Femtoliter and Attoliter Electrochemical Cells on Chips - American

Jan 27, 2010 - femtoliter volume cells, indicating the domination of a radical diffusion regime and a greatly improved signal/ background ratio. Quasi...
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Anal. Chem. 2010, 82, 1521–1526

Femtoliter and Attoliter Electrochemical Cells on Chips Tao Li,†,‡ Lei Su,† Wenping Hu,*,† Huanli Dong,† Yongfang Li,† and Lanqun Mao† Beijing National Laboratory for Molecular Sciences, Institute of Chemistry, Chinese Academy of Sciences, Beijing 100190, China, and Graduate University of Chinese Academy of Sciences, Beijing 100039, China Chip electrochemistry is one of the top ambitions of today’s electrochemistry. Here, a study for manufacturing electrochemical microcells on chips in a cost-effective, facile, and mass-producible way is presented. The ultrasmall, planar electrochemical cells, ranging from 140 femtoliter to 14 attoliter, can work independently as electroanalytical devices with embedded functional microelectrodes. Electrochemical responses of the miniaturized cells have been characterized by cyclic voltammetry. Ideal steady-state voltammograms were recorded with femtoliter volume cells, indicating the domination of a radical diffusion regime and a greatly improved signal/ background ratio. Quasi-thin-layer behavior was observed for attoliter volume cells, which exhibited a special capability of offering accurately confined domains for redox processes. Positive feedback effect of the cells indicated that interactions between the close-by working and reference/counter microelectrodes can be well developed and potentially utilized for trace level electroanalysis. This study vividly offers i) a new protocol of electrochemical chip for applications, ii) a new tool for trace electroanalysis, and iii) a more approachable insight for single molecule electrochemistry in the near future. Technologies of electrochemical microsystems1 have drawn much attention in the last 20 years with the progress of micromachining technology, electrochemical theory in microscopic domain, and trace analytical sensors with lower cost and higher sensitivity. Electrochemical microsystems possess all the advantages of microelectrodes, such as enhanced mass transport, improved faradic-to-capacitive current, and decreased deleterious effects of solution resistance.2-8 Moreover, radial (three-dimensional) diffusion regime resulted from a small-size effect produces * To whom correspondence should be addressed. E-mail: [email protected]. † Chinese Academy of Sciences. ‡ Graduate University of Chinese Academy of Sciences. (1) (a) Schultze, J. W.; Tsakova, V. Electrochim. Acta 1999, 44, 3605–3627. (b) Schultze, J. W.; Bressel, A. Electrochim. Acta 2001, 47, 3–21. (2) Bard, A. J.; Faulkner, L. R. Electrochemical Methods: Fundamentals and Applications, 2nd ed; John Wiley & Sons, Inc.: New York, 2001. (3) Wightman, R. M. Anal. Chem. 1981, 53, 1125A–1134A. (4) Montenegro, I. M.; Queiros, M. A.; Daschbach, J. L. Microelectrodes: Theory and Applications; Kluwer Academic Publishers: Dordrecht, The Netherlands, 1991. (5) Heinze, J. Angew. Chem., Int. Ed. Engl. 1993, 32, 1268–1288. (6) Forster, R. J. Chem. Soc. Rev. 1994, 23, 289–297. (7) Zoski, C. G. Electroanalysis 2002, 14, 1041–1051. (8) Arrigan, D. W. M. Analyst 2004, 129, 1157–1165. 10.1021/ac902681g  2010 American Chemical Society Published on Web 01/27/2010

