Development of a Pulsed Radio Frequency Glow Discharge for Three

Jan 23, 2007 - Gerardo Gamez, Steven J. Ray, Francisco J. Andrade, Michael R. Webb, and Gary M. Hieftje*. Department of Chemistry, Indiana University,...
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Anal. Chem. 2007, 79, 1317-1326

Development of a Pulsed Radio Frequency Glow Discharge for Three-Dimensional Elemental Surface Imaging. 1. Application to Biopolymer Analysis Gerardo Gamez, Steven J. Ray, Francisco J. Andrade, Michael R. Webb, and Gary M. Hieftje*

Department of Chemistry, Indiana University, Bloomington, 800 East Kirkwood Avenue, Bloomington, Indiana 47405

Glow discharge optical emission spectrometry has cemented itself as an important surface elemental analysis technique in part because of its superb depth resolution (on the order of single nanometers). However, very few studies have explored the ability of the glow discharge to provide laterally resolved elemental information. In the present study, an end-on-viewed pulsed radio frequency glow discharge is coupled to a monochromatic imaging spectrometer to provide lateral surface imaging. The performance of the technique is demonstrated with etched copper circuits on fiber-glass substrates, and it is shown how several operating parameters including pressure, pulsed mode operation, and time-resolved detection affect the lateral surface resolution. In addition, because a pulsed radio frequency glow discharge offers elemental information on nonconducting samples, the technique is applied to the three-dimensional elemental analysis of proteins on blotting substrates. Several alternative sample types are also examined, including photographic film and glass. Glow discharge optical emission spectrometry has become renowned in the surface analysis community for its ability to provide direct elemental analysis of solid samples.1-4 Among other attributes, the glow discharge offers a broad dynamic range of about 5 orders of magnitude, limits of detection in the low parts per billion range, simultaneous multielemental analysis, and excellent stability.3-6 However, one of its more useful characteristics is superb depth resolution, on the order of single nano* To whom correspondence should be addressed. E-mail: [email protected]. Phone: (812) 855-7905. Fax: (812) 855-0958. (1) Payling, R., Jones, D. G., Bengtson, A., Eds. Glow Discharge Optical Emission Spectrometry; John Wiley & Sons: New York, 1997. (2) Angeli, J.; Bengtson, A.; Bogaerts, A.; Hoffmann, V.; Hodoroaba, V.-D.; Steers, E. J. Anal. At. Spectrom. 2003, 18, 670-679. (3) Marcus, R. K.; Broekaert, J. A. C. Glow Discharge Plasmas in Analytical Spectroscopy; John Wiley & Sons: New York, 2003. (4) Jakubowski, N.; Bogaerts, A.; Hoffmann, V. At. Spectrosc. Elem. Anal. 2004, 91-156. (5) Nelis, T.; Payling, R. Glow Discharge Optical Emission Spectroscopy: A Practical Guide; Springer: Berlin, 2004. (6) Winchester, M. R.; Payling, R. Spectrochim. Acta, Part B 2004, 59B, 607666. 10.1021/ac061361l CCC: $37.00 Published on Web 01/23/2007

© 2007 American Chemical Society

meters.7 For example, Payling et al.8 used radio frequency glow discharge optical emission spectrometry (rf-GDOES) to show how the quantitative depth profile of galvanized steel surfaces changes within the first 12 nm. More recently, Shimizu et al.9 performed a depth profile analysis of a monolayer of thiourea adsorbed on a copper substrate. These authors showed how atomic emission peaks from the monolayer could be distinguished in the order of appearance according to the orientation of the thiourea molecule (where the sulfur faced the copper substrate). In particular, it was possible to observe the hydrogen emission first, followed by nitrogen and then sulfur and finally emission from the copper substrate.9 In view of this depth-resolution capability, it might seem surprising that very few efforts have been directed at exploring lateral resolution in GDOES.10-12 Winchester and Salit11 were able to obtain macroscale elemental composition maps by simultaneously collecting the emission from multiple direct current glow discharges located at different positions on the same sample. A single photomultiplier tube and Hadamard transform spatial imaging were used to achieve a spatial resolution on the order of millimeters.11 Further, Hoffmann and Ehrlich10 observed the lateral distribution of emission from a dc glow discharge with an 8 mm diameter sputter crater by using a point-by-point emission collection rastering system. They demonstrated that atomic emission from the plasma support gas and impurities is constant across the sputtered area while the emission peak for elements that are homogeneously distributed in the solid sample is maximum at the center of the discharge.10 However, when they analyzed an iron-plate sample containing a 1 mm inclusion of compressed copper powder, they observed that copper emission peaked at the position of the copper inclusion.10 In a more recent study, Webb et al.12 coupled a dc glow discharge to a monochromatic imaging spectrometer to obtain (7) Shimizu, K.; Habazaki, H.; Skeldon, P.; Thompson, G. E. Surf. Interface Anal. 2003, 35, 564-574. (8) Payling, R.; Michler, J.; Aeberhard, M. Surf. Interface Anal. 2002, 33, 472477. (9) Shimizu, K.; Payling, R.; Habazaki, H.; Skeldon, P.; Thompson, G. E. J. Anal. At. Spectrom. 2004, 19, 692-695. (10) Hoffmann, V.; Ehrlich, G. Spectrochim. Acta, Part B 1995, 50B, 607-616. (11) Winchester, M. R.; Salit, M. L. Spectrochim. Acta, Part B 1995, 50B, 10451058. (12) Webb, M. R.; Hoffmann, V.; Hieftje, G. M. Spectrochim. Acta, Part B 2006, 61, 1279-1284.

