Anal. Chem. 2001, 73, 2268-2272
Simultaneous Multianalyte Detection with a Nanometer-Scale Pore John J. Kasianowicz,* Sarah E. Henrickson, Howard H. Weetall, and Baldwin Robertson
NIST, Biotechnology Division, ACSL 227/A251, Gaithersburg, Maryland 20899-8313
It was recently shown that naturally occurring, genetically engineered or chemically modified channels can be used to detect analytes in solution. We demonstrate here that the overall range of analytes that can be detected by single nanometer-scale pores is expanded using a potentially simpler system. Instead of attaching recognition elements to a channel, they are covalently linked to polymers that otherwise thread through a nanometer-scale pore. Because the rate of unbound polymer entering the pore is proportional to its concentration in the bulk, the binding of analyte to the polymer alters the latter’s ability to thread through the pore, and the signal that results from individual polymer translocation is unique to the polymer type; the method permits multianalyte detection and quantitation. We demonstrate here that two different proteins can be simultaneously detected with this technique. Ion channels are proteinaceous nanometer-scale pores in phospholipid bilayer membranes. They play diverse roles in cells and organelles including the transport of specific ions and macromolecules, conduction of signals within and between cells, and antibiotic activity against organisms.1-10 Some ion channels are elements of cellular sensors: they transduce the concentration of an analyte into a change in channel conductance and, hence, in the transmembrane potential.1 The ability to measure picoampere currents permits trace and ultratrace chemical analysis. This low detection limit can be enhanced further by the use of nanometer-scale pores that can gate ∼1-100 pA ionic current by the binding of single molecules to them. As a result, relatively simple amperometric measurements provide single-molecule detection capability. * Corresponding author: (office) 301-975-5853; (fax) 301-330-3447; (e-mail)
[email protected]. (1) Hille, B. Ionic channels of excitable membranes, 2nd ed.; Sinauer Assoc.: Sunderland, MA, 1992. (2) Tsien, R. W.; Tsien, R. Y. Annu Rev. Cell Biol. 1990, 6, 716-760. (3) Crowley, K. S.; Liao, S.; Worrell, V. E.; Reinhart, G. D.; Johnson, A. E. Cell 1994, 78, 461-471. Simon, S. M.; Blobel, G. Cell 1991, 65, 371-380 (4) Prod′hom B.; Pietrobon D.; Hess P. Nature (London) 1987, 329, 243246. (5) Blachly-Dyson, E.; Peng, S. Z.; Colombini, M.; Forte, M. Science 1990, 247, 1233-1236. (6) Garcia, L. R.; Molineux, I. J. J. Bacteriol. 1996, 178, 6921-6929. (7) Gene Transfer in the Environment; Levy, S. B., Miller, R. V., Eds.; McGrawHill: New York, 1989. (8) Young, R. Microbiol. Rev. 1992, 56, 430-481. (9) Leippe, M. Parasitol. Today 1997, 13, 178-183.
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Until recently, only highly specific analyte types (e.g., neurotransmitters,1 anesthetics,11 and small ions4,12,13), which bind to some naturally occurring channels, could be detected using channels. However, with the goal of using channels as transducers for analyte detection, recognition sites have been genetically engineered inside the channel,14,15 adjacent to the channel’s mouth,16 or chemically linked to locations outside the channel’s lumen.17-19 We demonstrate here that two different proteins can be simultaneously detected with a simpler system. Instead of attaching the recognition elements to a channel, they are covalently linked to polymers that thread through a nanometer-scale pore. Because the signal that results from such a translocation is unique to the polymer type and the binding of analyte to the polymer alters the latter’s ability to traverse the pore, the sensor should permit simultaneous multianalyte detection and quantitation for a wide range of molecules. The potential for using channels in generalized analytical applications was realized when the stochastic current fluctuations induced in a solitary R-hemolysin (RHL) channel by the reversible binding of hydrogen ions4,12,13 and deuterium ions13 were observed. Frequency analysis of the analyte-induced stochastic current noise provided a direct measurement of the binding constant and the kinetic rate constants (kon and koff) for the fastest diffusioncontrolled chemical reactions in solution. In those studies, a spectral analysis of the current fluctuations permitted the identification of two isotopically different species that bind to the channel. Ion channels have either been genetically engineered or otherwise modified to bind specific types of analytes.14-17 As was shown with the reversible binding