steady-state voltammetric responses which are convenient for electrochemical analysis, including steady-state limiting current (id),9,10 half-wave potential (E1/2),11 and heterogeneous electron transfer rate (ks),12-15 etc. On the other hand, the unique superiorities such as low cost, small sample volume, less waste, portability, and the facile localization of redox reactions make electrochemical microsystems more outstanding. A vivid example to take those advantages of electrochemical microsystems is to utilize them as analytical tools for low volume or trace concentration samples, which is crucial in the research of medicine and biology.16-20 A key element for the fabrication of electrochemical microsystems locates at the microfabrication of electrodes, i.e., the working, reference/counter electrodes, which should be miniaturized and integrated in a small cell. Several ways have been previously described for electrochemical analysis, including positioning needle-type microelectrodes in picoliter microvials21,22 or microdroplets,23,24 preparing two- or three-electrode integrated microcell tips.25,26 Recently, microfabrication techniques of semiconductors enable preparing microelectrodes with well-defined (9) Oldham, K. B.; Zoski, C. G. J. Electroanal. Chem. 1988, 256, 11–19. (10) (a) Wang, G.; Zhang, B.; Wayment, J. R.; Harris, J. M.; White, H. S. J. Am. Chem. Soc. 2006, 128, 7679–7686. (b) Wang, G.; Bohaty, A. K.; Zharov, I.; White, H. S. J. Am. Chem. Soc. 2006, 128, 13553–13558. (11) Wong, D. K. Y.; Xu, L. Y. F. Anal. Chem. 1995, 67, 4086–4090. (12) Penner, R. M.; Heben, M. J.; Longin, T. L.; Lewis, N. S. Science 1990, 250, 1118–1121. (13) Scharifker, B.; Hills, G. J. Electroanal. Chem. 1981, 130, 81–97. (14) Mirkin, M. V.; Bulhoes, L. O. S.; Bard, A. J. J. Am. Chem. Soc. 1993, 115, 201–204. (15) Sun, P.; Mirkin, M. V. Anal. Chem. 2006, 78, 6526–6534. (16) (a) Bratten, C. D. T.; Cobbold, P. H.; Cooper, J. M. Anal. Chem. 1997, 69, 253–258. (b) Bratten, C. D. T.; Cobbold, P. H.; Cooper, J. M. Anal. Chem. 1998, 70, 1164–1170. (c) Cai, X.; Klauke, N.; Glidle, A.; Cobbold, P. H.; Smith, G. L.; Cooper, J. M. Anal. Chem. 2002, 74, 908–914. (17) Rossier, J. S.; Vollet, C.; Carnal, A.; Lagger, G.; Gobry, V.; Girault, H. H.; Michel, P.; Reymond, F. Lab Chip 2002, 2, 145–150. (18) Marchand, G.; Delattre, C.; Campagnolo, R.; Pouteau, P.; Ginot, F. Anal. Chem. 2005, 77, 5189–5195. (19) Baldwin, R. P.; Roussel, T. J.; Crain, M. M.; Bathlagunda, V.; Jackson, D. J.; Gullapalli, J.; Conklin, J. A.; Pai, R.; Naber, J. F.; Walsh, K. M.; Keynton, R. S. Anal. Chem. 2002, 74, 3690–3697. (20) Nyholm, L. Analyst 2005, 130, 599–605. (21) (a) Clark, R. A.; Hietpas, P. B.; Ewing, A. G. Anal. Chem. 1997, 69, 259– 263. (b) Clark, R. A.; Ewing, A. G. Anal. Chem. 1998, 70, 1119–1125. (22) Troyer, K. P.; Wightman, R. M. Anal. Chem. 2002, 74, 5370–5375. (23) Yum, K. S.; Cho, H. N.; Hu, J.; Yu, M. F. ACS Nano 2007, 1, 440–448. (24) Kashyap, R.; Gratzl, M. Anal. Chem. 1998, 70, 1468–1476. (25) Spaine, T. W.; Baur, J. E. Anal. Chem. 2001, 73, 930–938. (26) Gao, N.; Zhao, M. H.; Zhang, X. L.; Jin, W. R. Anal. Chem. 2006, 78, 231– 238.

