Applied Surface Science 253 (2007) 92099214 www.elsevier.

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AFM study of BSA adlayers on Au stripes

Michal Tencer a,c, Robert Charbonneau a, Nancy Lahoud a, Pierre Berini a,b,*
b a Spectalis Corporation, 610 Bronson Avenue, Suite 205, Ottawa, Ontario K1S 4E6, Canada University of Ottawa, School of Information Technology and Engineering, 161 Louis Pasteur Street, Ottawa, Ontario K1N 6N5, Canada c MST Consulting, 21F Banner Rd., Ottawa, Ontario, K2H 8T3, Canada

Received 2 November 2006; received in revised form 17 May 2007; accepted 23 May 2007 Available online 7 June 2007

Abstract Thin narrow Au stripes suitable for propagating long-range surface plasmonpolaritons were deposited by evaporation and lift-off on a thermal oxide layer on a silicon substrate, and modied by direct adsorption of bovine serum albumin (BSA). Atomic force microscopy (AFM) measurements reveal that BSA adsorbs onto the Au stripes from phosphate buffer solutions forming an adlayer having an average thickness of about 2 nm (surface mass density of about 2 ng/mm2). Comparisons with a simple adsorption model suggest the side-on adsorption of a single monolayer of BSA followed by denaturation and attening. The BSA-coated stripes have an increased surface roughness compared to a virgin stripe. # 2007 Elsevier B.V. All rights reserved.
Keywords: Surface; Plasmon; Waveguide; Protein; AFM

1. Introduction Long-range surface plasmonpolaritons (LRSPPs) are lowloss transverse magnetic surface waves guided at optical wavelengths by a symmetrically cladded thin metal lm [1] or stripe [25]. LRSPPs are surface sensitive, whereby changes in the dielectric region along the surface of the metal lm cause changes in the effective index or phase velocity of the LRSPP wave. The LRSPPs surface sensitivity and low loss offer great potential for sensing in chemical and biological systems where the analyte deposits onto or binds to the metal lm [6]. By an appropriate surface modication the sensing function can be rendered specic thus enabling many applications related to drug discovery, clinical analysis and biotechnology. LRSPPs propagating along thin narrow metal stripes [25] share the same attributes as LRSPPs propagating along metal lms, but additionally, are conned in the plane transverse to the direction of propagation. This enables optical signal processing geometries, such as splitters, couplers and Mach-Zehnder interferometers (MZIs), implemented as an arrangement of metal
* Corresponding author at: University of Ottawa, School of Information Technology and Engineering, 161 Louis Pasteur Street, Ottawa, Ontario K1N 6N5, Canada. E-mail addresses: berini@site.uottawa.ca, pierreberini@spectalis.com (P. Berini). 0169-4332/$ see front matter # 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.apsusc.2007.05.079

stripes operating in the LRSPP mode [79]. High sensitivity biosensors are created by arranging such stripes into a long optical interaction length MZI [10]. LRSPPs along metal stripes supported by an ultra-thin optically non-invasive dielectric membrane look particularly promising since they allow LRSPP propagation through any transparent uidic background [11]. Au is a good choice for the metal in LRSPP waveguides for biosensing applications since it has very good plasmonic properties [5], and a unique reactivity allowing a variety of surface modications through the formation of alkanethiol selfassembled monolayers (SAMs) [12]. Direct adsorption of protein on Au is also possible, although direct adsorption at the liquid solid interface is, in general, not very well understood, as highlighted in Ramsdens Puzzles and paradoxes in protein absorption [13]. Rather minor changes (pH, ionic strength, and temperature) strongly inuence both the thermodynamics and kinetics of protein adsorption [14]. The observed lack of t of the adsorption features with the theoretically predicted ones indicates a variety of adsorption mechanisms acting at the same time whose relative importance depends on the factors mentioned above as well as the structure and condition of the adsorbing surface. It may also point to the formation of multilayers in some conditions and/or denaturation of the protein upon contact with the surface. In fact, it is well-known that proteins denature upon direct contact with gold [15] and other surfaces [1618] while they retain their shape and function when

