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Tissue Bloreactor for Elimlnatlng Interferences in Flow Analysis Joseph Wang' and Najih Naser Department of Chemistry, New Mexico State University, Las Cruces, New Mexico 88003
INTRODUCT10N Immobilized enzyme reactors are gaining considerable importance in flow analysis.1-6 Such reactors serve mainly to facilitate the analyte conversion into a detectable species. Indeed, of all the conversion methods used in flow injection analysis (FIA), biocatalytic reactors are by far the most common. Three types of biocatalytic reactors have thus been used the packed bed, open tubular and single string reactors. Such reactors often require high enzyme loadings and very stable preparations, and may suffer from the build-up of backpressure under continuous use.1'2 Significant amounts of time and resources are thus being invested for optimizing enzyme reactors for maximum efficiency. The present note describes the utility of tissue bioreactors for the elimination of potential interferences in FIA. Enzyme reactors have been used previously for the removal of interferences in connection with electrochemical monitoring of flow stream.6,' Tissues possess several advantages over their enzymatic counterparts, including improved stability, higher biocatalytic activity, and low cost. Such advantages have received considerable attention in connection with the preparation of electrochemical biosensors.8*9 However, little attention has been given to the use of plant tissues (in place of isolated enzymes) as reactor columns in flow analysis. A kidney reactor was employed in flow measurements of glutamine, with a downstream potentiometric detection of the generated ammonia.10 Unlike substrate measurements (for which low conversion efficiencies are sufficient), the destruction of interferences requires high enzymatic loadings, as provided by cellular materials. In particular, on-line degradation of surface-active proteins and electrooxidizable interferants (e.g. ascorbic acid and acetaminophen) is accomplished in the present work by exploiting the rich papain, ascorbic acid oxidase (AAO), and polyphenol oxidase activities of the papaya, zucchini, and potato tissues, respectively. The optimization and characterization of these low-cost and yet highly efficient tissue-eliminator bioreactors are reported in the following sections.
EXPERIMENTAL SECTION Apparatus. Amperometric measurements were performed with an EG&G PAR Model 264A voltammetric analyzer, the output of which was displayed on a X-Y-t recorder (Bioanalytical Systems (BAS), Model RXY). The flow injection system consisted of a 50-mLsyringe/carrier reservoir,held by the syringe pump (Model 341B, Sage), interconnecting Teflon tubing, a Rheodyne Model 5020 injection valve (20-pL loop), the tissue reactor, and a carbon paste thin-layer detector (Model TL-4, BAS). The Ag/AgCl reference electrodeand the counter electrode were located in a downstream compartment (ModelRC-2A,BAS). The tissue reactor consisted of a polyethylene cartridge (usually of 5.0-cm length and 1.0-cm i.d.), filled with the desired tissue. (1) Bowers, L. D. Anal. Chem. 1986,58, 513A. (2) Cliffe, S.; Filippini, C.; Schneider, M.; Fawer, M. Anal. Chim. Acta 1992,256, 53. (3) Yao, T.; Akasaka, R.; Wasa, T. Electroanalysis 1989, 1, 413. (4) Masoom, M.; Townshend, A. Anal. Chim.Acta 1986, 179, 399. (6)Almuiabed, A. M.; Townshend, A. Anal. Proceedings 1989,26,56. (6) Adams, R. N.; Bradberry, C. W. Anal. Chem. 1983,55, 2439. (7) Risinger, L.; Yang, X.; Johansson, G. Anal. Chim. Acta 1987,200, 313. (8) Rechnitz, G. A. Science 1981, 214,287. (9) Wang, J. Electroanalysis 1991, 3, 255. (10) Mascini, M.; Rechnitz, G. A. Anal. Chim. Acta 1980, 116, 169. 0003-2700/92/0364-2469$03.00/0
A central hole (usually of 1.0- or 2.0-mm diameter) was made, with a cork borer, in the tissue cylinder to obtain a desired open tubular reactor configuration. Luer fittings (to the connecting Teflon tubing) served for the solution inlet and outlet. Reagents. All solutions were prepared with doubly distilled water. The supporting electrolyte was 0.05 M phosphate buffer (pH 7.4). Acetaminophen, dopamine, casein, bovine albumin (Sigma),potassium ferrocyanide, and ascorbic acid (Baker)were used without further purification. The plants used in this study were purchased from a local grocery store. Procedure. Amperometric detection proceeded under flow injection conditions. The working potential (usually +0.60 V) was applied, and transient currents were allowed to decay to a steady-state value. All measurements were performed at room temperature.