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and reproducible geometries,27,28 which provides effective ways to prepare microelectrodes and their arrays by photolithography,16,18,29-31 ion beam or plasma etching,17,32 laser ablation,33-35 nanoimprint lithography36 techniques, and so forth. Several distinguished pioneers have paid great efforts on the utilization of these techniques for the construction of electrochemical microsystems. Murray et al.30 first reported the fabrication of three-electrode electrochemical cells on Si/SiO2 wafers with the utilization of three coplanar gold-film electrodes as working, counter, and pseudoreference electrodes and demonstrated that such microcells could be considered as disposable electroanalytical devices. Cooper et al.16 then fabricated a series of miniaturized electrochemical sensors with subnanoliter-volume for the analysis of metabolic activity such as single cell, etc., and predicted that these methods may be generally applied in a variety of other fields, such as in more miniaturized electrochemical systems (e.g., in the femtoliter and attoliter volumes), although no report has addressed it due to the difficulty in fabrication and lack of corresponding theory to convincingly interpret the electrochemical process in such small domains.21b,24,33 High resolution, well-defined nanogap electrodes (namely, a pair of electrodes with nanometer gap width) have been prepared and used for the examination of material properties at nanometer scale, even at molecular scale.37,38 We have worked on nanogap electrodes for several years and used them effectively to study optoelectronic properties of molecular materials at nanometer and molecular scale.39 It is confirmed that nanogap electrodes provide highly confined systems with ultrahigh sensitivity. If the great advantages of nanogap electrodes could be advanced into electrochemical microcells, together with the mass-producible ability of photo- or electron-beam lithography for desirable configured devices, it is highly possible to introduce i) a new protocol of electrochemical chip for applications, ii) a new tool for trace (27) (a) Zoski, C. G.; Simjee, N.; Guenat, O.; Koudelka-Hep, M. Anal. Chem. 2004, 76, 62–72. (b) Zoski, C. G.; Yang, N. J.; He, P. X.; Berdondini, L.; Koudelka-Hep, M. Anal. Chem. 2007, 79, 1474–1484. (28) Lemay, S. G.; van den Broek, D. M.; Storm, A. J.; Krapf, D.; Smeets, R. M. M.; Heering, H. A.; Dekker, C. Anal. Chem. 2005, 77, 1911–1915. (29) Li, X.; Zhou, Y. L.; Sutherland, T. C.; Baker, B.; Lee, J. S.; Kraatz, H. B. Anal. Chem. 2005, 77, 5766–5769. (30) Morita, M.; Longmire, M. L.; Murray, R. W. Anal. Chem. 1988, 60, 2770– 2775. (31) Henry, C. S.; Fritsch, I. Anal. Chem. 1999, 71, 550–556. (32) Lanyon, Y. H.; De Marzi, G.; Watson, Y. E.; Quinn, A. J.; Gleeson, J. P.; Redmond, G.; Arrigan, D. W. M. Anal. Chem. 2007, 79, 3048–3055. (33) Rossier, J. S.; Roberts, M. A.; Ferrigno, R.; Girault, H. H. Anal. Chem. 1999, 71, 4294–4299. (34) Wittstock, G.; Grundig, B.; Strehlitz, B.; Zimmer, K. Electroanalysis 1998, 10, 526–531. (35) Ball, J. C.; Scott, D. L.; Lumpp, J. K.; Daunert, S.; Wang, J.; Bachas, L. G. Anal. Chem. 2000, 72, 497–501. (36) Sandison, M. E.; Cooper, J. M. Lab Chip 2006, 6, 1020–1025. (37) Reed, M. A.; Zhou, C.; Muller, C. J.; Burgin, T. P.; Tour, J. M. Science 1997, 278, 252–254. (38) (a) Kubatkin, S.; Danilov, A.; Hjort, M.; Cornil, J.; Bredas, J. L.; StuhrHansen, N.; Hedegard, P.; Bjørnholm, T. Nature 2003, 425, 698–701. (b) Moth-Poulsen, K.; Bjørnholm, T. Nat. Nanotech. 2009, 4, 551–556. (c) Chen, F.; Hihath, J.; Huang, Z.; Li, X.; Tao, N. J. Annu. Rev. Phys. Chem. 2007, 58, 535–564. (39) (a) Hu, W. P.; Jiang, J.; Nakashima, H.; Luo, Y.; Kashimura, Y.; Chen, K. Q.; Shuai, Z.; Furukawa, K.; Lu, W.; Liu, Y. Q.; Zhu, D. B.; Torimitsu, K. Phys. Rev. Lett. 2006, 96, 027801. (b) Hu, W. P.; Nakashima, H.; Furukawa, K.; Kashimura, Y.; Ajito, K.; Liu, Y. Q.; Zhu, D. B.; Torimitsu, K. J. Am. Chem. Soc. 2005, 127, 2804–2805. For a review of nanogap electrodes, see: (c) Li, T.; Hu, W. P.; Zhu, D. B. Adv. Mater. 2010, 22, 286–300.

