inlaid multi-walled carbon nanotube nanoelectrode arrays for electroanalysis

Review

Inlaid Multi-Walled Carbon Nanotube Nanoelectrode Arrays forElectroanalysis

Jun Li,* Jessica E. Koehne, Alan M. Cassell, Hua Chen, Hou Tee Ng, Qi Ye, Wendy Fan, Jie Han, M. Meyyappan

Center for Nanotechnology, NASA Ames Research Center, Moffett Field, CA 94035*e-mail: [email protected]

Recceived: May 17, 2004Final version: June 16, 2004

AbstractThe rapid development in nanomaterials and nanotechnologies has provided many new opportunities forelectroanalysis. We review our recent results on the fabrication and electroanalytical applications of nanoelectrodearrays based on vertically aligned multi-walled carbon nanotubes (MWCNTs). A bottom-up approach isdemonstrated, which is compatible with Si microfabrication processes. MWCNTs are encapsulated in SiO2 matrixleaving only the very end exposed to form inlaid nanoelectrode arrays. The electrical and electrochemical propertieshave been characterized, showing well-defined quasireversible nanoelectrode behavior. Ultrasensitive detection ofsmall redox molecules in bulk solutions as well as immobilized at the MWCNT ends is demonstrated. A label-freeaffinity-based DNA sensor has shown extremely high sensitivity approaching that of fluorescence techniques. Thisplatform can be integrated with microelectronics and microfluidics for fully automated microchips.

Keywords: Multi-walled carbon nanotubes, Vertically aligned nanoelectrode array, Chemical sensors, Biosensors,DNA sensors, Multiplex array, Microchips

1. Introduction

Ultramicroelectrodes (UMEs), whose critical dimension isless than the scale of the diffusion layer [1 – 3], haveattracted extensive attention since 1980s, for developingultrasensitive electrochemical sensors, analytical tools formeasuring kinetics of fast-electron transfer reactions, andprobes to detect species in microenvironments. With recentadvances in fabrication technologies, the size of UMEs canbe further reduced down to nanometer scale. Such nano-electrodes (NEs) have shown unprecedented temporal andspatial resolution as well as extremely high sensitivity [4 – 8].Nanoelectrode arrays (NEAs) are of particular interest forelectroanalysis due to the ease of use and higher reliabilitythan single NEs. The detection limit for small electroactivespecies has been demonstrated down to the nM regime [9]. Itwas found that the size and the spatial distribution arecritical to the performance of a NEA [9 – 11]. However, areliable method to mass-produce well-controlled NEAs at areasonable cost is still lacking.

Accompanying the rapid development in nanomaterialsand nanotechnologies, there is a strong interest to inte-grate the new nanoscale elements into electroanalyticaland bioanalytical systems for miniaturization and per-formance enhancement [12 – 20]. High aspect-ratio one-dimensional (1-D) nanomaterials such as carbon nano-tubes (CNTs) are of particular interest [16 – 21]. However,most of these studies [16 – 20] employ randomly stackedCNT films with a large 3-D surface area exposed insolutions. For many electroanalytical applications, theelectrode area needs to be minimized to reduce the

background noise. A more carefully designed platform isneeded to achieve this goal.

Recently, we have demonstrated, in a series of reports[22 – 26], that well-insulated NEAs can be fabricated usinga bottom-up approach, which combines the recentlydeveloped CNT nanotechnology with existing microfabri-cation techniques. The sidewall of the carbon nanotubes inthe vertically aligned array is encapsulated with SiO2,leaving only the very end exposed to the solution to forminlaid nanodisk electrode array. This technique is funda-mentally different from the carbon nanotube array report-ed by Gooding et al. [18] and Yu et al. [20], where carbonnanotubes form a forest-like structure with a large three-dimensional surface area and behave as a highly porousmacroscopic film instead of NEs. The capacitive charging-discharging current of such electrodes is too high (propor-tional to the effective electrode surface area), limiting thedetection sensitivity for electroanalysis. The goal of ourstudy is to develop a NEA showing well-defined NEbehavior with minimum surface area so that the sensitivityand temporal resolution can be improved. In this review, wesummarize the fabrication processes and thorough elec-trical and electrochemical characterization of CNT NEAs.It is shown that such CNT NEAs present well-defined NEbehavior, which can be directly used for measuring electro-active species in bulk solutions down to a few nM. Thegraphitic structure also provides a flexible surface chem-istry so that biomolecules can form covalent bonds selec-tively to the end of the CNTs. A specific oligonucleotideprobe is functionalized through covalent bonds for devel-oping affinity-based DNA sensors. The hybridization of

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Electroanalysis 2005, 17, No. 1 � 2005 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim DOI: 10.1002/elan.200403114

both oliogonucleotide probes and PCR amplicons isdirectly detected with an electrocatalytic method. High-degree multiplex devices can be fabricated with this methodfor integrating with microfluidics systems for electroanal-ysis.

2. The Fabrication of CNT NEAs

CNTs are a family of materials consisting of seamlessgraphitic cylinders with extremely high aspect ratios [27 –30]. The typical diameter varies from about 1 nm tohundreds of nm and the length spans from tens of nano-meters to hundreds of microns or even centimeters. A CNTmay consist of one graphitic layer, referred as single-walledCNTs (SWCNTs) [31], or multi graphitic layers, referred asmulti-walled CNTs (MWCNTs) [32]. A SWCNT normallypresents a smaller diameter (down to 7 �), whose electron-ics properties are strongly dependent on the helicity, namelythe (m, n) lattice vector in the graphitic sheet along which itis rolled into the tube [33]. About 2/3 of SWCNTs aresemiconducting with the chirality (m,n) satisfing m-n=3 xinteger and 1/3 of them are metallic with m-n¼ 3 x integer[27 – 30]. A MWCNT consists a random mixture of allpossible helicities in each shell and overall behaves as ametallic wire [34]. In this review, we focus on usingMWCNTs as metallic wires to form NEs.

