5484
Langmuir 2004, 20, 5484-5492
Biosensing Properties of Diamond and Carbon Nanotubes Wei Choong Poh and Kian Ping Loh* Department of Chemistry, National University of Singapore, 3 Science Drive 3, Singapore 117543
Wei De Zhang and Sudhiranjan Triparthy Institute of Material Research and Engineering, 3 Research Link, Singapore 11760
Jian-Shan Ye and Fwu-Shan Sheu Department of Biological Science, National University of Singapore, 14 Science Drive 4, Singapore 117543 Received April 9, 2004 The biochemical properties of boron-doped diamond (BDD), carbon nanofiber, fullerene, and multiwalled carbon nanotube (MWCNT) electrodes have been investigated comparatively. Physiochemical factors which affect the biosensing properties such as surface hydrophobicities, effective surface area, and intrinsic material properties are studied. Voltammetric responses of the as-grown thin film electrode and surfacemodified electrode to biomolecules such as L-ascorbic acid (L-AA), dopamine (DA), and uric acid are examined. As-grown MWCNT electrodes exhibit selective voltammetric responses to the different biomolecules and faster electron-transfer kinetics compared to BDD. The selective response is due to the considerably lower anodic potential of L-AA on MWCNT (-48 mV vs Ag|AgCl compared to 575 mV on BDD). This electrocatalytic response can be replicated on a nonselective carbon nanofiber electrode by coating it with gold nanoparticles. BDD has no intrinsic selective response to L-AA, and surface modification by anodic polarization is necessary for resolving L-AA and DA.
1. Introduction Boron-doped diamond (BDD) and multiwalled carbon nanotube (MWCNT) electrodes have unique electronic and structural properties that are useful for integrated biosensing and signal processing systems. BDD is attractive due to its wide electrochemical potential window and chemical inertness.1 MWCNT is special due to its array of conducting wiring networks that can provide a highsurface matrix for the entrapment of biomolecules and the mediation of electrocommunication between the biomolecule and the substrate.2 Currently, there are two camps of researchers who focus their research efforts in extolling the virtues of either one of these two materials for electroanalysis and biosensing.1-8 It is timely to perform a comparative study of BDD and MWCNT in terms of their selectivity and sensitivity for biosensing applications. These performance issues depend fundamentally on the structural and electronic properties of the substrate material, as these impact on the speed of electron transfer between the enzyme active site and the electrochemical transducer. Other important require* Corresponding author. E-mail:
[email protected] (K. P. Loh). (1) Granger, M. C. Anal. Chem. 2000, 72, 3793-3804. (2) Popa, E.; Notsu, H.; Miwa, T.; Tyrk, D. A.; Fujishima, A. Electrochem. Solid-State Lett. 1999, 2 (1), 49. (3) Lau, C. H.; Grehan, K. J.; Compton, R. G.; Foord, J. S.; Marden, F. Diamond Relat. Mater. 2003, 12, 590-595. (4) Fujishima, A.; Rao, T. N.; Popa, E.; Sarada, B. V.; Yagi, I.; Tryk, D. A. J. Electroanal. Chem. 1999, 473, 179. (5) Sotiropoulou, S.; Chaniotakis, N. A. Anal. Bioanal. Chem. 2003, 375, 103. (6) Rubianes, M. D.; Rivas, G. A. Electrochem. Commun. 2003, 5, 689. (7) Luo, H.; Shi, A.; Li, N.; Wu, Z.; Zhuang, Q. Anal. Chem. 2001, 73, 915-920. (8) Takashi, K.; Tanga, M.; Takai, O.; Okamura, H. Diamond Relat. Mater. 2003, 12, 572.