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Figure 1. Schematic view of ‘Type Ι’ electrochemical cells prepared by photolithography on Si/SiO2 wafer chips. Each cell (10 µm × 10 µm × 1.4 µm) has a pair of microband gold electrodes and the same size silver microelectrode on the vertical direction. The big pads (0.25 mm2) are exposed for electric connection.

electroanalysis, and iii) a more approachable insight for single molecule electrochemistry.40,41 Evoked by the above-mentioned enlightenments, in this study, a cost-effective, facile, and massproducible way is developed to prepare electrochemical microcells on chips based on nanogap electrodes with volumes ranging from femtoliter to attoliter (with micrometer or nanometer sized electrodes). Cyclic voltammetric results of the microcells suggested a new protocol of electrochemical chip for trace electroanalysis and theoretical research as well as the potential ability for single molecule electrochemical detection. EXPERIMENTAL SECTION Two types of electrochemical microcells are fabricated, and both of them utilize the resist film as insulating layer and framework of the cells. The cell volumes are calculated according to the portion of resist that is controllably etched away. One type is prepared by photolithography with 140 femtoliter volume and embedded three-finger microelectrodes (i.e., a pair of Au microelectrodes and an Ag microelectrode), referred to as ‘Type Ι’. The other is electron-beam lithographically fabricated with even smaller volume ranging from 200 to ∼14 attoliter and four-finger nanoelectrodes (i.e., a pair of Au nanoelectrodes and a pair of Pt nanoelectrodes), referred to as ‘Type Π’. Schematic drawing of device ‘Type Ι’ is illustrated in Figure 1. Arrays of electrochemical microcells comprised of central chamber, connecting tracks, and electrical connection pads on Si/SiO2 wafers were prepared by the Suss MA6 photolithography system. AZ 5214 photoresist was used for patterning the microelectrodes and acting as the framework of the central well. ‘Type Π’ devices were fabricated by electron-beam lithography instead of photolithography. A pair of Au electrodes and a pair of Pt electrodes were (40) (a) Fan, F. -R. F.; Bard, A. J. Science 1995, 267, 871–874. (b) Fan, F. -R.; Bard, A. J. J. Am. Chem. Soc. 1996, 118, 9669–9675. (c) Bard, A. J.; Fan, F. -R. Acc. Chem. Res. 1996, 29, 572–578. (41) Sun, P.; Mirkin, M. V. J. Am. Chem. Soc. 2008, 130, 8241–8250.