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Figure 1. (A) Schematic diagram of the rf glow discharge cell coupled to the monochromatic imaging spectrometer (a, rf connector; b, cooling water in; c, cooling water out; d, Macor restrictor; e, sample; f, collimating lens; g, monochromator; h, focusing lens; I, ICCD camera). (B) Monochromatic image (324.754 nm) of a 1951 USAF target obtained with the MIS system.

surface elemental maps via optical emission spectroscopy. The principle of the monochromatic imaging spectrometer has been described elsewhere.13,14 In short, a collection lens is used to collimate emission from the source; this light is then passed through a monochromator. A second lens is used to refocus the collimated light at the selected wavelength onto a camera. The result is a monochromatic image of the source. Webb et al.12 used a nickel alloy substrate with a solid copper inclusion to show that running the glow discharge in pulsed mode improved spatial resolution greatly as opposed to running the discharge continuously. This is because when the discharge is run continuously the sputtered analyte is allowed to diffuse throughout the plasma, thus emitting far from its original position and causing a loss in surface spatial information. The gas pressure and the pulse width and frequency were found to affect the lateral resolution.12 These authors showed that the full width at half-maximum of the emission profile above a 1 mm copper inclusion had a better than 0.1 mm accuracy under optimized conditions12 In those experiments, the lateral resolution limit was restricted by the sample. The limitation of a dc glow discharge, however, is that only conducting samples can be analyzed. Conveniently, this barrier can be overcome by using radio frequency (rf) powered glow discharges. Although previous studies have shown that the electron and ion number densities are lower in rf glow discharges, it has also been found that the electron energies are about an order of magnitude higher than in their dc counterparts.15-19 The aim of the present study is to employ a pulsed radio frequency glow discharge optical emission imaging system to perform two-dimensional surface elemental mapping of solid samples. Pulsed glow discharge operation is employed mainly to provide improved lateral resolution and not to influence ionization, excitation, or sputtering properties of the discharge.15,20 An rf glow discharge cell and its coupling to a monochromatic imaging (13) Olesik, J. W.; Hieftje, G. M. Anal. Chem. 1985, 57, 2049-2055. (14) Webb, M. R.; Hieftje, G. M. Appl. Spectrosc. 2006, 60, 57-60. (15) Marcus, R. K. J. Anal. At. Spectrom. 1993, 8, 933-943. (16) Pan, X.; Hu, B.; Ye, Y.; Marcus, R. K. J. Anal. At. Spectrom. 1998, 13, 11591165. (17) Gamez, G.; Huang, M.; Lehn, S. A.; Hieftje, G. M. J. Anal. At. Spectrom. 2003, 18, 680-684. (18) Gamez, G.; Bogaerts, A.; Andrade, F.; Hieftje, G. M. Spectrochim. Acta, Part B 2004, 59B, 435-447. (19) Bogaerts, A.; Gijbels, R.; Gamez, G.; Hieftje, G. M. Spectrochim. Acta, Part B 2004, 59B, 449-460. (20) Pan, C.; King, F. L. Anal. Chem. 1993, 65, 3187-31933.

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spectrometer are described. In addition, the effects of operating parameters of the glow discharge and detection system on the achievable spatial resolution are investigated. The system is demonstrated on etched copper patterns on fiberglass, silver deposited on glass slides, and photographic film sample targets. Finally, an application involving protein elemental mapping is developed for blotting membranes with dot- and Western-blotted proteins stained with silver-enhanced colloidal gold. EXPERIMENTAL SECTION rf Glow Discharge Imaging Instrument. Figure 1A shows a schematic diagram of the rf glow discharge imaging instrument. A 13.56 MHz generator (Cesar 1350, Dressler, Stolberg-Vicht, Germany) was used to supply the rf power. A wave synthesizer (Hewlett-Packard model HP 3326A, Palo Alto, CA) provided a signal for pulsing the rf power supply and triggered the ICCD camera described below. An in-house-built L-type impedancematching network (two 25-1000 pF variable capacitors and a 3 µH air-core inductor) was employed to couple the rf energy into the glow discharge cell. The rf glow discharge cell is based on the one previously described by Heintz and Hieftje.21-23 However, several modifications were made, mainly to the cathode assembly, to permit analysis of blotting membranes, etched copper circuits, and photographic film. The first part of the glow discharge cell (cf. Figure 1) was comprised of an rf connector and aluminum cooling block maintained in direct contact with either a flat solid sample or a sample holder. The sample holder consists of a cylindrical aluminum backing electrode (o.d. 33 mm) and a Delrin insulating casing which allows the vacuum to be sustained independently from the immobilization of the sample. This assembly was attached to a Macor insulator (with either an 8 or a 28 mm limiting orifice) placed between the cathode sample and the rest of the cell, which is grounded. The end-on-viewed glow discharge cell is constructed from stainless steel and has several ports: two for the vacuum, one inlet for the support gas, one for a pressure gauge, and a window for plasma-imaging purposes. An Edwards E2M2 high-vacuum pump (Wilmington, MA) and a (21) Heintz, M. J.; Hieftje, G. M. Spectrochim. Acta, Part B 1995, 50B, 11091124. (22) Heintz, M. J.; Hieftje, G. M. Spectrochim. Acta, Part B 1995, 50B, 11251141. (23) Heintz, M. J.; Hieftje, G. M. Spectrochim. Acta, Part B 1996, 51B, 16291646.