of hydrogen and deuterium (10) Jakes, K. S.; Kienker, P. K.; Slatin, S. L.; Finkelstein, A. Proc. Natl. Acad. Sci. U.S.A. 1998, 95, 4321-4326. (11) Barann, M.; Wenningmann, I.; Dilger, J. P. Toxicol. Lett. 1998, 101, 155161. (12) Bezrukov, S. M.; Kasianowicz, J. J. Phys. Rev. Lett. 1993, 70, 2352-2355. (13) Kasianowicz, J. J.; Bezrukov, S. M. Biophys. J. 1995, 69, 94-105. (14) Walker, B.; Kasianowicz, J. J.; Krishnasastry, M.; Bayley, H. Protein Eng. 1994, 7, 655-662. Kasianowicz, J.; Walker, B.; Krishnasastry, M.; Bayley, H. MRS Symp. 1994, 330, 217-223. (15) Braha, O.; Walker, B.; Cheley, S.; Kasianowicz, J. J.; Hobaugh, M. R.; Song, L.; Gouaux, J. E.; Bayley, H. Chem. Biol. 1997, 4, 497-505. (16) Kasianowicz, J. J.; Burden, D. L.; Han, L.; Cheley, S.; Bayley, H. Biophys. J. 1999, 76, 837-845. (17) Van Wie, B. J.; Davis, W. C.; Moffett, D. F.; Koch, A. R.; Silber, M.; Reiken, S. R.; Sutisna, H. U.S. Patent 5,736,342, 1998. (18) Gu, L. Q.; Braha, O.; Conlan, S.; Cheley, S.; Bayley, H. Nature (London) 1999, 398, 686-690. (19) Cornell B. A.; Braach-Maksvytis V. L. B.; King L. G.; Osman P. D. J.; Raguse B.; Wieczorek, L.; Pace, R. J. Nature (London) 1997, 387, 580-583. 10.1021/ac000958c Not subject to U.S. Copyright. Publ. 2001 Am. Chem. Soc.
Published on Web 04/11/2001
ions to the wild-type RHL channel,12,13 engineered RHL mutants exhibited analyte-induced single-channel current fluctuations that identified the chemical species of interest.15,16 Experiments using channels in supported bilayer membranes as a sensor were recently reported.19 This kind of robust setting for channels is significantly more stable than a planar lipid bilayer and appears suitable for measuring analyte concentration in relatively harsh environments. However, because that system uses a large number of channels, the characteristic kinetic information contained in the stochastic reactions between a single molecule and a solitary channel4,12,13,16,18,20 is lost. In addition, the ability of several molecules that bind to the RHL channel was recently reported.18,20,21 Among these molecules is β-cyclodextrin, which has a cavity that binds organic ligands. The latter effort may eventually help circumvent the problem of engineering binding sites for such ligands into a channel’s structure, but it did not address the problem of making a truly robust sensor with RHL. We show here that analyte quantitation can be accomplished using polymers that thread through a single nanometer-scale pore. By placing the analyte recognition site on a pore-permeant polymer instead of the nanopore, this single-molecule detection method has the advantage of being easily reprogrammed to detect different species (e.g., by replacing one class of pore-permeant polymer with another). It also permits simultaneous detection and quantitation of multiple analytes and should be realizable with robust, artificial nanopores, if they become available. SENSOR MODEL A generalized method of measuring analyte concentration can be accomplished using a polymer that threads through a single nanometer-scale pore. When an individual polymer is driven through a nanopore, it will sterically block the pore’s ionic conductance20-24 (Figure 1, top). The average repetition rate of the polymer entering the pore is directly proportional to the concentration of the polymer in the bulk solution.23 Analyte bound to the polymer will cause the latter either to become poreimpermeant (model I; Figure 1, middle) or to occlude the pore for a time that is commensurate with the mean lifetime of the analyte-polymer complex (model II; Figure 1, bottom). Each case leads to a distinct and measurable effect on polymer-induced nanopore conductance changes. EXPERIMENTAL SECTION We are using the ion channel formed by Staphylococcus aureus RHL as a model nanopore for sensor applications12-16,20-23 and biotinylated single-stranded DNA (bT-DNA) as the pore-permeant polymer. The method of reconstituting single RHL channels into solvent-free planar lipid bilayer membranes is described in detail elsewhere.13 Briefly, a membrane of diphytanoylphosphatidylcholine (Avanti Polar Lipids, Birmingham, AL) is formed on a small (20) Bezrukov, S. M.; Vodyanoy, I.; Brutyan, R. A.; Kasianowicz, J. J. Macromolecules 1996, 29, 8517-8522. (21) Bezrukov, S. M.; Kasianowicz, J. J. Eur. Biophys. J. 1997, 6, 241-246. (22) Kasianowicz, J. J.; Brandin, E.; Branton, D.;Deamer, D. W. Proc. Natl. Acad. Sci. U.S.A. 1996, 93, 13770-13773. (23) Henrickson, S. E.; Misakian, M.; Robertson, B.; Kasianowicz, J. J. Phys. Rev. Lett. 2000, 85, 3057-3060. (24) Akeson, M.; Branton, D.; Kasianowicz, J. J.; Brandin, E.; Deamer, D. W. Biophys. J. 1999, 77, 3227-3233.
