Anal. Chem. 1995, 67,4065-4070
Trace Measurements of RNA by Potentiometric Stripping Analysis at Carbon Paste Electrodes Joseph Wang,* Xiaohua Cai, Jianyan Wang, and Colleen Jonsson
Department of Chemistry and Biochemisfy, New Mexico State Universify, Las Cruces, New Mexico 88003 Emil Palei5ek
Institute of Biophysics, Academy of Sciences of the Czech Republic, 612 65 Bmo, Czech Republic
Remarkably low levels of ribonucleic acid (RNA) can be measured by coupling its adsorptive accumulation onto carbon paste electrodes with constant current potentiometric stripping analysis (PSA). The computerized PSA operation effectively addresses the high background response inherent to carbon surfaces, while the anodic pretreatment of the electrode greatly enhances the preconcentration aciency. The detection limit for tRNA (10 pg, 4 x mol) is substantially lower than that reported recently (PalaCek, E.; Fojta, M. Anal. Chem. 1994, 66, 1566) for analogous voltammetric measurements at mercury surfaces. Variables influencing the accumulation and stripping processes of RNA are explored and optimized. Results are reported for measurements of RNA in the presence of excess dsDNA, for PSA of synthetic polyribonucleotides,for hybridization of complementaty strands of synthetic polyribonucleotides, for enzymatic hydrolysis of RNA, and for flow injection operation. Such solid electrode experiments obviate the need for mercury electrodes or a deoxygenation step and open the door for modern RNA detectors and probes. Ribonucleic acid (RNA) is a biopolymeric constituent of the cell, which can be translated into protein sequences (in the case of "A), function as structural molecules (in the case of tRNA and rRNA), or serve as a biocatalyst during gene expression. Consequently, there is considerable interest in determining low levels of different kinds of RNA and in measuring RNA in the presence of DNA. Absorption 0spectroscopy is commonly used for measuring RNA and DNA in nucleic acid samples.' Yet, the optical procedure cannot differentiate between RNA and DNA, and its detection limit is -0.1 mg/L. Separation techniques, such as liquid chromatography? gel electrophoresis,3and capillary zone electrophre~is,~ are more often used for measuring RNA and RNA fragments. Relatively few studies have been devoted to electroanalysis of RNA, as compared to numerous ones dealing with DNA5-* These include polar~graphic,~ cyclic voltammetric,1° capacitance," and (1) Holden, M.; Pine, N. Biochim. Biophys. Acta 1955,16,317. (2) Preston, M. R. /. Chromatogr. 1983,275,178. (3) Chan, IC; Koutny, L.: Yeung, E. S. Anal. Chem. 1991,63,746. (4) Huang, X.; Shear, J.: Zare, R Anal. Chem. 1990,62,2049. (5) Palecek, E. Bioelectrochem. Bioeneg. 1986,15,275. (6)Palecek, E. Bioelectrochem. Bioeneg. 1988,170,421. (7) Palecek. E. In Topics in Bioelectrochemistry and Bioenergetics; Millazo, G . , Ed.: Wiley: Chichester, 1983; Vol. 5, p 65.
0003-2700/95/0367-4065$9.00/0 0 1995 American Chemical Society
adsorptive stripping'2 measurements. These schemes have commonly relied on the cathodic redox and interfacial processes of RNA at mercury drop electrodes. Solid electrode experiments, which may open the door to modern RNA probes and detectors, have not been intensively explored due to large background current contributions at these surfaces. This article describes an effective solid electrode protocol, based on potentiometric stripping analysis (PSA),for measuring low levels of RNA. PSA,originally developed for monitoring trace m e t a l ~ , ' ~couples J~ the effective preconcentration step (inherent to stripping analysis) with monitoring of the potential of the working electrode (as a function of time) during the stripping step. The time required for stripping the accumulated analyte is thus proportional to its solution concentration. While this relatively new stripping technique has been widely used for trace metal analysis,15it has not been applied for the quantitation or study of nucleic acids. In the following, we will illustrate that the effective accumulation of tRNA onto carbon paste electrodes can be followed by passage of a constant (anodic) current to yield a welldefked PSA peak over a nearly flat baseline. Picogram quantities of RNA can thus be conveniently detected without the need for a mercury surface or an oxygen removal step. Such coupling of solid-state sensors, simplilied operation, picogram detection limits, and microliter volumes is shown below to offer new opportunities for nucleic acids measurements and research. EXPERIMENTAL SECTION Apparatus. The TraceLab potentiometric stripping unit (psU20, Radiometer, Denmark), was used in connection with an IBM PS/2 55SX computer. In accordance with the TraceLab protocol, the potentials were sampled at a frequency of 30 kHz, the derivative signal (dt/dE) was plotted against the potential, and the peak area (following baseline fitting) served as the analytical signal. Voltammetric experiments were performed with a BAS 1OOA electrochemical analyzer. Most experiments were carried out in a BAS VC-2 cell, containing a 1.0 mL solution. The carbon paste working electrode, Ag/AgCl reference electrode model RE(8) Brett, C. M. A.; Brett, A M.; Serrano, S. /. Electroanal. Chem. 1994,366, 225. (9) Reynaud, J. Bioelectrochem. Bioeneq. 1976,3, 561. (10) Fojta, M.; Teijeiro, C.; Palecek, E. Bioelectrochem. Bioeneg. 1994,34,69. (11) Palefek, E.; Doskocil, J. Anal. Biochem. 1974,60,518. (12) Palefek, E.; Fojta, M. Anal. Chem. 1994,66,1566. (13) Jagner, D. Anal. Chem. 1979,51,342. (14) Jagner, D. Trends Anal. Chem. 1983,2 (3), 53. (15) Wang, J. Analytical Electrochemistry; VCH Publishers: New York, 1994.