Figure 2. Effect of discharge pressure on spatial resolution. (A) Monochromatic images (324.754 nm) of the etched copper target on fiberglass collected via rf glow discharge at different pressures. A photograph of the original copper target is shown in the inset to the right of the monochromatic images. (B) Measured improvement of valley resolution (%) with pressure (squares) and a corresponding fit to an x-0.25 function (dashed line). Other conditions: 80 W peak rf forward power, 5 W peak reflected rf power, 40 µs rf pulse width, 1 kHz pulse frequency, 20 µs ICCD gate width, and 20 µs ICCD gate delay. The RSD was below 5% for all measurements.

mass flow controller (1159A, MKS Instruments Inc., Wilmington, MA) were used to maintain the desired pressure (from 0.5 to 30 Torr, or from 66 to 4 × 103 Pa), which was monitored with a capacitance manometer (Baratron 122AA-00100AB, MKS Instruments Inc.). The plasma gas used for all experiments was argon (99.999%). The shape of the rf waveform was monitored at the cooling block of the glow discharge cell by means of a model P6013A high-voltage probe and a model TDS 2024 digital oscilloscope (Tektronix, Beaverton, OR). The monochromatic imaging spectrometer (MIS) was constructed from a collimating lens (plano-convex, fused silica, 250 mm focal length, 60 mm diameter), Czerny-Turner monochromator (model EU 700, Heath Co., Benton Harbor, MI; 2.0 nm/mm reciprocal dispersion), a focusing lens (plano-convex, fused silica, 150 mm focal lengh, 60 mm diameter), and an intensified CCD (ICCD) camera (PI-MAX, 512 × 512 pixels; Princeton Instruments Inc., Trenton, NJ). To determine the spatial resolution of this optical system, a United States Air Force (USAF) 1951 resolution target was put in place of the sample and back-illuminated with a Xe arc lamp. The 1951 USAF target is commonly used to determine spatial resolution in microscopic imaging techniques. Figure 1B shows the image of the USAF target obtained with the

MIS at 324.754 nm. The resolution is 3.56 line pairs/mm in the horizontal direction and 8.98 line pairs/mm in the vertical direction. This means that in the vertical direction it is possible to resolve lines that are 0.0557 mm wide and separated by 0.0557 mm; the same applies to the horizontal direction but with 0.141 mm wide lines. The difference is due in part to the nature of the imaging system, in which the height (12 mm) of the monochromator slits is much greater than the width (0.75 mm) and in part to distortion introduced by the grating, where the width of the reconstructed image varies with the wavelength because of the difference in incident and diffraction angles. This latter phenomenon has been described in detail elsewhere14 but is not a fundamental limitation of the design because it can be corrected mathematically or instrumentally. In fact, this relatively minor distortion could be overcome if desired by using a different wavelength isolation system such as an interference filter. The procedure described by Monnig et al.24 was used to perform flatfield correction of all images. Images of copper targets were collected at 324.754 nm and silver target images at 328.068 nm. (24) Monnig, C. A.; Gebhart, B. D.; Hieftje, G. M. Appl. Spectrosc. 1989, 43, 577-579.

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Figure 3. Effect of rf glow discharge operating parameters on achievable spatial resolution. The copper target shown in Figure 2 was employed, and resolution is expressed as the valley (%) of the resulting images. Key: (A) gas flow rate (11 Torr, 80 Wf, 5 Wr, 40 µs pulse width, 1 kHz pulse frequency, 20 µs ICCD gate width, 20 µs ICCD gate delay); (B) forward power (conditions same as in (A) except variable forward power); (C) rf pulse separation time (conditions same as in (A) except 13 Torr and variable pulse frequency); (D) rf pulse width (conditions same as in (A) except 13 Torr, 60 Wf, and variable pulse width); (E) ICCD gate width (conditions same as in (A) except ICCD variable gate width); (F) ICCD gate delay (conditions same as in (A) except variable gate delay). The RSD was below 5% for all measurements.

Images for background correction were obtained at 322.0 nm for the copper targets and at 2 nm above and below 328.068 nm for the silver-containing samples. A total of 10 CCD frames were averaged for each image, where each frame also had 10 accumulated pulses. Sample Preparation. Several different sample types were examined. An aluminum alloy substrate with a 1 mm diameter hole filled with silver powder was used to study the effect of operating parameters on the spatial pattern of silver emission. A series of etched copper on fiberglass targets were prepared in the same fashion as etched copper circuits (0.025 mm thick copper target on a 0.075 mm thick fiberglass substrate) to characterize the effect of operating parameters on the spatial resolution of the rf glow discharge imaging system. Samples of glass slides with silver patterns deposited through vacuum evaporation were also prepared. These samples were made to mimic a 1951 USAF target such that several lines of solid analyte (copper or silver) of a desired width were separated by a distance equal to the width of each line. In addition, a 1951 USAF target was used as a mask to prepare similar samples on photographic film (ILFORD FP4 PLUS). In this fashion, a negative image of silver embedded in the film was obtained by exposing the film to light through the mask and then developing the film in the customary fashion. 1320 Analytical Chemistry, Vol. 79, No. 4, February 15, 2007