successively patterned onto the wafer substrate with twolayered resist, EP50G (JSR corp.)/PMGI (Micro Chem Inc.), by electron-beam lithography. A single layer of EP50G was used for insulating the electrodes and forming the central well. After that, the resultant resist pattern was irradiated by deep UV and then treated at 473 K for 1 h, making the insulating layer insoluble in common solvents, such as acetone, ethanol, etc. All electrochemical characterizations were carried out with a CHI 660C Electrochemical Workstation (CH Instrument). A CHI200 Picoamp Booster (CH Instrument) was added to ensure current down to a few picoamperes detectable. The electric connection between the ‘Type Ι’ electrodes and the electrochemical analyzer was made by a Micromanipulator 6150 probe station under optical microscope, the shielded and grounded box of which served as a Faraday cage. As for ‘Type Π’ cells, thin Au wires (20 µm diameter) and conductive silver paste were used for linking the connecting pads of the microelectrodes and the electrode clips (see Figure S1). The device was mounted on a glass slide and tested in CHI202 Faraday cage (CH Instrument). All electrochemical experiments were carried out at room temperature. Scanning electron microscopy (SEM) images were obtained on a Hitachi S4300-F (Hitachi Inc., Japan). Ferrocenecarboxylic acid (FcCOOH, Aldrich), reagent grade KCl, KH2PO4, NaCl, and Na2HPO4 · 7H2O were used as received. 0.1 M HCl solution was prepared by diluting 37.5% concentrated hydrochloride acid with deionized H2O. 0.01 M phosphatebuffered solution (PBS) was prepared by dissolving 8.0 g of NaCl, 0.2 g of KCl, 2.16 g of Na2HPO4 · 7H2O, and 0.24 g of KH2PO4 in 800.0 mL of deionized H2O, adjusting pH to 7.4, and then filling up to 1000.0 mL. All aqueous solutions were prepared with 18 MΩ•cm water supplied by a Milli-Q purification system and degassed with N2 for 30 min prior to all experiments. RESULTS AND DISCUSSION A schematic view of device ‘Type Ι’ is shown in Figure 1. A pair Au electrodes and the same size Ag electrode are integrated at the bottom of the microcell. Four cells are designed as a group to save space, and arrays of these groups are patterned on the Si/SiO2 chip substrates. Ninety-six cells can be fabricated once a time on a 2-in.-diameter Si/SiO2 wafer, and almost every cell can be reliably utilized, representing a high production yield. The depth of the chamber is about 1.4 µm, which was determined by means of atomic profiler. The volume of the chamber is calculated to be as small as 140 femtoliter (10 µm × 10 µm × 1.4 µm) (SEM image of a cell as shown in Figure S2). It can be seen that the chips are well-defined and easy handling, with individually addressable Au and Ag microelectrodes resting at the bottom of the well in the desired pattern. The coplanar configuration and small thickness of the microband electrodes basically prevent the other two electrodes interfering with the mass transport of electrolyte species to the working electrode. The pseudoreference electrode is placed in close vicinity to the working electrode and therefore further minimizes uncompensated solution resistance (besides the merit of microelectrodes). Also, positions of all the three electrodes are fixed, which alleviates variations in the experimental process and is favorable to get reproducible results. The dimension of the microelectrodes is generally under 4 µm, which guarantees radical (three-

Figure 2. SEM images of ‘Type Π’ cells with designed parameters (cell volume and nanoelectrode diameter) as (A), (B) (magnified image) 200 attoliter, 200 nm; (C) 50 attoliter, 100 nm; (D) 32 attoliter, 80 nm; (E) 18 attoliter, 60 nm. Electrodes on the horizontal and vertical direction are made of Au and Pt, respectively.

dimensional) diffusion regime under potential scan rates ν < 1 V/s. All potentials relevant to the cells are referred to as Ag pseudoreference electrode. The configuration of ‘Type Π’ devices is shown in Figure 2, with a pair of Au nanoelectrodes and a pair of Pt nanoelectrodes. The depth of the cells H is at ∼200 nm. The side length of the square chamber, L, and the diameter of the quasi-semisphere microelectrodes, d, are designed as variable parameters. Four kinds of cells with different sizes are fabricated, and the volumes of the chambers can be calculated to be 200, 50, 32, and 18 attoliter, respectively (Figure 2B-E, Table S1). The depth of the cells and the thickness of the electrodes are characterized by an atomic force microscopy (AFM). For example, the depth of the cell and the thicknesses of the metal leads are 200 and 30 nm for the cells of 200 attoliter (see Figure S3). It is noteworthy that drilling the well in the desired pattern gets more and more difficult as the cell becomes smaller. The most distinguished point is the smallest chamber with designed volume of 18 attoliter shown in Figure 2E. It can be clearly seen that the opening is circular rather than square with the diameter equal to the designed side length. In this case, the actual volume is calculated to be about 14 attoliter. In the case of facile utilization, four-end configuration is designed. On a 4-in.-diameter wafer at least 80 ‘Type Π’ devices can be fabricated, suggesting the mass-producible ability of this method for manufacturing electrochemical microcell chips. Usually, cyclic voltammograms obtained with microelectrodes in bulk solutions exhibit a sigmoid shape, and the steady-state limiting current offers a straightforward and reliable access to Analytical Chemistry, Vol. 82, No. 4, February 15, 2010

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Figure 3. (A) Typical cyclic voltammogram of ‘Type Ι’ electrochemical cells recorded in 1.5 mM FcCOOH in 0.01 M PBS at the scan rate of 0.05 V/s. (B) Voltammetric response to supporting electrolyte (PBS, pH 7.4) only, 0.05 V/s.