Analytical Chemistry, Vol. 67, No. 22, November 15, 1995 4065
1, BAS), and platinum wire auxiliary electrode joined the cell through holes in its Teflon cover. The carbon paste [made of 70/30 w/w graphite powder (Acheson 38, Fisher)/mineral oil (Sigma; free of DNase, RNase or protease)] was housed in a Teflon body to give a 3.5mmdiameter disk surface. Electrical contact to its inner side was made with a stainless-steel screw. Small (microliter) volume experiments were performed with a twoelectrode system, involving a 06"-diameter carbon paste electrode (housed in a micropipet tip) and a Ag/AgCl wire (placed/coiled on the working electrode tip body). A copper wire served as a contact to the microcarbon paste electrode. The flow injection system consisted of a carrier reservoir, a micropump (BAS Model 1001), an injection valve (Rainin Model 5041) with a 2o;UL sample loop, interconnecting Teflon tubing, and a thin-layer carbon paste detector (BAS Model TLA). All glassware, containers, and the cell (with the exception of the electrodes) were sterilized by autoclaving for 30 min. The electrodes were thoroughly rinsed with sterilized water prior to use. The W absorption of the RNA was measured with a diode array spectrophotometer (Model 8452A, Hewlett Packard). T e m perature control (during the RNase experiment) was achieved with a digital temperature controller (Model 9101, Fisher). Reagents. The following chemicals were obtained from S i a and were used as received transfer RNA (tRNA, from bakers' yeast, lyophilized powder; Catalog No. R8759), doublestranded calf thymus DNA (dsDNA, activated and lyophilized; Catalog No. D4522), polyguanylic acid (poly(G), potassium salt; Catalog No. P4404), polyuridylic acid ( p o l y o , potassium salt; Catalog No. P9528), polycytidylic acid (poly(C), potassium salt; Catalog No. P4903), polyadenylic acid (poly(A), potassium salt; Catalog No. P9403), ribonuclease (RNase, EC 3.1.27.5, Type X-A, from bovine pancreas), and diethyl pyrocarbonate OEPC). The RNase stock solution was prepared with 10 mM Tris buffer, pH 7.16 Total RNA from human lung tissue was prepared using RNAs01 (precipitated and suspended in DEPC-treated water). All aqueous media used for preparing the RNA solutions were treated with 0.1%w/w DEPC for 12 h at 37 "C and were then autoclaved for 30 min,16while all other solutions were prepared with sterile doubly distilled water. A 1000 mg/L RNA solution was diluted before use, as needed for the speci6c experiment. The RNA and DNA concentrations were vedied by W measurements at 260 nm. A 0.2 M acetate buffer solution (PH5.0) served as supporting electrolyte. Procedure. The smoothed carbon paste surface was pretreated prior to each measurement by applying a potential of +1.7 V for 60 s, using the electrolyte solution. The accumulation of RNA proceeded from a stirred solution for different times (depending on its level), using a potential of +0.5 V. After completion of the accumulation, the potentiostat was disconnected, and the preconcentrated RNA was oxidized by applying a constant oxidizing current (usually 4 PA). Stripping voltammetric experiments were carried out using a similar accumulation step, followed by a 5s rest period, and a positivegoing squarewave potential scan. For small-volume experiments, the combined working/ reference electrode assembly was immersed in a drop (5-100 pL) of the RNA solution on a parafilm. The solution was vibrated to facilitate the accumulation (by using a stirrer to vibrate the plastic support). Details of the flow injection operation are given (16) Maniatis, E.; Sambrook, J. Molecular Cloning: A Laboratoy Manual; Cold Springs Harbor Laboratory: New York, 1982; p 190.