Due to the well-defined graphitic structure, MWCNTspresent similar chemical properties as conventional carbonelectrodes, with a wide potential window, flexible surfacechemistry, and good biocompatibility. However, the electro-chemical properties of MWCNTs are highly anisotropic: theopen end of a MWCNT has fast electron transfer rate (ETR)similar to a graphite edge-plane electrode while the sidewallpresents a very slow ETR and low specific capacitancesimilar to the graphitic basal plane [35]. Therefore, theproper construction and orientation of the electrode iscritical for its electrochemical properties. Ideally, a well-defined vertical array is desired which can transportelectrons from an active open end to the other end that isdirectly attached to the measuring circuit, while the sidewallis insulated to reduce the background.

As illustrated in Figure 1, a novel bottom-up approachcan be employed to fabricate desired MWCNT NEAs on ametal film by the combination of micro- and nano-lithography with catalytic CNT growth techniques. Thefabrication scheme consists of the following steps:

a) Metal film deposition: A metal film (typically ca. 200 nmthick Cr or Pt) is deposited on a Si wafer covered with ca.500 nm SiO2 or Si3N4. The metal film serves as theelectrical contact connecting MWCNT NEs to themeasuring circuitry. The metal film can be designed asindividually addressed microelectrode pads using UV-lithograpgy if necessary.

b) Catalyst deposition: A Ni film of ca. 10 to 30 nm thicknessis deposited on top of the metal film to serve as thecatalyst to promote the CNT growth.

c) Plasma enhanced chemical vapor deposition (PECVD)for CNT growth: A forest-like vertically alignedMWCNT array is grown on Ni catalyst film by PECVDusing a DC biased hot-filament CVD system [36 – 38].The electrical field normal to the surface helps to alignMWCNTs vertically on the substrate surface and formfree-standing arrays.

d) Dielectric encapsulation: A SiO2 film is deposited by alow-pressure thermal CVD (LPCVD) process usingtetraethoxysilane (TEOS) at a vapor pressure of ca.400 mTorr and at a temperature of 715 8C in a quartz tubefurnace. SiO2 forms a conformal film filling the spacebetween MWCNTs as well as covering the substratesurface.

e) Planarization: The dielectric film dramatically increasesthe mechanical strength of the CNT array so that achemical mechanical polishing (CMP) process can beapplied to planarize the surface. Excess SiO2 and part ofthe MWCNTs are removed so that the very end of someMWCNTs is exposed. We control this process so that onlya small number of MWCNTs are exposed to form a low-density inlaid NEA.

f) Electrochemical characterization: Electrochemicalmeasurements are carried out with a three-electrodeconfiguration. The embedded MWCNT NEA is used asthe working electrode (w.e.) defined with a 3 mm i.d. O-ring sealed in a Teflon cell. A Pt coil is used as the counterelectrode (c.e.) and a saturated calomel electrode (SCE)is used as the reference electrode (r.e.).

Figures 2a and b show SEM images of an as-grown MWCNTarray. Clearly, each MWCNT is well aligned vertically at thesubstrate surface and separated from its neighbors. Theaverage diameter of the MWCNTs is ca. 80 nm in thissample, but generally can be varied from 20 to 100 nm bytuning the growth conditions. The MWCNTs are randomly

Fig. 1. The processing procedure for fabricating MWCNT nano-electrode arrays with metal film deposition, catalyst deposition,plasma enhanced chemical vapor deposition (PECVD), tetrae-thoxysilane (TEOS) chemical vapor deposition (CVD), chemicalmechanical polishing (CMP), and the setup for electrochemical(EC) characterization [24].

16 J. Li et al.

Electroanalysis 2005, 17, No. 1 � 2005 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

distributed with an average spacing of about 200 to 300 nm,i.e., a density of ca. 2� 109 MWCNTs/cm2. The averagelength is controlled at about 5 mm. TEM images inFigures 2c – e indicate that these CNTs have multiplegraphitic shells, i.e., MWCNT structures, and are veryuniform in diameter along the axis. There is ca. 20%variation in the length over the forest-like MWCNT array.The Ni catalyst forms pear-shaped particles and stays at the

tip of each MWCNT. The high-resolution TEM image inFigure 2e indicates a series of bamboo-like closed graphiticshells along the tube axis, which is due to the fact that thegraphitic layers are not perfectly parallel to the tube axis asillustrated in Figure 2f. This type of bamboo-like structure isbetween an ideal MWCNT and a carbon nanofiber [39],which consists many graphitic edge at the outer surface. As aresult, the bamboo-like MWCNT behaves as a nanorodelectrode with a large active surface area [24]. The closedgraphitic shells, despite causing higher electrical resistance[40, 41], are advantageous for NEs since they preventelectrolytes from accessing the hollow channel at the centerand thus keep the background noise at the minimum.

Figures 3a and b show that the exposed MWCNTstypically protrude by about 30 to 100 nm over the SiO2

matrix after CMP, presumably due to the strong mechanicalresilience of MWCNTs. Each MWCNT retains its positionheld by surrounding SiO2 matrix. The protruded part mayconsist of some defects due to the mechanical damageduring CMP, which is removed by electrochemical etching in1.0 M NaOH [22]. This process creates an inlaid NEAslightly recessed in the SiO2 matrix so that the backgroundnoise is minimized. Figures 3c and d show a high-density (ca.2� 109 electrodes/cm2) and a low-density (ca. 7� 107

electrodes/cm2) NEA, respectively, after electrochemicaletching.

