Modification of a Capillary for Electrophoresis by Electrostatic Self-Assembly of an Enzyme for Selective Determination of the Enzyme Substrate
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Modification of a Capillary for Electrophoresis by ElectrostaticSelf-Assembly of an Enzyme for Selective Determination of theEnzyme SubstrateStephanie E. Hooper, Mark R. Anderson*
Department of Chemistry, Virginia Polytechnic Institute and State University, Blacksburg, VA 24061-0212, USA*e-mail: firstname.lastname@example.org
Received: November 9, 2006Accepted: December 28, 2006
AbstractThe integration of a separation capillary for capillary electrophoresis (CE) with an on-column enzyme reaction forselective determination of the enzyme substrate is described. Enzyme immobilization is achieved by electrostaticassembly of poly(diallydimethylammonium chloride) (PDDA) followed by adsorption of a mixture of the negativelycharged enzyme glucose oxidase (GOx) and anionic poly(styrenesulfonate) (PSS). The reaction of glucose with theGOx produces hydrogen peroxide which migrates the length of the capillary and is detected amperometrically at thecapillary outlet. The enzyme reaction occurs during a capillary separation, allowing selective determination of thesubstrate in complex samples without the need for pre- or post-separation chemical modification of the analyte. Theenzyme reactor is found to have an optimal response to glucose when a 5 :1 mixture of PSS:GOx is used. Under theseconditions the limit of detection for glucose is found to be between 5.0 104 and 1.3 103 M, dependent upon theinner-diameter of the capillary. The apparent Michaelis-Menten constant for the enzyme reaction was determined tobe 0.047 (0.001) M and 0.0037 (0.0007) M for a 50 and 10 mm inner-diameter capillaries, respectively. These resultsindicate that the enzyme reaction is efficient, having enzyme kinetics similar to that of a reaction occurring in solution.This enzyme immobilization method was also applied to another enzyme, glutamate oxidase, yielding similar results.
Keywords: Capillary electrophoresis, Amperometric detection, Enzyme reactor, Ionic self-assembly, Capillarymodification
The high-resolution and microscale capabilities of capillaryelectrophoresis make it a powerful tool for the determina-tion of molecules of biological interest. The primarylimitation of CE in bioanalysis is in the detection of theanalyte. Many biological species do not have a UV/visiblechromophore nor do they have easily accessible redoxchemistry to allow direct detection [1 3]; consequently,these species are frequently chemically modified prior to orafter the separation to generate a species that can bedetected by spectroscopic or electrochemical measurement.While effective, this chemical modification introduces addi-tional complexity to the analysis .An enzyme reaction coupled with CE can be used to
convert the analyte of interest into a detectable species.Nashabeh et al. and Mechref et al. developed a capillaryenzyme reactor that introduces an enzyme reaction prior tothe separation [5, 6]. Here, enzymes were covalentlyimmobilized inside a short length of a capillary that wascoupled to the sample introduction endof theCE separationcapillary. Gravity flow of the sample allowed the analyte toreact with the enzyme prior to the sample being subjected tothe separation force of the capillary electrophoresis. While
thismethod is still a pre-separation chemicalmodification ofthe analyte, it is conducted with small sample sizes that arecharacteristic of a capillary separation. This technique wassubsequently extended by covalent immobilization of theenzyme along the inner wall of the separation capillary,eliminating the need for a separate capillary microreactor[7 9].Because enzymeshave anet chargebasedon thepHof the
solution, they may also be confined to an interface usingelectrostatic interactions. LEvov et al. demonstrated thefeasibility and reproducibility of ionic self-assembly byalternately depositing layers of polyions and chargedbiological molecules on planar surfaces [10, 11]. Recently,Tang and Kang ionically immobilized the negatively charg-ed angiotensin-converting enzyme ACE along the innerwall of a separation capillary . This modified capillarywas subsequently used in an investigation of an enzymeinhibitor. Their results demonstrate that enzymes confinedto the walls of a separation capillary maintain their activity.While enzyme immobilization by electrostatic assembly hasbeen used for interfacial modification in a number ofapplications, its use with capillary separations is relativelyunexplored.
