Anal. Chem. 1990,62,755-759 (3) Prabhu. S. V.; Baldwin, R. P. J . Chromatogr., in press. (4) Miller, E. J . €/ecffochem. Soc. 1969, 776, 1675-1680. (5) AW El Haleem, S. M.; Ateya, E. G. J . Electroanel. Chem. Interfacial Electrochem. 1981, 777, 309-319. (6) Pyun, C.-H.; Park, S A . J . Electrochem. SOC. 1986, 733, 2024-2030. (7) Drogowska, M.; Brossard, L.; Menard, H. Corrosion (Houston) 1987, 43, 549-552. (8) The Merck Index, 9th ed.; Merck & Co.: Rahway, NJ, 1976; pp 345-347. (9) Van Effen, R. M.; Evans, D. H. J . ElecffoamI. Chem. Interfacia/€/ectrochem. 1978, 703, 383-399.
755
(10) Watanabe, N.; Inoue, M. Anal. Chem. 1983. 55, 1016-1019. (11) Kok, W. Th.; Hanekamp, H. E.; Bos, P.; Frel, R. W. Anel. Chim. Acte 1982, 742, 31-45. (12) Kok, W. Th.; Brinkman, V. A. Th.; Frei, R. W. J . Chromatogr. 1983, 256, 17-26.
RECEIVED for review November 13, 1989. Accepted January 8, 1990. This work was supported by the National Science Foundation through EPSCoR Grant 86-10671-01 and by the University of Louisville College of Arts and Sciences.
Fiber-optic Glucose Sensor with Electrochemical Generation of Indicator Reagent Hari Gunasingham,* Chin-Huat Tan, and Jimmy K. L. Seow Department of Chemistry, National University of Singapore, Kent Ridge, Singapore 0511
An electrochemically generated tetrathiafuivaiene redox m e diator provides a convenient and reversible indicator reagent for a glucose fiber-optic sensor. The fabrication and characterization of this sensor based on a thin-layer cell conflguration are described. The performance of the fiber-optic sensor is compared with that of an amperometric enzyme electrode in flow-Injection and steady-state analyses. The upper limit of the linear range in the flow-injection mode is about 12 mM, whereas in the steady-state mode it is about 3 mM. The sensor Is less prone to interfering species and dissolved oxygen. It can be used continuously for 2 days without signHicant loss of activity.
INTRODUCTION Although the efficacy of fiber-optic sensors has been demonstrated in many analytical applications (1-3),they do suffer from a number of practical limitations that have prevented their more widespread use. One significant limitation is the difficulty of finding suitable, long-lasting, and reversible indicator reagents. Most colorimetric and fluorometric reagent systems are inherently irreversible because the analytical reaction invariably involves the formation of a tightly bound complex or an irreversible adduct. The requirement of a suitable reversible reagent systems is a particular limitation when developing enzyme-based fiber-optic sensors. Consequently there have been only a few practical examples. There have been essentially three approaches to the development of such sensors: (i) The fiber-optic sensor directly measures enzymatic generation of a spectrophotometrically detectable product (4, 5). (ii) The product of the enzymatic reaction is detected through a secondary reaction resulting in a change in the optical properties of the indicator reagent. For example, a fiber-optic sensor for glucose has been developed where the principle of measurement is the change in the fluorescence of an oxygen-sensitive dye where fluorescence is quenched by oxygen (6). Another example is also a sensor for glucose determination based on chemiluminescence generated by reaction of peroxyoxalate with hydrogen peroxide formed in the enzymatic reaction (7). 0003-2700/90/0362-0755$02.50/0
(iii) A dehydrogenase enzyme is immobilized a t the end of a fiber-optic probe with fluorometric detection of consumed. or generated nicotinamide adenine dinucleotide for measurement of lactate or pyruvate (8). Here a fresh supply of nicotinamide adenine dinucleotide has to be provided in the external sample solution. Another route to developing an enzyme-based fiber-optic sensor that has not been hitherto explored is to make use of a redox mediator dye as the indicator reagent. Such dyes have been well developed in conventional enzymatic analysis (9) and dry chemistry systems (IO). The fundamental problem in regard to their application to fiber-optic sensors, however, is the fact that most redox dyes are irreversible. Consequently, it is necessary to provide a large reagent reservoir or provide a means of renewing the reagent. The subject of this paper concerns the fabrication and characterization of a reversible enzyme-based fiber-optic sensor for glucose. Reversibility is afforded through the electrochemical regeneration of an optically active redox mediator. In this work, use is made of tetrathiafulvalene, which is a good redox mediator for glucose oxidase and also possesses desirable optical properties (11). The usefulness of the glucose fiberoptic sensor is demonstrated in steady-state and flow-injection analysis.