Through an iterative process it was found that a more homogenously distributed discharge over the whole sample surface could be obtained if the sample was attached to the backing electrode with double-sided conducting copper tape. Subsequently, all samples where mounted in this way. Protein samples were prepared by a conventional blotting technique. Poly(vinylidene fluoride) (PVDF) blotting membranes were used for this purpose because of their common use in blotting procedures and their endurance when they were exposed to the glow discharge (discussed in detail below). Figures of merit were obtained with dot blots of bovine serum albumin (BSA) with an in-house-built dot-blotting apparatus. Solutions of BSA dissolved in Tris buffer saline (pH 7.5) were applied to the blotting membranes (PVDF, 0.45 µm pore size; Pierce, Rockford, IL) to have dot blots of 5.8 mm in diameter. In addition, a protein standard mixture (MultiMark multicolored standard, Invitrogen Corp., Carlsbad) containing myosin, phosphorylase, glutamic dehydrogenase, carbonic anhydrase, myoglobin blue, myoglobin red, lysozyme, aprotinin, and insulin was separated via gel electrophoresis (NuPAGE, 10% Bis-Tris gel, XCell SureLock MiniCell CE Mark, Invitrogen Corp.) followed by a Western blot (XCell II Blot Module CE Mark, Invitrogen Corp.) onto a PVDF membrane (Invitrolon, 0.45 µm pore size, Invitrogen Corp.).

Figure 4. The rf glow discharge 3D elemental mapping technique is suitable for a wide variety of sample types, shapes, and sizes. Photographs of several sample instances, each with its corresponding surface elemental emission image, are depicted here. Key: (A, E) etched copper on fiberglass targets in the shape of the Indiana University logo; (B, F) low-density microdot array; (C, G) microscopy glass slide with two silver metal lines on its surface; (D, H) developed photographic film with a negative image of the 1951 USAF target.

Both blotting procedures were followed by staining with silverenhanced colloidal gold (Enhanced Colloidal Gold Total Protein Detection Kit, Bio-Rad Laboratories, Hercules, CA). The blotted proteins were equilibrated in colloidal gold solution for 105 min, followed by the enhancement solution (5.6 mM silver lactate and 77 mM hydroquinone in citrate buffer at pH 3.7) for 2.5 min, and finally washed with fixing solution for 3.5 min. The stained blots were then dried overnight and analyzed. RESULTS AND DISCUSSION Effect of Operating Parameters on Spatial Resolution. It was previously found with a dc glow discharge that operating the source in pulsed mode (on/off cycles) improved the lateral spatial resolution.12 This improvement arises because in pulsed mode the analyte atoms have a limited time to travel from the site from which they are sputtered before being excited and subsequently emitting. In addition, pulsing the discharge reduces cathode heating and thereby simplifies analysis of thermally labile samples. Consequently, all experiments in this study were performed with the rf glow discharge operated in pulsed mode. To evaluate the effect of operating parameters on spatial resolution, the etched copper on fiberglass targets were used along with a Macor restrictor of 8 mm diameter (resulting cathode area of 50.3 mm2). Figure 2 shows a photograph of one of these targets. The shape of the targets was chosen to enable measurement of the lateral resolution in a manner analogous to that of the use of a 1951 USAF target. Each copper strip is 0.75 mm wide and separated by 0.75 mm from the next copper strip. Figure 2A shows a series of background and flat-field-corrected monochromatic (25) Parker, M.; Hartenstein, M. L.; Marcus, R. K. Spectrochim. Acta, Part B 1997, 52B, 567-578. (26) Ye, Y.; Marcus, R. K. Spectrochim. Acta, Part B 1996, 51B, 509-531.

images obtained for copper emission (324.754 nm) from the glow discharge under different conditions of pressure. Other operating parameters were 80 W peak rf forward power, 5 W peak reflected rf power, 40 µs rf pulse width, 1 kHz pulse frequency, 20 µs ICCD gate width, and 20 µs ICCD gate delay (with respect to the rising edge of the driving trigger). It is evident that as the discharge pressure is increased from 3 to 30 Torr (4 × 102 to 4 × 103 Pa) the lateral resolution of the atomic emission image is greatly improved. To quantify the spatial resolution, an intensity profile across the three copper stripes was extracted and the valley resolution (%) obtained. Here, the valley resolution is defined as the ratio of the peak value to the value at the midpoint of the peaks, multiplied by 100. The average valley resolution (obtained from triplicate measurements of valley resolution at three different positions within one sample and averaged for three independent samples) across the stripes as a function of pressure is plotted in Figure 2B. Not surprisingly, resolution improves as the pressure is raised. As the pressure is raised, the mean free path drops and sputtered analyte atoms have less time to travel from their original site before being excited and emitting. This hypothesis is supported by previous studies in which it was shown that relative emission yields in rf glow discharges go up with pressure25 because of increases in electron and ion number densities.26 Figure 2 also shows that the improvement in resolution becomes less at higher pressures with a trend that follows closely a P-0.25 dependence (where P is pressure). Webb et al.12 observed a P-1 dependence of the spatial resolution and pointed out that the mean diffusion distance should follow a P-0.5 dependence with pressure in accordance with the F-0.5 dependence of the mean diffusion rate (where F stands for density). Of course, this dependence could be distorted by the initial velocity imparted to released analyte Analytical Chemistry, Vol. 79, No. 4, February 15, 2007