ments include resistance to fouling by the oxidation products, long-term stability, and biocompatibility. The unique structure of MWCNT allows the entrapment of small proteins both in the inner channel and on the outer wall by hydrophobic or electrostatic interactions. In contrast, BDD provides a two-dimensional, structurally compact surface for the attachment of biomolecules. Recently, Takahashi and co-workers reported that the diamond substrate provides the highest density of DNA chip per unit area and exhibits high stability and reusability for preservation of gene samples.8 In this work, we compare the biosensing properties of diamond, MWCNT array electrodes, carbon nanofibers, and fullerenes for the voltammetric detection of biomolecules that are natural interferents in the human body. Low levels of dopamine (DA) have been found in patients with Parkinson’s disease. A major problem encountered in voltammetric detection is the coexistence of interfering compounds such as ascorbic acid (L-AA) and uric acid (UA).9,10 Generally, voltammetric differentiation of these biomolecules is not possible on bare metal or carbon electrodes due to the overlap of oxidation voltages for these species. Major efforts in biosensor research essentially involve elaborate surface modification steps to impart perm-selection or electrostatic selection on these electrodes toward DA, L-AA, and UA.9-12 The research effort here is to understand the physiochemical properties of diamond and MWCNT which influence their selective response to the three biomolecules. (9) Wang, Z. H.; Liang, Q. L.; Wang, Y. M.; Luo, G. A. J. Electroanal. Chem. 2003, 504, 129. (10) Raj, C. R.; Ohsaka, T. J. Electroanal. Chem. 2001, 496, 44. (11) Olivia, H.; Sarada, B. V.; Shin, D.; Rao, T. N.; Fujishima, A. Analyst 2002, 1572. (12) Zhang, Z.; Lei, C. H.; Deng, J. Analyst 1996, 121, 971.
10.1021/la0490947 CCC: $27.50 © 2004 American Chemical Society Published on Web 05/20/2004
Diamond and Carbon Nanotubes
Langmuir, Vol. 20, No. 13, 2004 5485
Figure 1. SEM images and associated Raman spectra of the BDD and MWCNT electrodes. MWCNT#1 is constructed by growing a dense vertical array of CNT directly on tantalum. MWCNT#2 is constructed by pasting bulk-synthesized CNT powder on a Pt working electrode.
2. Experimental Section 2.1. Preparation of Electrodes. The BDD electrode was grown on a 1 mm diameter Niobium disk using conventional CH4/H2/diborane chemistry, with a 2.45 GHz commercial microwave chemical vapor deposition (CVD) system. Pinhole-free, wellfaceted polycrystalline diamond could be obtained with a Raman signature at 1331 cm-1 characteristic of diamond. Hydrogentermination of the BDD was achieved by subjecting the BDD electrode to hydrogen-plasma treatment at 700 °C in the microwave plasma CVD system. MWCNT#1 is a multiwalled carbon nanotube array grown directly on tantalum (Figure 1b). The nanotubes (10 µm thick, vertically oriented) were grown in a quartz furnace using either ethylenediamine or acetylene on 1 mm diameter tantalum substrates seeded with cobalt nanoparticles. The as-grown films
were attached via the backside of the metal substrate to a Pt working electrode (CH instrument) using silver paste. The peripheral regions were then encapsulated in epoxy glue to expose only the working surface. MWCNT#2 was prepared by impregnating a Pt working electrode with multilayers of MWCNT. Ten milligrams of acidtreated MWCNT (powder) was dispersed in 10 mL of N,Ndimethylformamide (DMF) with the aid of ultrasonic agitation. From 10 mg/mL of the saturated MWCNT-DMF (black) solutions, 50 µL was introduced onto the surface of the Pt working electrode substrate (at 10 µL intervals) using a dropper, and the solvent was evaporated under an infrared heat lamp. Scanning electron microscopy (SEM) visualization of the surface (Figure 1d) shows that multilayer CNT could be deposited on the electrode
5486
Langmuir, Vol. 20, No. 13, 2004
Poh et al.