RESULTS AND DISCUSSION The use of tissue reactors as effective matrix isolation tools in flow analysis offers the advantages of high activity and stability, self-supported rigidity, and extreme simplicity.Such unique application was examined and demonstrated in connection with amperometric measurementa a t a thin-layer detector. Passage of the samples through the biocatalytic reactors was used to alleviate two major interferences characteristic of amperometric monitoring, including biofouling and overlapping signals. For example, tissue reactors can circumvent protein passivation effects and hence impart high stability during amperometric detection. Such prevention of biofouling is illustrated in Figure 1 utilizing a papaya reactor. The papain enzyme, present in the papaya reactor, rapidly cleaves proteins into smaller peptide fragmentsl1J2 that do not passivate the detecting electrode. When the protein-rich ferrocyanide (A) and acetaminophen (B)solutions bypass the papaya reactor (top), a rapid decrease of the flow injection response of these analytes is observed (up to 68 and 6076,respectively). This passivation problem is eliminated by passing the samples through the papaya reactor (bottom). No loss of the amperometric response is observed throughout these prolonged series (RSD of 1.7 (A) and 1.2% (B)).Note also that the speed inherent to the flow injection operation is not compromised by the use of the tissue reactor (Le. sharp peaks with no dispersion). Ascorbic acid is an easily oxidizable endogenous compound which exists in relatively high concentrations in most biological fluids. Hence, amperometric assays of such fluids oftan suffer from a large ascorbic acid response which masks the peaks of interest. The high enzymatic (AAO) activity of the zucchini tissue13 can greatly facilitate the elimination of the ascorbic acid interference, in accordance to eq 1:
-
AAO
ascorbic acid + '/,02 dehydroascorbic acid + H,O (1) Figure 2 (bottom) demonstrates the interferant-removal efficiency obtained with the zucchini reactor. It shows amperometric peaks for a 1 X M ascorbic acid solution obtained with (B) and without (A)passage through the reactor. (11) Hill, R. In Hydrolysis of Proteins; Anfinsen, C. B., Anson, M. L., Edsall, J. T., Richard, F. M., Eds.; Advances in Protein Chemistry; Academic Press: New York, 1965; Vol. 20. (12) Wang, J.; Wu, L. H.; Martinez, S.;Sanchez, J. Anal. Chem. 1991, 63, 398. (13) Wang, J.; Naser, N.; Ozsoz, M. Anal. Chim. Acta 1990,234, 315.
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ANALYTICAL CHEMISTRY, VOL. 64, NO. 20, OCTOBER 15, 1992
1 Figure 1. Amperometric response to repetitive injections of 1 X lo-' M ferrocyanide (A) and 4 X lo4 M acetaminophen (B)solutions
contalnlng 500 ppm casein and albumln, respectively, without (a)and with (b) passage through the papaya reactor. Operating potential, +0.50 V; flow rate, 1.0 mL/min; carrier/electrolyte,phosphate buffer (pH 7.4). Reactor length and inner dlameter, 5 cm and 1 mm, respectively.
A
I
2
3
FLOW RATE /cm3min-l
TIME
I
TIME
I
TIME
Figure 2. Bottom: flow Injection amperometric peaks for 1 X M ascorblc acid, with (B)and without (A) passage through the zucchini M ascorbic acid solutions, reactor. Top: response to 1 X M (c),and 3 X M (b), 2 X containing 0 (a), 1 X M (d)
dopamine, wkh @)andwithout (A) passage throughthe zucchini reactor. Operating potential, +0.60 V; reactor length and inner diameter, 4 cm and 1 mm, respectively. Carrierlelectrolyte and flow rate, as in Fig. 1.
The short (4-cm) tissue reactor results in more than 90% diminution of the ascorbic acid response. The biocatalytic degradation capability is maintained throughout this prolonged series of 30 repetitive injections. Following this operation, the zucchini reactor was bypassed, and the resulting peaks were similar to those recorded in the beginning of the experiment. The bioanalytical utility of the ascorbic acid destruction process is also illustrated in Figure 2 (top),using the common interference of ascorbic acid on measurements of catecholamine neurotransmitters. This figure displays flow injection peaks for ascorbic acid solutions containing increasing level of dopamine. An additive response, that precludes the measurement of dopamine, is observed without passage through the plant reactor (A). In contrast, the 94% removal of ascorbic acid when the sample mixture is flowing through the reactor permits a nearly selective quantitation of dopamine
(B). Crucial to the successful use of a tissue-eliminator reactor is appropriate optimization of experimental conditions for maximum conversion efficiency (R, i.e., the fraction of substrate consumed). Figure 3A shows the dependence of R upon the volume flow rate for different reactor lengths. As the residence time of an element of solution within the zucchini
2
4
OMYeTER/mm
Flgure 9. Dependence of the degree of conversion upon
the volume
flow rate (A) and tube diameter (E). Injections of a 1 X Mascorbic acid solution. Zucchini reactor of 1-mm tube diameter (A) and 3-cm length (B); flow rate (B), 1.OmLlmin. Carrierleiectrolyte and potential,
as in Flgure 2.