3. Physical Characterization of the EmbeddedMWCNT Array

For the application as an electrode, each MWCNT needs tobe metallic with high conductance. The electrical propertiesneed to be examined to ensure that MWCNTs are notdamaged during thermal CVD of TEOS and CMP. Currentsensing atomic force microscopy (CSAFM) and electricalI – V measurements were reported in our previous studies[40, 41]. The topography, deflection, and current images ofthe top surface of a planarized embedded MWCNT arraycan be simultaneously measured with a CSAFM at a voltagebias of �5 mV between the Pt-coated AFM probe and themetal film underneath the MWCNTs. The protrudedMWCNT tip in topography is confirmed to correlate wellwith the conductive spots in the current image. MWCNTsremain separated at the original positions similar to those ofSEM images in Figures 3c and d. A two-terminal I – Vmeasurement can be carried at each conductive spotcorresponding to an individual MWCNT as shown inFigure 4a. Clearly, a linear curve is obtained within theinstrumental limit (þ /� 10 nA), corresponding to a resist-ance of 300 kW at each MWCNT. The linear I – V is furtherconfirmed with a semiconductor analyzer with a much largerdynamic range as shown in Figure 4b. More than onehundred MWCNTs are parallel connected to two 10�10 mm2 Cr contact pads deposited on the surface of anembedded MWCNTarray. The resistance of MWCNTarrayunder each Cr pad is only ca. 20 W. Clearly, these MWCNTs

Fig. 2. SEM images of a) a 458 perspective view and b) a topview of an as-grown MWCNT array, side-view TEM images of anas-grown MWCNT array c) at the region near the tip, d) at asingled-out spot on the surface, and e) with a high magnificationon one of the MWCNTs. f) schematic of the bamboo-likestructure of the MWCNT. The scale bars in (a) – (e) are 500 nm,500 nm, 100 nm, 200 nm, and 50 nm, respectively.

Fig. 3. a) A SEM image at 458 perspective angle and b) a TEMimage at the cross section of an embedded MWCNT nano-electrode array right after chemical mechanical polishing. Afterelectrochemical etching, the MWNTs are slightly recessed to forman inlaid nanoelectrode array. c) and d) show the surface of ahigh-density MWCNT nanoelectrode array (ca. 2� 109 electrodes/cm2) and a low-density one (ca. 7� 107 electrodes/cm2), respec-tively. The scale bars in (a) – (d) are 200 nm, 50 nm, 500 nm, and500 nm, respectively.

17Inlaid Multi-Walled Carbon Nanotube Nanoelectrode Arrays


are metallic wires with ohmic contact with the underlyingmetal film and are highly conductive.

Since each MWCNT is a simple ohmic resistor, theaverage resistance under a contact pad is inversely propor-tional to the density of exposed MWCNTs. One can use theelectrical resistance between two spots at the sample surfaceas a parameter to estimate the MWCNT NE density. Asshown in Figure 4c, the electrical resistance decreasesexponentially from ca. 560 W to a stabilized value at ca.80 Wat the final stage of CMP, corresponding to the increasein the density of exposed MWCNTs (from ca. 1� 107

electrodes/cm2 to ca. 2� 109 electrodes/cm2) [23]. Thechange in resistance correlates with the number of exposedMWCNTs caused by the varaiation in MWCNT length inthe array, which can be used to monitor the CMP process. Bystopping CMP at the proper resistance value, a MWCNTNEA with the density lower than ca. 1� 107 electrodes/cm2

can be easily fabricated without using expensive nanolitho-

graphic facilities, making this technology more affordablefor electroanalytical applications.

4. Electrochemical Properties of MWCNT NEAs

The electrochemical properties of MWCNT NEAs can becharacterized with benchmark species in bulk solutions(such as 1.0 mM K4Fe(CN)6 in 1.0 M KCl) as well asfunctionalized at the surface (such as a ferrocene (Fc)derivative, Fc(CH2)2NH2, binded to CNTs). A MWCNTNEA post CMP procedure typically shows a large peakseparation over 500 mV in cyclic voltammetry (CV) meas-urements with K4Fe(CN)6 solutions. After electrochemicaletching with 1.0 M NaOH, this value can be reduced to ca.100 mV and intrinsic properties of NEA start to appear.Figures 5a and b show CV results in 1.0 mM K4Fe(CN)6 and1.0 M KCl with the two MWCNT NEAs shown in Figures 3cand d, respectively. The CV curve of the high-density array issimilar to a solid macroelectrode due to the heavy overlap ofthe diffusion layer from each CNT electrode. The peakseparation is about 96 mV, indicating that the reaction isquasi-reversible at the CNT electrode, similar to a carefullyprepared conventional carbon electrodes [35]. The back-ground charging-discharging currents are almost negligiblerelative to the redox signals. As the density is lowered, theCV feature dramatically changes to a sigmoidal shape,indicating the presence of a steady-state diffusion-limitedcurrent at each MWCNT since the length of the diffusionlayer is smaller than the average separation betweenneighboring MWCNT NEs. The small non-steady-statecomponent and a significant hysteresis between the forwardand backward scans are attributed to the partial overlapof the diffusion layers at some locations in the randomlydistributed array where NEs are closer to each other[11].