4066 Analytical Chemistty, Vol. 67, No. 22, November 15, 1995
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POTENTIAL (V) Figure I.Repetitive cyclic voltammograms for 2 mg/L tRNA at the untreated (a) and pretreated (b) carbon paste electrodes, after a 2-min stirring at +0.1 V. Scan rate, 100 mV/s; electrolyte, acetate buffer (pH 5.0; 0.2 M, 1.O mL). Pretreatment (b) for 60 s at +1.7 V.
below. All accumulation and stripping steps were carried out at room temperature (23 f 0.5 OC). RESULTS AND DISCUSSION The anodic pretreatment of carbon paste greatly enhances the adsorptive accumulation of RNA. Figure 1 displays repetitive cyclic voltammograms for 2 mg/L tRNA at the untreated (a) and pretreated (b) carbon paste electrodes after 2 mins stirring at +O. 1 V. A very small anodic peak (Ep= f1.02 V) is observed at the untreated surface. In contrast, this peak increases substantially (> 15fold) after the electrode is activated. No peaks are observed in the cathodic branch. Subsequent scans exhibit substantially smaller peaks, corresponding to the response of the solution-phase RNA (and hence indicating rapid desorption of the product). Similar small peaks were observed at the treated electrodes without prior accumulation (not shown). Notice also the substantially larger background current contribution and envelope following the surface activation. According to Adams and cow o r k e r ~ the , ~ ~electrochemical pretreatment produces a more hydrophilic surface state and a concomitant removal of organic layers. Such a change in the surface state appears to facilitate the interfacial accumulation of RNA The similar oxidation peak potentials (before and after the treatment) indicate that the treated surface has no electrocatalytic activity. Of the nucleic acid bases, only guanine and adenine can be oxidized at carbon e l e c t r o d e ~ . ~Our ~ J ~experimental data, and those of others,16suggest that the cyclic voltammetric anodic peak of RNA corresponds to the oxidation of the guanine residue. For example, similar cyclic voltammetric profiles were observed for analogous experiments using the synthetic ribonucleotide poly (G), which contains only guanine residues (not shown). In addition, guanine and adenine displayed defined oxidation peaks at f0.9 and +1.2 V, respectively. The oxidation of these monomeric bases commonly occurs at potentials -0.2 V lower that those of the bases bound in the polynucleotide.ls In view of the large solvent decomposition current, it is more dficult to evaluate the signal of the bound adenine residue [at the solution pH (5) examined]. (17) Rice, M.; Galus, Z.; Adams, R N. J. Electroanal. Chem. 1983,143, 89. (18) Brabec, V.; Koudelka, J. Bioekcfrochem. Bioezeq. 1980,7, 793. (19) Kafil, J.; Cheng, H. Y.; Last, T. Anal. Chem. 1986,58, 285.
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Effect of accumulation potential (A) and stripping current (6)on the PSA response for 1 mg/L tRNA. Accumulation for 30 s; stripping current, 4 PA. Other conditions as in Figure 2B. Figure 3.
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Square-wave voltammetric (A) and PSA (B) peaks for 1 mg/L tRNA following different accumulation times: 2 (a), 30 (b), 60 (c),120 (d), and 240 (e) s. Precocentration potential, 0.5 V; stripping current (B), 8 PA; amplitude and frequency (A), 10 mV and 40 Hz, respectively;stirring rate (during accumulation),400 rpm. Electrolyte and pretreatment, as in Figure 1. (C) displays the resulting timedependent plots. Figure 2.