Table 1. Physiochemical Properties of Various Electrodes
electrodes
effective surface area/ cm2
electrochemical potential window/V
BDD#1 BDD#2 MWCNT#1 MWCNT#2 MWCNT (w/o N) carbon nanofiber gold-modified carbon nanofiber C60
4.11 × 10-1 4.73 × 10-1 12.30 × 10-1 11.20 × 10-1 8.35 × 10-1 4.22 × 10-1 8.51 × 10-1 1.95 × 10-1
2.55 2.41 2.24 2.31 1.91 1.97 2.36 1.94
anodic peak potential of biomolecules/mV L-AA UA DA 575.6 447.0 91.33 -48.0 -30.7 209.3 6.1 467.6
441.3 439.0 380.0 390.0 401.9 342.8 327.2 446.1
232.0 355.0 125.7 216.7 220.1 188.6 191.3 318.7
separate voltammetric signals for L-AA, UA, and DA
contact angle of water droplet/deg
× × x x x × x ×
96.4 87.2 145.0 105.7 127.4 70.3 128.2 77.7
surface with good adhesion. Similar procedures were repeated for impregnating the working electrode with fullerene. The effective surface area of the MWCNT and BDD electrodes was estimated by cyclic voltammetry using 4 mM K3[Fe(CN)6]/ 0.1 M KCl as a probe at various scan rates. The slope of the straight line of the plot Ipc versus v1/2 can be substituted into the equation
Ip ) (2.69 × 105)n3/2AD1/2v1/2C0 to calculate the effective surface area A. This is tabulated in Table 1 for the various electrodes after normalizing against the area of the base substrate. The reported current values in the cyclic voltammograms have been normalized against the effective surface area of the electrodes in this study. 2.2. Equipment and Measurements. For voltammetric studies, a Gamry potentiostat (Femtostat) was used with a Pt wire counter electrode and a 3 M KCl-Ag|AgCl reference electrode (CH instruments, E ) 0.208 V vs standard hydrogen electrode). All voltages were referenced to that of the Ag|AgCl reference electrode. All the measurements were performed in solutions which were thoroughly deaerated with high-purity nitrogen. Cyclic voltammetry (CV) was typically performed at a scan rate of 100 mV s-1 unless stated otherwise. The contact angle of water introduced by a microsyringe on the sample was measured by a contact angle meter and recorded by a video.
3. Results 3.1. Structural Characterization of Electrodes. Figure 1 shows the SEM images of the BDD electrodes used in this study. Two samples are used here which differ in their grain sizes and boron doping levels. BDD#1 consists of diamond with grains in the range of several micrometers, while BDD#2 consists of submicron grains. The associated Raman spectra of both, in addition to the diamond signature peak at 1331 cm-1, show an impurityrelated peak at 1220 cm-1 associated with Raman scattering from boron-induced impurity bands. The impurity band is stronger in BDD#1 due to the heavier doping to induce metallic conduction. MWCNT#1, as shown in Figure 1c, is characterized by multiwalled nanotubes growing in a dense, vertical array fashion on a tantalum substrate. The tantalum substrate, at the base of the MWCNT array, is passivated by a carbon film. MWCNT#2, as shown in Figure 1d, is fabricated by impregnating a Pt working electrode with bulk-synthesized MWCNT. The associated Raman spectra of both reveal the presence of D-band resonance (1350 cm-1) and G-band resonance (1584 cm-1) peaks, which are characteristic of graphitic materials. MWCNT#2 has been pretreated with acid etching to remove metallic catalyst and to open the nanotube edges. Transmission electron microscopy (TEM) analysis of the MWCNT#2 verifies that the majority of the nanotubes are catalyst-free and consist of open-ended tubes, as shown in Figure 2. Although MWCNT#1 consists of well-aligned carbon nanotubes oriented vertically, the nanotubes collapse once these are immersed in aqueous solution. For MWCNT with tube
Figure 2. TEM images showing the catalyst-free nature of the multiwalled carbon nanotubes used for constructing the MWCNT#2 electrode.
lengths shorter than 5 µm, SEM visualization of the surface after removal from the solution reveals that the MWCNT collapses into regular conical bundles due to capillary forces, as shown in Figure 3. Longer MWCNTs are found to collapse into a random mass. The collapsed nanotubes expose only the exterior conical surface and exclude the permeation of aqueous reactants into the hydrophobic core. Consequently, the effective surface area for charge transfer will be reduced significantly. This explains why the effective surface area of the MWCNT nanotube array as evaluated by cyclic voltammetry, as shown in Table 1, is not several orders larger than that of diamond, as would be expected if the physical surface area of MWCNTs correlates with their effective surface area for charge transfer. For the same reason, no significant differences in effective surface area existed between electrodes fabricated by growing vertically oriented MWCNT directly on the working electrode (MWCNT#1) and electrodes that were fabricated by
Diamond and Carbon Nanotubes
Langmuir, Vol. 20, No. 13, 2004 5487
Figure 3. SEM images of a multiwalled carbon nanotube array (a) before insertion into PBS and (b) after insertion into PBS.