Figure 1. Analytical sensor based on nanometer-scale pores and polymers. In the absence of analyte, polymers with analyte binding sites covalently linked to them partition freely into the pore and cause short-lived blockades (i.e., downward transients) in the ionic current (top). The frequency of blockades is proportional to the free polymer concentration. The polymer’s physical properties change when it is bound to analyte, rendering the polymer either undetectable by the pore (model I) or able to occlude the pore for a time commensurate with the mean time the analyte is bound to the polymer (i.e., 1/koff; model II). If the system exhibits model I behavior, the analyte concentration is deduced from the decrease in the number of polymerinduced blockades per unit time. In a model II system, the analyte concentration is determined from the mean time to the first long-lived nanopore current blockade that occurs after the electric field (which is indicated by the + and - symbols) is applied.
orifice (∼50-100 µm diameter) in a Teflon partition (∼17 µm thick) that separates two identical Teflon chambers. Each chamber contains ∼1.5 mL of electrolyte solution (1 M KCl, Mallinckrodt, Paris, KY; 10 mM HEPES, Calbiochem, La Jolla, CA; pH 7.5). Less than 1 µg of RHL is added to one chamber, herein called cis, and excess protein is immediately removed after a conductance increase heralds the formation of a single channel. A potential of -120 mV is applied across the membrane via two Ag-AgCl electrodes, and the current is converted to voltage using an Axopatch amplifier with a high-impedance headstage (Axon Instruments, Foster City, CA). This signal was digitized using a National Instruments AT-MIO16 A/D board (Austin, TX), an Axon Instruments Digidata 1200B, or a Digidata 1320A interface. The data were acquired using software either from our laboratory or from Axon Instruments and subsequently analyzed using in-house software. A negative potential drives anions from the cis to the trans chamber. Unless otherwise noted, DNA homopolymers (Midland Certified Reagants), NeutrAvidin (Pierce, Rockford, IL), StreptAvidin (Pierce), or sheep anti-bromodeoxyuridine polyclonal antibody (Fitzgerald Industries, Int., Inc., Concord, MA) are added to the cis chamber. The biotinylated homooligonucleotides were synthesized by Midland Certified Analytical Chemistry, Vol. 73, No. 10, May 15, 2001
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Figure 2. Experimental verification of the two sensor models illustrated in Figure 1. The leftmost recording shows the step increase in current that flows through a single RHL ion channel in response to an applied potential of -120 mV. The middle recordings demonstrate that adding 250 nM short (10-mer) or 30 nM long (50-mer) singlestranded 5′-biotinylated poly(dA) causes transient current blockades. Most of these transient blockades correspond to the transport of an individual DNA strand through the channel.22,23 Adding excess avidin (2.6 µM for bT-(dA)10 or 670 nM for bT-(dA)50) to bind virtually all of the bT-poly(dA) caused the polymer-induced current blockades either to disappear or to remain until the potential was reversed. The dotted lines indicate the zero current level. Both effects were observed more than 10 times each in experiments with single channels reconstituted into fresh lipid membranes.