The high-density NEA is very similar to nanoelectrodeensembles (NEEs) fabricated by filling nanoscale one-dimensional (1-D) channels in filtration membranes [10],which may be interesting for many studies. However, thelow-density NEAs are more interesting for ultrasensitiveelectroanalysis. Particularly, a much lower detection limitcan be obtained for surface immobilized redox species.Since the MWCNTends are dominated by carboxylic groupsafter the electrochemical etching [35], a ferrocene (Fc)derivative, Fc(CH2)2NH2, can be selectively functionalizedat the tube ends through amide bonds facilitated by thecoupling reagents dicyclohexylcarbodiimide (DCC) and N-hydroxysuccinimide (NHS) [42] as illustrated in Figure 6.CV curves of the two MWCNT NEAs after Fc functional-ization are shown in Figure 5c and 5d, respectively. In thehigh-density sample, a pair of waves can be seen centeredaround 0.16 V (indicated by the arrows) while it presentsalmost a flat background in Figure 5d with the low-densitysample. The peak separation is about 30� 10 mV in Fig-ure 5c, indicating a quasi-reversible surface adsorbed proc-ess consistent with the nature of the electrode indicated byCV in Figure 5a. The integrated peak area in Figure 5c

Fig. 4. I – V curve of a) a single MWCNT measured with currentsensing AFM (CSAFM) probe and b) multiple MWCNTs inparallel connection with two 10� 10 mm2 Cr pads deposited on thesurface of an embedded array, measured with a semiconductoranalyzer in a wider range. c) The calibration curve for controllingthe density of exposed MWCNTs by stopping CMP at the properstage. The electrical resistance decays exponentially vs. the time ofCMP. [23]. Insets illustrate the configuration of I – V and electricalresistance measurements.

18 J. Li et al.


corresponds to ca. 2.6� 1011 Fc/mm2 on the electrode, i.e.,ca. 9000 Fc per MWCNT and ca. 90 �2/Fc (by assuming aMWCNT diameter of ca. 100 nm). The number of Fc perMWCNT is rather high, likely because some molecules arefunctionalized at the sidewall of the protruding tubes,particularly since the CNTs in our study have bamboo-likedefective fiber structure. The roughness at the MWCNTendmay be quite large, producing a larger effective surface area.In any case, it is certain that the functionalization efficiencyis sufficiently high.

The NEA, particularly the low-density one, also presentsa much lower cell time constant, i.e., a smaller RC factor,which is proportional to the radius of individual electrodes[43]. Thus fast electrochemical techniques such as ACvoltammetry (ACV) and differential pulse voltammetry(DPV) can be applied to improve the sensitivity. Figures 5eand f show ACV curves in 1.0 M KCl obtained with the twoMWCNT NEAs functionalized with Fc, respectively. Clear-ly, a peak corresponding to the faradaic signal is observed atca. 0.16 V with both samples. The background baseline isstable and can be subtracted with a linear curve indicated bythe dash line. The extracted peak height is approximatelyproportional to the number of Fc(CH2)2NH2 molecules on

the electrode surface, but deviates from the ideal equationdue to the quasireversible nature of the electrode. However,it appears that the functionalization sites are simply propor-tional to the density of MWCNTs, which gives ca. 8.7�109 Fc/mm2 in the low-density sample.

5. Electrochemical Impedance Spectroscopy (EIS)

The quality of the SiO2 insulation is critical for NEA�sperformance. This can be examined with EIS [44]. Par-ticularly, the data obtained in the absence of redox speciesdirectly reflects both the electronic and ionic insulatingproperties of SiO2 prepared by TEOS CVD, which wasreported to present microdefects [45]. EIS measurementswith AC waves from 10 kHz to 1 MHz are carried out in1.0 M KCl to derive the basic equivalent circuit. Figure 7shows the EIS data obtained with a ca. 1 mm2 highlyoriented pyrolytic graphite (HOPG) edge-plane electrode(HOPG-edge), a high-density MWCNT NEA (h-NEA,with ca. 1.3� 108 electrodes/cm2), and a low-densityMWCNT NEA (l-NEA, with ca. 2 – 5� 107 electrodes/cm2). The data normalized to the geometric surface area is

Fig. 5. a), b) CV measurements in 1 mM K4Fe(CN)6 and 1.0 M KCl; c), d) CV measurements of Fc derivative functionalized MWNTarray electrodes in 1.0 M KCl solution; and e), f) ACV measurements at 50 Hz and an amplitude of 50 mV on the staircase DC rampfrom �0.05 V to 0.45 V. a), c), e) are measured with the high-density MWCNT NEA (with ca. 2� 109 electrodes/cm2) and b), d), f) aremeasured with the low-density one (with ca. 7� 107 electrodes/cm2), respectively. All CV measurements are taken with a scan rate of20 mV/s. The arrows indicate the position of the redox waves of the Fc derivative functionalized on the high-density CNT arrayelectrode. The dotted lines and dash lines indicate the baselines of the background current. [22]



presented as Bode plots and Nyquist plot, where Zrepresents the total impedance with Z’ and Z’’ correspond-ing to its real and imaginary part, respectively.

Clearly, the data set shifts systematically to higherfrequency in Figure 7a following the trend of HOPG edge(þ), h-dNEA (open circles), and l-dNEA (filled circles). Alldata can be nicely fit with a simple Randles circuit shown inthe inset of Figure 7b with a constant phase element (CPE)[44] in parallel with a faradaic reaction resistance (Rf) inaddition to the ohmic resistance of the solution (Rs). Theimpedance of a CPE is given by

Z¼ 1/(Y0iw)a (1)

where w is the angular frequency (w ¼ 2pf) and f is theconventional frequency in Hz. The exponent a is typicallybetween 0.8 to 1.0 for solid electrodes and the CPE behavesas a capacitor when a¼ 1.0. Table 1 lists all the fittingparameters.

CPE is necessary since EIS data of all three electrodes inNyquist plot (as shown in Figure 7c) are depressed invertical axis from an ideal semicircle, which cannot be fitwith ideal capacitors. The parallel resistance Rf indicates theexistence of a small faradaic reaction likely due to theelectroactive oxygen-containing groups at the broken edgeof graphitic sheets. The Nyquist plot in Figure 7c shows aclear trend that the impedance increases with a decrease inNE density.