impregnation with externally synthesized carbon nanotubes (MWCNT#2). MWCNTs synthesized under different environments show some differences in their hydrophobicities and effective surface areas. For electrodes fabricated from MWCNT grown using ethylenediamine as the precursor, the N content in the sample assayed by elemental analysis is about 1%. For electrodes fabricated from MWCNT grown using acetylene, there is no N incorporation. The Nincorporated MWCNT incorporates some polar character in the MWCNT, and it is found to be more hydrophilic compared to the pure MWCNT, with a slightly larger effective surface area as a result. Other than that, no significant differences occur in the biosensing of the different biomolecules. However, we found that if the growth conditions were changed to produce carbon nanofibers rather than tubes (by increasing the concentration of acetylene), the desired biosensing properties were degraded. Figure 4a,b shows the SEM and TEM images of the vertically aligned carbon nanofibers. Structurally, each carbon nanofiber is a bundle of individual defective, bamboo-like nanotubes adjoined side-to-side, and therefore the carbon nanofibers are thicker than individual MWCNTs. The catalyst-encapsulated, bamboo-like segments of the individual nanotubes in the bundle can be seen in the TEM image. These defective tubes differ from crystalline carbon nanotubes because the internal structure is made of interconnected chains of carbon nanocages rather than continuous walled tubes. The internal structure arises from the fluidlike capillarity of the metal catalyst which traces out segmented growth. We found that carbon nanofibers do not have the intrinsic electrocatalytic property of high-quality multiwalled carbon nanotubes.
Figure 4. (a) SEM images of carbon nanofibers grown under acetylene-rich conditions. (b) TEM image showing that each carbon nanofiber is a bundle of defective, bamboo-like nanotubes.
Table 1 summarizes important parameters for the biosensing and physiochemical properties of various carbon electrodes, ranging from BDD, MWCNT, and carbon nanofibers to fullerenes. Wettability studies reveal that BDD is consistently less hydrophobic than MWCNT. This may be due to presence of surface oxygen on BDD compared to MWCNT. Even for a freshly hydrogenplasma-treated surface, oxygenation of the surface proceeds to some extent once the surface is exposed to the ambient, as verified by X-ray photoelectron spectroscopy (XPS). Figure 5 shows the contact angle of a water droplet on the surface of BDD#2 and MWCNT#1. We can see very clearly that the contact angle is smaller on BDD (87.2°) compared to MWCNT (145°), indicating a better wettability of water on BDD. The effective surface for charge transfer as verified by CV is found to be largest on nitrogen-doped MWCNT; this is about 3 times larger than that of BDD. The electrochemical potential windows of the BDD, MWCNT, carbon nanofibers, and fullerene in phosphate-buffered saline (PBS) are shown in Figure 6. BDD has the widest electrochemical potential window and the narrowest background current of all, followed by MWCNT, carbon nanofibers, and fullerene. The electrochemical potential window of fullerene is the smallest. It is well documented that C60 is redox active and undergoes multiple electrontransfer steps.14 The wider potential window of BDD is especially advantageous in the detection of nucleic acids due to their high positive oxidation potential.15-16
5488
Langmuir, Vol. 20, No. 13, 2004
Poh et al.
Figure 5. The contact angle of a water droplet on the surface of BDD#2 (left) and MWCNT#1 (right).
Figure 7. The CV of 1 mM DA in pH 7 PBS on BDD #1 at three scan rates.
Figure 6. Electrochemical potential window of various electrodes in pH 7 PBS. From top to bottom: boron-doped diamond (BDD), multiwalled carbon nanotube (MWCNT), carbon nanofiber (CNF), and fullerene.
3.2. Voltammetric Sensing of L-AA, DA, and UA. 3.2.1. Comparing the Anodic Peak Potentials of Biomolecules. The peak potential for the oxidation of the various biomolecules is indicative of the physiochemical properties of the electrode surfaces.1 Table 1 lists the oxidation potential of L-AA, UA, and DA on the various as-grown electrodes. The anodic peak potentials of the individual biomolecules provide clues on whether selective detection is possible on the untreated, as-grown electrodes. For example, to resolve L-AA from DA and UA, L-AA should be first oxidized before DA and UA in the anodic scan direction. One reason is that the oxidation of DA is a twoelectron-transfer process and the oxidation products interfere with the detection of L-AA. If the oxidation of DA occurs before that of L-AA in the CV scan and if the (13) Troupe, C. E.; Frummond, I. C.; Graham, C.; Grice, J.; John, P.; Wilson, J. I. B.; Jubber, M. G.; Morrison, N. A. Diamond Relat. Mater. 1998, 7, 575. (14) Sherigara, B. S.; Kutner, W.; D’Souza, F. Electroanalysis 2003, 15, 753. (15) Prado, C.; Flechsig, G.-U.; Gundler, P.; Foord, J. S.; Marken, F.; Comptom, R. G. Analyst 2002, 127, 329. (16) Gu, H.; Su, X.; Loh, K. P. Chem. Phys. Lett. 2004, 388, 483.