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In this research, the inner walls of a separation capillaryare modified by ionic assembly of the polycation poly(-diallydimethylammonium chloride) (PDDA) and the neg-atively charged enzyme, glucose oxidase (GOx) onto theinterior walls of the capillary. The immobilized GOxcatalyzes the oxidation of glucose to gluconic acid andhydrogen peroxide, and the hydrogen peroxide is detectedamperometrically using an end-capillary electrochemicaldetector . The feasibility of combining the enzymereaction with a CE separation is evaluated. Followingoptimization and characterization of the immobilizedenzyme system with glucose oxidase, electrostatic assemblyis used with another FAD-type of enzyme, glutamateoxidase (GlutOx), to illustrate the general utility of thismodification and detection method.
d-Glucose, glucose oxidase from Aspergillus Niger (146units per mg), l-glutamate oxidase from Streptomyces (5units per mg), l-glutamic acid, catechol, 3-hydroxytyraminehydrochloride (dopamine), poly(diallydimethylammoniumchloride) (PDDA), and poly(styrenesulfonate) (PSS) wereobtained from Sigma-Aldrich (Milwaukee, WI, USA) andused as received. All solutions were prepared using 18 MWdeionized water (Barnstead Nanopure system).Fused silica capillaries cut to a 50 cm lengthwereobtained
from Polymicro Technologies (Phoenix, AZ, USA). Allcapillaries had a 360 mm outer-diameter and inner-diame-ters of either 50, 20, or 10 mm. Capillaries were initiallyconditioned by flowing 0.1 M NaOH through the capillaryfor 15 minutes using static pressure. The capillaries werethen rinsed with the running buffer for 15 minutes. Therunning buffer consisted of 0.01 M NaH2PO4, 0.01 M Na2HPO4, and 0.05 M KCl. Once conditioned with NaOH, a5.0 104 M solution of the cationic polymer PDDA ispumped through the capillary for one hour, followed by a 10minute rinse with the running buffer. This is followed byflowing a solution containing amixture of the polyanionPSSand negatively charged GOx through the capillary, againfollowed by a 10minute rinsing with the running buffer. ThePSS:GOx solutions used in this capillary preparation stepcontain 5.0 105 M GOx and different amounts of PSS(ranging from 0.002 M to 5.0 105 M) in the pH 7 buffer toestablish the desired concentration ratio in the adsorptionsolution. If the capillary is not immediately used, it is storedat 4 8C with the capillary filled with the running buffer andboth capillary ends under solutions of the running buffer.The same adsorption procedure was utilized with GlutOx.For theGlutOx experiments only a PSS:GlutOx ratio of 5 to1 was used.Capillary zone electrophoresis is performed using a
Spellman CZE1000R high voltage power supply. Theseparation voltage was applied along the length of thecapillary with the sample introduction at the high voltageend and detection occurring at the ground end of thecapillary. The high voltage electrode is isolated by an
interlocked Plexiglass box. Sample was introduced to thecapillary electrokinetically by placing the high voltageelectrode and capillary end into the sample reservoir for afixed time.Amperometric detection is performed at the ground end
of the capillary with a 1 mm diameter platinum workingelectrode aligned with the capillary opening in a wall jetarrangement using a previously described detector cell .The working electrode is manually positioned at a distanceof ca. 10 mmoutside the opening of the capillary with the useof an XYZ positioner under observation with a stereo-microscope . A constant potential of 700 mV vs. Ag/AgCl was applied to the working electrode for the ampero-metric detection. The potential applied to the indicatorelectrode was controlled by a Princeton Applied Researchmodel 273A potentiostat (Ametek, Oak Ridge, TN).
3. Results and Discussion
Capillaries modified by electrostatic assembly with aPDDA/GOx bilayer were initially evaluated using flowinjection analysis to determine if the GOx confined to thecapillary walls was able to produce detectable amounts ofhydrogen peroxide by the enzyme reaction as small samplesof glucose were introduced to the flowing stream. Capil-laries with an inner diameter of 250 mm were modified asdescribed previously and were found to produce a meas-urable response that varied with the concentration ofglucose introduced (Figure 1).When smaller inner-diameter capillaries were similarly
modified and used in the electrophoresis system, however,no analyte zones were detected for up to 45 minutes aftersample introduction. This result could be due to eitherinsufficient electroosmotic flow (EOF) needed to transportthe material the length of the capillary, or to inefficiency inthe enzyme reaction. Because H2O2 was detected when
Fig. 1. Glucose concentrations of 0.010, 0.005, 0.001, and0.0005 M were injected three times into a 10 mL sample loopthat was connected to a 250 mm inner-diameter capillary. Bufferwas pumped through the system at a flow rate of 0.5 mL/min.Detection occurred at a 1 mm Pt working electrode that was set toa potential of 700 mV vs. Ag/AgCl.
