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Journal of Chromatography A, 1135 (2006) 122–126

Glucose microfluidic biosensors based on immobilizing glucose oxidasein poly(dimethylsiloxane) electrophoretic microchips

Qing Zhang, Jing-Juan Xu, Hong-Yuan Chen ∗Key Lab of Analytical Chemistry for Life Science (MOE), School of Chemistry and Chemical Engineering,

Nanjing University, Nanjing 210093, China

Received 20 July 2006; received in revised form 7 September 2006; accepted 14 September 2006

bstract

Here we reported a novel microfluidic biosensor with an on-column immobilized enzyme microreactor. The fabrication approach of this biosensors simple and the enzyme microreactors with controlled sizes can be placed at any desired position on the microchip. Taking glucose oxidase (GOx)s an example, electroosmotic flow (EOF) as a driving force and amperometry as a detection method, the performance of biosensors were modulated

y changing the length of enzyme reactor from 0.5 cm to 3 cm, and the linear ranges were changed from 0–8.0 mM to 0–30.0 mM with the detectionimits from 42 �M to 6.5 �M. The enzyme reactor remained its 65% activity after 23 days storage. It also showed good anti-interference abilitynd was used to quantify glucose in human serum samples.

2006 Elsevier B.V. All rights reserved.

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eywords: Microfluidic biosensor; Glucose oxidase; Poly(dimethylsiloxane) m

. Introduction

Enzyme-based microchip (microfluidic-based) devices haveather significant attention in recent years [1–10]. Among theseevices, the electrophoretic microchips based on immobilizednzymes have their special advantages [11–13] such as elimina-ion of the influence from interference by electrophoresis, fastransformation of analytes measured difficultly to easily measur-ble form, improved storage stability of enzymes and the abilityo avoid the contamination of product with enzymes. In spitef their advantages, there are several issues that restricted theirpplications. One of the most serious issues is the difficulty inontrolling the position and the size of the patch of immobilizednzymes. The few successful strategies mainly include photoat-achment chemistry [14] and photopolymerization [15,16].

Previously, we have reported a simple approach for pattern-ng microbeads on poly(dimethylsiloxane) (PDMS) microchip

nd its application for binding proteins in microchannels[17].t makes use of the elastic properties of PDMS and is availableo achieve the region selective immobilization of enzymes on

∗ Corresponding author. Tel.: +86 25 83594862; fax: +86 25 83594862.E-mail address: hychen@nju.edu.cn (H.-Y. Chen).

021-9673/$ – see front matter © 2006 Elsevier B.V. All rights reserved.oi:10.1016/j.chroma.2006.09.052

chip

microchips. In this paper, we developed a convenient and reli-able microfluidic glucose biosensor by immobilizing GOx on themicrochips based on this approach. This biosensor was appliedto determine glucose in human serum and the results showedgood agreement with the hospital assay results. Moreover, theperformance of the biosensor can be conveniently modulated bychanging the length of enzyme reactor.

2. Experiments

2.1. Materials and reagents

PDMS prepolymer (Sylgard 184) was from Dow Corning(Midland, MI, USA). GOx, ascorbic acid, sodium cyanoboron-hydride and fluorescein isothiocyanate-labeled bovine serumalbumin (FITC-BSA) were purchased from Sigma (St. Louis,MO, USA). Glucose was from Shanghai Bio Life Science &Technology (Shanghai, China). Spherical amino (NH2)-silica(5 �m) was from Akzo Nobel (Bohus, Sweden). Glucose stocksolutions were allowed to mutarotate overnight before use. All

other chemicals were of analytical grade and used withoutfurther purification. All solutions were prepared with doublydistilled water and passed through a 0.22 �m cellulose acetatefilter.

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.2. Apparatus

The microchip was placed on a homemade Plexiglas holderntegrated with a precisely three-dimensional adjustor [18]. Aomemade power supply was used to provide high voltage.oles with a diameter of 3 mm were punched into PDMS chips

nd acted as reservoirs. Platinum electrodes were inserted intohe reservoirs for the application of the high voltage. Ampero-

etric detection was performed with an Electrochemical Work-tation 660A (CH Instruments, Austin, TX, USA). A carbonbre cylindrical electrode with a diameter of 8 �m was used asworking electrode, combining an Ag/AgCl reference electrodend a Pt wire auxiliary electrode to construct a three-electrodeystem (Fig. 1). The working electrode was placed close to theutlet of the separation channel and the position was adjustednder a stereoscopic microscope equipped with microruler.

.3. Microfabrication process

The fabrication procedure of masters and immobilizednzyme reactors were performed according to a slight modi-cation of our previous works [17,19].

.3.1. Fabrication of mastersBriefly, the outline of microchannels was designed with a

raphic software, and then printed out on a transparency film byn HP 4050 laser printer. Then the transparency film was cut touitable size and baked at 105 ◦C for 20 min. The layout of theicrofluidic network was shown in Fig. 1 with a sample channel

150 �m wide, 16 mm long) and a separation channel (200 �mide, 35 mm long). The height of the channels was identical for

a. 10 �m.

ig. 1. Schematic diagram of the experimental setup: (PS) potentiostat, (HV)igh voltage, (PC) personal computer, (WE) working electrode, (CE) counterlectrode, (RE) reference electrode, (GE) ground electrode, (BR) buffer reserv-or, (SR) sample reservior, (SW) sample waste, (S1)–(S3) adjusting screws inlexiglass. The scale using for the channel length in the figure was in millimeters.