Figure 8. The voltammetric peaks of 2 mM L-AA, 1 mM DA, and 2 mM UA in pH 7 PBS using BDD#1 as the sensing electrode.
electrolyte contains a mixture of DA and L-AA, DA would be oxidized first, and the oxidized form of DA will subsequently catalyze the oxidation of L-AA at a potential close to that of DA, resulting in a single, ill-resolved peak for both analytes due to interference. Looking at Table 1, the differences in the anodic peak potentials of UA appear to be relatively insensitive to the type of electrode, while L-AA exhibits the largest range of differences, with DA showing responses intermediate between those of L-AA and UA. In the case of L-AA, for example, the window of differences in the peak potential is as wide as 500 meV on different electrodes. The remarkable feature of MWCNT stands out because the anodic peak potential of L-AA is at the most negative in the anodic scans and occurs before that of DA and UA. Subsequent tests verified that MWCNT could be selective for L-AA when the electrolyte was premixed with DA, UA, and L-AA. In contrast, the anodic peak potentials of L-AA on other untreated electrodes such as BDD, carbon nanofibers, and fullerene occur at a higher potential (more positive direction) than those of DA, and
Diamond and Carbon Nanotubes
Langmuir, Vol. 20, No. 13, 2004 5489
Figure 9. (a-d) High-resolution XPS showing the changes in the C 1s profile as a function of the anodic polarization voltages on BDD: (a) 0-0.5 V, (b) 0-1.0 V, (c) 0-1.5 V, (d) 0-2.0 V.
subsequent experiments verified that resolving the two was not possible. One method of lowering the anodic peak potential of L-AA with respect to DA is to introduce catalytic gold nanoparticles on the surface of the untreated electrodes. We found that the presence of gold nanoparticles lowered the anodic peak potential of L-AA with respect to DA, affording selective detection. 3.2.2. Biosensing on BDD. The physiochemical response of BDD electrodes depends critically on the surfaceterminating groups. These electrodes give the most stable electrochemical response when the surfaces are freshly terminated by hydrogen after a hydrogen-plasma treatment. From contact angle measurement, we found that the hydrogen-plasma-treated surface was the most hydrophobic surface. A hydrogen-plasma treatment is preferred to polishing the BDD surface with alumina grit because the latter approach produces a surface that is readily fouled by the oxidation products. Generally, for the hydrogenated BDD surface, a well-defined, diffusioncontrolled voltammetric signal can be detected for the oxidation of DA, UA, and L-AA. Figure 7 shows the CV of DA on the BDD#1 at three scan rates. For all three biomolecules, linear relationships between the peak current and the square root of the scan rate indicate a
diffusion-controlled process on BDD. The hydrogenated BDD surface prepared by microwave hydrogen plasma treatment displays Epa ) 232 mV versus Ag|AgCl for DA and a peak-to-peak separation (∆Ep) between Epa and Epc of 140 meV. Previously Popa reported a ∆Ep of 500 meV for the oxidation of DA on as-received diamond.2 We found that a wider ∆Ep occurs for BDD whenever the surface is oxidized or after secondary treatment like polishing with alumina grit. A hydrogen-plasma treatment improves the electron-transfer kinetics dramatically by removing surface oxygenated species. The oxidation of L-AA occurs at 575.6 mV on BDD and is irreversible. The process has been proposed to follow a two-electron-transfer two-proton pathway accompanied by cyclization and water exchange steps.18 For UA, the anodic peak occurs at 441.3 mV. The voltammetric peaks for L-AA, DA, and UA are shown together in Figure 8. We found that resolving these species voltammetrically was not possible for BDD#1, because only an ill-resolved peak (17) Notsu, H.; Yagi, I.; Tatsuma, T.; Tryk, D. A.; Fujishima, A. Electrochem. Solid-State Lett. 1999, 2, 522. (18) Akkermans, R. P.; Wu, M.; Bain, C. D.; Fidel-Suarez, M.; Comptom, R. G. Electroanalysis 1998, 10, 613 and references therein.
