Teflon membrane, Millipore MF™ membrane filters, type 0.45 |im HA.

17. Hot-plate stirrer.

18. Cysteamine (2-mercaptoethylamine) (Sigma, Sydney, Australia).

19. Microperoxidase MP-11 (Sigma).

20. Phosphate buffer (0.3 M NaCl, 5 mM phosphate from KH2PO4 and K2HPO4, pH 7.0).

21. 0.01 M HEPES buffer solution, pH 7.5.

23. Argon gas.

3. Methods

3.1. Preparation of Gold Surfaces

1. Polish the gold electrodes to a mirror finish by forming a paste with the alumina powder mixed with Milli-Q water starting with the 1.0-| paste, then the 0.3-| alumina, and finishing with the 0.05-| powder. Polish with each powder for about 5 min by gently drawing figure eights.

2. Place the polished electrodes in a container half filled with Milli-Q water; then place in a sonicator for 10 min.

3. After sonication, fill a clean electrochemical cell with 0.05 M sulfuric acid; place the polished gold electrode, a reference electrode, and a counterelectrode; and connect to the potentiostat.

4. Perform a cyclic voltammogram between -300 and 1500 mV with repeated cycling for at least 20 min or until the cyclic voltammogram becomes stable (see Note 2).

3.2. Cutting of SWCNTs

1. Weigh 2 mg of uncut nanotubes into a sample tube.

2. Add 10 mL of the prepared acid mixture to the nanotubes.

3. Sonicate the sample tubes for the desired time (see Note 3).

4. Pour the sonicated solution into a beaker and dilute to 500 mL with Milli-Q water.

5. Collect the nanotubes via vacuum filtration with a Buchner funnel using a 0.45-|im pore-size Teflon membrane.

6. Discard the acid and filter 1.5 L of Milli-Q water through the nanotubes to reduce their acidity.

7. Test the pH of the filtrate with a universal indicator to ensure a minimum pH of 5.0 before proceeding. Continue washing with Milli-Q water if the pH <5.0.

8. Immerse the membrane containing the shortened nanotubes in 10 mL of ethanol, sonicate for 15 s (any longer and the membrane will begin to break up), and remove the membrane from the solution with tweezers.

9. Sonicate the ethanol-nanotube solution for 20 min.

10. Pipet 1 mL of the ethanol-nanotube solution into a clean sample tube.

11. Heat the solution to evaporate most of the ethanol, leaving a thin film of ethanol to make the redispersion of the shortened nanotubes easier.

12. Add 10 mL of DMF (0.2 mg mL-1) and shake gently to redisperse the nanotubes. The shortened CNTs are now ready for use.

3.3. Characterization of Lengths of Cut SWCNTs

1. Disperse the original and shortened SWCNTs with different cutting times in ethanol.

2. Place a drop or two of this dispersion onto 3-mm-diameter copper grid.

3. Allow the drop or two to evaporate at room temperature overnight.

4. Insert the sample in the transmission electron microscope and take several pictures.

5. Determine the length of each shortened SWCNT prepared at any cutting time simply by manually measuring the length from the transmission electron microscope images. Use at least 100 individual ropes from the images for the distribution determination (see Notes 3 and 4).

3.4. Assembly of SWCNTs on Electrode Surfaces

3.4.1. Assembly of Aligned Nanotubes

1. Prepare a 1 mM cysteamine solution in 75% aqueous ethanol.

2. Pipet 200 |L of the cysteamine solution into a 500-mL Eppendorf tube.

3. Place a gold electrode (cleaned immediately prior to modification) into the Eppendorf tube so that the gold layer is in the solution.

4. Leave for 5 h for the cysteamine monolayer to self-assemble.

5. Remove the electrode and rinse thoroughly with ethanol.

6. Sonicate the electrode in ethanol for 15 s to remove possible surface contaminants.

7. Pipet 200 ||L of the shortened nanotubes dispersed in DMF (0.2 mg mL-1) into an Eppendorf tube and add 0.5 mg of DCC (to give a concentration of approx 2 mM).

8. Immerse the cysteamine-coated electrode in the nanotube solution for the desired time (typically 4 h) during which the tubes will covalently attach to the cysteamine coated electrode.

9. Remove the shortened SWCNT-modified electrode and wash in DMF, ethanol, and then phosphate buffer (see Notes 5 and 6).