The adsorption of RNA onto the treated carbon paste electrodes can be used as an effective preconcentration step prior to the measurement of the surface species. In this way, highly sensitive measurements of RNA can be achieved by means of adsorptive stripping analysis. Figure 2 displays the voltammetric (A) and potentiometric @) stripping responses for 1mg/L tRNA for increasing accumulation periods [ranging from 2 (a) to 240 (e) SI. The squarewave voltammetric response, while increasing with the preconcentration time, is poorly defined and superimposed on a rising background current. In contrast, a sharper and well-defined response, coupled with a nearly flat background, is observed in the PSA operation. Convenient measurements are thus feasible following very short accumulation periods. Yet, the longer the time, the more RNA is adsorbed, and the larger the peak area is observed. As indicated also from the resulting peak area versus accumulation time plot, the peak rises rapidly at first and then more slowly. For a 60-s accumulation (curve c), the response is about 15 times that attained with a 2-s accumulation (curve a). Overall, the data of Figure 2 clearly demonstrate the advantage of the potentiometric stripping mode over its voltammetric counterpart for trace measurements of RNA. As will be illustrated later, such sensitivity advantage becomes even more pronounced for monitoring lower (ultratrace) levels of RNA. The stripping step, coupled with the subsequent conditioning one, result in a complete desorption of the accumulated RNA. Hence, a single carbon paste surface can be used repetitively without affecting the precision (see data below). Various procedure parameters, such as the accumulation potential or stripping current, have a profound effect upon the
PSA response for RNA (Figure 3). For example, while the peak area is only slightly affected by increasing the accumulation potential between 0.0 and 0.6 V, it decreases rapidly at higher potentials, approaching the peak potential (8. The RNA response decreases sharply upon raising the stripping current between 2 and 10 pA, and then decays more slowly (B). Such larger time signals for small stripping currents reflect the reduced oxidation rates. A large background noise accompanied the peaks for stripping currents smaller than 4 pA. A potential of f0.5 V and a current of 4 pA were thus selected for most subsequent quantitative work. The solution pH can affect the peak area and potential. For example, the response increased rapidly upon increasing the pH from 4 to 5, and decreased gradually above 5.3, e.g., to 60%and 40% of its maximum value at pH 7.4 and 9, respectively. The peak potential decreased linearly, from $1.07 to +0.82 V, upon raising the pH from 4 to 9 (not shown; BrittonRobinson buffer solutions; 5 mg/L tRNA and 3@saccumulation). An acetate buffer solution (PH 5.0) was used in all subsequent work, as it yielded the most favorable background response (compared to sodium chloride or phosphate buffer solutions). Carbon pastes containing 70% w/w graphite yielded the most favorable signal-to-backgroundcharacteristics . With lower graphite contents, it was difficult to resolve the RNA response from the background one, while higher ones (>70%w/w) were not suitable for the paste binding. We also assessed the effect of the pretreatment time and potential and found that 60 s at +1.7 V yields the most favorable conditions (not shown), Similar signals were observed by carrying out the pretreatment in the presence and absence of the target RNA. Such in situ pretreatment capability (in the presence of the analyte) greatly benefits the practical utility of the sensor. The bioanalytical utility is based on the correlation between the stripping response and the RNA concentration. A series of five concentration increments, from 0.5 to 2.5 mg/L tRNA, was used to evaluate the linearity. Figure 4A displays potentiograms for this series following a 30-s accumulation. The well-defined Analytical Chemistty, Vol. 67,No. 22,November 15, 1995
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Figure 4. (A) Potentiograms obtained for solutions of increasing tRNA concentrations from 0.5 to 2.5 mg/L tRNA (b-f), along with that for the blank solution (a). Also shown (6)are the resulting calibration (a) and another plot over the range 50-500 pg/L (b). Accumulation for 0.5 (a) and 2 (b) min; stripping current, 4 pA; other conditions as in Figure 26.
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POTENTIAL (V) Figure 5. Voltammetric (A) and potentiometric (6,C) stripping response curves for 1Opg/L (trace b in panels A, 6)and 5 pg/L (trace b in panel C) tRNA, along with the corresponding background response (traces a). Preconcentration for 5 (A, 6)and 10 (C) min; stripping current, 3 (6)and 0.3 (C) pA; solution volume, 1 mL (A, 6) and 5 pL (C). Other conditions as in Figure 2.
