Analytica Chimica Acta 506 (2004) 127–132

Direct immobilization in poly(dimethylsiloxane) for DNA,protein and enzyme fluidic biochips

Christophe A. Marquette∗, Loı̈c J. Blum

Laboratoire de Génie Enzymatique et Biomoléculaire, UMR 5013-CNRS, Université Claude Bernard Lyon 1, Bˆat. CPE,43 Boulevard 11 Novembre 1918, 69622 Villeurbanne, Cedex, France

Received 20 January 2003; received in revised form 23 October 2003; accepted 7 November 2003

Abstract

A new arraying method is presented based on the properties of poly(dimethylsiloxane) (PDMS) polymer to entrap beads bearing biologicallyactive compounds. It is shown that such beads could be spotted and dried at the surface of a poly(vinyl chloride) master and subsequentlytransferred at the PDMS interface by direct moulding of the polymer on the mask. Moreover, the use of the PDMS-assisted-immobilizationenables the development of either a low density array (100 spots) or a micro-channel biochip with a direct incorporation of the sensing elementin a fluidic system for the quantitative detection of enzyme substrates, antigens and oligonucleotides, depending on the immobilized sensingelement. All biochip formats were revealed by a chemiluminescent reaction detected with a charge coupled device camera.

As a result, arrays of beads bearing active enzymes, antibodies and oligonucleotides were successfully obtained and enabled the achievementof biochips for the chemiluminescent detection of enzyme substrates, protein antigens and oligonucleotides sequence with detection limit of1�M, 1.5 × 107 molecules and 108 molecules, respectively.© 2003 Elsevier B.V. All rights reserved.

Keywords:Biochip; Chemiluminescence; Enzyme; Fluidic; Low density array; Nucleic acid; PDMS; Protein

1. Introduction

The development of biochips for the detection of nu-cleic acids, proteins or enzyme–substrate interactions hasexperienced a large increase since those tools were foundto provide to researchers and diagnostic companies a rapidsample’s screening[1,2]. Nevertheless, one of the mainhandicap for the development in this area appeared to bea lack of generic immobilisation procedure for those bio-logically active compounds; a lack linked in one hand tothe obligatory effort to keep active the immobilized biolog-ical molecules and in the other hand to the diversity of de-tection/transduction systems used. The early immobilisationprocedure on silicon and glass support based on silane tech-nology, still in use for protein and DNA immobilisation[2–5]is now in competition with thiol-disulfide techniques[6–9]on gold surface, beads array[10,11], micro-contact printing[12] and particle lithography[13]. Each of these techniques

∗ Corresponding author. Tel.:+33-472-44-8214;fax: +33-472-44-7970.

E-mail address:christophe.marquette@univ-lyon1.fr (C.A. Marquette).

are used in conjunction with a detection system, most of thetime intimately dependent of the immobilisation procedure.

The present paper proposed a new approach to the de-velopment of a general immobilisation procedure useful forenzymes, proteins and oligonucleotides biochip design. Thistechnique, based on the entrapment of micrometric beads ina poly(dimethylsiloxane) (PDMS) material—a powerful toolfor designing micro-fluidic systems[14]—open a path to thedevelopment of directly integrated active fluidic systems.

2. Materials and methods

2.1. Reagents

Biotin labelled anti-human IgG (�-chain specific), bi-otin labelled oligonucleotides d(T)22, glucose oxidase (Gox,grade I, EC 1.1.3.4, fromAspergillus niger), human IgG,luminol (3-aminophtalhydrazide), peroxidase labelled strep-tavidin, Sepharose beads bearing human IgG, Sepharosebeads bearing d(A)22 were purchased from Sigma (France).DEAE (diethylaminoethyl) Sepharose Fast Flow and IDA

0003-2670/$ – see front matter © 2003 Elsevier B.V. All rights reserved.doi:10.1016/j.aca.2003.11.015

128 C.A. Marquette, L.J. Blum / Analytica Chimica Acta 506 (2004) 127–132

(imidodiacetic acid) Sepharose Fast Flow were obtainedfrom Pharmacia. PDMS precursor and curing agent (Sylgard184) were supplied by Dow Corning. All buffers and aque-ous solutions were made with distilled demineralized water.