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using a similar system with pressure driven flow, we believethat this result is due to the poor EOF found with capillariesmodified with PDDA/GOx bilayers. This interpretationwassupported by adding hydroquinone to the analyte solutionas a neutral molecule that should 1) emerge from thecapillary at a rate equal to the EOFand 2) is oxidized at thepotential used for H2O2 detection. Again, no detectorresponse was observed for samples containing hydroqui-none.At the pH of the running buffer (ca. 7.00), GOx is
negatively charged . The negative charge of the enzymeallows GOx to adsorb to the positively charged PDDAconfined to the capillary wall, as indicated by the flowinjection results. This is also demonstrated by Hodak et al.who form multilayers with a polycation and GOx byelectrostatic assembly onto gold electrode surfaces .The interfacial negative charge density after electrostaticdeposition of the enzyme onto the capillarywall, however, isapparently not sufficient to support EOF in the capillarywhen potential is applied. Immobilization of the polycationPDDA by itself is known to reverse the interfacial chargeand support electroosmotic flow in the direction oppositethat seen with the unmodified capillary . By co-immobilizing an anionic polymer with a relatively highcharge density, such as PSS, with the enzyme, we hope tomaintain the enzyme activity while also establishing suffi-cient negative charge density along the capillary wall tosupport EOFof the bulk solution toward the ground end ofthe capillary when a positive separation voltage is applied.Solutions containing different mixtures of PSS and GOx
were used during the capillary modification procedure, andthese modified capillaries were used to determine the effectof the inner wall composition on (1) the system response toglucose and (2) the electroosmotic mobility (me) of glucose(Table 1). For these measurements, five second electro-kinetic injections of a 0.1 Mglucose solution (correspondingto approximately 2 pmol of glucose) were introduced into a50 mm inner-diameter capillary, and the amperometricresponse of the hydrogen peroxide produced by the enzymereaction was monitored as it emerged from the capillaryoutlet. The electroosmotic mobility is determined by theelution time, and the system response by the peak currentfrom the H2O2 detected.As the relative amount of GOx in the anionic layer
increases, the me of glucose decreases (Table 1). This result isconsistent with a decreasing charge density along thecapillary walls. When the PSS:GOx ratio is 5 :1, the me is90% of that found with a 40 :1 ratio. A 2 :1 PSS:GOx ratio
has an me that is only 77% of that found with a 40 :1 ratio. Asthe relative amount of PSS in the anionic layer continues todecrease, theme falls off rapidly to the pointwhere there is nomeasurableEOFwhen theGOx is in higher proportion thanthe PSS in the interfacial assembly (as evidenced by the lackof an H2O2 response). The decrease in the electroosmoticflow rate with the increasing proportion of GOx confined tothe capillary wall is consistent with the interpretation thatGOx by itself does not have sufficient charge density tosupport electroosmotic flow.Because the amount of glucose introduced to the capillary
during each of these trials is constant, the detector responseis a measure of the efficiency of the enzyme reaction. Evenwhen the amount of confined PSS is relatively high (40 :1),enough hydrogen peroxide is generated by the enzymereaction to be detected (Table 1). As the relative amount ofGOx in the anionic layer increases, both the residence timeof the glucose within the capillary (due to the decreasing me)and the detector response increase. This result is consistentwith the increased opportunity for the substrate (glucose) tointeract with the confined enzyme (GOx) and producehydrogen peroxide. This trend continues up to a 5 :1 ratio ofPSS :GOxwhere the detector current is 2.4-times that foundwith a 40 :1 ratio.As the amount of GOx in the anionic layer increases
beyond the 5 :1 ratio, however, the detector responsedecreases. This change in the detection trend coincideswith the significant decrease in the me of glucose, andsuggests a decrease in the enzyme reaction efficiency. Thesignificantly slower migration rate lowers the efficiency ofthe separation resulting in broader, more diffuse analytezones for detection. The glucose zone-width does increase(Table 1) with decreasing me, however, the increase in thepeak width does not by itself account for the decreaseddetector response.Based on the capillary preparation procedure, the enzyme
should be homogeneously distributed throughout the lengthof the capillary.Optimization of the electroosmoticmobilityof glucose and the system response to glucose assumed thatthe enzyme reaction was occurring the entire length of thecapillary and was not isolated to a small volume at thebeginning of the capillary. To test this assumption, theresponse of three identically modified capillaries havingdifferent lengths was investigated. The average peak-current response to 5-second injections of 0.10 M glucosewas found to be 3.47 (0.76) 108, 5.40 (0.70) 108,and 9.00 (0.15) 108 A for capillary lengths of 40, 50, and65 cm, respectively. If the enzyme reaction were confined to
Table 1. System response to changes in the PSS:glucose oxidase composition.