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adsorbed to SAMs [19]. Interestingly, the structure and biological activity are often retained when proteins are adsorbed to metals in the form of nanoparticles [20]. Protein adsorption to SAM modied surfaces is better behaved than direct adsorption, so SAM modied surfaces generally form the basis of biosensors. Nonetheless, for verication purposes, it is important to be able to correlate the surface sensitivity of the LRSPP deduced from optical measurements [10] with a known amount of a single protein as determined by another analytical technique. In view of the extreme sensitivity of adsorption isotherms to the experimental conditions, it is imperative that identical conditions be used for the protein deposition on the samples characterized and that the samples be very similar to each other. Most of the analytical methods currently available to determine protein surface coverage develop limitations when applied to thin narrow Au stripes. Quartz crystal microbalance (QCM) requires a dedicated, relatively large quartz crystal covered with Au, likely having surface properties that are different from those of a waveguide stripe. Radioactive labeling will not distinguish the protein adsorbed on the waveguide stripe from that adsorbed on the surrounding area. Conventional ellipsometry has two important limitations: one is the lateral resolution of the technique and the other is its sensitivity to underlying layers which can limit the accuracy since a priori assumptions of refractive index and thickness are required. The technique of choice here appears to be atomic force microscopy (AFM) which suffers from relatively less limitations when applied to our Au stripes. Hence, the purpose of this study is to investigate via AFM, bovine serum albumin (BSA) adlayers deposited onto thin narrow Au stripes designed for propagating LRSPPs, and used in recent surface sensing experiments [10]. Three different BSA deposition methods are considered: (i) sample immersion, (ii) droplet deposition and evaporation, and (iii) droplet connement and positioning using a hydrophilic guide [21]. Section 2 gives the experimental details, Section 3 gives the results, Section 4 discusses the results, presents a simple adsorption model explaining our observations and makes comparisons with results reported in the literature, and Section 5 gives a brief summary and concluding remarks. 2. Experimental 2.1. Materials BSA fatty acid free and globulin free (A0281), and phosphate buffer solution 0.1 M, pH 7.5 (P5244) were purchased from SigmaAldrich Canada and used as received. De-ionized (DI) water (0.22 mm, 10 MV) was received from Fisher Scientic (Protocol1 brand), and 2-propanol semicon ductor grade (Puranal1) was obtained from Riedel-de Haen. 2.2. Fabrication of Au stripes and structures Si wafers with a 15 mm thick thermally oxidized SiO2 layer were used. A contact aligner and bi-layer photoresist were then

used in a lift-off process to dene Au stripes and features. Evaporation was used to coat the wafers, rst with 2 nm of Cr as an adhesion layer, followed by 23 nm of Au, for a total metal stack thickness of 25 nm. The metal stack is polycrystalline and annealing was not performed. Various structures such as straight waveguides, couplers and MZIs were fabricated. 2.3. BSA solutions and deposition methods The BSA solutions were prepared as follows: 1 mg of BSA was dissolved in 1 g of 0.1N phosphate buffer yielding Solution A (15 mM BSA in 0.1N buffer). Solution A was diluted 10 with either 0.1N phosphate buffer yielding Solution B (1.5 mM BSA in 0.1N buffer), or with DI water yielding Solution C (1.5 mM BSA in 0.01N buffer). The gold surfaces were degreased with 2-propanol, rinsed with de-ionized water and placed in a Novascan PSD-UV UV/ ozone cleaner (5 min UV irradiation followed by 20 min ozone action). The clean Au surfaces were hydrophilic as expected [22]. Three BSA deposition methods were used. (i) Method 1: A die with waveguide stripes and features was immersed overnight (16 h) in Solution B. The following day the die was washed thoroughly with DI water and dried. (ii) Method 2: 0.2 ml droplets of Solution A or B were deposited on various Au features including 100 mm 100 mm pads and 5 or 10 mm wide waveguide stripes as sketched in Fig. 1. The droplets were allowed to dry and the sample was rinsed in a jet of DI water and dried. (iii) Method 3 was a new microspotting method based on using a hydrophilic guide used to conne and position a solution droplet onto a specic area [21]. Here, the guide was an AFM probe holder available commercially from nanosensors which consisted of a rectangular piece of silicon of dimensions 3400 mm 1600 mm with beveled corners. The guide was placed parallel to and 60 mm above the sample surface with the help of a micropositioner. A 0.4 ml droplet of Solution C was deposited onto the sample next to the guide using a micropipette and then pulled in below the guide via capillary forces. (Solution C was used here because it is less viscous than Solutions A and B which were leaving behind smudges when moved with the guide.) The guide and conned solution were

Fig. 1. LRSPP waveguide stripes and structures used for the AFM study. The stripe widths are 5 and 10 mm and have a total thickness of 25 nm (23 nm Au on 2 nm Cr). The circles denote areas with deposited BSA using droplets of Solutions A and B.