2.2. Bioactive beads preparation

Glucose oxidase supporting beads were prepared by in-teraction of Ni-charged IDA-Sepharose with Gox modifiedwith histidine. The histidine grafting on the Gox carbo-hydrate moiety was performed using a method previouslydescribed for horseradish peroxidase modification[18].Briefly, 5 mg of Gox dissolved in 200�l of distilled waterand 80�l of a 0.1 M NaIO4 solution were added and mixedunder stirring for 20 min. Then, 40�l of a 0.2 M histidinesolution in 0.1 M carbonate buffer, pH 9, and 480�l of0.1 M carbonate buffer, pH 9, were added to the activatedglucose oxidase and incubated 2 h under stirring. At thattime, the reaction was complete and 20�l of freshly pre-pared 0.1 M NaBH4 solution in water were added and mixedfor 15 min in order to stabilize the bounding of histidine onthe activated Gox.

The Gox-His was then separated from non-reacted speciesby desalting on a Sephadex G25-M column. Afterwards,500�l of IDA-Sepharose beads were equilibrated with30 mM Veronal Buffer, 30 mM KCl, pH 8.5, and perfusedwith 100 mM NiCl2 solution. On the Ni-IDA-Separosecolumn thus obtained, 5 mg of the Gox-His desalted as de-scribed above were deposited. The column was then washedwith 10 ml of equilibrating buffer and stored at 4◦C.

Luminol was immobilized on DEAE-Sepharose beadsby electrostatic interactions. Five hundred microliters ofDEAE-Sepharose were packed from ethanol slurry into aplastic column, equilibrated with 10 ml of 30 mM Veronalbuffer, 30 mM KCl, pH 8.5, and perfused with 1 ml of 10 mMluminol solution in equilibrating buffer. The column was

Fig. 1. Scheme for the fabrication of the low density array. (A) The Sepharose beads bearing bioactive compounds are arrayed as 0.3�l drop onto a flatPVC master. (B) A PDMS solution is poured onto the spotted array. (C) The PDMS based array is peeled off from the master and ready to use. (D)Micrograph of a typical low density array revealed by chemiluminescence. Each particular spot contains d(A)22 charged beads and were hybridized withbiotin labelled d(T)22 at a concentration of 0.5 nM.

then washed with 25 ml of equilibrating buffer and stored at4◦C.

2.3. Biochips preparation

The biochips were prepared by arraying spotting solutionsas 0.3�l drops. The spotting solutions were prepared bymixing 45�l of an aqueous solution composed of glycerol2.5%, 0.05 M NaCl with either 5�l of wet beads bearingd(A)22, 5�l of human IgG bearing beads, or 5�l of glu-cose oxidase bearing beads added of 5�l of luminol bearingbeads.

To design the low density array, 0.3�l drops were de-posited on a flat poly(vinyl chloride) (PVC) substrate ev-ery 3 mm and dried under a tungsten lamp during 30 min.The presence of glycerol in the spotting solution enables theachievement of homogeneous and smooth dry beads spots.The dry bead spots were then transferred to the PDMS in-terface by pouring a mixture of precursor and curing agentonto the PVC substrate and cured for 90 min under a tung-sten lamp. The biochip preparation was then terminated bypeeling off the PDMS polymer (Fig. 1).

In a similar way, the active fluidic biochip was preparedby depositing 0.3�l drops of the different spotting solu-tions (see above) on a PVC master composed of 12 channels(w:2.5 mm, h:1 mm, l:15 mm), prepared by sealing differ-ent PVC parts together with epoxy glue, and following thesame protocol as presented above. Peeling off the PDMSsubstrate and covering it with a flat PVC surface leads tothe achievement of 12 active macro channels (Fig. 3).

2.4. Nucleic acid assay

As a model oligonucleotide hybridization process, ad(T)22–d(A)22 couple was used. The low density arraysand the active macro-fluidic systems were assayed with

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the same general protocol. Briefly, the biochip or a partic-ular channel were saturated during 30 min by a solutionof buffer A (30 mM Veronal (diethylbarbiturate), pH 8.5containing 30 mM KCl, 0.2 M NaCl, 0.1% tween and 1%BSA), then incubated for 1 h with biotinylated d(T)22 ata particular concentration in buffer A. The biochip or aparticular channel were then washed 10 min with bufferB (30 mM Veronal, pH 8.5 containing 30 mM KCl, 0.2 MNaCl), afterwards saturated with buffer A during 20 minand then incubated with peroxidase-labelled streptavidinat a 1�g/ml concentration during 30 min. The array or aparticular channel were then washed 20 min with buffer Band was then ready to be measured.

2.5. Protein biochip assay

In a way similar to that used for the nucleic acid biochip,the protein biochip was saturated during 30 min by a solu-tion of buffer A, then incubated for 1 h with biotinylatedanti-IgG antibody at a particular concentration in buffer A.The biochip was then washed 10 min with buffer B, saturatedwith buffer A during 20 min and then incubated with perox-idase labelled streptavidin at a 1�g/ml concentration during30 min. The array was then washed 20 min with buffer Band was then ready to be measured.