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. A 1135 (2006) 122–126 123

.3.2. Fabrication of microchips with immobilized enzymeeactors

Briefly, a slurry of microbeads (6 mg microbeads in 200 �lethanol) was ultrasonicated for 20 min. The suspension was

ropped on the master to cover the desired position of theoner, and the master was then put in an oven at 105 ◦C formin. Then the master was taken out and carefully washedith water. A mixture of PDMS monomer and its cure agentas cast on the master and cured for 1 h at 80 ◦C. Then theDMS microchip peeled off from the master was ultrasoni-ally washed orderly with ethanol and doubly distilled waterach for 10 min, and dried under infrared lamp. After that, 5%v/v) of glutaraldehyde in 20 mM phosphate buffer (pH 7.0) wasropped on the PDMS microchip, covering the region of immo-ilized amino-functionalized microbeads. After a 2 h reaction atoom temperature, the chip was carefully washed with phosphateuffer to remove the excess glutaraldehyde. Then a GOx solution8 mg/ml) were dropped on and incubated overnight at 4 ◦C inhe presence of sodium cyanoboronhydride. After the reaction,he chip was washed and dried at room temperature. Finally, itas sealed with a clean PDMS substrate at room temperature

nd stored in buffer solution at 4 ◦C before use. To observe themmobilized microbeads on PDMS under a DMIRE2 invert fluo-escence microscope (Leica, Germany), FITC-BSA was bondedo the microbeads through the same process described above.

.4. Electrophoresis procedure

The running buffers used in the experiments were phosphateuffer (20 mM, pH 7.0) and sodium tetraborate buffer (20 mM,H 8.5). Before detection, the enclosed PDMS chip was filledith the running buffer by vacuum and flushed under an elec-

ric field until the separation and injection currents leveled off.ampling mode was simple crossing. For enzymatic assays,lucose solutions were added in sample reservior and electroki-etically injected into the microchannels. For serum analysis, theerum should be treated to remove proteins before it was injectednto the microchip since proteins can be strongly adsorbed onDMS surface. The procedure is as follows: Blood samples wasixed with acetonitrile (V/V, 1:3) and centrifuged at a speed

f 6000 r/min for 8 min, then the supernatant serum was trans-erred to a vial. After acetonitrile was removed by N2 stream,he remnant was dissolved in running buffer and filtered through0.22 �m cellulose acetate filter. Then it was kept at 4 ◦C beforese.

. Results and discussion

.1. Fabrication of the immobilized enzyme reactors

The fabrication approach profits from the elastic propertiesf PDMS and the easily functionalized surface of silica beads.he location of entrapped beads acting as the support of enzymes

an be easily controlled by spotting the slurry of beads at desiredosition on the separation channel. The length of the immobi-ized beads region was determined by the diameter of the spot,he low limitation of which is 0.5 mm using a 10 �l pipette tip.

124 Q. Zhang et al. / J. Chromatogr. A 1135 (2006) 122–126

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ig. 2. (a) Optical (left, A and C) and corresponding fluorescence images (righoncentrations in A and C were 1 mg/ml and 30 mg/ml, respectively. The white dicrobeads bonded with FITC-BSA. (b) Optical image of microchannels with

nd the width of the region was controlled by the width of theeparation channel (200 �m).

The concentration of microbeads slurry controlled the cov-rage degree of microbeads immobilized on the surface oficrochannels. The coverage degree increased with the concen-

ration of the slurry (Fig. 2). When the concentration is higherhan 30 mg/ml, the coverage of microbeads reached a saturatedtate. As we mentioned above, the present method is very facileo immobilize enzymes to a desired position and available touild multiple reactors in a single channels. Fig. 2b showed aicrochannel integrated with four reactors.

.2. Performance of the immobilized enzyme reactors

When a sample plug of glucose passed the reactor, it wasnzymatically oxidized with dissolved oxygen in the bufferolution and formed hydrogen peroxide, which can be detectedt a carbon fiber electrode. A typical electropherogram oflucose was shown in Fig. 3a. Biosensors with four differentength of enzyme reactors were fabricated. The linear rangesnd detection limits were compared in Table 1. From the resultst can be seen that the increase in reactor length results in the

ncrease of both sensitivity and linear region. Considering annzyme-immobilized region with the same length of a samplelug as a unit microreactor, the whole immobilized enzymeeactor is a sum of many unit microreactors with identical

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able 1ummary of the properties of biosensors with different long enzyme reactor

eactor length (cm) Linear range (mM) Detection limit (�M

.5 0–8 42

.0 0–15 22

.0 0–20 7.9

.0 0–30 6.5

ondition: separation voltage: 650 V; sample injection: at 200 V for 3 s.

nd D) of the immobilization of microbeads with different concentrations. Theoptical images and the green dots in fluorescence photos were the immobilizedreactors. The white part in the image represents the enzyme reactors.

oncentration of enzyme and oxygen. The reaction time oflucose in the whole microreactor, therefore, is dependentn the length of the whole reactor (l) and the electroosmoticelocity (νeo): t = l/νeo, where νeo = L/teo. L is the length of theeparation channel and teo the migration time of the EOF. Thenhe reaction time can be rewritten: t = lteo/L. It is increasedith the length of the microreactor, and the value is listed inable 1.