estimate the apparent radius of electrodes. Generally, the steadystate limiting current id can be given as the following equation id ) anFDC*r

(1)

Here, C* and D are the bulk concentration and diffusion coefficient of the redox species, n is the number of electrons transferred per molecule, F is Faraday’s constant, r is the apparent radius of the microelectrode, and a is a constant relative to the dimensional and topological access of redox solution to the microelectrode (e.g., 4 for disk-shaped microelectrodes). For ‘Type Ι’ cells, the shielded and grounded box of a Micromanipulator 6150 probe station was taken as a Faraday cage. Two Au microelectrodes were used as working and counter electrodes, respectively, and Ag microelectrode acted as a pseudoreference electrode. A droplet of ∼1 µL redox solution was positioned onto the aperture of the cell using a micropipet under an optical microscope. Ferrocenecarboxylic acid (FcCOOH) was selected as the redox probe. Glycerin (5% (v/v)) was added into the solution to eliminate the solution evaporation, and it guaranteed no visible evaporation for about 10 min. As the sample volume was 6 orders larger than the chamber, it was suitable for the study of voltammetric responses of microelectrodes in bulk conditions. The cyclic voltammetric response of the cell, recorded in 0.01 M PBS containing 1.5 mM FcCOOH at a scan rate of 50 mV/s, is shown in Figure 3A. It can be seen that the voltammogram exhibits a well-defined sigmoid shape with little hysteresis between the forward and reverse scans, which confirms completeness of steady state attributing to the enhanced mass transport rate and a good insulating condition between the photoresist and electrode fraction. Little disparity in voltammograms recorded at scan rates between 1 and 100 mV/s was observed, which is also the characteristic of radial diffusion to the microelectrodes. Beyond 1524

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Figure 4. (A) Cyclic voltammograms of ‘Type Ι’ electrochemical cells obtained at the scan rate of 0.05 V/s in PBS containing 1.5 mM, 1.0 mM, 0.5 mM, 0.2 mM, and 0.08 mM FcCOOH, respectively; (inset) voltammogram recorded in 0.08 mM FcCOOH with magnified current axes. (B) Linear relationship between id and electrolyte concentration with a regression coefficient higher than 0.999.

100 mV/s, the voltammograms tend to be distorted with a distinct gap between the forward and reverse tracks due to an increase of capacitance current at higher scan rates. CV detections in blank solution containing 0.01 M PBS only demonstrated that no faradic current was present. Figure 3B shows an example at the scan rate of 50 mV/s. The magnitude of capacitance current was on the average of 4 pA, to the point of undetectable in practical experiments. In this case, the capacitance of the device can be calculated to be about 80 pF, attributing to the greatly reduced active area of the microelectrodes. This value is low enough for the capacitive charging current not to interfere with the cyclic voltammetry signals for scan rates below 1 V/s. The improved signal/noise ratio potentially endows the chip devices with capability for highly sensitive electroanalysis. Considering a and r are both constants for a given working microelectrode, the magnitude of the limiting current id should be linear with the bulk concentration of redox species under ideal conditions. A series of steady-state voltammetric curves of a given microcell as a function of FcCOOH concentration at a scan rate of 50 mV/s is depicted in Figure 4A. It is obvious that the voltammograms with various FcCOOH concentrations exhibited a uniform sigmoid shape. The limiting current recorded apparently increased with increasing FcCOOH concentration and showed a good linearity with a regression coefficient higher than 0.999 (Figure 4B). The detected limit of FcCOOH can be calculated to be 0.035 mM, based on a signal-to-noise ratio of 3. These results demonstrate the capability of the reusable chip devices in developing analytical applications. As shown in Figure 4A, the half-wave potential of voltammograms with various FcCOOH concentrations retains an average value of 0.262 V (±10 mV), indicating that the Ag electrode is