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then moved over the Au structures to be treated and maintained in position for several minutes. Then the guide was removed and the sample was rinsed in a jet of DI water and dried. Die 1, bearing Au stripes and features similar to those shown in Fig. 1, was processed according to Method 1. Die 2 was processed according to Method 2 using droplets of either Solution A or B deposited as sketched in Fig. 1. Die 3, bearing Au structures similar to those shown in Fig. 1, was processed using Method 3. 2.4. Atomic force microscopy The samples were analyzed in a PSIA XE-100 AFM instrument. The AFM tip had radius of 10 nm and the imaging force was $5 nN. 3. Results 3.1. Virgin Au surfaces AFM measurements were conducted on an uncoated sample bearing narrow stripes identied as Die 0 and fabricated as described in Section 2.2. The metal stack thickness of Die 0 was measured as 25 nm, which is equal to the nominal thickness, and the root mean square (RMS) surface roughness was 0.77 nm measured over a large scanned area. Au stripe widths as narrow as 0.9 mm were reliably obtained and Au structures and features such as those shown in Fig. 1 were well dened. The roughness values of this die are summarized in the rst row of Table 1. AFM measurements were also conducted on an uncoated Au region of Die 2, yielding lower roughness values as noted from the second row of Table 1. The resulting AFM scan is shown in Fig. 2(a). 3.2. BSA-coated Au surfaces Fig. 2(b)(d) gives the AFM scans obtained on BSA-coated Au stripes on Dies 1 and 2, rendered as 3D plots. The scans given in Fig. 2(a)(d) were obtained over 500 nm 500 nm areas and are plotted using the same scale in order to facilitate comparisons. The vertical scale ranges from 0 to 8 nm (deep red to white). The last four rows of Table 1 summarize the roughness values measured for the BSA-coated dies, including those obtained for Die 3.
Table 1 Measured surface roughness of the samples investigated Sample RMS roughness (nm) 0.77 0.38 1.20 0.65 0.78 1.14 Average roughness (nm) 0.61 0.30 0.79 0.50 0.58 0.82 Peak-tovalley (nm) 1.79 5.80 3.37 3.70 8.93 Fig. 2. AFM scans of (a) Die 2, uncoated Au, (b) Die 1, BSA coated with Method 1, Solution B, (c) Die 2, BSA coated with Method 2, Solution A, (d) Die 2, BSA coated with Method 2, Solution B. The AFM scans were conducted over 500 nm 500 nm areas and the color scale of the scans ranges from 0 to 8 nm (deep red to white). (For interpretation of the references to color in this gure legend, the reader is referred to the web version of the article).

Uncoated Au (Die 0) Uncoated Au (Die 2) BSA-coated Au (Die 1) BSA-coated Au (Die 2-A) BSA-coated Au (Die 2-B) BSA-coated Au (Die 3)