2.6. Biochips measurement

The ready-to-measure nucleic acid and protein biochipswere bring into contact with a buffered solution (30 mMVeronal, 30 mM KCl, pH 8.5) containing in addition200�M luminol, 500�M hydrogen peroxide and 20�Mp-iodophenol. At that time, the arrays were introduced inthe charge coupled device (CCD) light measurement system(Intelligent Dark Box II, Fuji Film) and the emitted lightintegrated during 3 min.

The enzyme substrate biochip was used in a similar way;a buffered solution (30 mM Veronal, 30 mM KCl, pH 8.5)containing a particular concentration of glucose and 200�Mof the triggering agent (1,3-dichloro-5,5-dimethylhydantoin)was injected in each channel just before the introductionof the biochip in the measurement system, and the signalintegrated during 3 min.

The biochip pictures obtained were quantified with a Fujifilm image analysis program (Image Gauge 3.12).

3. Results

3.1. Low density arrays

Low density arrays were prepared by spotting aqueous so-lution composed of glycerol 2.5%, NaCl 0.05 M containing1:10 (v/v) of wet modified Sepharose beads at the surfaceof a PVC substrate, drying the spots under a tungsten lampand pouring a PDMS solution on it. After the removal of the

solidified PDMS, bead emergence appeared on the surfaceof the polymer corresponding to the spots deposited (Fig. 1).No beads residue were observed on the PVC master after theremoval of the PDMS layer, demonstrating the high transferefficiency of the beads to the PDMS interface. Those beadislands were then able to anchor active bio-molecules at theinterface, through the Sepharose beads (Fig. 2).

The low density array format was used to design pro-tein and oligonucleotide biochips based on Sepharosebeads bearing human IgG or d(A)22 homo oligonucleotide.Fig. 1 presents the immobilisation process and a mi-crograph of a 100 d(A)22 spots array revealed with astreptavidine–peroxidase conjugate after an hybridizationof the entire biochip with a biotin labelled d(T)22. A 8%standard deviation of the chemiluminescent signal wasfound within the array. Similar results were obtained whenincubating the protein array, composed of immobilized hu-man IgG, with biotin labelled anti-human IgG antibodies.The present biochip preparation procedure appeared thento enable the achievement of homogenous array of the twomost studied types of bio-molecules. Moreover, changingthe properties of the beads used could lead to a larger scaleof immobilized bio-molecule. Thus, oligosaccharide[15] orkinase[9] substrate could be easily immobilized and usedas analytical tools.

The non-specific signal generated by the non-specific ad-sorption on either the PDMS or the Sepharose material wereevaluated by preparing a biochip with unmodified beads(not bearing any bioactive compound) and running the pro-tein or nucleic assay in the presence of a maximum amountof labelled antibody or labelled nucleic acid (100�g and7 pmol, respectively). An extraordinary low non-specific sig-nal, close to the limit of the instrumentation sensitivity, wasfound.

The PDMS assisted biochip demonstrates here its greatpotential capabilities in array technology, since a low stan-dard deviation and an almost non-existing background wereobserved.

3.2. Quantitative measurement with the active fluidicbiochip

The macro-fluidic biochips were prepared by spottingthe bioactive bead solution on a three-dimensional masterleading to the achievement of 2.5/1/15 mm (w/h/l) channels(Fig. 3). The different solutions used in each assay formatswere manually introduced in the channels under vacuumpressure, and stand to react during a stop flow duration (asdescribed in theSection 2).

A first study was performed to evaluate the ability ofthe biochip to quantitatively detect nucleic acid. Sampleswith different biotin labelled d(T)22 concentrations were in-jected in each particular channel, hybridized and revealed.Fig. 4A presents the evolution of the chemiluminescent sig-nal obtained as a function of the oligonucleotide amount in-troduced in each channel. As a matter of fact, the nucleic

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Fig. 2. The different possibilities of the active PDMS biochips. (A) A Sepharose bead bearing oligonucleotide emerges from the PDMS surface. Abiotinylated tracer is hybridized to the immobilized nucleic acid and revealed with a peroxidase labelled streptavidin. (B) A protein modified Sepharosebead is included in the PDMS polymer. A biotin labelled antibody reacts with the immobilized antigen and is revealed with a peroxidase labelledstreptavidin. (C) An IDA-Sepharose bead bearing glucose oxidase (Gox) and a luminol-charged bead emerge from the PDMS surface. The Gox oxidizesthe glucose present in the reaction medium, generating H2O2 which in turn, reacts with the immobilized luminol to produce light.

