Feature Article

Micromachined Separation Chips with Post-Column EnzymaticReactions of ™Class∫ Enzymes And End-Column ElectrochemicalDetection: Assays of Amino AcidsJoseph Wang,*a Madhu Prakash Chatrathi,a Alfredo Iba¬nƒez,a and Alberto Escarpab

a Department of Chemistry and Biochemistry, New Mexico State University, Las Cruces, NM 88003, USA; e-mail: joewang@nmsu.edub On leave from: Departmento de Quimica Analitica, Universidad de Alcala, Alcala de Henares, Madrid, Spain

Received: August 22, 2001Final version: September 21, 2001

AbstractMicrochip devices integrating eletrophoretic separations, post-column enzymatic derivatization reactions, andamperometric detection, have been developed. The performance of the new integrated microfabricated chip system isdemonstrated for on-chip assay of amino acids based on their electrophoretic separation, post-column reaction withamino-acid oxidase and amperometric detection of the hydrogen peroxide product. Factors influencing the responseare examined and optimized, and the analytical performance is characterized. The concept can be extended todifferent target analytes based on the post-column reactions of other ™class∫ enzymes.

Keywords: Capillary electrophoresis, Amino acids, Microchip, Derivatization, Amperometric detection

1. Introduction

Because of their versatility, efficiency, speed and ability tohandle nanoliter volumes, microfluidic devices have proventhemselves as ideal vehicles for enzymatic assays [1 ± 5].Enzyme-based microchips combine the analytical powerand reagent economy of microfluidic devices with theselectivity and amplification features of biocatalytic reac-tions. Such on-chip enzymatic assays commonly rely onmixing and reaction of the substrate and enzyme, in additionto capillary-electrophoretic (CE) separations. Ramsey×sgroup described microchip separation devices for perform-ing enzyme (galactosidase or acetylcholinesterase) inhib-ition assays in connection to fluorescence detection [1, 2].Since numerous oxidase and dehydrogenase enzymes gen-erate oxidizable products (hydrogen peroxide or NADH,respectively), our team has focused on interfacing enzyme-based biochip assays with various amperometric detectors[3 ± 5]. Such electrochemical detectors offer additionaladvantages for CE microchips, including compatibilitywith micromachining technologies, miniaturization of boththe detector and control instrumentation, and high sensi-tivity and selectivity [6, 7]. On-line pre-column and on-column reactions of glucose oxidase and alcohol dehydro-genase have thus been employed for selective measure-ments of glucose (in the presence of ascorbic acid and uricacid) [3] and for the simultaneous measurements of glucoseand ethanol (in connection to electrophoretic separation ofthe peroxide and NADH products) [4]. However, to ourknowledge, there are no reports on the use of ™class∫enzymes in connection to on-chip separations of theindividual substrates.

In this article we exploit the versatility of lab-on-a-chipdevices for carrying out post-column derivatization reac-tions of ™class∫ enzymes after electrophoretic separation ofthe corresponding substrates. Such use of ™class∫ enzymescan expand the scope of biochips to multiple substrates ofclinical or environmental significance. The new integratedseparation/bioreactor/electrochemical-detection microchipconcept is illustrated for the post-column reaction of aminoacids with amino-acid oxidase (AAOx). Such on-chip assayof amino acids relies on their electrophoretic separation,post-column reaction with AAOx, and amperometric de-tection of the resulting peroxide product. Separationmicro-chips have been combined previously with chemical(phthaldialdehyde, OPA) derivatization of amino acids[8 ± 10], but not to enzymatic reactions. Themicrofabricateddevice used in the present work has an electrophoreticseparation channel, a post-column bioreactor, and an end-column amperometric detector (Figure 1). Its optimization,characterization, and attractive performance characteris-tics, are reported in the following sections.

2. Experimental

2.1. Reagents

�-Amino acid oxidase (D-AAOx) was purchased fromAerBio Ltd (Bloomington, IN) while �-arginine, �-alanine,�-phenylalanine, �-isoleucine, and flavin adenine dinucleo-tide (FAD) were obtained from Sigma. The gold atomicabsorption standard solution (1000 mg/L) was purchasedfrom Aldrich. Sodium borate was purchased from J. T.Baker (Phillipsburg, NJ). All chemicals were used without

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further purification. A borate buffer (20 mM, pH 10.0)solution served as the run buffer. The ™post-column∫enzyme solution consisted of 15 U/mL AAOx in 20 mMborate buffer (pH 9.3). FAD was added to stabilize theenzyme [11]. Sample solutionswere preparedbydiluting thecorresponding stock solutions with the electrophoresisbuffer. All the solutions were filtered daily using 0.45 �mGelman Acrodisc filters.