EXPERIMENTAL SECTION Chemicals and Solutions. Tetrathiafulvalene (TTF)(>99%) was obtained from Fluka Chemie AG, Buchs/Switzerland. Glucose oxidase (GOX) (EC 1.1.3.4 Type I1 from Aspergillus niger),bovine serum albumin (BSA) (fraction V 9f3-99%albumin), glutaraldehyde, (GLA) (25% aqueous), and D-(+)-glUCOSewere from Sigma Chemical Co. (St. Louis, MO). All other reagents used were of AnalaR grade. All aqueous solutions were made up in 0.1 M pH 7.4 sodium phosphate buffer solution containing 0.01% (w/v) sodium azide prepared with Millipore Milli Q grade water. Glucose solutions were allowed to mutarotate overnight before use. Construction of Enzyme Electrode. Working electrodes were made from disks cut from 3 mm diameter platinum (Johnson Matthey, England) or glassy carbon (Tokai, Japan) rods. Electrical contact was made with a copper wire connected to one side of the disk with silver-loaded epoxy resin. The disk was then press fit into a Teflon holder and sealed with epoxy resin so that only a circular plane was exposed. Electrodes were polished with abrasive paper and then mirror polished with 0.3-fimdiamond paste (K-5000, Kyoto, Japan). Prior to use, electrodes were 0 1990 American Chemical Society
756
ANALYTICAL CHEMISTRY, VOL. 62. NO. 7. APRIL 1. 1990
Figure 2. Exploded vlew of *in-layer ceik (a)enzyme electrode: (b) potyester spacer: (c) wunter elecbode: (d) solution inlet; (e)bifucated fiber-optic bundle: (f) Perspex salt brMga f u reference electrode: (9) Perspex body; and (h) solution outlet.
Flgm 1. Schematic &gram of the 0pticaCelecaOChemicaIsystem.
cleaned s u d v d y with ethanol and mtric acid and UltraMniCally in distilled water. The electrodes were then oven-dried at 80 "C for 1 h. Enzyme electrodes were prepared hy evaporating droplets (2 X 5 rL) of "F/acetone solution onto the electrode surface. Ten microliters of 4% g l u m oxidaee solution was depasited over the TTF layer and then dried at 4 "C overnight. Finally, 2.5 p L of freshly prepared 1:l mixture of 5% BSA and 2.5% glutaraldehyde wm depasited on top of the dried g l u m oxidase and then caved with a polycarbonate membrane (0.03 rm pore size from Nudepore, Pleasanton, CAI. The membrane was held tightly in place with a Teflon cap. The enzyme electrode so made was allowed to set at 4 OC overnight. Optical System. A schematic diagram of the experimental setup is given in Figure 1. The light source is a halogen lamp (12 V, 103W,Halo Star, Watan, Germany) operated with a current stabilized power supply (Bentham, Model 505). The source light was modulated with an optical chopper (Bentham, Model 218) at a frequency of 200 Hz. The chopper frequency provided the reference input to the lock-in amplifier (LIA) (Princeton Applied Research, Model 5104). A monochromator (Bentham, Model M300EA) utilizing a hclographic grating (Bentham G324HOu24,2400 lines/mm) coupled to a huiltin stepping motor drive (Bentham, Model SMD38/ IEEE) was used to obtain a wavelength range between 220 and 620 nm with a resolution of 0.5 nm. The light from the monochromator was passed through one arm of the bifurcated fiheroptic bundle (Quentron, Model 777-1, Australia). The signal arising from specular reflection at the electrode is then passed through the other arm of the bifurcated fiber-optic bundle to the photomultiplier tube deteetor (Bentham,DH3 0 ) powered by a high-voltage supply (Bentham, Model 215). The enhanced signal from the photomultiplier is delivered to the programable current amplifier (Bentham, Model 265HF)and then to the LIA. The entire system was under the control of a computer (Model HP9826, Hewlett-Packard) via the HPIB (IEEE/488) interface, The control software written in HPBASIC 3.0 enahled the measurement of intensity against time or wavelength. Prior to each measurement, the reference and analytical signals were brought into phase at the input of the LIA's phase-sensitive detectar. Optical messurements were then carried out at this fixed phase. Thin-Layer Cell. Steady-state and flow-injection measurements were made with a modified thin-layer cell that enahled the fiber-optic probe