The deviation from Helmholtz parallel plate capacitor toCPE has been attributed to the surface roughness orinhomogeneous reactivity in the literature [46, 47], both ofwhich may be valid for MWCNT NEAs. First, the surface ofthe SiO2 matrix in NEAs has a roughness of ca. 10 nm, whichis larger than normal solid electrodes. Second, the surface ofMWCNTs may consist of several types of groups such as�OH and�COOH, resulting in inhomogeneous activities atdifferent sites. However, since EIS can be nicely fit with asimple equivalent circuit, it is certain that both the electronicand ionic insulating properties of SiO2 deposited by TEOSCVD are comparable to well-known compact SAMs on Auelectrodes [48 – 50].

6. MWCNT NEAs as Ultrasensitive ChemicalSensors

The signal-to-noise ratio is critical for trace analysis of smallredox molecules. Since the background noise in electro-chemical measurements is normally due to the charge-discharge current, in proportion to the active surface area,low-density MWCNT NEAs may give extremely highsensitivity. Figures 8a and 10b each show three sets of dataobtained with CVand DPV in 13 nM, 1.6 mM, and 40 mM K4

[Fe(CN)6] solution, respectively, using a low densityMWCNT NEA (with ca. 1� 106 electrodes/cm2). Both CVand DPV show a clear signal of Fe(CN)4�

6 at the concen-

Fig. 6. The scheme for functionalizing an amine-terminated ferrocene derivative to MWCNT ends by forming an amide bond with thecarboxylic group facilitated with carbodiimide coupling reagents. [22]

Table 1. Fitting parameters for the EIS data of three electrodes using equivalent circuit shown in Figure 7b.

Electrode Rs (W cm2) Rf (MW cm2) CPE

Y0 (� 10�5) a

HOPG-Edge 0.579� 0.012 0.381� 0.031 17.19� 0.27 0.879� 0.002h-NEA 18.79� 0.16 1.038� 0.077 1.014� 0.018 0.822� 0.001l-NEA 31.70� 0.65 2.184� 0.082 0.0951� 0.0023 0.820� 0.002

20 J. Li et al.


tration as low as 13 nM. All CV curves present a sigmoidalshape with almost a constant charge-discharge backgroundcurrent at ca. 9� 10�10 A/mm2.

The steady-state signal, id in CV and the peak height ip inDPV, obtained in a series of concentration (C0) varied overabout six orders of magnitude are summarized in Figures 8cand d, respectively. Both the CVand DPV data indicate thesame trend, with a steep linear plot at high C0 and a linearplot with a much smaller slope at low C0. The two linear plotscross at C0,t � 1 mM. At C0> 1 mM, the slope is about 1.0,corresponding to

id/C0 in CV, (2)

and

ip/C0 in DPV, (3)

which is consistent with macroelectrodes in electroanalyt-ical applications. For C0< 1 mM, the plot of log i vs. log C0

has an interesting slope a� 0.20, which is clearly differentfrom macroelectrodes. Experiments with different NEAdensities show that C0,t decreases as the density of NEAincreases.

In general, the curve at low concentration can bedescribed as

id/C0a (4)

and

ip/Ca0 (5)

with a< 1, indicating that it likely involves a surfaceadsorption process where the adsorbates have large surfacemobilities. As a result, the local concentration is enhancedcomparing to the bulk concentration C0, particularly if C0 isvery low. The surface adsorption is more evident for low-density NEAs since the surface area per MWCNT, i.e., thenumber of adsorbates supplied to each MWCNT, is muchbigger. This explains why it can measure Fe(CN)4�

6 speciesdown to a few nM. Such low-density MWCNT NEA may beemployed for trace analysis of redox species in bulksolutions, such as monitoring the level of toxic metal ions,pesticides, hormones, and neurotransmitters. The detectionlimit at the level of 10�9 M is about 100 to 1000 times lowerthan conventional electrodes (typically with a limit above10�7 M) [43, 51]. In addition, the surface could be modifiedto enhance the adsorption and gain chemical selectivity asdemonstrated by Martin et al. with NEEs [52].

7. MWCNT NEAs for Ultrasensitive Biosenosrs

The embedded MWCNT array minimizes the backgroundnoise from the sidewalls and leaves only well-definedgraphitic open ends with abundant �COOH groups atisolated spots on the surface. This allows the selective

Fig. 7. The electrochemical impedance spectroscopy of a ca.1 mm2 HOPG edge plane electrode (shown in þ ), a high-densityMWCNT nanoelectrode array embedded in SiO2 (with ca. 1.3�108 electrodes/cm2, shown in open circles), and a low-densityMWCNT nanoelectrode array embedded in SiO2 (with ca. 2 – 5�107 electrodes/cm2, shown in black dots). All measurements weredone at the open circuit potential in 1.0 M KCl with 10 mVsinusoidal waves whose frequencies vary from 10 kHz to 1 MHz.a) is Bode plot of the logarithm of the absolute impedance ( jZ j ,in W-cm2) vs. the logarithm of the frequency f (in Hz), b) is Bodeplot of the negative phase (in degree) vs. the logarithm of thefrequency f (in Hz), and c) Nyquist plot of the real (Z’) andimaginary (Z’’) parts of the impedance, respectively. Solid linesrepresent the fitting curves to the experimental data using thesimple Randles equivalent circuit shown in the inset in (b). Theinset in c) shows the enlarged plots in the impedance amplitudesranging from 0 to 2.5� 105 (W cm2). [25]