peaks allow convenient quantitation of these concentrations. The resulting calibration plot, shown in Figure 4B,graph a, is highly linear (correlation coefficient, 0.991). A longer (2-min) accumulation period was used in connection with smaller concentration increments over the 50-500 pg/L range. As shown in Figure 4B, graph b, this experiment also yielded a linear calibration plot (correlation coefficient, 0.998). The coupling of the efficient adsorptive accumulation and a microprocessor-controlled potentiometric stripping results in extremely low detection limits. Figure 5 (parts A and B, trace b) displays the voltammetric and potentiometric stripping responses for a 10 pg/L tRNA solution following a 5min accumulation. No response is observed for the square-wave voltammetric stripping operation. In contrast, a well-defined peak is obtained in the PSA experiment. The limit of detection-calculated from 3 times the noise (of Figure 5B, trace b)-was found to be 3 pg/L, or 3 ng in the 1-mL solution used. The corresponding value for the squarewave voltammetric operation is about 150 pg/L, Le., 50 times 4068
Analytical Chemistry, Vol. 67, No. 22, November 15, 7995
higher (not shown). Substantially lower PSA mass detection limits can be achieved by decreasing the volume requirement. Such a decrease (to the microliter domain) is also important, in view of the volumes employed in analogous electrophoretic measurements of RNA, Figure 5C displays the PSA response for 5 pg/L tRNA in a 5pL droplet. The sample was vibrated to facilitate the preconcentration. A sharp peak (over a flat baseline) is observed following a 10-min accumulation. A detection limit of around 2 pg/L can be estimated on the basis of the signal-to-noise characteristics of these data. This means that 10 pg of tRNA (Le., -4 x 10-l6 mol based on an average molecular weight of 2.6 x 104) can be detected in the 5pL solution used. Such a remarkably low detection limit compares favorably with that (100 pg) reported recently for analogous stripping voltammetric measurements at a hanging mercury drop electrode.12 A substantially higher detection limit, 1 mg/L, was estimated for analogous PSA measurements of total RNA which consists primarily of rRNA (10. min accumulation; not shown). Apparently, the preconcentration efficiency is higher for smaller-size RNAs. Repetitive measurements using 5 p L volumes require a new sample drop for each accumulation/stripping cycle (due to partial evaporation of the sample), yet such repetitive runs yielded reproducible results. Larger drops (100 pL) allow repetitive runs in the same solution. For example, a series of 10 repetitive measurements of 0.1 mg/L tRNA (in a 100-pL sample) resulted in a mean peak area of 7.8 ms and a relative standard deviation (RSD)of 8.0%(2-min accumulation and 0.3-pA stripping current; not shown). Better precision (RSD of 3.1%)was obtained for 10 repetitive measurements of 1 mg/L tRNA in a stirred 1-mL solution (1-min accumulation). Low molecular weight interferences that may be present in RNA samples can be eliminated by transferring the electrode (with the accumulated nucleic acid) to a blank electrolyte medium.Iz Such an adsorptive/transfer stripping protocol has been traditionally performed manually, by rinsing the electrode (after the accumulation) and dipping it in the blank solution.12 Our data indicate that the RNA-modified carbon paste electrode gives similar signals in the sample and blank solutions. We also developed a more elegant and simplified protocol for adsorptive/ transfer stripping experiments based on the medium-exchange character of flow injection systems.*O In flow injection analysis PIA),the preconcentration period can be started as the sample plug arrives in the detector and terminates after its passage through. The actual potentiometric measurement of the accumulated RNA can thus be performed after the arrival of the carrier blank solution. Figure 6 (traces b-d) displays the flow injection PSA response for repetitive injections of 20-pL samples containing 5 mg/L (Le., 100 ng) tRNA. Well-defined and reproducible peaks are observed, along with a favorable background response (potentiogram a). A flow rate of 125 pL/min and an accumulation time of 150 s were employed to ensure an effective accumulation while performing the stripping in the presence of the carrier solution (based on the dispersion profile/residence time of the sample plug). Such a FWPSA operation thus results in an injection rate of about 20 samples/h and a reproducible delivery of microliter RNA samples to the carbon paste detector. Compared to earlier batch operations, the FWPSA protocol should also facilitate the automation of RNA assays, as desired (20) Wang, J.; Freiha, B. Anal. Chem. 1983,55. 1285.
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POTENTIAL (mV) Figure 8. Adsorptive/transfer PSA hybridizationexperiment.Traces a: Response of poly(A)- and poly(G)-modifiedelectrodes in the blank electrolyte (panels A and B, respectively). Traces b: As in traces a but after transfering the modified electrodes to electrolyte solutions containing 20 mg/L poly(U) or 2 mg/L poly(C) (panels A and B, respectively). Conditions: accumulation for 1 min using electrolyte solutions containing 10 mg/L poly(A) or 1 mg/L poly(G) (A and B, respectively). Stripping started after 10 s-stirring at f 0 . 5 V, using currents of 12 (A) and 8 (B) FA; electrolytes, (A) 0.1 M BrittonRobinson buffer, pH 7; (6) acetate buffer, pH 5.