sufficiently stable to be used as a pseudo reference. In a control experiment, an Ag/AgCl wire (diameter, 0.5 mm; dark brown) was prepared by polarizing a Ag wire at +0.6 V (vs Ag/AgCl filled with 3 M KCl) in 0.1 M hydrochloride acid for 15 min as reference/counter electrode. The end of the Ag/AgCl wire was maneuvered into the microdroplet with the probe manipulator, while the working electrode was still the Au microelectrode in the cell. Typical cyclic voltammogram of FcCOOH using Ag/AgCl wire as the reference/counter electrode is shown in Figure S4, which also exhibits a sigmoid shape. The average value of the half-wave potential was 0.282 V (±1 mV), consistent with the results reported in the literature.32 There is one thing noteworthy that in the control experiment, the limiting plateau current was about 140 pA for 1.0 mM FcCOOH, which was apparently smaller than the voltammetric response of the ‘Type Ι’ cell in the same concentration of FcCOOH (430 pA), and it can be explained as follows. As we know, when an oxidation reaction occurs at the working electrode, there must be a reduction reaction of equal extent at the counter electrode. For a regular bulk electrochemical system, the counter electrode is far away from the working electrode. Thus regularly the reaction products on the counter electrode would not interfere with the redox process on the working electrode. While in our case, the working and counter electrodes were very close to each other (∼4 µm), so the oxidized species (O) generated on the surface of a working electrode would very easily get to the surface of a counter electrode by diffusion. The transit time for a molecule to diffuse between two electrodes is about d2/2D. Taking d ) 4 µm and D ) 5.3 × 10-6 cm2 · s-1, it would only take a molecule about 16 ms to go over the shortest distance between the two electrodes. Then, the regenerated reduced species (R) at the counter electrode will diffuse back, contributing to a larger flux of the redox species on the working electrode and a higher current value observed in the experiment. The whole process was very similar to the situation of cross-talk in the collector/generator mode between closely spaced electrodes, when the regenerated species at the collector electrode diffuse back to the generator electrode and participate in the redox cycle again, and the recycling or feedback between the paired electrodes results in an amplified value of the generator current.42 So by substituting the Ag/ AgCl wire for the reference/counter microelectrodes, the mass communication between the functional elements in the cell was absent, resulting in a smaller value of limiting plateau current. It would be advantageous to make use of this positive feedback characteristic of the cell for trace level electroanalysis. Compared with ‘Type Ι’ cells, with an Au nanoelectrode as the working electrode and a Pt nanoelectrode as the pseudoreference/ counter electrode, peak-shaped curves, rather than sigmoid voltammograms, were obtained with ‘Type Π’ cells. Figure 5 shows the typical CVs recorded in a bulk drop of 1 mM FcCOOH (∼1 µL) with the 200-attoliter cell at scan rates of 50 mV/s. Electrochemistry in microscopic droplets,24 channels,33 or sealed microcavities21b were previously investigated and reported. Voltammetric responses similar to thin-layer cell behavior,2 rather than sigmoid signals, were observed. Our experimental results resemble the literature, although in our case, CVs were not (42) Fosset, B.; Amatore, C. A.; Bartelt, J. E.; Michael, A. C.; Wightman, R. M. Anal. Chem. 1991, 63, 306–314.

Figure 5. Cyclic voltammograms recorded in a bulk drop of 1 mM FcCOOH (∼1 µL) with the 200-attoliter cell at 0.05 V/s. The arrows indicate the sequencing of repetitive potential scans; (inset) voltammogram recorded by substituting an Ag/AgCl wire for the Pt nanoelectrode in the 200-attoliter cell.