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Figure 5. Evaluation of two alternative blotting membranes for use in rf glow discharge imaging of proteins: (A) time required to sputter through different blotting membranes as observed by a rise in silver emission (328.068 nm) from the backing electrode; (B) background emission from different blotting membranes around 328.068 nm. PVDF ) poly(vinylidene fluoride), and NC ) nitrocellulose. The RSD was below 8% for all measurements.

atoms during the sputtering process, by thermalization of the sputtered atoms, and/or by temperature and density gradients in the discharge.12 For example, before the sputtered atoms start to diffuse, they become thermalized, and the spatial profile of this process also depends on pressure (at lower pressures thermalization occurs at greater distances from the cathode).27 In terms of discharge regions, when the pressure is raised, the axial regions are compressed toward the cathode, which means the cathode dark space and negative glow (where excitation and emission take place) will be closer to the cathode. The discrepancy in pressure trends between our results and those of Webb et al.12 might be the result of differences in operating conditions and cell geometry. If our trend line is extrapolated, raising the pressure to 100 Torr (13.3 × 102 Pa) would result in a resolution improvement to a 43% valley of the present target, if all other operating parameters are fixed. However, this extrapolation might not be valid nor feasible because of the higher power that is required at elevated discharge pressures. In the foregoing experiments, cell pressure was adjusted by stepping the input argon flow from 0.16 to 0.98 L/min. To isolate the contribution of the gas flow rate independently, a series of experiments was performed in which the pressure was held constant and the argon flow was varied. A constant cell pressure (27) Bogaerts, A.; Straaten, M. v.; Gijbels, R. J. Appl. Phys. 1995, 77, 18681874.

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Figure 6. Use of the pulsed rf glow discharge imaging instrument for the evaluation of dot blots. (A) Photographic image of a BSA dot blot (10 µg/cm2) after staining with silver-enhanced colloidal gold. The dot-to-dot variation was measured by densitometry to be 4.5%. (B) Spatially resolved silver emission (328.068 nm) from the dot-blot sample in (A) obtained via rf glow discharge imaging. When the topleft blot was discarded because of a discharge artifact, the variability among the remaining three monochromatic images was 6.1%.

was achieved by changing the argon mass flow controller while a restrictor valve attached to the vacuum line was simultaneously adjusted. The rest of the operating conditions were kept the same as above, and the pressure was maintained at 11 Torr (1.47 × 102 Pa). It can be observed in Figure 3A that the flow rate does not affect noticeably the measured resolution over the range from 0.1 to 0.7 L/min (the flow rate range was constrained by limitations of the restrictor valve). Thus, it can be confidently concluded that the argon flow rate does not contribute measurably to the variation in observed spatial resolution in the pressure study. Although Webb et al.12 employed a very different gas-flow geometry, they arrived at a similar conclusion, further support that gas flow does not have a great impact on achievable spatial resolution. Figure 3B shows the effect of rf forward power on the lateral spatial resolution. As with argon flow, forward power does not play a significant role in governing spatial resolution. Nevertheless, sputtering rates have been shown to climb linearly with applied power density.28,29 Although higher sputtering rates do not (28) Van Straaten, M.; Gijbels, R.; Vertes, A. Anal. Chem. 1992, 64, 1855-1863. (29) Parker, M.; Hartenstein, M. L.; Marcus, R. K. Anal. Chem. 1996, 68, 42134220.

Table 1. Limits of Detection of Common Total Protein Stains

total protein stain

blot limit of detection (ng of protein/spot)

Ponceau-S red Amido black 10B Coomassie brilliant blue R-250 India ink colloidal gold Spyro Ruby silver-enhanced colloidal gold (densitometry) silver-enhanced colloidal gold (rfGD imaging)

500034 100034 50034 10034 434 235 0.5a 0.05b

a Under the present conditions, for a 1 mm2 spot. b Bio-Rad Enhanced Colloidal Gold Total Protein Detection Kit instruction manual, catalog number 170-6517, Bio-Rad Laboratories, Inc., Hercules, CA.

Figure 7. Quantitative performance of the rf glow discharge imaging instrument: (A) photgraphic image of a BSA dot-blot series of increasing concentrations (0.01, 0.1, 1.0, and 10.0 µg/cm2) after staining with silver-enhanced colloidal gold; (B) spatially resolved silver emission (328.068 nm) map from the dot-blot sample in (A) collected with the rf glow discharge imaging system.