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BSA-coated and uncoated Au stripes were scratched with a probe tip at a large applied force (200 nN) and the scratched areas were subsequently imaged via AFM. The resulting AFM scans are shown in Fig. 3. 4. Discussion 4.1. AFM measurements We can see from Fig. 2(b)(d) that the BSA-coated surfaces show higher features than the uncoated one shown in Fig. 2(a), clearly due to the deposition of BSA. Indeed, from Table 1, it is noted that all BSA-coated stripes have a higher measured roughness than the virgin surface of Die 2. Although all of the roughness measures are increased, the most affected measure is the peak-to-valley roughness reecting the non-uniformity of BSA deposition on the surface, possibly due to the formation of aggregates in some areas. Although the effect of BSA deposition on AFM scans is clearly visible, it is not possible to separate individual BSA molecules from features in the Au stripe due primarily to the polycrystalline morphology of the evaporated stripe. In order to observe BSA molecules directly, an atomically at surface is required, as in [23,24] for example, where protein was adsorbed on the 1 1 1 surface of Au [23,24] or on Si [24]. Hence, in order to positively identify and quantify the presence of BSA on the Au surface, coated and uncoated samples were scratched with a probe tip at a large applied force (200 nN) and the scratched areas were subsequently imaged via AFM yielding the scans shown in Fig. 3. The scratch tests reveal the removal of a large amount of material from the BSA-coated Au stripes as shown in Fig. 3(b) and (c), while hardly any material has been removed from the uncoated Au stripe as shown in Fig. 3(a). In Fig. 3(b), the surface along the stripe edge was scratched from top to bottom, and material piled about 75 nm high has accumulated along the bottom of the scratched area. In Fig. 3(c), the surface in the middle of the stripe was scratched from right to left, and an accumulation of material, piled about 50 nm high, is apparent along the left of the scratched area. It is surmised that the accumulated material in both cases consists of removed BSA molecules. The difference in the average height of the green (pristine) and blue (scratched) regions of Fig. 3(b) is 2.1 nm. Likewise, the red region within the scratched area of Fig. 3(c) is 2.1 and 1.8 nm lower than the green and blue regions of the adjacent pristine areas, respectively. On the other hand, scratching the uncoated Au in the same manner damages the surface slightly, as shown in Fig. 3(a), but does not produce any measurable lowering of the surface. 4.2. BSA adsorption model A simple adsorption model is suggested as follows to account for the measured adlayer thickness. From hydrodynamic considerations a molecule of BSA in solution is considered to have an oblate ellipsoid (cigar) shape with dimensions of 14 nm 4 nm [25], as shown in side and end
Fig. 3. (a) AFM scan of the uncoated Au surface scratched (left to right) at an applied force of 200 nN. (b) AFM scan of the BSA-coated Au surface, Die 2 (Solution B), scratched top to bottom at 200 nN. An accumulation of material, piled about 75 nm high, is apparent along the bottom of the scratched area. (c) AFM scan of the BSA-coated Au surface, Die 3, scratched right to left at 200 nN. An accumulation of material, piled about 50 nm high, is apparent along the left of the scratched area (For interpretation of the references to color in the citation of this gure, the reader is referred to the web version of the article).

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Fig. 4. Sketch of a BSA molecule in (a) side and (b) end views.

views in Fig. 4(a) and (b), respectively. The molecular weight of BSA is MW = 66 430, based on the amino-acid sequence [26]. There are two extreme orientations for the adsorption of BSA molecules onto a surface: side-on and end-on. In side-on adsorption the occupied footprint is 4 14 = 56 nm2. Assuming monomolecular side-on coverage, the maximum number of BSA molecules deposited on a 1 mm2 (=1012 nm2) surface is 1012/56 = 1.79 1010. Since the mass of one molecule is 66 430/6.02 1023 = 1.104 1019 g, the maximum surface mass density of BSA adsorbed in this orientation is 1.79 1010 1.104 1019 = 1.975 109 g/mm2 = 1.975 ng/mm2 (1.975 mg/m2). In end-on adsorption, the occupied footprint is 4 4 = 16 nm2, and an analogous reasoning leads to the maximum monomolecular surface mass density of 6.91 ng/ mm2 (6.91 mg/m2). A monomolecular layer having mixed BSA adsorption orientations would have a surface coverage between these extreme values. The volume of an ellipsoid is V = (4/3)pr1r2r3 = (1/6)pd1d2d3, where r1, r2, and r3 are the lengths of the semiaxes, and d1 = 2r1, d2 = 2r2 and d3 = 2r3, as dened in Fig. 4. In side-on adsorption the footprint area is A = d1d2 so the average height of coverage h1, given by h1 = V/A = (1/6)pd3, is h1 = 2.09 nm since d3 = 4 nm. A similar reasoning for end-on adsorption leads to the average height h2 = (1/6)pd1 = 7.33 nm since d1 = 14 nm in this case. The average height h1 or h2 is interpreted as meaning that the protein attens/denatures upon adsorption lling the unused spaces between molecules. This adsorption model shows that a close-packed denatured BSA monolayer adsorbed side-on should have an average thickness of 2.09 nm and a surface mass density of 2 ng/mm2. The measured average thickness of the BSA adlayers obtained from the scratch tests are in good agreement with this thickness: 2.1 nm from Fig. 3(b), and 1.8 and 2.1 nm from Fig. 3(c). Hence, our experimental results clearly suggest that our deposition methods produce a close-packed side-on monolayer of denatured BSA on Au. 4.3. Comparisons with the literature Results reported in the literature regarding the thickness of albumin adlayers, adsorbed directly onto various surfaces,