5490
Langmuir, Vol. 20, No. 13, 2004
Figure 10. Only a single voltammetric peak is obtained on untreated BDD#1 when DA and UA are added in the same buffer: (solid line) 0.11 mM DA + 1.78 mM UA in pH 7 PBS. Oxygenated BDD#1 can resolve DA and UA as two separate peaks: (dashed line) 0.09 mM DA + 1.82 mM UA and (dotted line) 0.11 mM DA + 1.78 mM UA. All measurements were done using 0.1 M HClO4 as the supporting electrolyte. Scan rate, 100 mV/s.
Poh et al.
Figure 12. Untreated BDD#2 is able to resolve DA and UA: (solid line) 0.33 mM DA + 1.33 mM UA; (dashed line) 0.5 mM DA + 1 mM UA; (dotted line) 0.4 mM DA + 0.8 mM. All measurements were done using pH 7 PBS as the supporting electrolyte. Scan rate, 100 mV/s.
Figure 13. Comparison of CV for 1 mM DA in pH 7 PBS using MWCNT#1 and BDD#1. ∆Ep(BDD) ) 0.14 V and ∆Ep(MWCNT) ) 0.08 V. Scan rate, 100 mV/s. Figure 11. The voltammetric differentiation of 0.2 mM DA and 1.6 mM L-AA by oxygenated BDD#1 using 0.1 M HClO4 as the supporting electrolyte (solid line). Scan rate, 100 mV/s. The background (dashed) lines show the peaks when DA and L-AA are sensed individually.
was obtained whenever any two of the three species were added together. One strategy first proposed by Fujishima and coworkers2,17 involves generating negative surface functional groups on the surface such as COO- or OH- to afford electrostatic selection toward these biomolecules. DA exists as cationic species at neutral pH, while L-AA and UA exist as anionic species. An oxygenated surface was prepared by anodically polarizing the BDD surface for 30 min between 0.5 and 2 V. The chemical phase on the BDD surface was investigated by high-resolution XPS. The XPS spectra of the electrode surface following anodic polarization at different voltages are plotted in Figure 9. The inset shows the spectrum for the hydrogenated BDD surface, which shows a C 1s peak with a narrow full width at half-maximum. After anodically polarizing the surface, the C 1s peak broadens considerably and several chemically shifted components start to manifest in the spectra. It can be seen that there is a clear trend between the anodic oxidation voltages and the intensities of the chemically shifted peaks assignable to the CdO species. At an anodic voltage of +2.0 V, chemically shifted peaks associated with the carboxylic groups are produced. These components have a stronger intensity than those of the original bulk C 1s, suggesting that the near-surface region has been converted to a matrix of carboxylated groups following electro-oxidation.
The electrochemical activity of the oxidized BDD surface was more sluggish compared to that of the hydrogenated surface. There is a concomitant shift of Epa for all biomolecules to higher anodic voltages for diamond pretreated by higher anodic polarization voltages, with a consequent widening of the ∆Ep, which indicates that the oxygenated surface has a slower electron-transfer kinetics compared to the hydrogenated surface. The negatively charged oxygenated surface has some form of electrostatic interaction with the charged analyte. In perchloric acid, DA is protonated and is attracted to the negatively charged oxygenated surface, while UA is anionic and repelled. This allows the DA peak to be resolved from UA as two separate peaks when both UA and DA are added into the 0.1 M perchloric acid, as shown in the CV plot in Figure 10. Resolving L-AA from DA has also been demonstrated to be possible on the oxygenated surface, as shown in Figure 11. However, the drawback is that this method works only in an acidic buffer and the voltammetric peaks are broad. Moreover, the oxidized BDD surface can at most resolve a two-component system, such as UA and DA or L-AA and DA, but not a three-component system where UA, L-AA, and DA are present together because of the broad individual peaks. The ability to separate DA from UA was found to depend on the nature of the BDD electrode. Although untreated BDD#1 cannot resolve UA and DA, we found that these could be separated as two voltammetric peaks on untreated BDD#2, without the need to preanodize the electrode, as shown by the voltammogram in Figure 12. The peak potential of DA is sensitive to the band continuum below the Fermi level for doped samples. For BDD#2, which is less heavily doped with respect to BDD#1, the over-
Diamond and Carbon Nanotubes
Langmuir, Vol. 20, No. 13, 2004 5491
Figure 14. Comparison of multiple CV scans for 2 mM L-AA in pH 7 PBS using BDD#1 (left) and MWCNT#1 (right) electrodes.