2.2. Apparatus

The biochip glass chip was fabricated by Alberta Micro-electronic Company (AMC, Model MC-BF4-001, Edmon-ton, Canada) based on our custom design. The layout isdifferent from that employed previously [12] in that itpermits post-column reactions (Figure 1). The chip con-sisted of a sample reservoir, a waste reservoir, and a run-buffer reservoir connected through 5 mm long ™arms∫ to afour-way injection cross.A77.7 mm long separation channelfollowed the injection cross. An additional 77.2 mm-longchannel (referred to as ™post-column∫) joined the end of theseparation channel, leaving 10 mm for the reaction beforethe detector. All channels were 50 �mwide and 20 �mdeep.The fabrication of a Plexiglas holder, supporting the

separation chip and housing the screen-printed electrodedetector and the reservoirs, was described elsewhere [12].The distance between the channel outlet and electrodesurface was 50 �m. Electrical contact with the solutions wasachieved by placing platinum wires into the individualreservoirs. A ™home-made∫ power supply, containingmulti-ple voltage terminals, was used for applying the selecteddriving voltage (between 0 and �4000 V) to a givenreservoir and for switching between the ™postcolumn/separation∫ and ™injection∫ modes.The screen-printed working electrodes were fabricated

with a semi-automatic printer (Model TF 100, MPM,Franklin, MA), and using an Acheson carbon ink Electro-dag 440B (Acheson Colloids, Ontario, CA). Details of theprinting processes and preparation of gold-plated screen-printed electrodes were described previously [12].Ampero-metric detection was performed with an electrochemicalanalyzer 621 (CH Instruments, Austin, TX). The electro-pherograms were recorded with a time resolution of 0.1 swhile applying �1.0 V detection potential (vs. Ag/AgClwire). Sample injections were performed after stabilizationof the baseline. The raw data of electropherograms weredigitally filtered by the ™built-in∫ 15 point least-squaresmoothing option of the CH electrochemical analyzer(Software CHI Version 2.05).

2.3. Electrophoresis Procedure

The post-column enzyme/buffer solution contained 1 mg/mL FAD and had a pH 9.3, as needed to maintain highenzymatic activity [11]. The ™Sample∫ reservoir was filledwith 75 ± 80 �L of the sample mixture solution while the

™Post-column∫ reservoir was filled with 80 �L of the 15 U/mLD-AAOx/FAD buffer solution. The other two ™Buffer∫reservoirs were filled with 80 �L of the borate run buffer(20 mM, pH 10.0). The detection/waste reservoir at thechannel outlet side was also filled with the run buffersolution.After the initial loadingof the sample in the samplearm, the sample was injected using �1500 V for 3 s to thesample reservoir with the detection reservoir grounded andother reservoirs floating. Separation was usually achievedby applying �1000 V to both ™Run Buffer∫ and ™Post-column∫ reservoirs, with the detection reservoir groundedand the other reservoirs floating. Details of the ampero-metric detection protocol were described elsewhere [12].

Safety Considerations: The high voltage power supply andassociated open electrical connections should be handledwith extreme care to avoid electrical shock.

3. Results and Discussion

Post-column enzymatic derivatizations are particularlysuitable for ™class∫ enzymes in connection to the separationof the individual substrates. The new microchip integrateson-line post-column biocatalytic reactions ofAAOx and theamino-acid substrates with effective CE microseparationsand amperometric detection on a microchip platform(Figure 1). Each of these elements was optimized.Hydrodynamic voltammograms (HDV)were constructed

for assessing the effect of the detector potential. Figure 2shows such HDV for isoleucine (�) and phenylalanine (�),obtained by changing the potential of the gold-coatedscreen-printed electrode detector over the �0.3 ±�1.0 Vrange, while injecting this sample mixture. The peroxideproducts of the corresponding post-column enzymaticreactions display similar voltammetric profiles. The oxida-tion signal starts above�0.5 V, increases gradually between�0.6 and �0.9 V, and decreases slightly thereafter. Allsubsequent work employed a potential of �0.9 V thatoffered the most favorable signal/background character-istics.The successful operation of the new enzyme/CE micro-

chip requires proper attention to the level of AAOx.Figure 3 examines the influence of the D-AAOx activity,upon the response for amixture 3� 10�4 M of isoleucine (�)

Fig. 1. Schematic of the post-column reaction chip used in thisstudy. RB: run buffer, B: unused buffer reservoir, S: samplemixture, E: ™reagent∫ (enzyme) reservoir, and D: detector. Seetext for exact dimensions and details.