Fig. 5. AFM probe tip interacting with BSA on the surface (a) when individual protein molecules retain their original shape, and (b) when the protein denatures.

vary over a large range with our results located somewhere at the midpoint. It was estimated by optical methods that 1.2 nm thick adlayers of BSA and 1.6 nm thick adlayers of HSA (human serum albumin) form directly on silica/titania surfaces [27,28]. Other BSA adlayer thicknesses include 3.23.7 nm on silica, depending on the BSA concentration in solution [16] and the pH [29], and 0.8 nm on Au as measured by ellipsometry [30]. Adsorption on conductors depends also on the conductor potential, ranging from 1 nm at 800 mV, though 2 nm at the open circuit voltage, to 4 nm at +700 mV (pH 7) [31]. Despite the known sensitivity of protein adsorption to solution properties and process conditions [13,14], it is interesting to note that the deposition methods used herein (open droplet: Method 2 with Solution B, and conned droplet: Method 3 with Solution C [21]) produced BSA adlayers with no measurable difference in thickness, even though these two methods differed in the contact time of the BSA solution with the Au surface and in the ionic strength of the solution. Although the kinetics might be different, the end protein coverage achieved appears to be similar. There are, however, some potential problems with the reliability of AFM measurements on protein adlayers which should be mentioned. One problem is the resolution achievable with a given tip diameter. If the diameter is too large then some surface texture might not be resolved properly. In our case, the tip diameter was 20 nm so surface texture on the scale individual BSA molecules probably cannot be resolved, as suggested by the sketch of Fig. 5(a). Another problem is that proteins are soft so they may be deformed by the AFM probe, producing an apparent layer thickness that would be lower than the actual one. Hence, we cannot positively distinguish between the adsorption cases sketched in Fig. 5(a) and (b). Nevertheless, our model and experimental results support the interpretation that directly adsorbed BSA forms a monomolecular layer, likely in the side-on orientation, which denatures and attens on the surface. The fact that the BSA adsorption is irreversible further conrms the denatured character of the adsorbed protein.

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5. Summary and concluding remarks The purpose of this study was to investigate via AFM, BSA adlayers deposited onto thin narrow Au stripes designed for propagating LRSPPs, and used in recent surface sensing experiments. Different BSA deposition methods were considered including droplet deposition and evaporation, and droplet connement and positioning using a hydrophilic guide. The AFM results reveal that the BSA-coated stripes have higher roughness measures (RMS, average and peak-to-valley) than a similar virgin Au surface (Die 2). The most affected roughness measure is the peak-to-valley roughness reecting the non-uniformity of BSA deposition on the surface, possibly due to the formation of aggregates. Scratching BSA-coated stripes using a probe tip resulted in the removal of a large amount of material surmised to be BSA molecules. The difference in the average height of pristine and scratched areas was then measured via AFM and found to be 1.82.1 nm. The step height measured in this manner is taken as the average thickness of the BSA adlayer. A simple adsorption model shows that a close-packed denatured BSA monolayer adsorbed side-on should have an average thickness of 2.09 nm and a surface mass density of 2 ng/mm2. The measured average thickness of the BSA adlayers is in very good agreement with this thickness. The AFM measurements and adsorption model suggest that the adsorbed BSA is attened and denatured although this is not absolutely certain due to the probe tip diameter being large compared to the BSA molecules and the fact that the molecules could be compressed by the probe tip during the measurements. Nevertheless, our interpretation and the thicknesses measured for the BSA adlayers are consistent with the literature. Acknowledgment The authors would like to thank Dr. Heng-Yong Nie of Surface Science Western, University of Western Ontario, Canada, for his diligent help with the atomic force microscopy measurements. References
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