Reagants using 5′-bT-phosphoramidite (Glen Research, Sterling, VA) and standard solid-phase oligodeoxyribonucleotide synthetic, deprotection, and purification techniques. RESULTS AND DISCUSSION Single-Channel Current in the Absence and Presence of DNA. Previous experiments in our laboratory showed that singlestranded DNA and RNA cause transient blockades in the ionic current that flows through a solitary RHL channel.22,23 Two lines of experimental evidence demonstrated that the current blockades are caused by the translocation of polynucleotide molecules through the pore. First, the lifetime of the polymer-induced channel current blockades is proportional to the polynucleotide’s length. Second, polymerase chain reaction experiments demonstrated that single-stranded, but not blunt-ended double-stranded DNA (i.e., without overhangs) traverses the pore. The current through a single RHL channel is large and quiescent (Figure 2, leftmost recording). Adding relatively short biotinylated single-stranded DNA (10-nucleotide-long poly(deoxyadenylic acid) biotinylated at the 5′-end; bT-poly(dA)10, Figure 2, top middle) or relatively long bT-poly(dA)50 (Figure 2, bottom middle) to the aqueous phase bathing one side of the channel causes transient decreases in the open channel current. Analyte Alters Polymer-Induced Nanopore Current Blockades. Avidin binds strongly to biotin. Subsequent addition of excess avidin (i.e., a concentration much greater than that of the bT-poly(dA)) causes the polymer-induced current blockades to disappear if the polynucleotide is relatively short (e.g., bT-poly(dA)10, Figure 2, top right). In contrast, excess avidin complexed to the longer bT-ssDNA occludes the channel for times that are 2270 Analytical Chemistry, Vol. 73, No. 10, May 15, 2001
much longer than occlusion by bT-poly(dA)50 (Figure 2, bottom right). Results qualitatively similar to both of these outcomes are obtained with relatively short and long strands of bT-poly(dC) (poly(deoxycytidilic acid)) (four trials; data not shown). Avidin25-27 itself does not cause single-channel current blockades, because it is too large to partition into the RHL channel’s pore 20,21,28 and remain there for a time comparable to or greater than the electronic detector’s bandwidth (i.e., the very short-lived channel blockades caused by the random collision of avidin with the pore’s entrance apparently occur on a time scale that is much faster than the current amplifiers’ temporal resolution). However, avidin alters the ability of biotinylated homopolymers of ssDNA to traverse the RHL channel (Figure 2) as suggested in the sensor models illustrated in Figure 1. Quantitative Analysis. The sensor systems described here can be used to quantitate analyte concentration and to characterize the type of analyte that binds to the polymer. The number of polynucleotide-induced current blockades per unit time is proportional to the free polymer concentration.23 Thus, it follows for a model I system (Figure 1, middle), analyte binding to free polymer will decrease the number of current transients per unit time. The concentration of bound polymer can be determined from the decrease in the time-averaged frequency of transient current blockades. By the principle of mass action, for a completely irreversible reaction between analyte and sensing polymer, the blockade rate should decrease linearly with analyte concentration. If the reaction is reversible, as the free polymer concentration approaches zero, the blockade rate will decrease asymptotically. Avidin reduces the frequency of current blockades caused by bT-poly(dA)10 in a graded manner (Figure 3). The results suggest that, under the conditions used here, the reaction between avidin and biotin attached to a short polynucleotide does not proceed with a 4:1 (biotin/avidin) stoichiometry. The initial slope of the blockade rate versus avidin concentration is ∼2-fold less steep than one would predict if all four binding sites per avidin molecule were equally accessible to biotin. Nevertheless, as is shown in Figure 3, a calibration determines empirically the stoichiometry between the analyte and polymer. With this information, the analyte concentration can easily be determined. For a model II detector, the analyte concentration is deduced from the mean time that it takes the nanopore to be first occluded by the analyte-polymer complex after the electric field is applied. This was verified experimentally by monitoring the kinetics of RHL channel occlusion by the complex of avidin-bT-poly(dA)50 (data not shown). Alternative Detector Schemes. The method described here could be used to detect any analyte that alters a polymer’s ability to partition into or completely traverse the pore. A model I sensor could also work in the reverse of the manner illustrated in Figure 1. For example, if an analyte converts a pore-impermeant polymer into a pore-permeant form, then the number of blockades per unit time will increase with analyte concentration, (i.e., exactly opposite (25) Pugliese, L.; Coda, A.; Malcovati, M.; Bolognesi, M. J. Mol. Biol. 1993, 231, 698-710. (26) Livnah, O.; Bayer, E. A.; Wilchek, M.; Sussman, J. L. Proc. Natl. Acad. Sci U.S.A. 1993, 90, 5076-5080. (27) Green, N. M. Adv. Protein Chem. 1975, 29, 85-133. (28) Song, L.; Hobaugh, M. R.; Shustak, C.; Cheley, S.; Bayley, H.; Gouaux, J. E. Science 1996, 274, 1859-1866.