conducted in a completely sealed environment. In a control experiment, by substituting the Ag/AgCl wire described above for the reference/counter electrodes in the cell, with the Au nanoelectrode still acting as the working electrode, sigmoid voltammograms were obtained, shown in the inset of Figure 5. The magnitude of limiting current (∼45 pA) roughly agreed with the value calculated for hemispherical electrodes in bulk solutions (a ) 2π in eq 1). The difference of Figure 5 and Figure 5 inset can be assigned to the difference in diffusion modes in the two cases. Based on the experimental observations, we propose a model to clarify the difference in voltammetric responses. When an external counter/ reference electrode is used, oxidation and reduction processes occur inside and outside the cell, respectively. The concentration gradient facilitates the mass transfer between the working electrodes and the outside bulk solution, and disruption of radial diffusion pathways by the side walls does not play a significant role. The radial diffusion mode becomes dominant, and the condition resembles what is known about microelectrode voltammetry in bulk solution (Figure 5, inset). In contrast, as the Type II cells are at least 3 orders of magnitude smaller than Type I cells, a more apparent effect of spatial constrains on voltammetric behavior appears for the former. On one hand, the nanoscale aperture of the cell would act as a resistant factor that makes it difficult for the redox species to diffuse into/out of the cell; on the other hand, when both the working and counter/reference electrodes are acted by the nanoelectrodes integrated at the bottom of the vials, the reactant would be more inclined to undergo redox process back and forth in between the two electrodes. The result is that the reaction domain of interest is mostly confined inside the cell, and the diffusion profile is bounded by the walls of the cell to a great extent, like a quasi-thin-layer cell. In this sense, the use of nano counter/reference electrode distinguishably alters the diffusion pathways to the surface of working electrode, and plannar diffusion is dominated. Larger contributions from planar diffusion result in peak-shaped voltammograms (Figure 5). Furthermore, cross-talk between the working and counter/reference electrodes results in a positive feedback effect that contributes to a larger current. The arrows in Figure 5 indicate the sequencing of repetitive potential scans, from which we can see a negative shift of the reference potential and a gradual increase of reduction peak current. Based on the Nernst equation, the negative shift of the reference potential could be interpreted Analytical Chemistry, Vol. 82, No. 4, February 15, 2010

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due to a more reducing environment provided by the adjacent counter electrode on which the reduced species were regenerated.2 The increase of peak current would be attributed to a small extent of solvent evaporation during the potential scan process. There is one thing noteworthy that there is not much difference in experimental setup between Type I and Type II cells, while the voltammetric responses are different. This means for this novel kind of devices, by down scaling the cell from micrometer to nanometer range, the space confinement effect would gradually dominate the diffusion behavior, from mostly spherical to planar diffusion. Also, the responses recorded with ‘Type Π’ cells were not so stable and reproducible as that of ‘Type Ι’, and nearly 40% of the devices failed to response probably due to the small dimension of the apertures or the hydrophobic nature of the insulating layer. Furthermore, nanoelectrodes in ‘Type Π’ cells were much more fragile, especially the 32- and 14-attoliter ones. Special care must be taken in case of a breakdown of the metal electrodes. CONCLUSIONS A cost-effective, facile, and mass-producible method of lithographically manufactured electrochemical microcells with “builtin” working, reference, and counter electrodes is presented. These simple-structured on-chip microsystems were easily fabricated with volumes as small as 140 femtoliter to 14 attoliter and can independently serve as an electrochemical cell. Cyclic voltammetry has been employed for characterization of the microcells. For ‘Type Ι’ cells with 140-femtoliter volume, well-defined steady-state responses were recorded proving the dominant role of radical diffusion regime. While for ‘Type Π’ cells with volume on the

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attoliter level, peak-shaped voltammograms were observed due to the altered diffusion patterns and spatial constrains effect. Crosstalks between the close-by working and reference/counter microelectrodes contributed to a positive feedback effect, which would be advantageous for trace level electroanalysis. This method offers a new protocol for design and fabrication of ultrasmallvolume electrochemical cells with integrated functional elements on chip, which appear very suitable for trace electroanalysis. They can also find applications in making electrical contacts to nanostructures or even single molecules benefiting from their restricted spaces and well-defined nanogap electrodes. ACKNOWLEDGMENT The authors are grateful to Prof. Royce Murray (Univ. North Carolina), Prof. Jon Cooper (University of Glasgow), Prof. Pingwen Zhang and Ms. Xiaolin Wang (Peking University) for profound discussion, Prof. Ming Liu, Dr. Liwei Shang (Institute of Microelectronics, Chinese Academy of Sciences), Dr. Yoshiaki Kashimura, and Keiichi Torimitsu (NTTBRL, Japan) for experimental help. The authors appreciate the financial support from National Natural Science Foundation of China (20721061, 50725311), Ministry of Science and Technology of China (2006CB806200, 2006CB932100) and Chinese Academy of Sciences. SUPPORTING INFORMATION AVAILABLE Figures S1-S4 and Table S1. This material is available free of charge via the Internet at http://pubs.acs.org. Received for review November 23, 2009. Accepted January 18, 2010. AC902681G