PSS:GOx ratio Current response (A) Peak width (s) Electroosmotic mobility (cm2/Vs)
40 to 1 2.39 (0.12) 108 17.8 (1) 4.40 (0.07) 10410 to 1 3.65 (0.17) 108 27.9 (1.6) 4.16 (0.04) 1045 to 1 5.62 (0.11) 108 34.3 (0.6) 3.94 (0.02) 1042 to 1 4.70 (0.26)108 33.8 (0.8) 3.37 (0.04) 1041 to 1 3.95 (0.24) 108 32.6 (1.5) 2.80 (0.02) 104
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a small volume at the beginning of the capillary, the totallength of the capillary should have no affect on the detectorsignal. Similarly, if all the glucose were consumed by theenzyme reaction, the current response would be expected tobe the same for the different length capillaries. Because thedetector response increases with capillary length, theenzyme reaction appears to continue the entire length ofthe capillary. The detector response, however, does not scalelinearly with length, being 2.5 times larger for the 65 cmcapillary than is found with the 40 cm capillary.Results from these measurements suggest that the
optimum system parameters establishes a balance betweenthe rate of the enzyme reaction and the residence timeof theglucose to maximize the system response. This is furtherillustrated by changing the value of the separation voltage(Table 2). As the separation voltage increases from 4 kV to10 kV, the migration time (residence time of glucose)decreases and the detector current increases slightly. Theincreased detection current is likely a reflection of theincreased mass-transport of analyte to the wall-jet electro-chemical detector. If separation voltage is increased furtherto 13 kVand 16 kV, the migration time of glucose continuesto decrease, but the detector current under these conditionsdecreases as well. Because the mass-transport of analyteincreases under these conditions, the decreased detectorcurrent at the higher separation voltages reflects a de-creased efficiency of the enzyme reaction under theseconditions. These results are consistent with the interpreta-
tion that there is an optimum migration-rate of the glucosethrough the capillary for themost efficient enzyme reaction.Using the optimized separation conditions, the enzyme
kinetics for this capillary system were evaluated by mon-itoring the system response to different concentrations ofglucose (Figure 2). The response is linear up to a glucoseconcentration of approximately 0.010 M, and then deviatesfrom linearity at higher concentrations. From the concen-tration data, the apparent Michaelis-Menten constant, KmE,was found to be 0.047 (0.001) Mwhen using a 50 mminner-diameter capillary. This value is consistent with KmE valuesfor the glucose/GOx enzyme reaction reported in theliterature .The efficiency of the enzyme reaction is a function of the
ability of the enzyme and the substrate to interact. For ourconfined system, we can influence the ability of the flowingsolution with the wall of the capillary by changing the inner-diameter of the capillary. Three capillaries having inner-diameters of 50, 20, and 10 mm (all 50 cm in length) weremodified with a bilayer of PDDA/PSS:GOx, and theresponse of the confined enzyme to varying concentrationsof glucose was determined. The values of the limit ofdetection (LOD) and limit of quantification (LOQ) deter-mined for glucose for these capillary inner-diameters are5.0 104 M and 4.0 103 M, respectively, and do not varysignificantly for the different capillary sizes.Apparent KmE values of 0.047 (0.001), 0.0093 (0.001),
and 0.0037 (0.0007) M are determined for the 50, 20, and10 mm inner-diameter capillaries, respectively. For thesecapillaries, the circumference to cross-sectional area of thecapillary increases by an amount inversely proportional tothe inside radius of the capillary. The larger surface area tovolume of the smaller capillaries affords more opportunityfor analytes in the flowing stream to interactwith thewalls ofthe capillary and react with the confined enzyme. Thedecreasing KmE values for the smaller capillary inner-diameter is consistent with a more efficient enzymereaction. The 10 mm ID capillary has a ten-fold improve-ment in its KmE value compared with a capillary having a
Table 2. System response to changes in the separation voltage.