Figure 3. Measuring analyte concentration by detecting the rate of blocking of single-channel ionic current by pore-permeant polymers. In the absence of polymer, a steady current flows through a single RHL channel (Figure 2, leftmost recording). Adding 400 nM singlestranded DNA (bT-poly(dA)10) to the cis side causes transient blockades of the current (Figure 3, upper left current recording). Increasing the analyte concentration (NeutrAvidin) decreases the frequency of channel blocking because the analyte binds to the biotinylated polymer and prevents it from entering the pore. The blocking rate is proportional to the unbound polymer concentration.23 The solid curve is a fit function and can be used as a calibration curve for the sensor. The resolution of an analyte concentration measurement by the sensor, e.g., at 120 nM, is just the inverse slope (∼1.5 nM/count for a 1-min average) multiplied by (1 count. This equals (1% of reading and is the minimum resolution for a 1-min average. Similarly the resolution is (5% at 20 nM and at 240 nM. This defines the analyte measurement range for 400 nM polymer. The range is changed by changing the polymer concentration. The resolution can be improved 10-fold by averaging for 10 min. The results shown here represent the mean and standard deviation for the blockade rates of five experiments on a single RHL channel. The error bars represent the standard deviation of four consecutive measurements performed on the same single channel at the same analyte concentration. Virtually identical results were observed for at least five out of five similarly prepared experiments with different membranes containing single RHL channels. The small tail in the blockade rate at high avidin concentration may be due to a relatively small amount (∼10%) of the single-stranded DNA not being biotinylated.
to that suggested in Figure 1, middle, and shown in Figure 3). Although not strictly analogous, a similar principle was demonstrated by the cleavage of RNA homopolymers (e.g., poly(U)) into more numerous and shorter polymers by ribonuclease A.22 A model II sensor could also be used to detect a wide variety of analytes if the applied potential across the pore is sufficiently low that the analyte-polymer complex is not rapidly torn asunder by the electrical force that drives the sensing polymer into the pore. In this case, the rate constants for the association and dissociation of an analyte with a recognition site on a polymer, kon and koff, could be directly measured because the potential holds the complex in the pore until dissociation of the analyte and sensing polymer occurs. Specifically, the pore will be partially occluded by the analyte-polymer complex for a mean time t ∼ 1/koff. This additional kinetic information may help reduce error caused by false positive signals. Multianalyte Detection. Because single-stranded DNA homopolymers thread through the RHL channel at ∼1-10 µs/base, and at relatively low concentration of short polymers, the channel is almost always polymer-free (Figure 2, top middle recording). Thus, the probability that two polymers would simultaneously
Figure 4. Transport of different DNA homopolymers through the RHL channel, Transport causes unique single-channel current blockade signatures that could be used to simultaneously detect multiple analytes with one pore (for a model I sensor). Transient blockades induced by 100-nucleotide-long homopolymers of poly(dT), poly(dC), or poly(dA) have markedly different lifetimes. In addition, poly(dT)induced blockades are clearly distinguishable from those caused by either poly(dA) or poly(dC) because of the pronounced double-step pattern. Homopolymers of poly(dG) were not studied because they are essentially insoluble, difficult to produce, and form more complicated structures than do homopolymers of dT, dC, and dA. The characteristic current blockade patterns and relative blockade lifetimes were observed in more than 10 different experiments for each polymer type. They were also observed for shorter lengths of each of these homopolymers.