and enzyme electrode to be mounted opposite each other. Figure 2 shows an exploded view of the thin-layer cell. The reference electrode was Ag/AgCl and the counter electrode was graphite. The working volume of the cell could be adjusted by varying the number of polyester film spacers (each of 125 pm thickness measured by a micrometer). The spacers had a central hold of 1 an diameter. The entire cell was blackened to keep out ambient light. Sample solutions were injected by means of a four-way manual injector (Valve V-7, P h m a c i a Fine Chemicals, Sweden). Sample solutions were delivered with a peristaltic pump (Eyela Model MP-3, Tokyo Rikakikai, Tokyo, Japan). The pump was calibrated prior to use. For the steady-state measurements, a large volume loop (1mL) was used and the flow rate was kept at 0.4 mL/min. For the cyclic voltammetric studies, a continuous delivery of glucose solution was
provided at 0.4 mL/min In thisway, a steady-state flux of g l u m could be achieved over an extended period. For flow-injection analysis (FIA),a 50 rL volume loop was used which afforded the characteristic peak profiles. Cyclic voltammetry and ampemmetric studies were performed with a PAR petentiostat/galvanostatModel 273. Voltammograma e 8 recorded with an XY recorder (Graphtec, and current-time m Model WX2400). RESULTS AND DISCUSSION Principle of Operation. TTF has been shown to serve as a redox mediator for glucose oxidase (12). The redox mediation takes place via a homogeneous electron exchange between the fmt oxidation state, W , and the enzyme active center. The glucose oxidase catalyzed reaction with glucose occurs according to the following scheme: glucose + GOXO' glnconolactone + GOX" (1)
GOXd
+ 2"F+
--
GOXO'
+ 2TTF0 + 2H+
(2)
"F+ can he regenerated at the electrode surface 2TTF" e 2WF+ 2e(3) For practical pur-, we can define a reaction layer lying between the electrode surface and membrane wherein reactions 1and 2 take place. 'ITF is regenerated at the electrode surface and diffuses into the reaction layer. By monitoring the generation or consumption of TTF+in the reaction layer it is feasible to derive an analytical response that can be related to the glucose concentration. Higher glucose concentrations result in faster rates of ?TF+ reduction and lower 'TIT' levels. A steady-state flux of TTF+ is established when the rate of ita regeneration at the electrode is counterhalanced by the rate of ita loss from the reaction layer. The reaction scheme represented by eqs 1-3 indicates that the analytical signal is not dependent on the oxygen concentration. However, there is a possibility that there is competition between TTF+and oxygen for oxidation of GOX. Experiments were carried out in the air-saturated and deaerated solutions to verify this. It was found that the air-saturated solution resulted in a reduction in the steadystate amperometric signal by about 5 % , whereas it had a negligible effect on the fiber-optic signal. The reduction in the ampemmetric signal is probably more due to the negative current arising from the reduction of H,02 than an effect on the TTF+ concentration. This is confirmed by injecting a solution containing 2 mM H202 which gave no significant fibs-optic signal hut a reduction current for the amperometric signal. In terms of ita spectra characteristics, TTF+ can be distinguished from the neutral 'I""F0 by ita ability to absorb in the visible region between 540 and 580 nm. This is shown in Figure 3, which records reflectance spectra at various applied potentials. As can be seen, the reflectance intensity around this wavelength range is greatly diminished with increasing potential due to the formation of TTF+. Figure 4a further shows the decrease in the reflectance intensity (at 570 nm) with the formation of TTF+ as the
+
ANALYTICAL CHEMISTRY, VOL. 62, NO. 7, APRIL 1, 1990
0012
0
L
k
-
Z
-
-
5i0
540
SA0
580
d
2
0
Figure 3. Specular reflectance spectra at R/TF-GOX enzyme electrode for variow applied potentials: (a) 0 mV, (b) 100 mV, (c) 200 mV, (d) 250 mV, (e) 300 mV; spacer thickness, 250 pm; flow rate, 0.4 mL min-‘.