necessarily translate into better excitation conditions,25 they can affect depth resolution. Thus, a compromise may have to be reached between optimizing for signal and depth resolution. Parts C and D of Figure 3 show that the pulse characteristics7 of the rf power supply have a substantial impact on the achievable lateral resolution. In general, spatial resolution improves as the duty cycle is reduced. In the pulse repetition rate study (Figure 3C), the pulse width was fixed at 40 µs and the pressure at 13 Torr (1.73 × 102 Pa) while the rest of the operating conditions were maintained as in the pressure study. Here, spatial resolution improves as the separation between applied rf pulses becomes longer. This initially surprising result arises because atoms that were sputtered from a preceding glow discharge pulse can be excited by a following pulse. These atoms, which are not completely flushed out of the cell between pulses, can travel a significant distance before being excited by a following pulse. Importantly, above pulse separation times of 750 µs the improvements in spatial resolution are modest, so repetition frequencies in the kHz range can conveniently be used. Webb et al.12 reported a similar transition at an interpulse spacing of 80 µs. The difference between these two values might be due to cell geometry, which will influence the flush times. In general, the trends of spatial resolution with power, pulse frequency, and pulse width are in agreement with the results of Webb et al. in their dc glow

discharge imaging study.12 In the pulse width study (Figure 3D), the repetition frequency was maintained at 1 kHz while the rest of the operating conditions were the same as in the pulse repetition rate study. Pulse widths shorter than 40 µs were not investigated because they caused the discharge to become unstable. From Figure 3D, longer pulses degrade spatial resolution, probably because they afford sputtered analyte atoms a longer time to diffuse before their emission is captured. Although the overall observed trends in spatial resolution as a function of operating conditions agree in general with the results of Webb et al.,12 detailed differences might be due not only to cell geometries but also to the ignition process, which differs between radio frequency and direct current glow discharges. Briefly, in the case of the radio frequency operated glow discharge, the plasma is generated first and then it takes a finite time before sample polarization occurs and for the dc bias voltage to build up. In contrast, in a direct current glow discharge polarization is accomplished directly by application of the external voltage.30 This dissimilarity will also affect the lower limits of the pulse width in the radio frequency glow discharge. Further studies to assess disparities between rf and dc operated glow discharges with respect to lateral spatial resolution are under way. Given the importance of capturing emission from sputtered analyte atoms before they drift too far from their original position, a series of studies on the benefits of time-resolved detection were performed. Parts E and F of Figure 3 show the effects of ICCD gating on the measured valley resolution. A fixed rf pulse width of 80 µs and a forward power of 60 W were used, while the rest of the operating conditions were the same as in the studies described above. Although its effect is not as dramatic as that of the operating conditions discussed above, the ICCD gate width (cf. Figure 3E) should be kept as short as possible, as long as high signal-to-noise ratios can be maintained. It is only reasonable that the ICCD captures only emission from sputtered analyte atoms that are close to their original position when its gate width is short. Also, having a narrower detection gate provides a more selective detection window within the excitation pulse width as is covered in detail below. The ICCD gate delay study (with respect to the driving trigger) was performed with a 40 µs pulse width and a 20 µs ICCD gate (30) Nelis, T.; Aeberhard, M.; Hohl, M.; Rohr, L.; Michler, J. J. Anal. At. Spectrom. 2006, 21, 112-125.

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Figure 8. Photographic image of a section of a Western blot of a standard protein mixture separated by gel electrophoresis and stained with silver-enhanced colloidal gold. The spatially resolved silver emission (328.068 nm) as measured by rf glow discharge imaging is depicted at the bottom.

width. It should be noted that there is a 10 µs inherent delay between the rising edge of the driving trigger and the time when the rf envelope starts to increase. This delay is followed by a rise time on the order of 10 µs. The fall time of the rf envelope was measured to be ∼2 µs. Figure 3F shows how the lateral resolution improves as the ICCD gate delay is narrowed from 40 to 20 µs. This behavior is consistent with the hypothesis that resolution is best when sputtered analyte atoms have less time to drift away from their original position. On the other hand, at a short gate delay of 10 µs the spatial resolution is compromised. This finding is possibly due to sputtered atoms that remain from the previous pulse and which are excited by the present, monitored pulse. Overall, the best lateral resolution achieved with etched copper targets on a fiberglass substrate was 0.67 line pairs/mm at 50% valley resolution. This resolution was obtained under optimized conditions of 29 Torr (3.86 × 103 Pa), 250 Hz pulse repetition frequency, 40 µs pulse width, 20 µs ICCD gate width, and 20 µs ICCD gate delay, which are in keeping with the results above. A forward power of 80 W peak was used to obtain stronger signals without damaging the samples. These values were used for all subsequent investigations. It is important to recognize that this new imaging technique is not restricted to a certain kind of sample. Figure 4 illustrates several sample types (A-D) in which the method is effective (EH). The elemental imaging of an etched copper pattern on fiberglass is extended to different sizes and shapes: Figure 4A in the shape of the Indiana University logo exemplifies targets of larger dimensions (28 mm diameter cathode or 615 mm2 total sputtering area), and Figure 4B in the shape of an array of 10 µm copper spots separated by 2.5 mm depicts targets of different shapes. The atomic emission map of the surface of a glass slide substrate with vacuum-deposited silver lines (Figure 1324