occupy the pore is virtually nil. It follows that a model I nanopore sensor can accommodate a large number of polymer types that produce unique blockade patterns. For example, the transient blockades induced by identical length homopolymers of poly(dT) (poly(deoxythymidilic acid)), poly(dC), or poly(dA) are easily distinguished from each other on the basis of their distinctive current blockade patterns or different lifetimes (Figure 4). Thus, in the absence of analytes, a model I sensor with N different homopolymers (differing either by nucleotide type or contour length, or both), each designed to bind a specific analyte, will produce an ensemble of N unique current blockade patterns. Adding an analyte that binds to one of the polymers will cause a reduction in the number of transient blockades caused by its corresponding polymer, as was shown for one analyte and its corresponding polymer (Figure 2). The single-channel recordings in Figure 5 demonstrate proofof-concept for multianalyte detection with this nanopore sensor illustrated in Figure 1. Specifically, the results show that two different polymers (a relatively short strand of 5′-biotinylated-poly(dC) and a longer strand of 5′- bromodeoxyuridine-poly(dT)) with unique current blockade signatures and with unique analyte binding sites can be simultaneously detected. Moreover, the blockades caused by the shorter bT-poly(dC) polymer disappear after the addition of an excess concentration of the analyte that binds to it (i.e., avidin). Subsequent addition of a second analyte (i.e., an R-BRDU antibody) that binds to the longer polymer causes the second polymer to occlude the pore for time intervals that are much longer than the blockades caused by the polymer itself (i.e., model II behavior). Obviously, if more than two analytes are to be detected, an ensemble of short polymers that only exhibit model I behavior should be used. Although the signals caused by the polymers in Figures 4 and 5 are easy to differentiate by eye, a wide variety of statistical measures (e.g., mean, variance, mean lifetime, autocorrelation or Analytical Chemistry, Vol. 73, No. 10, May 15, 2001
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Figure 5. Simultaneous detection of two analytes with two different polymers and a single nanopore. The upper leftmost single-channel recording shows the step increase in current that flows through a single RHL ion channel in response to an applied potential equal to -120 mV in the absence of single-stranded DNA and analytes. Adding ∼400 nM bT-poly(dC)10 to the cis chamber causes transient current blockades. The leftmost expanded view shows one of the characteristic poly(dC)10 events. Subsequently adding ∼400 nM BRDU-poly(dT)50 (i.e., 5′-bromodeoxyuridine-poly(dT)50-3′) to the cis chamber increases the total number of blockades. The central expanded view shows that characteristic poly(dT)50 events, with the double-step pattern, are now clearly present in addition to the poly(dC)10 events. Adding ∼600 nM streptavidin eliminates virtually all the bT-poly(dC)10 events, shown by the reduction in event frequency and in the rightmost expanded view, which is expected for a model I sensor (Figure 1). Adding ∼240 nM R-BRDU polyclonal antibody (Fitzgerald Industries) causes single-channel current blockades that are much longer lived than those induced by BRDU-poly(dT)50, many remaining until the potential is reversed, as expected for a model II sensor (Figure 1).
spectral analysis12,13,20-24 of the current blockades, etc.) could be used to automatically distinguish current blockades caused by different types of polymers. One of the challenges is to design a large number of suitable and unique polymers that will enable combinatorial levels of analyte detection with the sensor systems described herein. SUMMARY We have demonstrated that a single nanoscale pore can provide the basis for a novel sensing scheme that locates analyte binding sites on pore-permeant polymers. This method offers several important advantages over detection schemes that affix the recognition sites directly to an ion channel. First, the two sensor mechanisms illustrated in Figure 1 are more flexible in the type and number of analytes that can be simultaneously measured (e.g., the specificity and/or concentration range of the sensor can be changed by replacing the polymer). Second, the analytes to be detected need not fit inside the pore or inside an even smaller molecular adapter,18 thus permitting the detection of large
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macromolecules. Third, the sensor can be used to simultaneously detect multiple analytes. Fourth, this method utilizes a single nanopore and detects individual molecules, which preserves the kinetic information of the reaction between the analytes and the sensing polymers. Finally, the method described here is not restricted to the use of biological channels. It will work with artificial nanopores, if they become available. ACKNOWLEDGMENT We thank Mr. Sean Lee for writing some of the computer programs used in the data acquisition. Supported in part by the NIST Advanced Technology Program and the National Academy of Sciences/National Research Council (J.J.K.).
Received for review August 14, 2000. Accepted March 1, 2001. AC000958C