0 100 zoo Potential. mV, vs Ag/AgCI
LJ
Time 1s)
0
100
200
;
0
‘Wavelength inm)
-io0
757
300
Figure 4. Cyclic voltammograms and corresponding reflectance lntensity at R/TF-GOX enzyme electrode in (a) phosphate buffer pH 7.4 (b) wlth 2 mM glucose solution or (c) wlth 10 mM glucose solution: cyclic Voltammetry scan rate, 1 mV/s; reflectance wavelength, 570 nm; spacer thickness, 250 pm; flow rate, 0.4 mL min-’.
electrode potential is scanned to more positive values. The decreased reflectance intensity arises because of the increased absorption of light as the TTF+concentration increases. In the reverse scan (to more negative potentials) the reflectance intensity increases due to the reduction of TTF+back to the neutral state, indicating the reversible nature of the redox mediator. In the presence of 2 mM glucose, the amount of TTF+ in the reaction layer is diminished via reactions 1and 2. Figure 4b thus shows the reflectance intensity to be higher at more positive potentials compared to Figure 4a. In the presence of excess glucose, however, the reflectance intensity of Figure 4c appears to be relatively unaffected as the potential is scanned to more positive potentials. This is
02
04
06 Cell ihiCkne55
08
10
12
14
lmml
Figure 5. Effect of cell thickness on relative reflectance Intensity: glucose concentration, 6 mM glucose; operating potential, 250 mV vs Ag/AgCI; sample volume, 50 pL. Other conditions are the same as in Figure 4.
because all the TTF+ in the reaction layer is converted to WFO via the enzymatic reaction or lost by diffusion through the membrane. In this case TTF+generated at the electrode surface is immediately reduced as it diffuses into the reaction layer. In contrast with the response of the TTF coated enzyme electrode described above, it was found that a glucose oxidase immobilized electrode without “Floading gave no significant response in the wavelength range between 400 and 620 nm. As might be expected, the reflectance signal from the glassy carbon electrode was generally found to be lower (by about a factor of 10) and noisier than the absolute reflectance signal from the platinum electrode. While this reduced the sensitivity of the detector, the response time and upper linear range were unaffected. Effect of Cell Thickness. Increasing the cell thickness was found to enhance the reflected light intensity, although the response time was slightly increased. The effect of cell thickness on the relative intensity (with reference to the base line prior to the injection of glucose) on flow-injection peak is shown in Figure 5; the increase in intensity is due to two factors: First, it is due to an increase in the effective area of the electrode (or reaction layer) exposed to the incident light from the fiber-optic probe. Also, when a bifurcated fiber-optic bundle is used, up to a point, the further the reflector is away, the greater the overlap between the “cone of light” emerging from the fiber (from the source) and the “cone of light” accepted by the fiber (to the detector). The enhancing effect of increasing the cell thickness, however, reaches a limit a t around 1.2 mm, presumably where maximum overlap of the “cones of light” occurs. Second, the enhancement of the reflected light intensity is due to the increase in residence time in the cell as the cell thickness increases. The longer the residence time, the larger the time scale and the more TTF’ that is consumed. FIA Response. Figure 6a shows reflectance light intensity (with reference to the base line prior to injection of glucose) versus time plots for injections of increasing concentrations of glucose in the FIA mode. Before a sample injection, the concentration of TTF+ in the reaction layer is a maximum and, consequently, the base-line reflectance intensity-time response is a minimum. When the glucose sample is injected into the cell, the reflected light changes as the TTF+ concentration in the reaction layer changes in relation to the glucose concentration profile as the sample plug passes through the cell. It is of interest that the corresponding current-time plots for amperometric detection are significantly sharper than the
758
ANALYTICAL CHEMISTRY, VOL. 62, NO. 7, APRIL 1, 1990
I
00'
15
I
Reia t ive Intensity =0004
1OmM
k
200
0
L
mb'
14\
600
4% Time
800
- Q
C *
I
--,
20
C.
Amwnl
0'
TTF
bC
pg)
Figure 8. Effect of TTF loading on electrode surface on steady-state reflectance: electrode. platinum; steady-state reflectance measured with 2 mM glucose solution; working potential, 250 m V cell thickness, 500 pm.
1000
+
(5)
Flgure 6. FIA peak profiles for (a) fiber-optic sensor and (b) amperometriC sensor: operating potentiel, 250 m V Row rate, 0.4 Wmin; sample volume, 50 P;spacer thickness, 500 pm; l l F loading, 25 pg.
;030*-
0323
-
I
I
P
,/1
i
/'
8 GIUCOSC
2 CaPcenIrcl on
6
04
@e. Flow
2 Rote
16
20
Imllmin)
Figure 9. Effect of flow rate on relative reflectance intensity. Conditions are the same as in Figure 7. Glucose concentration was 6 mM.