The sensitivity of the biosensors is not only dependent on theensitivity of the working electrode to H2O2 but also dependentn the reaction time, which determine the amount of H2O2.nder the same experimental conditions, the sensitivity of theorking electrode to H2O2 is almost constant. Thus whethercertain concentration of glucose is detectable is dependent onhether it can produce enough H2O2. For a certain concentrationf glucose, it can be oxidized to produce more H2O2 throughlonger reactor due to the corresponding longer reaction time.herefore, the lower detection limit of the biosensor decreasesith the increase of the microreactor length. On the other hand,

he longer reactor can support more glucose to be oxidized toroduce H2O2, which induces the increase of the upper detectionimit, thus the linear range.

It should be pointed out that the length of microreactor onlylightly changes the EOF (Table 1). Since glucose and hydrogeneroxide are neutral molecules, their migration time is the sames that of the electroosmotic velocity. This property favors the

) Migration time (s) Reaction time in microreactor (s)

76 1178 2283 4790 77

Q. Zhang et al. / J. Chromatogr. A 1135 (2006) 122–126 125

Fig. 3. (a) Electropherogram of 1 mM glucose passing the enzyme immobilizedmicrochip. Conditions: separation voltage: 650 V; sample injection: at 200 Vfor 3 s; the detection potential: 1.0 V; running buffer: phosphate buffer (20 mM,pH 7.0). (b) Electropherogram of a mixture of 1 mM glucose (Glu) and 0.4 mmasp

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Table 2Concentration of glucose in human serums

Sample Concentration of glucosein serum (mM)

Reference valuea

(mM)

1 8.1 8.52 6.2 5.33 4.6 5.14

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scorbic acid (AA) passing the enzyme immobilized microchip. Conditions:eparation voltage: 1250 V; sample injection: at 200 V for 3 s; the detectionotential: 1.0 V; running buffer: sodium tetraborate buffer (20 mm, pH 8.5).

se of microreactors with different length and avoids the peakroadening in long microreactors.

.3. Reproducibility and stability

The operational stability of the immobilized enzyme wasnvestigated by consecutively injecting a glucose solution overh. There is no obvious activity loss of the immobilized enzymefter 50-times assays. The storage stability of the immobilized

nzyme was also examined by injecting a glucose solution. Theurrent response remained about 85% of its original value afterhe chip was stored in buffer at 4 ◦C for 1 week, and remained5% activity after they were stored for 23 days. The chip-to-chipeproducibility was 5.3% (n = 3).

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a Reference values are cited from those obtained with ONETOUCH SureStreplus in the second hospital affiliated to Nanjing Medical University.

.4. Interference

The interference from ascorbic acid, the main interferent inerum, can be eliminated by electrophoretic procedure becauset exist as an anion form in neutral or alkaline solution and hasifferent electrophoretic mobilities from glucose. The electro-herogram of a mixture of ascorbic acid and glucose using anlkaline buffer solution (sodium tetraborate buffer (20 mm, pH.5)) was shown in Fig. 3b. It can be seen that the migration timef ascorbic acid was much longer than that of glucose. In a neu-ral buffer solution (phosphate buffer (20 mM, pH 7.0)), ascorbiccid even cannot be transported to the working electrode, sincehe lower pH decreased the electroosmotic flow, which was notast enough to carry ascorbic acid to the detection cell.

.5. Application for glucose determination in human serum

The microchip electrophoretic biosensor was used to deter-ine the glucose concentration in human serum to investigate its

easibility for biological sample analysis. Serum samples fromour persons were analyzed. The glucose in the serum samplesas quantified by comparing versus a calibration curve and theata were the mean of three assays (RSD = 5.4%). The resultsere shown in Table 2, which were in agreement with thosebtained using a commercial glucose meter.

. Conclusion

We developed a novel microfluidic biosensor for enzyme-ase assays, using glucose oxidase as a model enzyme. Thisiosensor is easy to be fabricated as well as stable and durableo be used. And since the microchip with immobilized enzymeeactor was constructed based on a cheap polymer material,ts cost was very low and can be used one-off. Moreover, theength of the immobilized enzyme reactor can be controlledonveniently, thus biosensors with different linear ranges can bebtained. The application of this biosensor was demonstratedy using it for quantitative determination of glucose in humanerum. The results have shown that it is an attractive alterna-ive for clinical bioassays. The studies using other enzymes arender going.

cknowledgement

The authors are greatly thankful for the financial sup-ort of the National Natural Science Foundation of China

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NSFC, No. 20475025, 20575029, 20635010, 20521503,0435010).

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