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4C) is shown in Figure 4G. Finally, Figure 4H shows the elemental (silver emission) map of a developed photographic film previously exposed to light through a mask in the shape of a 1951 USAF target (Figure 4D). In Figure 4H, 1.12 line pairs/mm can be observed. However, it should be noted that these lines have a spatial resolution poorer than 50% valley. The spatial resolution attainable even at the present stage of development of the new method could be applied to several existing problems. For example, the elemental homogeneity in forged metals available as standard reference materials could be examined. As pointed out by Winchester and Salit,11 the needed spatial resolution for such samples may be on the order of millimeters to centimeters. An additional illustration would be the elemental mapping of mixtures separated by thin-layer chromatography. Another example is that of biopolymer elemental analysis. From parts F and G of Figure 4 it is not hard to imagine this technique being applied to biopolymer macroarrays or even low-density microarrays. These applications are currently being pursued by the authors. In the following section the elemental mapping of separated proteins on blotting membrane substrates will be demonstrated. Surface Elemental Imaging of Blotted Proteins. Application of the rf glow discharge elemental surface imaging technique to blotted proteins is especially fitting because gel electrophoresis produces two-dimensional maps of separated biopolymers. Furthermore, gel electrophoresis is still by far the most popular separation procedure in biochemical analyses for the resolution and purification of components within complex mixtures.31 Although there are several ways to detect such proteins after gel (31) Hames, B. D. Gel Electrophoresis of Proteins; Oxford University Press: New York, 1998.

electrophoresis is completed, they are not without their limitations. For example, in densitometry radiation absorption in the gel contributes to the background, in fluorescence the dyes are prone to photobleaching or quenching effects, and in autoradiography special care is needed for handling radioactive isotopes; moreover, the film response is nonlinear. Atomic spectrometry has also made its way into protein detection because it has been recognized that trace elements play vital roles in biopolymer structure and function. In addition, determining the ratio of elemental concentrations within a protein can give an indication of its constitution. Some of the most popular techniques for elemental analysis of proteins include X-ray spectrometry as well as liquid sample introduction atomic absorption spectrometry, atomic fluorescence spectrometry, and inductively coupled plasma mass spectrometry (ICPMS).32 Recently, laser ablation ICPMS has been successfully applied to protein elemental mapping in gels and blotting membranes.32 However, all these techniques can be time-consuming, and the instrument components can prove quite expensive. There are many other roles that metals and other elements play in protein detection and quantification on which atomic spectrometry can capitalize. For example, metal colloids have been and are currently used for improved total and specific protein detection by densitometry. Because the number of metal atoms per protein is very high, an enhancement factor arises that improves limits of detection. In addition, metal chelate dyes and element-coded affinity33 can be used to perform simultaneous specific-protein detection. Protein analysis can benefit from rf glow discharge 3D elemental mapping not only because of the spatial resolution or the quantitative advantages offered by a glow discharge (low limits of detection, wide dynamic range, accurate quantitation in a wide range of matrixes) but also because it allows direct analysis of solid samples, thus obviating the need for digestion, spot excision, or electroelution. In addition, a new information dimension is introduced with the ability to perform elemental analysis. Ideally, one could obtain rapidly and simultaneously elemental composition maps of complete electropherograms. A set of preliminary experiments were performed to evaluate the characteristics and robustness of blotting membranes exposed to an rf glow discharge. To test the endurance of each membrane, an aluminum plate with a silver powder insert was used as a backing electrode and an untreated membrane was immobilized on it (with no conductive adhesive). The 28 mm Macor restrictor was used for all the following experiments. The rf glow discharge was then sustained for 20 min or until emission from the silver insert in the backing electrode was observable. This latter event would indicate that the membrane had been punctured by the discharge. It is important to determine how long it takes to sputter through the membrane because this will determine the amount of time available for analysis under selected conditions. It is clear from Figure 5A that under the same conditions a PVDF membrane survives for a longer time in the glow discharge than does a (32) Ma, R.; McLeod, C. W.; Tomlinson, K.; Poole, R. K. Electrophoresis 2004, 25, 2469-2477. (33) Whetstone, P. A.; Butlin, N. G.; Corneillie, T. M.; Meares, C. F. Bioconjugate Chem. 2004, 15, 3-6. (34) Dunn, M. J. Methods Mol. Biol. 1999, 112, 319-329. (35) Haugland, R. P. Handbook of fluorescent probes and research products, 9th ed.; Molecular Probes, Inc.: Eugene, OR, 2002.