functionalization of primary amine-terminated biomole-cules through amide bonds with�COOH groups, which hasbeen employed in developing nucleic acid sensors [22 –26].Similar NEAs or nanoelectrode ensembles (NEEs)have been used by Lin et al. to develop glucose sensors[53], using a spin-coated epoxy layer instead of TEOS CVDSiO2 as the insulating dielectrics. The wide electropotentialwindow of carbon is of particular interest for nucleic acidsensors since it is possible to directly measure the oxidationsignal of guanine bases without labeling target moleculeswith other redox moieties. As schematically illustrated inFigure 11, an oligonucleotide probe with a sequence[Cy3]5’ – CTIIATTTCICAIITCCT – 3’[AmC7-Q] is cova-lently attached to the open MWCNT end through amidebonds facilitated with carbodiimide reagents. This DNAsequence is related to the wild-type allele (Arg1443stop) ofcancer-related BRCA1 gene [54]. It is estimated that thereare about a hundred probe molecules functionalized to asingle MWCNTwith ca. 100 nm diameter. Target moleculeswith a complementary sequence, i.e., [Cy5]5’-AG-GACCTGCGAAATCCAGGGGGGGGGGG-3’ areused to demonstrate the feasibility of direct electrochemicaldetection. A 10 base polyG is attached to the complemen-tary sequence as the major signal moieties. The guaninebases in the probe are replaced with nonelectroactiveinosines to eliminate the redox background from probes.After incubation and rigorous washing, only fully matchedDNA targets are expected to stay at the MWCNT ends dueto the strong specific hybridization and provide an oxidationcurrent that can be measured.

However, the oxidation current is proportional to thenumber of guanine bases, which is extremely small due tothe limited DNA molecules to be detected. Direct CVmeasurements cannot pick it out from the background noise[22]. This problem can be solved by introducing Ru(bpy)2þ

3

mediators to amplify guanine oxidation based on anelectrocatalytic mechanism [55, 56] as illustrated in Fig-ure 9b. The mediators can efficiently transfer electrons fromthe guanine bases to the electrode even when they are not indirect contact. This mechanism can utilize all the guaninebases within the hemispherical diffusion layer of Ru(bpy)2þ

3

mediators and thus generate a much larger signal. Inaddition, ACV measurements can be used to furtherimprove the signal-to-noise ratio. Combining the MWCNTNEA with Ru(bpy)2þ

3 mediated guanine oxidation mecha-nism and ACV method, we demonstrated that the hybrid-ization of less than a few attomoles of oligonucleotidetargets can be easily detected with a 20� 20 mm2 electrode[22], with orders of magnitude improvement in sensitivitycompared to previous EC based DNA detections [57].

For clinical diagnostics, particularly molecular diagnos-tics, there is a strong need to reduce the cost and time.MWCNT NEAs combined with mediator amplified guanineoxidation mechanism provide a platform with both desiredultrahigh sensitivity and minimum sample preparationrequirements. Ideally, it is desired that the inherent guaninebases in PCR amplicons can be directly detected with simpleelectrochemical methods without going through the time-consuming labeling process, which was also demonstratedby us [23].

Fig. 8. a) Cyclic voltammetry at a scan rate of 20 mV/s and b) differential pulse voltammetry at a pulse amplitude of 25 mV with aninterval time of 1.2 s and a modulation time of 50 ms. The measurements are carried out with a low-density MWCNT nanoelectrodearray in 1.0 M KCl with K4[Fe(CN)6] concentration at 13 nM (dark line), 1.6 mM (dashed line), and 40 mM (grey line), respectively. c) andd) are the logarithm of the extrapolated diffusion-limited current id in CV and peak current ip in DPV, respectively, vs the logarithm ofthe concentration of K4[Fe(CN)6], which is varied over about six orders of magnitude from 13 nM to 8 mM. [24]

22 J. Li et al.


Figure 10a shows three consecutive ACV scans of aprobe-functionalized MWCNT nanoelectrode array afterhybridization with a specific PCR amplicon with ca. 300bases. A rigorous washing procedure is again employed toremove nonspecific adsorption at the electrode surface andleave only fully hybridized target molecules. Well-definedpeaks are observed around 1.04 V, with the 1st scan (red line)clearly higher than the 2nd (blue dotted line) and the 3rd

(black line) scans, while the latter two are nearly super-imposed on each other. The baselines are flat and stable. Theanodic current peaked around 1.04 V can be approximatelyattributed into two parts:

I¼ Imediatorsþ IamplifiedG (6)

Where Imediators is from the oxidation of Ru(bpy)2þ3 mediators

in the bulk electrolyte solution, and IamplifiedG is the anodiccurrent associated with the oxidation of guanine bases intarget DNA molecules through specific hybridization withthe probe molecules. These two reactions occur at almostthe same potential, i.e., 1.04 V. IamplifiedG is approximatelyproportional to the number of guanine groups at the surface.However, guanine oxidation is irreversible so that IamplifiedG isonly observed in the first scan while Imediators is always presentand appears to be very stable. This makes it possible toderive the IamplifiedG by subtracting the data of the 2nd scanfrom that of the 1st scan, i.e.,

IamplifiedG� I1 – I2/ [G] (7).

Hence, the difference between the 1st and 2nd scans carriesthe quantitative information approximately proportional tothe number of guanine bases presented at the electrodesurface. Figure 10b shows the differential curve (DI1,2¼ I1 –I2) after subtracting the 2nd scan from the 1st one, which gives

a well-defined positive peak (red line), i.e. DIp1,2¼ Ip1 – Ip2>0, where Ip is the peak current of a scan. In contrast, thedifferential current between the 2nd and 3rd scans, i.e. (Ip2 –Ip3), is within the baseline noise. Therefore, the 1st scancarries the information from target molecules while thesubsequent scans can be used as control experiments, whichgreatly simplifies the process for data extraction andnormalization.