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for routine bioanalytical work. Batch PSA-transfer experiments may be more useful for fundamental studies aimed at elucidating structural transitions of RNA or its interactions with various agents. Measurements of low RNA levels in the presence of DNA is a challenging and important task.1°J2 In view of the high sensitivity of the present procedure toward tRNA (as compared to analogous measurements of dsDNA), the latter has a very small effect upon the RNA response of interest. This was examined in measurements of lmg/L tRNA in the presence of increasing levels of dsDNA (over the 0.5-20 mg/L range). Only %, 13%,17%,and 20%increases in the RNA signal were observed in the presence of 2-, 3-, l@,and 2@fold excess, respectively, of dsDNA (not shown).
The presence of RNase in the sample matrix does not interfere in the measurement of RNA This allows use of the PSA procedure for monitoring the progress of enzymatic hydrolysis reactions. For example, the time dependence of such reactions was investigated by analyzing the tRNMRNase mixture at different times intervals (1 mg/L tRNA, 0.5 mg/L RNase, 37 "C; not shown). The PSA peak increased rapidly (up to 45%of its original value) within the first 10 min and then started to level off (for samples taken over the entire 60 min experiment). A similar profile, but with a 110%signal enhancement, was observed using a 1.0 mg/L RNase solution. Analogous cyclic voltammetric/ hydrolysis experimentsat the mercury electrodeyielded a similar temporal profile, but with a decrease (rather than increase) of the RNA response.10 This difference may be due to diflerent conditions of the RNA hydrolysis that result in different chain lengths of the RNA cleavage products. Figure 7 displays the PSA response for the synthetic polyribonucleotide poly(G). As expected from its high guanine content, poly(G) yields a well-defined PSA peak (at a potential similar to that of RNA) . This peak increases rapidly upon increasing the accumulation time (A) or the poly(G) concentration (B). Convenient quantitation of low milligrams per liter levels of the synthetic polyribonucleotide can thus be accomplished following a 3@saccumulation. As expected, no response was observed for analogous measurements of poly(U) or poly(C). Similar measurements of poly(A) yielded no response at pH 5 (due to the large background) but a defined peak at pH 7 (associated with the lowered oxidation potential). Batch adsorptive/transfer PSA experiments are particularly attractive for following hybridization reactions between complementary strands of polyribonucleotides. For example, in Figure Analytical Chemistry, Vol. 67, No. 22, November 15, 1995
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8 traces a are potentiograms obtained after transfering the poly(A)- and poly(G)-modiiied electrodes to the background electrolyte solution (panels A and B, respectively). Well-defined and stable peaks are observed for the polyribonucleotide-modifedelectrodes in the blank solution. Substantially smaller (65% and 35%) and yet stable peaks are observed at these electrodes upon repeating the experiment in the presence of the complementary strand [polyor poly(C)] in the exchange solution (traces b, panels A and B, respectively). The decrease of the signal in the presence of the complementary strand was proportional to its concentration in the exchange solution, as was indicated for different levels of this strand (not shown). Such dependence holds great promise for the design of sequence-selective sensors. In conclusion, we have demonstrated that PSA can be used for measuring ultratrace levels of RNA following its adsorptive accumulation onto carbon paste electrodes. Such PSA operation effectively addresses the large background contribution inherent to carbon electrodes. The new adsorptive PSA procedure offers substantial lowering of the detection limit, not only compared to analogous voltammetric measurements but also in comparison to stripping voltammetry at mercury electrodes. Such application of solid electrodes, and the elimination of the deoxygenation step,
4070 Analytical Chemistty, Vol. 67, No. 22, November 15, 1995
may open the door to new RNA probes and detectors. For example, the RNA-modified carbon paste electrode can be used for investigating structural transitions and interactions of RNA. Pretreated carbon paste or fiber electrodes may be useful for amperometric detection for liquid chromatography or capillary zone electrophoresis. The new FIA operation may also facilitate the assay and study of nucleic acids. Similar results for singleor double-stranded DNA will be reported elsewhere in the near future. The new coupling of solid-state sensors, picogram detection limits, microliter volumes, and simplified operation should expand the role of electroanalysis in nucleic acids research. ACKNOWLEDGMENT
The technical assistance of L. Partin, M. Balakrishnan, and L. Chen is greatly appreciated. Received for review May 30, 1995. Accepted July 25, 1995.a AC950520Q Abstract published in Aduance ACS Abstracts, October 15, 1995.