nitrocellulose (NC) membrane. The NC membrane lasted for only about 5 min at 1 Torr (133 Pa) and 100 W forward power; however, in these experiments, the pulse width was 500 µs and the pulse frequency was 1 kHz. It is not unreasonable to expect that the membranes would survive longer at lower frequencies and shorter pulse widths. In addition, in an rf glow discharge, the power available at the surface of the sample changes according to the sample characteristics. Thus, it will be different for these experiments than in ones in which the membrane is mounted on the backing electrode with conductive copper tape. Membranes used in these latter experiments exhibited an even longer endurance at much higher powers and pressures (see below). Figure 5B shows the background emission of PVDF and NC unadulterated membranes from 322 to 332 nm. The wavelength resolution demonstrated in Figure 5 is a result of a necessary compromise with the spatial resolution of the imaging system. On one hand, improved spectral resolution will be achieved with narrower slit widths (greater selectivity), while, on the other hand, better spatial resolution will result from having wider slits (a wider aperture in the Fourier domain). Some of the spectral features might be associated with emission from species linked to the membranes (NH, OH, etc.) and will therefore not be the same due to the different materials. Although NC membranes produced a somewhat lower background than the PVDF membranes, subsequent experiments were performed on PVDF membranes because of their robustness, ease of use, and higher binding capacity. The same backing electrode with the silver powder insert described above was analyzed at different pressures and powers to characterize the silver emission at 328.068 nm (data not shown). In general, the signal-to-background ratio increased with both power and pressure. This behavior is not surprising; higher powers have been shown to raise sputtering rates, and higher pressures are known to create better excitation conditions in an rf glow discharge.25 Figure 6A shows the photographic image of a set of 10 µg/ cm2 BSA dot blots (5.8 mm diameter, separated by 10 mm from center to center) stained with silver-enhanced colloidal gold. These blots were performed to measure the reproducibility of our technique. The original dot-to-dot variability was measured by densitometry to be 4.5% RSD. The monochromatic image of silver emission from the same dot-blot sample obtained with the rf glow discharge 3D elemental analysis instrument is shown in Figure 6B. These membranes were mounted with double-sided conductive copper tape, and the experimental conditions where set at 11 Torr, 300 W forward power, 10 W reflected power, 125 µs pulse width, 250 Hz pulse frequency, 100 µs ICCD gate width, and 20 µs ICCD gate delay. Silver emission from each dot blot is clearly well resolved. The dot-to-dot variation in silver emission was determined to be 11.9% RSD, decidedly poorer than the densitometry results for the original stained blots. However, a very consistent, stable, and diffuse arclike “streamer” was observable with one of its ends at the glow discharge cell and another at the sample close to the top-left dot blot. This streamer elevated the emission slightly for that dot. When the top-left blot was not taken into account, the RSD improved to 6.1%. A dot-blot series (Figure 7A) with increasing BSA concentrations (0.01, 0.1, 1.0, and 10.0 µg/cm2) was prepared to construct a calibration curve. Figure 7B shows a false-color image (to clarify Analytical Chemistry, Vol. 79, No. 4, February 15, 2007

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the emission intensity) of silver emission at 328.068 nm obtained via the rf glow discharge from the same dot-blot sample shown in Figure 7A. The resulting calibration curve (obtained by integrating the emission over the area of each dot blot of this image) is linear over the lower 3 orders of magnitude (0.01, 0.1, and 1.0 µg/cm2) but becomes nonlinear at the highest concentration (10.0 µg/cm2). This nonlinearity is probably due to several factors; for instance, silver deposition during enhancement is not linear over a very broad dynamic range. Also, the silver concentration for the 10.0 µg/cm2 BSA spot might be high enough to cause self-absorption. The equation for the calibration curve obtained for the first three BSA concentrations is y ) 4350x + 160, with R2 ) 0.997 over 3 orders of magnitude. The detection limit according to the 3σ definition is 1.25 ng of BSA (0.005 µg/cm2). For a 1 mm2 spot, roughly the spatial-resolution limit of the current system, this detection limit corresponds to a mass of 0.05 ng, or 1 fmol, of BSA. Moreover, this already impressive value is not the ultimate LOD for this method. One could elevate the amount of silver deposited on each dot blot by modifying the silverenhancement procedure, which would lead to better LODs. Nevertheless, this scheme is already competitive with or better than the most sensitive total protein detection methods, as shown in Table 1. Finally, the rf glow discharge elemental imaging technique was demonstrated on a Western blot of a standard protein mixture separated by one-dimensional gel electrophoresis (Figure 8). Several lanes of the gel were used with the same biopolymer mixture to obtain a larger 2D map of separated proteins. The monochromatic silver emission images from two sections of the Western blot containing separated glutamic dehydrogenase and carbonic anhydrase are shown in Figure 8. It is evident that the resolution and sensitivity provided by the rf glow discharge imaging technique are sufficient for these samples. The different parts of the blotting membrane were analyzed separately because the current glow discharge cell is not large enough to accommodate the whole membrane. A larger glow discharge cell to accommodate bigger samples is currently being designed by the authors.

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CONCLUSIONS An rf glow discharge optical emission imaging instrument has been developed that allows one to obtain laterally resolved elemental quantitative and qualitative information directly from solid conductive or nonconductive samples. In the present study a depth profile of the samples was not demonstrated or required. However, depth profiling is a well-established ability of glow discharge analysis; thus, the ability to perform three-dimensional analysis with an rf glow discharge is now possible with the newly developed instrument. Even though the present lateral resolution is on the order of hundreds of micrometers, it is suitable for several applications including biopolymer analysis of macroarrays, blots, and gel electrophoresis separations, analysis of thin-layer chromatography plates, and spatial analysis of forged-metal standard reference materials. Several current limitations could be overcome by instrumentation modifications and improvements. For example, use of a wider aperture wavelength selection system, such as an interferometer or filter, would provide better signalto-background ratios and spatial resolution. On the other hand, a rapid-scanning monochromator, driven by a galvomotor, would offer faster sequential multielemental analysis. Also, a glow discharge cell featuring a larger sputtering area would enable analysis of larger samples. In addition, better lateral spatial resolution might be obtained by improving the discharge stability so shorter pulses could be applied. Several of these research avenues are currently being pursued by the authors. ACKNOWLEDGMENT This research was supported in part by the U.S. DOE through Grant No. DE-FG02-98ER14890 and by the Indiana Metabolomics and Cytomics Initiative (METACyt) at Indiana University.

Received for review July 25, 2006. Accepted December 8, 2006. AC061361L