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Electroanalysis 2002, 14, No. 6

and alanine (�). The response peak increases rapidly withthe D-AAOx activity up to 15 U/mL, then more slowly anddecreases slightly above 20 U/mL. All subsequent workemployedD-AAOxactivity of 15 U/mL, in viewof the risingbackground noise at higher enzyme levels. Such level

corresponds to a negligible consumption over repetitiveruns (considering the actual flow rates at these CE micro-chip platforms).The optimal pH for the enzymatic reactions may not

necessarily be the favorable one for the separation ordetection processes.A tradeoff between the efficiencyof thereaction, separation, and detection processes may berequired in selecting the pH of the running buffer. Theeffect of the pHof the run buffer is depicted in Figure 4 for amixture of four amino acids [1.5� 10�3 M arginine (a), 3.0�10�4 M each of isoleucine (b), alanine (c), and phenylala-nine (d)] in connection to various pHs [9.3 (A), 9.6 (B), and10.0 (C)] and pH 9.3 of the ™Reagent∫ (enzyme) solution.The response of the arginine peak is nearly independent ofthe pH (as these pH are above its pKa value). The isoleucineand alanine response peaks are fully overlapped at pH 9.3; apeak splitting is observed at pH 9.6. A run buffer of pH 10.0results in a baseline separation and defined peaks for thefour amino acids. Note that the pKa values of these aminoacids are lower than 10.Since the separation continues between the enzyme

mixing and detection points, these are actually on-columnreactions preceded by a separation step. The differentmobilities of the reactants and products (in the reaction™zone∫) results in increased band broadening. A balancemust be sought for obtaining efficient mixing withouthindering the separation efficiency. The influence of theseparation and post column voltages upon the efficiency of

Fig. 2. Hydrodynamic voltammograms for a mixture of 3.0�10�4 M isoleucine (�) and phenylalanine (�). Separation andpost-column voltages, 1000 V; injection voltage, 1500 V; injectiontime, 3 s; run buffer, 2.0� 10�2 M borate (pH 10.0); ™Reagent∫solution, 25 U/mL D-AAOx (2.0� 10�2 M borate buffer, pH 9.3).

Fig. 3. The effect of the AAOx level upon the response for 3.0�10�4 M of isoleucine (�) and alanine (�). Detection potential,�0.9 V; run buffer, 2.0� 10�2 M borate buffer (pH 10.); enzymesolution in 2.0� 10�2 M borate buffer (pH 9.3). Other conditions,as in Figure 2.

Fig. 4. Influence of pH upon the response for a mixture of 1.5�10�3 M arginine (a), 3.0� 10�4 M each of isoleucine (b), alani-ne (c), and phenylalanine (d). Detection potential �0.9 V; en-zyme solution 15 U/mL in 2.0� 10�2 M borate buffer (pH 9.3).The pH of the running buffer was varied: pH 9.3 (A), pH 9.6 (B),and pH 10.0 (C). Other conditions, as in Figure 2.

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the reaction is examined in Figure 5A using a samplecontaining 1.5� 10�3 M arginine (1), 2.5� 10�4 M isoleu-cine (2), and 3.0� 10�4 M phenylalanine (3). As expected,increasing the separation andpost-columnvoltages between1000 and2500 V(a tod) decreases themigration times of thesubstrates and reaction products. While pushing the sub-strates faster through the separation channel, higher fieldstrengths also shorten the residence/reaction time of thereactants, and hence impair the efficiency of the enzymaticreactions. For example, the peak current for isoleucinedecreases from 1.6 (a, 2) to 0.4 (d, 2) nA. To examine theinfluence of these effects upon the overall separationefficiency, the corresponding electrophoretic currentswere measured (and plotted in Figure 5B). This wasaccomplished by varying the post-column voltages [0.5 (a),1.0 (b), 1.5 (c), 2.0 (d), 2.5 (e), and 3.0 (f)] at a fixedseparation voltage. Maximum mixing is observed when theseparation and post-column voltages are similar, as indi-cated by the maximum current resulting from minimumresistance to the flow. These results are in consistence withthe observations of Harrison×s group [9]. While a voltage of3000 Voffered themost efficientmixing, a voltage of 1000 Vwas used for all subsequent work, as it provided the mostfavorable balance between sensitivity, resolution, and iso-lation from the detection circuitry.