ZC
!
ma
Flguro 7. Calibration plots for glucose-based enzyme electrode: (0) steady-state mode, (0)FIA mode; operating potential, 250 mV; flow rate, 0.4 mL/min; spacer thickness, 500 pm; TTF loading, 5 pg.
reflectance-time plots of the fiber-optic sensor. Closer examination of these plots shows that the contribution to band broadening for the fiber-optic sensor response arises mainly in the trailing edge of the peak. By way of comparison, for the 15 mM glucose sample, the time taken to reach the peak apex was 50 and 65 s for amperometric and fiber-optic detection, respectively. In contrast, the time taken to reach the base line from the apex to the trailing edge was 80 and 180 s, respectively. The particularly slow response in the trailing edge is due to the fact that regeneration of TTFi in the reaction layer (via electrochemical oxidation and diffusion) takes place relatively slowly. There is also a lag time due to the time taken for the TTF+ to diffuse in the channel. The latter can be reduced by using smaller spacers. Linear Range. Figure 7 compares the linear range for the fiber-optic sensor in the steady-state and FIA modes. As in Figures 5 and 6, the reflectance intensity is measured with reference to the base-line reflectance intensity prior to in-
jection of glucose. In general, the upper limit for the former is about 3 mM, whereas for the latter it is about 12 mM. The generally lower linear range can be attributed to the fact that, under steady-state conditions, the glucose concentrations in the cell are higher. Consequently, most of the TTF+ in the reaction layer is converted to TTFO. The way of overcoming this is by increasing the concentration of TTF+ in the reaction layer. This can be done to a limited extent by increasing the T T F loading on the electrode. A study of the effect of TTF loading was made by recording relative intensity with reference to the base line prior to the injection of 6 mM glucose at +250 mV against T T F loading as shown in Figure 8. Increasing the loading appears to initially improve the relative response reaching a limiting value after a loading of about 10 pg. At this level of loading the linear range can be extended to about 15 mM. Effect of Flow Rate. Figure 9 shows the effect of flow rate on the FIA response. The relative intensity with reference to the base-line intensity varies approximately as the inverse square root of flow rate, which is related to the reduced residence time of the glucose sample. Stability. The stability of the electrode mostly depends on the availability of TTF. For this reason, the lifetime of the fiber-optic electrode will be substantially reduced if the applied potential on the electrode is continously held at a positive value resulting in the loss of TTF as TTF+. In our work, we have used the fiber-optic electrode continously for 2 days with only a slight reduction in the response. Comparison of Fiber-optic Electrochemical Detection and Amperometric Detection. Table I compares the performance of fiber-optic electrochemical detection and am-
759
Anal. Chem. 1990, 62. 759-766
Table I. Comparison of Fiber-optic a n d Amperometric Detectiona fiber-optic steady state FIA detection limit
Linear Range 4 mM 12 mM 0.2 mM
Interfering SpeciesC 1.6 ascorbic acid paracetamol N.D.b uric acid N.D. lysine N.D. leucine N.D. N.D. N202 effect of oxygene N.D.
amperometric 20 mM 40 mM 0.005 mM 2.4 0.1 0.3 N.D. N.D. -1.2d -5 %
"Electrode = platinum. Operating potential = 250 mV. Cell thickness = 500 pm. TTF loading = 5 pg. Flow rate = 0.4 mL/ min. Reflectance wavelength = 570 nm. bN.D., no signal detected for species. eData for interferences presented are given in terms of the ratio of the signal for 2 mM interfering species to the signal of 2 mM glucose solution. Negative sign arises because H,Opgives a reduction current at this potential. eData presented are the relative signal of air-saturated solution to the deaerated solution 2 mM elucose.
perometric detection based on the TTF-mediated enzyme electrode. The linear range for fiber-optic electrochemical detection was lower than that for amperometric detection. The detection limit for amperometric detection was found to be about 0.005 mM, compared to fiber-optic electrochemical
detection, which has a detection limit of about 0.2 mM. Table I also compares the effect of various chemical species and oxygen on the response of the fiber-optic and amperometric detector. It is of interest that the fiber-optic sensor appears to be relatively less sensitive to these species. Also, no significant effect on oxygen of the fiber-optic response is observed.