potentials for the oxidation of DA and UA are shifted toward the positive direction. However, the difference in the anodic peak potentials between DA and UA is ∼250 meV for both types of BDD electrodes despite the shifts in the individual peaks, so the differences in their biosensing properties cannot be explained by invoking the presence of selective electrocatalytic response. We speculate that the differences may arise from the different grain sizes of BDD#1 and BDD#2 which influence their physical interaction with different biomolecules. Further research on nanocrystalline diamond with nanograins may throw some light on this. 3.2.3. Biosensing on MWCNT. The biosensing properties of the MWCNT are independent of the manner of alignment of the respective nanoubes on the substrate; that is, no significant differences in biosensing are observed for MWCNTs which are vertically aligned or lying unoriented with respect to the substrate face. From Table 1, we can see that generally, the overpotentials for the oxidation of L-AA, UA, and DA are reduced for MWCNTs compared to BDDs; the oxidation currents for these peaks are also higher on MWCNTs, suggesting electrocatalytic activity due to the increase in electrontransfer rate from these biomolecules to MWCNT. Figure 13 compares the CV for DA on MWCNT and BDD. The ∆Ep for DA is 80 mV compared to 140 mV on diamond at a scan rate of 100 mV/s, indicating faster electron-transfer kinetics on MWCNT. Two important differences exist for MWCNT compared to BDD for the oxidation of L-AA. First, the anodic peak potential for L-AA is shifted 200 mV lower than that of DA. This is significant because if L-AA is oxidized before DA in the anodic scan, then the byproducts from the oxidation of DA will not interfere with its detection, and thus separation between the two should be possible. Second, the anodic peak for L-AA on MWCNT is observed to be sharper compared to that with BDD for the same molar concentration. Figure 14 shows the multiple CV scans for L-AA using MWCNT and BDD electrodes, respectively. We can see that the anodic peaks for L-AA are sharper on MWCNT, while on BDD, the peaks are broader, evidencing faster electrontransfer kinetics on CNT. However, the lower background current on BDD is noteworthy, suggesting that it may behave better than CNT in applications requiring highly sensitivie amperometric detection if the selectivity problem to L-AA can be solved first. The oxidation of L-AA proceeds via ascorbate radicals, and the byproducts are known to readily attach to metal electrode surfaces and foul the electrodes. Both BDD and MWCNT electrodes show good fouling resistance. We found that the voltammetric separation of L-AA and DA by as-grown MWCNT can be achieved when the two
Figure 15. CV of various biomolecules on MWCNT. Top: 2 mM L-AA. Middle: 1 mM DA. Bottom: 0.11 mM DA + 1.78 mM L-AA. Scan rate, 100 mV/s in PBS buffer.
Figure 16. Voltammetric differentiation of L-AA, DA, and UA in the form of three distinct anodic peaks on MWCNT: (dashed line) 2 mM L-AA only; (dotted line) 0.11 mM DA + 1.78 mM L-AA; (solid line) 0.07 mM DA + 1.07 mM L-AA + 0.8 mM UA. Scan rate, 100 mV/s.
biomolecules were added (DA/L-AA ) 1:8 molar ratio) in the same buffer at neutral pH. In Figure 15, the first CV is due to L-AA alone, while the second CV was run with L-AA and DA added in the same buffer solution. A clear differentiation of L-AA and DA is possible with a separation between the peaks of more than 200 mV. In addition, MWCNT is able to resolve the three-component system of L-AA, DA, and UA. Figure 16 shows voltammetric differentiation of L-AA, DA, and UA in the form of three distinct anodic peaks on MWCNT. To our knowledge, this superior property of MWCNT is unsurpassed, as no electrode material, in its “as-grown” form, can show resolvable voltammetric peaks for all three biomolecules in the same mixture.