The amperometric detector displays a well-defined con-centration dependence. Such dependence was examined byrecording the electropherograms for sample mixtures con-taining increasing levels of isoleucine, alanine, and phenyl-alanine in steps of 1.0� 10�4 M (Figure 6). Defined peaks,proportional to the analyte concentration, are observed.The resulting calibration plots for isoleucine, alanine, andphenylalanine (shown in the inset) are highly linear over theconcentration range studied, with resulting sensitivities of7.8, 5.1, and 5.3 nA/mM, and correlation coefficients 0.999,0.987, and 0.998, respectively. The sensitive response iscoupled to a low noise level, and hence to detection limits of20, 35 and 30 �M (based on three standard deviations of thenoise in assays of a mixture containing 1.0� 10�4 M ofisoleucine, alanine, and phenylalanine).Good precision is another attractive feature of the new

enzymatic/separation microchip protocol. A series of8 electropherograms obtained during repetitive assays of a3.0� 10�4 M isoleucine and phenylalanine solution mixtureresulted in a highly reproducible response (not shown).Relative standard deviations (RSD) of 3.9 and 4.3% wereestimated for the peak currents for isoleucine and phenyl-alanine, respectively. The migration times were also veryreproducible with RSD of less than 0.5%. Such goodprecision reflects the high reproducibility of the separa-tion/reaction/detection processes, and indicates a negligibleelectrode fouling or enzyme adsorption onto the channelwalls.

Fig. 5. A) Influence of separation and post-column voltagesupon the response for a mixture containing 1.5� 10�3 M argi-nine (1), 2.5� 10�4 M isoleucine (2), and 3.0� 10�4 M phenylala-nine (3). Separation and post-column voltage, a)� 1000, b)�1500, c)� 2000, and d)� 2500 V. B) Dependence of the electro-phoretic current upon the separation voltage at different post-column voltages (a, �) �500, (b, �) �1000, (c, �) �1500, (d, �)�2000, (e, �) �2500, and (f, �) �3000 V. The electrophoreticcurrents were measured at a fixed separation voltage whilevarying the post-column voltage. Other conditions, as in Figure 2.

Fig. 6. Portions of the electropherograms depicting the concen-tration dependence for a) isoleucine, b) alanine, and c) phenyl-alanine using successive increments of 1.0� 10�4 M (A±E). Alsoshown (in the inset) are the resulting calibration plots. Otherconditions, as in Figure 2.

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Electroanalysis 2002, 14, No. 6

4. Conclusions

We have demonstrated a new biochip strategy for perform-ing post-column reactions of ™class∫ enzymes on microchipplatforms. Such biochip assays were demonstrated for rapidand sensitive measurements of amino acids in connection toa post-column biogeneration of electroactive hydrogenperoxide. The ability to rapidly separate and quantitateamino acids on a microchip platform should find importantclinical and biotechnological applications. While the con-cept of integrated separation/bioreactor/amperometric-de-tection has been illustrated in connection to amino-acidsubstrates, it can be readily expanded to other enzymes/substrates pairs. For example, post-column additions oftyrosinase or laccase could be employed for analogousmeasurements of phenolic compounds. We are also explor-ing a multi-channel operation, involving different combina-tions of enzymes, for high-throughput measurements ofnumerous substrates. The utility of post-column enzymaticreactions in connection to on-chip enzyme immunoassays isalso being investigated. Ultimately, these (and similarefforts) will lead to self-contained and disposable chips fordecentralized testing.

5. Acknowledgements

This project was supported by the NASA, ONR andNational Institute of Health (NIH grant RO1 RR14173-02). A. E. acknowledges financial support from NATOfellowship from The European Community.

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