The reliability of this method was tested further with acontrol experiment after incubating the probe-functional-ized MWCNT NEA in an unrelated PCR amplicon with ca.400 bases followed by a rigorous washing procedure. Asshown in Figure 10c, the 1st scan gives a smaller ACV peakcurrent, resulting in a negative peak in the differential curve,i.e., DIp1,2¼ Ip1 – Ip2 < 0 at ca. 1.04 V in Figure 10d. Thisconfirms that a positive DIp1,2 can only be attributed toguanine bases from the specifically hybridized targets. Thestringent washing is sufficient to remove unmatched DNAmolecules from the electrode surface. Further controlmeasurements with a clean MWCNT NEA, a probe-functionalized MWCNT NEA before and after incubationin 10 mM 20 mer polyG solutions all gave consistent negativevalue in (Ip1 – Ip2). The variation of the NE density in eacharray can be normalized by dividing Ip1. Figure 10e summa-rizes the mean value and the standard deviation of (Ip1 – Ip2)/Ip1 obtained from 21 experiments at five different conditions,with at least 3 experiments repeated at each condition. Eventhough the density of NEA varies over about two orders ofmagnitude, the value of (Ip1 – Ip2)/Ip1 is rather consistent, withpositive results only appearing upon the hybridization of thespecific PCR amplicon. Therefore, the value of (Ip1 – Ip2)/Ip1

can be directly used as the criteria for diagnostics [23, 26]. Itis not necessary to precisely control the NE density duringfabrication processes.

The detection limit of PCR amplicons with ca. 300 baseswas estimated to be less than 1000 target molecules usinglow-density MWCNT NEAs on a microelectrode contact of20� 20 mm2 [23]. This already approaches the detectionlimit by laser fluorescence techniques in DNA microarrays.Compared to the most sensitive electrochemical resultwhich was reported by Thorp et al. [57] using the samemediator amplified guanine oxidation mechanism with asilane-modified 200 mm diameter indium tin oxide (ITO)electrode, the detection limit is at least lowered by morethan 30 times.

The MWCNTs in the above-discussed DNA sensorssimply behave as highly conductive molecular wires inter-connecting DNA molecules with the electronic circuits.Such embedded nanowire structure can be also employedfor electrical communication between proteins that attach-ed at the exposed end of MWCNTs and the underlyingcircuit [58]. The size of MWCNTs is of tens of nanometers,close to that of proteins, making it possible to directlyinteract with single proteins. With a proper functionaliza-tion scheme, one can selectively interact with the specificsite of the protein. It is also possible to etch back SiO2 toexpose MWCNT array protruding over the surface of SiO2

matrix with a controlled length and use them as nanoscale

Fig. 9. Schematic of the mechanism for nucleic acid sensing onan inlaid MWCNT nanoelectrode combined with Ru(bpy)2þ

3

mediator amplified guanine oxidation. The short curved linesrepresent oligonucleotide probe molecules functionalized at theopen end of MWCNTs and the longer ones represent targetmolecules hybridized with the probe molecules, respectively. Thehemisphere represents the diffusion layer of Ru(bpy)2þ

3 mediators.[24]



needles to probe the physiological environment within aliving cell [59] with only minimum disturbance to the cellmembrane. Both intracellular and extracellular communi-cations can be studies using such NEAs.

8. Selective Passivation / Functionalization Scheme

In the low-density inlaid MWCNT NEA presented above,more than 99% of the surface is covered with SiO2 and onlyless than 1% is MWCNTs. It is known that biomoleculeshave strong nonspecific adsorption at SiO2 surface. As aresult, a rigorous stringent washing has to be applied to

remove them after incubation. The sample is washed inthree saline sodium citrate (SSC) buffers (1) 2� SSC with0.1% sodium dodecyl sulfate (SDS), (2) 1� SSC, and (3)0.1� SSC, respectively, at 40 8C for 15 minutes at each step.This significantly slows down the process and hinder theapplication of MWCNT NEAs in practical diagnostics. It isdesired to immobilize probe molecules only at the end ofMWCNTs while keeping the SiO2 surface passivated withmoieties resisting non-specific adsorptions. A method isdeveloped as shown in Figure 11 to achieve such highlyselective functionalization and passivation.

One unique property of electronic techniques is that it canbe highly localized, which makes it possible to control the

Fig. 10. a) Three consecutive ACV measurements (1st: red line, 2nd: blue dotted line, and 3rd: black line) and b) the differential curvesbetween the 1st and the 2nd scans (DI1,2¼ I1 – I2): red line, and that between the 2nd and the 3rd scans (DI2,3¼ I2 – I3): blue dashed line, of aCNT nanoelectrode array functionalized with BRCA1 probes and hybridized with the specific PCR amplicon. c) and d) are similarmeasurements after incubating with the unrelated PCR amplicon. The black dash-dot lines in (b) and (d) represent the backgroundcurrent. The measurements were carried out in 5 mM Ru(bpy)2þ

3 and 0.20 M NaOAc (pH 5.2) with an AC sinusoidal wave of 10 Hz and25 mV amplitude on top of a staircase DC ramp. e) Summary of the mean value and the standard deviation of (Ip1 – Ip2)/Ip1 from 21measurements at five different conditions: 1) probe-functionalized MWCNT nanoelectrode array incubated in specific PCR amplicontarget, 2) unfunctionalized clean MWCNT nanoelectrode array, 3) probe-functionalized MWCNT nanoelectrode array alone, 4) probe-functionalized MWCNT nanoelectrode array incubated in a 20 mer polyG solution, and 5) probe-functionalized MWCNTnanoelectrode array incubated in unrelated PCR amplicon. [23]