5492
Langmuir, Vol. 20, No. 13, 2004
Poh et al.
Figure 17. (left) CV of 2 mM L-AA, 1 mM DA, and 2 mM UA recorded individually on a carbon nanofiber electrode. Note the overlap of the L-AA and DA peaks. (right) The inset shows an unresolved peak when L-AA and DA are mixed together. The bigger plot shows that DA and UA can be resolved on carbon nanofiber when mixed, 0.50 mM DA + 1.00 mM UA. Scan rate, 100 mV/s.
Figure 18. (left) CV of 2 mM L-AA, 1 mM DA, and 2 mM UA recorded individually on a carbon nanofiber electrode. Note that the addition of gold lowers the anodic potential of L-AA with respect to DA. (right) CV showing the successful separation of the biomolecules as three distinct voltammetric peaks on gold-modified carbon nanofiber: (solid line) 1.07 mM L-AA + 0.07 mM DA + 0.80 mM UA; (dashed line) 1.78 mM L-AA + 11 mM DA + 2.00 mM L-AA. Scan rate, 100 mV/s.
Is this enhanced electrochemical property intrinsic to the structure of carbon nanotubes? To confirm this, we evaluated the biosensing properties for related sp2 carbon nanophase materials such as carbon nanofibers and fullerene. Our studies show that carbon nanofibers and fullerene (data not shown) behave similarly to BDD#2; they can resolve DA and UA but not L-AA from the rest. The CV for untreated carbon nanofibers (CNF) is shown in Figure 17, where a broad ill-resolved peak for L-AA is detected when it is mixed with DA. We can see that the overpotentials for L-AA and DA overlapped. As mentioned earlier, to resolve L-AA from DA, it is essential that the oxidation of L-AA occurs at a lower anodic potential before DA. MWCNT shows catalytic oxidation of L-AA compared to other carbon electrodes such as BDD, carbon nanofibers, and fullerenes because the overpotential for the oxidation of L-AA is shifted to negative values. The enhanced catalytic response of MWCNT can be replicated on the nonselective carbon nanofiber by evaporating about 3-4 nm of gold onto the carbon nanofibers. The CV results plotted in Figure 18 confirmed that with the assistance of catalytic nanogold particles, the overpotential for the oxidation of L-AA is lowered by 100 mV in the negative direction, such that L-AA, DA, and UA could be resolved similar to the results obtained from MWCNT. Slow electrode reactions of L-AA result in sluggish kinetics and ill-resolved voltammetric signals for most electrodes; the acceleration of such kinetically hindered reactions by catalysts permits the analysis of these analytes at less extreme potentials, because catalyzed electrode reactions usually occur near the formal redox potential of the catalyst. Since the MWCNT we used
(MWCNT#2) is catalyst-free, we can conclude that the electrocatalytic property is an intrinsic property of the multiwalled carbon nanotubes. The high-density, nanoscale crystalline tubular walls, allied with a high densityof-states near the Fermi level, facilitate fast electrontransfer kinetics from the biomolecules. 4. Conclusions The biosensing properties of BDD, MWCNT, carbon nanofibers, and fullerenes have been studied. MWCNT is the only material which in its untreated state can show selective responses to L-AA, UA, and DA, due to its intrinsic electrocatalytic properties for the biomolecules. We found no advantages in using well-aligned MWCNT arrays in biosensing since these would collapse in solution due to capillary action, and the effective surface area is similar to that of the randomly oriented MWCNT. The latter can be fabricated readily by a simple drop-and-dry process involving bulk-synthesized CNT powder. BDD exhibits the widest electrochemical potential window and lowest background current, suggesting its use in high-sensitivity amperometric sensors. BDD, carbon nanofiber and fullerene electrodes can resolve DA and UA but not L-AA from the rest due to the slow electron-transfer kinetics of L-AA which results in overlapped voltammetric peaks. Introducing gold nanocatalysts on carbon nanofibers can replicate the electrocatalytic properties of CNT by lowering the overpotentials for the oxidation of L-AA with respect to DA. Surface modification of BDD by generating an oxygenated phase on the surface can impart partial selectivity for any two of the three biomolecules. LA0490947