reactor increases (i.e. slower flow rates or longer reactors) the conversion efficiency increases. For the 1-5-cm-long reactors, at 0.33-3.0 mL/min rates, the conversion efficiency ranges between 0.76 and 0.99. The attainment of such high efficiencies (at moderate flow rates or reactor lengths) is attributed to the rich AAO activity of the tissue. (It should be pointed out that the use of the pure enzyme for the same task would be extremelyexpensive,as 1mg of AAO, containing only 700 units, costs $70!). The inner diameter of the opentubular reactor is another parameter affecting its efficiency. Figure 3B shows the dependence of R upon the diameter for a 3-cm-long reactor. R increases (from 0.88 to 0.94) by increasing the reactor diameter between 1and 5 mm. Note also the broadeningof the FIA peaks (Le. increased dispersion) associated with the increasing diameter. Hence, longer (5 cm) reactors of 1-mm diameter were employed for achieving effective interferant elimination while maintaining high sample frequencies. The high biocatalytic conversion efficiencies are maintained over relatively long periods. No apparent mechanical stability problems were observed with "soft* tissues, e.g. zucchini. Indeed, the same zucchini reactor was operated for a period of 10 days, with no apparent loss in its efficiency (as was indicated daily from the effective removal of 1 X 10-6 M ascorbic acid). Such performanceis attributed to the inherent stability of the enzyme within its own natural environment. Considering the broad availability and extremely low cost of plant tissues, they can be easily replaced when needed. The reactor-to-reactor reproducibility was estimated by using six different sections of the zucchini plant (conditions 88 in Figure 2). The mean conversion efficiency found was 0.91, with a range of 0.89-0.93 and a relative standard deviation of 2.6 7%. Effective elimination of phenolic interferants can be accomplished utilizing a potato reactor, which is rich with polyphenol oxidase. In particular, the common analgesic acetaminophen (paracetamol) is easily oxidized and often interferes in amperometricmeasurementsin the anodicregion. As illustrated in Figure 4 (bottom),passage of acetaminophen solutions through the potato reactor results in 92% diminutions of the drug response. The potato polyphenoloxidase can facilitate also the biodegradation of catecholamines;ca. 90% decrease of the response for dopamine is indicated from the flow injection peaks displayed in Figure 4 (top). Obviously, the detection of numerous nonphenolic compounds shouldbenefit from such destruction of phenolic interferences. In conclusion, it has been shown that tissue reactors can be used for the removal of interfering substances, hence imparting high stability and selectivity on flow amperometry. High conversion efficiencies are coupled to extremesimplicity
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with amperometricdetection for flow injection eystems,tissue reactors may benefit other analytical flow systems and detection modes. Additional advantages may be gained by couplingseveraltissue reactors (for the simultaneous removal of various interferants), or by employing flow reversal schemes (for quantitative conversion with shorter columns). The conversion efficiency should be adjusted to meet the requirement of each sample (with 100% conversions used for a large excessof the interfering species). In addition, periodic calibrations should be employed to detect possible losses in the efficiency. Besides their great analytical utility, tissue reactors may find important environmental applications, i.e., low-cost cleanup of water streams from pollutants (such as phenols or peroxides).
ACKNOWLEDGMENT Fbwr 4. Fbw Injectionamperometrlc peaks for 1 X M dopamine (top) and 1 X lo4 M acetamlnophen (bottom), with (B) and wlthout (A) passage through the potato reactor ( k m length, l-mm tube dlameter). Other condltbns, as in Flgure 2.
and low cost (compared to analogous applications of immobilized enzyme reactors). The diversity of cellular materials holds great promise for the removal of other target interferants. While the concept has been illustrated in connection
This work was supported by the US. Environmental Protection Agency (Grant No CR-817936-010). Mention of trade names or commercial products does not constitute endorsement or recommendation by the US. EPA.
RECEIVED for review April 30, 1992. Accepted July 20, 1992.