Separation voltage (kV) Current response (A)
4 3.60 (0.17) 1087 3.70 (0.20) 10810 4.00 (0.35) 10813 3.20 (0.27) 10816 1.93 (0.42) 108
Fig. 2. Series of two consecutive injections of 0.1, 0.05, 0.025, 0.010, 0.005, and 0.001 M glucose onto a PDDA/PSS:GOx modified 50 mminner-diameter capillary. The applied separation voltage was 10 kV and a detection potential of 700 mV vs. Ag/AgCl was utilized. Inset:The corresponding peak-current response curve for multiple injections of glucose ranging in concentration from 0.001 M and 0.1 M wasgenerated using the same capillary.
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50 mmID.Because the surface area to volume ratio for thesecapillaries only increases by a factor of 5, the improvementin the KmE is not simply due to the surface area increase.With decreasing capillary inner-diameter, the rate ofEOF
also decreases. For these measurements, the EOF for the50 mm inner-diameter capillary is 1.5 times that of the 10 mminner-diameter capillary. With the slower EOF rate, theglucose has a longer residence time on the capillary andmore opportunity to interact with the confined enzyme. Thelonger residence time combinedwith the larger surface-areato volume of the smaller inner-diameter capillaries mayaccount for the significant improvement in the KmE valuesfound with the 10 mm capillaries.To demonstrate that glucose could be separated from a
mixture while the enzyme reaction was occurring, separa-tion of a mixture containing 0.001 M dopamine, 0.025 Mglucose, and 0.001 M catechol was performed (Figure 3).This separationwas conducted with two different capillaries one with and one without the electrostatically confinedglucose oxidase. When using the unmodified capillary, onlythe dopamine and catechol zones are detected (Figure 3).With the enzyme-modified capillary, however, the separa-tion has an additional feature due to the detection of thehydrogen peroxide produced during the enzyme reaction.In the separation of this mixture, the neutral glucose/
hydrogen peroxide and catechol zones emerged from thecapillary at slightly different times (687 and 710 s, respec-tively), with the hydrogen peroxide feature preceding thecatechol zone. We had anticipated that the glucose/H2O2would be slightly retained by the stationary enzymeduring the enzyme/substrate reaction and would have alonger elution time than a neutral species with no chemicalinteraction with the confined enzyme. This was not ob-served, and the glucose/H2O2 elutes with the electroosmoticflow. Catechol is weakly acidic (pKa1 9.4) and is 0.4% inthe first conjugate base form at the pH of the running buffer(pH 7). The acid/base properties of catechol may contributeto its slightly longer elution time.
Because the glucose/H2O2 elutes with the electroosmoticflow, glucose zones will overlap with other neutral species inthe sample and possibly introduce interferences in theanalysis. This is illustrated in Figure 4. The combination ofthe capillary enzyme reactor system along with the electro-chemical detection, however, establishes enough selectivityto quantitatively differentiate glucose from other neutralanalytes. The additional selectivity can be accomplishedeither by appropriate choice of the detection potential, or byconducting the separation in thepresence andabsenceof theconfined enzyme.A two week study was performed to evaluate the stability
of the capillary enzyme reactor. During these studies, asolution of 0.1 M glucose was electrokinetically introducedsix times and the average current response for the generatedhydrogen peroxide was determined. The same experimentwas performed again for three consecutive days and thenevery three days over a period of two weeks. The capillarywas stored at 4 8Cunder solutionwhen it was not being used.After two weeks, the average system response had onlydecreased by 2%, indicating that the capillary enzymereactor maintained stability and could be reused for manydays after initial assembly.The general utility of electrostatic assembly of an enzyme
reactor within an electrophoresis capillary was evaluatedusing glutamate oxidase (GlutOx). GlutOx will deaminatel-glutamic acid to produce a-ketoglutaric acid and hydro-gen peroxide. For this system, the GlutOx was electrostati-cally deposited from a 5 :1 PSS :GlutOx solution followingthe procedure outlined previously. Various concentrationsof l-glutamic acid were injected three times each onto the50 mminner-diameterGlutOxmodified capillary to producethe current responses observed in Figure 5. At the pH of thebuffer, the glutamic acid is in the conjugate base form. Thedetector response to glutamic acid is markedly reduced incomparison to the analogous system for glucose determi-
Fig. 3. Electropherograms of a test solution containing 0.001 Mdopamine, 0.025 M glucose, and 0.001 M catechol introducedelectrokinetically to a modified and unmodified 50 mm inner-diameter capillary with a separation voltage of 10 kV and adetection potential of 700 mV vs. Ag/AgCl.