acid biochip enables the quantitative detection of biotiny-lated oligonucleotide amounts in the range 58 pM–23 nM.Compared to the detection limits obtained with fluorescence[8,11] or radiolabeled-based[3] systems, involving gener-ally highly complex micro array designs, the present methodexhibits a slightly lower sensitivity but a larger detectionrange. The present detection limit of 58 pM corresponds toan amount of 1010 molecules in a 300�l assay volume.Nevertheless, when using the biochip in a low density array

Fig. 3. Scheme for the fabrication of the active fluidic biochip. Sepharose beads bearing bioactive compounds are arrayed as 0.3�l drop onto a PVCmaster. (B) A PDMS solution is poured onto the spotted array. (C) The PDMS based array is peeled off from the master and ready to use. (D) Micrographof a typical active fluidic biochip revealed by chemiluminescence. Each particular channel is incubated with biotin labelled d(T)22 at a concentration of(a) 20 nM, (b) 10 nM, (c) 5 nM, (d) 1 nM, (e) 0.5 nM, (f) 0.1 nM, (g) 0.05 nM.

format, a 3�l sample volume could be used, lowering thenumber of nucleic acid molecules detected to 108, which isclose to the best results obtained in previous works[3,8,11].

In a next study, the performance of the protein array todetect free antigen in a competition format was evaluated.Samples containing human IgG at different concentrationswere incubated in a particular channel, together with biotinlabelled anti-IgG antibodies. The chemiluminescent readingof the biochip leads to the obtaining of the competition curve,

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Fig. 4. Performances of the PDMS-based biochip. Dose–response andmicrograph of (A) the nucleic acid biochip (B) the protein biochip and (C)the enzyme biochip. The biochips were measured during 3 min and theimage quantified with a Fuji film quantification program. B0 representsthe light intensity obtained in the absence of free IgG and B the lightintensity obtained in the presence of a particular IgG concentration.

which a linearization by the “logit” function is presented onFig. 4B.

logit = lnB

B0 − B

whereB is the signal obtained at a fixed free antigen con-centration andB0 the signal obtained in the absence of freeantigen.

The protein biochip allows the detection of free antigenwith a detection limit of 12 ng/ml and a detection range overfour decades at least. This amount corresponds to 1.5× 109

molecules in the active fluidic format and could be lowered,as above, in the macro array format to 1.5× 107 molecules.

To demonstrate all the potentialities of the active macrofluidic biochip, an enzyme-based system was designed(Fig. 1), in which chelating beads bearing glucose oxidase[15] were co-entrapped at the PDMS interface with luminolcharged beads. In the reaction sequence, the glucose oxi-dase catalyses the oxidation of glucose and the productionof hydrogen peroxide which reacts in the presence of a trig-gering agent[17] with the immobilized luminol to generatelight. It has been previously shown that this configuration,with a different immobilisation system[16], enabled the re-alization of sensitive biosensor array without contaminationproblem between the different spots. Samples containingglucose at different concentrations were injected in thebiochip and the light intensity produced was measured con-comitantly on the entire biochip. The obtained calibrationcurve is presented inFig. 4C. Again, the performances ofthe PDMS biochip appeared to be competitive with regardto the sensitivity and the linear range presented in previousreports dealing with chemiluminescent detection of enzymesubstrate[16,18–21]. Moreover, we demonstrated in a pre-vious study that six different oxidase enzymes could be usedat the same time under the same experimental conditions,allowing to obtain a six-parameter biosensor. The presenttechnology could then be transferred in a close future to thedesign of PDMS based enzyme substrate biochip for thedetection of multiple parameters.

4. Conclusion

PDMS as an immobilisation matrix has been successfullyused to design multi-purpose biochips i.e. for either nu-cleic acids, proteins or enzymes. It is shown that the abilityof the PDMS-assisted immobilisation to generate homoge-neous spot arrays exhibiting very low non-specific adsorp-tion of biological compounds is one of the key path for ageneric biochip production. Indeed, the present spotting pro-cedure enables the use of a large scale of immobilizationprocedures and chemistries, and of chemical synthesis onbeads, prior to the spotting.

Moreover, the use of PDMS material allows to considerthe design of micro-fluidic systems integrating some sens-ing elements directly trapped at the macro-channel/flowingsolution interface. A first step to those systems has beenpassed with the presented macro-fluidic systems integratingnucleic acids, proteins or enzymes. The performances of thepresent analytical systems are competitive with previouslydescribed methods[3,8,11,16,18–21].

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