24 J. Li et al.


functionality at active MWCNT sites in the NEA. Asdiscussed above, electrochemical etching has been used toproduce hydroxyl and carboxylic acid at MWCNT ends.Aminopropyltriethyoxysilane is used first to react with thehydroxyl groups on both SiO2 surface and MWCNT endsand produces a monolayer with primary amine surfacefunctionalities [60]. 2-(2-Methoxyethoxy)acetic acid is thenapplied to form an amide bond with the –NH2 groupfacilitated by coupling reagents 1-ethyl-3(3-dimethyl ami-nopropyl) carbodiimide hydrochloride and N-hydroxysul-fo-succinimide [61]. This generates a surface terminatedwith ethyleneglycol (EG) moieties as shown in Figure 11c,which is known to resist non-specific adsorption of biomo-lecules [62]. Finally, molecules at the MWCNT ends can beremoved by electrochemical etching in 1.0 M NaOH, whichregenerates a well-defined MWCNT surface dominated by�OH and�COOH groups. Primary amine terminated DNAprobes or other biomolecules can then be attached to thesespecific sites through amide bonds as discussed above.Fluorescence measurements demonstrated that nonspecificmolecules can be easily removed from the surface of aselective passivated/functionalized MWCNT NEA by quickrinsing with PBS buffer, while the specific target molecules

remain attached to the probes. This method ensuresbiomolecules to be immobilized at desired sites for efficientbiosensing.

9. Integration into Miniaturized and MultiplexDevices

For many applications in chemical and biological sensors,miniaturized and multiplex electrodes are required forintegration with other sample preparation and separationtechniques such as microfluidics devices, capillary electro-phoresis, etc.. MWCNT NEAs are fully compatible withother microfabrication processes and microelectromechan-ical systems (MEMS). MWCNTs can be precisely grown atspecific locations down to a few nanometers depending onthe effort in lithography [22,41,63]. Figure 12 demonstratessuch capability with a 3� 3 multiplex array. Each micro-electrode pad in Figure 12a is a metal film deposited on aSiO2-covered Si wafer and individually wired to the externalcircuit through a metal line. The size of the microelectrodepad is about 200� 200 mm2 , which can be easily reduced to afew microns with UV-lithography. MWCNT array is grownon each microelectrode pad as shown in Figure 12b – d. Inprevious sections, the MWCNT arrays are randomly dis-tributed forest-like structure. For more precise control, theycan be fabricated into regular arrays of either microbundlesusing UV-lithography (Fig. 12c) or individual MWCNTsusing e-beam lithography (Fig. 12d). Figures 12e and f showcorresponding regular inlaid MWCNT NEAs post TEOSCVD and CMP processes. Clearly, MWCNT NEAs can befabricated as arrays in high-degree multiplex individuallyaddressed arrays. Such array-in-array architecture improvesthe statistical reliability at each microelectrode pad throughNEA instead of a single NE. The ultrahigh sensitivity canstill be maintained. Similarly, a MWCNT NEA can befabricated as the detector, which is integrated with othercomponents as a multifunctional microchip.

10. Conclusions

We have demonstrated that vertically aligned MWCNTarrays can be precisely fabricated on metal electrodes usingcatalytic PECVD. This process is compatible with currentmicrofabrication processing. Benefiting from this newtechnology, we were able to develop a bottom-up approachto reproducibly fabricate MWCNT NEAs. The sidewall ofthe MWCNTs and underlying metal lines are insulated inSiO2 matrix leaving only the graphite edge-plane like tipexposed at the surface to form an inlaid NEA. IndividualMWCNTs have shown highly conductive ohmic electricalproperties. Well-separated low-density MWCNT NEAsshow characteristic quasireversible NE behavior. Ultra-sensitive electrochemical detection has been demonstratedfor both trace redox species in bulk solutions as well ascovalently attached at the end of MWCNTs. This platform isof particular interest for developing ultrasensitive electro-

Fig. 11. The scheme for the surface passivation and selectivefunctionalization of biomolecules to the embedded MWCNTnanoelectrode array. a) A nonpassivated sample with the end ofMWCNTs dominated by�OH and�COOH groups after electro-chemical etch in 1.0 M NaOH. b) An amine-terminated self-assembled monolayer formed through silanization with –OHgroups on the surface. c) An ethylene glycol surface generatedthrough amide bonds for reducing non-specific adsorption. d)Selective functionalization of biomolecular probes through amidebonds at electrochemically regenerated MWCNT ends. [25]



chemical biosensors. The SiO2 surface can be passivatedwith moieties to reduce nonspecific adsorptions while theend of MWCNTs is selectively functionalized with a specificbiomolecular probe. A DNA sensor was demonstrated usinga mediator amplified guanine oxidation mechanism, whichcan directly measure the hybridization of both polyG taggedoligonucleotide targets and label-free PCR amplicons. Thedetection sensitivity can reach below 1000 DNA moleculesat each microelectrode pad, approaching the limit of thedetection sensitivity with conventional laser fluorescencetechniques used in DNA microarrays. The MWCNT NEAscan be directly integrated with microelectronics and micro-fluidics devices for the development of fully automatedmultiplex microchips. They can be also used as the detectorsin chromatography, electrophoresis, and other analyticalinstruments. Many of these potentials have just begun to beexplored.

11. Acknowledgements

A. M. C., H. T. N., and J. H. are with the University Affili-ated Research Center at NASA Ames operated by Uni-versity of California, Santa Cruz. H. C., Q. Y., and W. F. arewith ELORET Corporation. This work was supported byNASA under contract #NAS2-99092.

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inlaid multi-walled carbon nanotube nanoelectrode arrays for electroanalysis

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