Fig. 4. Three electropherograms of a test solution containing 1)0.005 M dopamine and 0.005 M acetaminophen, 2) 0.005 Mdopamine, 0.005 M acetaminophen, and 0.05 M glucose, and 3)0.005 M dopamine and 0.05 M glucose introduced electrokineti-cally to a modified 50 mm inner-diameter capillary with aseparation voltage of 10 kV and a detection potential of 700 mVvs. Ag/AgCl.
656 S. E. Hooper, M. R. Anderson
nation. This result is expected as the GlutOx has feweractive enzyme units per unit mass than the GOx (5 units permg for GlutOx compared to 146 units per mg forGOx). TheLOD and LOQ values for glutamic acid were found to be0.004 and 0.012 M, respectively, and an apparent KmE valueof 0.085 (0.006) M for the GlutOx/glutamic acid reaction.ThisKmE value improved by a factor of 5 to 0.017 (0.002)Mwhen a 20 mm ID capillary was used.l-Glutamic acid was also separated from positively
charged dopamine in the enzyme modified separationcapillary (Figure 6). The same injection was performed onan unmodified capillary as well. A peak for glutamate wasonly observed with the capillary modified with the immo-
bilized GlutOx, confirming that the glutamate/hydrogenperoxide being detected is generated solely by the enzymereaction itself.
Ionic adsorption of PDDA/PSS:GOx along the inner wall ofa separation capillary effectively transforms the capillaryinto an on-column enzyme reactor without loss in activity ofthe immobilized enzyme and without compromising theseparation properties of the CE separation. The PSS in theanionic layer is required to ensure enough negative chargeto fully reverse the interfacial charge and support EOFtoward the ground electrode when using a positive separa-tion voltage. During CE separation, the enzyme reactionconverts the glucose into hydrogen peroxide which isdetected as it emerges from the capillary by constantpotential amperometry. Optimization of the separationparameters show that a PSS:GOx ratio of 5 to 1 and anapplied separation voltage of 10 kV maximizes the enzymeresponse while maintaining suitable separation conditions.The system response varies linearly with glucose concen-trations up to 0.010 M, but deviates from linearity at higherconcentrations. This behavior is consistent with enzymekinetics, and apparent Michaelis-Menten constants for theconfined system were found to be 0.047 (0.001) M for a50 mm inner-diameter capillary, and 0.0037 (0.0007) M fora 10 mm inner-diameter capillary. TheseKmE values indicatethe glucose/glucose oxidase enzyme reaction has improvedefficiency with smaller inner-diameter capillaries. Becauseelectrochemical detection does not scale with sample size,small inner-diameter capillaries are optimal for bothelectrochemical detection and an efficient enzyme reaction.Although this work focused on confining glucose oxidase
to the capillary wall as a proof of concept, the electrostaticassembly method for generating capillary enzyme reactorsis applicable to other FAD-dependent enzymes, as shownwith glutamate oxidase. Future studies will be focused onpreparing capillaries with other enzymes and on usingmixtures of confined enzymes to determine several analytespecies in a single separation.
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Fig. 5. Series of three consecutive injections of 0.1, 0.075, 0.060,0.050, 0.040, 0.030, 0.020, 0.010, and 0.005 M l-glutamic acid ontoa PDDA/PSS:GlutOx modified 50 mm inner-diameter capillary.The applied separation voltage was 10 kV and a detectionpotential of 700 mV vs. Ag/AgCl was utilized. Inset: Thecorresponding peak-current response curve for multiple injectionsof l-glutamic acid ranging in concentration from 0.005 M and0.15 M was generated using the same capillary.
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657Selective Determination of the Enzyme Substrate
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658 S. E. Hooper, M. R. Anderson