3.4.2. Attachment of MP-11

1. Prepare a microperoxidase MP-11 solution at a concentration of 0.5 mg mL-1 in HEPES buffer at pH 7.5.

2. Place the shortened SWCNT-modified electrode in the MP-11 solution held at 4°C and incubate overnight.

3. Remove the electrode and rinse in HEPES buffer and then phosphate buffer to remove loosely bound enzyme.

3.5. Atomic Force Microscope Imaging of Aligned SWCNTs

1. Freshly prepare the molecularly smooth gold substrates by template stripping as described previously (46).

2. Prepare the aligned nanotubes as described in Subheading 3.4.1.

3. Use the tapping mode for imaging using commercial Si cantilevers/tips (Olympus) at their fundamental resonance frequencies, which typically varies from 275320 kHz.

4. Record both height and phase images.

5. Measure the height and length of the SWCNTs using the cross-section analysis using Digital Instruments off-line software (see Note 7).

4. Notes

1. The cleanliness of the gold surface is all important in determining the quality of the SAM formed on the surface. Hence, even evaporated gold surfaces were cleaned prior to assembly of the modified electrode surface. In the case of poly-crystalline gold surfaces, it is important that the epoxy resin used to seal the gold into the glass tube be capable of withstanding the acid conditions used in cleaning. Most epoxy resins will soften in acidic or basic conditions. The epoxy resin that we used was EPON 825 with EPI-CURE 3271 curing agent from Shell Australia, which is resistant to both acidic and basic conditions.

2. The electrochemical cleaning process involves etching away part of the gold surface by oxidizing the surface as the potential is swept anodically (positive). As the potential is swept back negative, the gold oxide that is formed is reduced and a pronounced stripping peak is observed. The additional benefit of the elec trochemical cleaning procedure is that the area under this stripping peak can be used to determine the electrochemically accessible area of the gold electrodes. The area of the stripping peak gives the charge passed, and then electrode area can be found using the conversion factor of 480 mC cm-2 (47).

3. The longer the nanotubes are sonicated, the shorter the length distribution but the thicker the bundles the shorten tubes form. The length and width distribution can be determined using either high-resolution transmission electron microscopy or scanning tunneling microscopy in which the tubes are coated onto molecularly smooth gold surfaces fabricated as described previously (46). Either technique gives the same length distribution, as expected.

4. We have also observed a relationship between the length of the tubes and the mass of the tubes that can be dispersed into various solvents such as DMF. Although we have yet to quantify the length relationship, the amount of tubes that can be dispersed into a given solvent can be monitored via UV-VIS absorption.

5. From this point, they can be used as electrodes or further modified to attach the redox enzymes or other species. The carboxylic acids at both ends of the shortened SWCNTs are activated by the DCC to a carbodiimide that is susceptible to nucleophilic attack from amines. Therefore, to attach species to the other ends of the aligned SWCNTs assembled on the electrode surface simply requires a species with free amines. In our research, we have attached microperoxidase MP-11, glucose oxidase, propylamine, and ferrocene methylamine, synthesized according to the procedure of Kraatz (48). With the enzymes, amino acids with amine side chains provide the nucleophilic amines for covalent attachments to the SWCNTs. In each case, the attachment procedure is the same with the exception of the solvent to which the molecule to be attached is dissolved. The procedure in Subheading 3.4.2. is presented for the attachment of microperoxidase MP-11.

6. With the short chain alkanethiols used in this study, a potential problem is that they are easily oxidized in the presence of light. The result of this oxidation is that the gold-thiolate bond is converted into a gold-sulfinate or gold-sulfonate bond (49). Both the gold-sulfinate and gold-sulfonate bonds are much less stable than the gold-thiolate bond. Consequently, when performing an electrochemical control with the SAM-modified electrode alone, via cyclic voltammetry, an oxidation peak, owing to the conversion of the sulfinate to the sulfonate, is sometimes observed at approx +0.3 V vs Ag/AgCl. This peak is suppressed on attachment of the tubes. The presence of such a peak also serves as a good guide to the quality of the SAM prior to tube assembly. If the SAM has been prepared such that there is little or no oxidation of the gold-thiolate bond, then no oxidation process will be observed at such low anodic potentials.

7. An important observation from the preliminary experiments is that, although the transmission electron microscope measurements show that length distributions with a mean of approx 100 nm for 4 h cutting, the heights of the tubes in the atomic force microscope (AFM) images are only 10 to 20 nm from the surface. We believe this anomoly is an artifact of the AFM image. Note that the longer the cysteamine-modified gold surface is incubated in the nanotubes, the higher the density of tubes on the surface. There is also a concomitant increase in mean length of the aligned tubes. These observations provide good evidence that the AFM images are of aligned nanotubes. Further evidence comes from images as shown in Fig. 2 in which bundles of tubes standing vertically and lying horizontally can be observed.


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