ACKNOWLEDGMENT We acknowledge the assistance of Ishak bin Ismail in constructing the thin-layer cell. LITERATURE CITED Seitz, W. R. CRC Crit. Rev. Anal. Chem. 1988, 79, 135. Seitz, W. R. Anal. Chem. 1984, 56, 16A. Peterson, J. I.; Vureck, G. G. Science 1984, 224, 123. Arnold, M. A. Anal. Chem. 1985, 57. 565. Wolfbeis, 0. S. Anal. Chem. 1986, 58, 2876. Trettnak, W.; Leiner, M. J. P.; Wolfbeis, 0. S. Analyst 1988, 773, 1519. Abdel-Latif, M. S.; Guilbault, G. G. Anal. Chem. 1988, 6 0 , 2671. Wangsa, J.; Arnold, M. A. Anal. Chem. 1988, 60, 1080. Blick, K. E.; Liles. S . M. Principles of Clinical Chemisby; J. Wiiey and Sons: New York, 1985; Chapter 10. Walter, B. Anal. Chem. 1983, 55, 499A. Kaufman, F. B.; Schroeder,A. H.; Engler, E. M.; Kramer, S. R.; Chambers, J. Q. J . Am. Chem. SOC. 1980, 102, 483. Albery, W. J.; Bartlett, P. N.; Craston, D. H. J . Electroenal. Chem. 1985, 794, 223.
RECEIVED for review April 10,1989. Accepted December 13, 1989. This work was supported by the award of grants from the National University of Singapore and the Singapore Science Council.
Poly(vitamin B,,)-Modified Carbon Electrodes Used as a Preconcentration-Type Sensor for Alkylating Agents Beat Steiger, Annette Ruhe, and Lorenz Walder* Institute of Organic Chemistry, University of Berne, Freiestrasse 3, CH-3012 Berne, Switzerland
A multlple-use electrochemlcal sensor for alkylatlng agents based on a poly(vltamln Blz) chemlcally modified electrode (BlzCME) Is described. Preconcentratlon Is swltched on and off depending on the appllrd potentlal, Le. the presence of Co(1). It Is related to oxldatlve addltlon of RX from dllute solutlons to the lmmoblllzed vltamln BlZr (Co( I ) ) yleldlng surface-conflned alkylcobalamins (Co( II 1)-R). With cycllc voltammetry the sololutlon concentration of RX (prlmary alkyl lodldes and bromldes) can be correlated wlth the reduction current of the alkylcobalamin. The detectlon llmlt ([RX],) depends on preconcentratlontlme ( T ) and the homogeneous = 4.3 reactlvlty of vltamln BlZ1 (Co(1)) toward RX ([CH,I], X lo-' M for T = 5 mln). Concomitant wlth a measurement the B,&ME Is regenerated. Photocurrents are observed at the Blz-CME In the presence of alkylatlng agents when It Is illuminated wlth vlsible light. The potentlaldependentformatlon and cleavage of an orgametaHlc adduct between CME sltes and the analyte are consldered a new sensor prlnclple sulted for organlc analytes.
In recent years chemically modified electrodes (CMEh) have found broad applications as sensors in electroanalysis (1,2). The concepts used for increasing the current response of an
electroactive analyte on a CME are based on (i) the catalysis of the otherwise sluggish electron transfer, (ii) membrane barrier effects for interfering substrates, and (iii) preconcentration of the analyte in the modifying layer. Most reports on the sensor abilities of CME's deal with the determination of inorganic metal ions, but only a few studies are directed toward organic analysis. Classified according to the three concepts these are,(i)catalysis of sulfhydryl (3,4), NADH (5,6), carbohydrate (7,8), and ascorbic acid oxidation (6), (ii) size selective membrane barriers for electroactive substrates in the kilodalton range (91,and (iii) preconcentration based on ion exchange phenomena and/or hydrophobic-hydrophilic interactions at polymer modified electrodes used for the detection of viologens (lo), protonated aromatic amines (11, 12), and phenolic compounds (13),as well as phenolic analytes at a lipid-modified electrode (14). The closely related detection of vitamin K,, K2,and E on a monolayer derivatized electrode has been described in terms of molecular recognition (15). Preconcentration based on covalent bond formation between modifier and analyte has been reported in one case only (16). The preconcentration method (iii) consists of a sequential procedure:accumulation detection regeneration. A certain time delay is used to achieve a detectable concentration of the andyte at the CME by any kind of attractive interaction
-
0003-2700/90/0362-0759$02.50/00 1990 American Chemical Society
-
