Ž .Applied Surface Science 164 2000 246–251www.elsevier.nlrlocaterapsusc

Use of microtechnology for DNA chips implementation

E. Souteyrand), J.P. Cloarec, J.R. Martin, M. Cabrera, M. Bras,J.P. Chauvet, V. Dugas, F. Bessueille

Laboratoire d’Ingenierie et Fonctionalisation des Surfaces ‘‘IFOS’’, UMR 5621 du CNRS Ecole Centrale de Lyon,´36 AÕenue de Collongue, 69130 Ecully Cedex, France

Keywords: DNA chips; Silicon; Hybridisation; Microtechnology

1. Introduction

The nucleus of every living cells holds chromo-Ž .some constituted of Desoxyribo Nucleic Acid DNA

and proteins. The DNA molecules bear the wholegenetic information of the individual species troughthe genes. For instance, human genome concernsabout 80 000 genes and access to genetic informationis ultimately limited by the ability to screen DNAsequence. A great program called ‘‘human genomesequencing project’’ would ending in the year 2000.To decipher base per base of the human genome is amilestone in the knowledge of molecular genomics,but next advances will require studies about geneexpression monitoring, genotyping or sequence anal-ysis. In this context, DNA chips are considered aspowerful tools, allowing massively parallel geneanalysis. Thanks to these devices, the ability toobtain rapid identification of human, animal or plantpathogens or to detect genetic disorders is now real-istic. These studies find broad applications in differ-ent fields such as biomedical research, diagnostic,pharmacogenomics, pharmacology, environment, orfood industry. One challenge for the implementation

) Corresponding author. Tel.: q33-472-18-62-35; fax: q33-478-33-15-77.

Ž .E-mail address: souteyra@ec-lyon.fr E. Souteyrand .

of these devices is to gather various competenciesissued from molecular biology, surface science,chemistry, microtechnology, and data processing.

2. The DNA world

The DNA is a macromolecule made of two poly-deoxynucleotide chains rolled in a helical structureaccording to the Watson and Crick model. The twostrands are constituted of a great number of nu-

Ž .cleotides. Fig. 1 . Each nucleotide is formed with aphosphoric acid and a desoxyribose sugar on which

Ž .is attached one of the four bases: A Adenine , TŽ . Ž . Ž .Thymine , C Cytosine and G Guanine . Thesebases are complementary by two, through to threehydrogen bonds in the C–G base pair and two in theT–A base pair. As a consequence, the two singleDNA strands are associated thanks to the comple-mentary base pairing principle: this is the hybridisa-tion process.

Genetic information is contained in the successionof different nucleotides. A sequence of nucleotideresponsible for character is called gene. Each gene ischaracterised by its own sequence and its location onthe DNA strand.

In fact, oligonucleotides are currently built inporous glass column owing to a synthesiser. Forinstance, from the phosphoramidite chemistry, DNA

0169-4332r00r$ - see front matter q 2000 Elsevier Science B.V. All rights reserved.Ž .PII: S0169-4332 00 00343-3

( )E. Souteyrand et al.rApplied Surface Science 164 2000 246–251 247

Ž . Ž .Fig. 1. a The double helix structure of DNA. b Principle ofbase pairing.

synthesis process includes six steps wherein four arerepetitive steps:

Ø First step consists to fix the 3X-OH from terminalnucleoside on the substrate via an ester or succi-

Žnate function surface of solid support have been.previously treated to react .

Ž .( a Deprotection step aims to remove protect-ing group to active nucleotide for next cou-pling. Classically, this deprotection is obtainedunder chemical treatment: for instance, DMTgroup largely used as protection can be taken

Ž .off by TCA TriChloroAcetique acid treat-ment.Ž . X

( b Coupling step concerns the addition of 3activated phosphoramidite for reacting on 5X

end fixed nucleotide. As a function of cycleand the sequence to be built, nucleosides differfrom one another in carried base. One cyclecorresponds to introduction of one type ofbase.Ž .( c Capping step is useful to prevent defect inthe oligonucleotide building. If some nucleo-sides have not reacted during coupling step,growth of these sequences must be stopped,and an acetylation process hinders them toreact in next cycles. These sequences are thusaborted.Ž .( d Oxydation step aims to stabilise the inter-nucleotide phosphodiester bond by transform-ing unstable trivalent phosphor into stable pen-tavalent phophor.

Ø A total deprotection step with NH4OH removesall protecting groups on heterocyclic bases and

yields free the 5X and 3X OH terminals. Oligonu-cleotides are then ready for eventual hybridisationprocess.

Synthesis of oligonucleotides of 25-base lengthŽŽ . Ž . Ž . Ž ..requires 25 cycles of the a , b , c , d steps and

only one total deprotection. At the end of synthesis,oligonucleotides are cut off the column and thenselected by electrophoresis.

3. What are DNA chips?

DNA chips are devices associating the specificrecognition properties of two DNA single strandsthrough hybridisation process with the performancesof the microtechnology. The use of microtechnologyand robotics allows obtaining more reproduciblemassively parallel analysis device for lower cost.

DNA chips are constituted of solid supports onwhich well-defined areas are designed. Each area

Žcontains a type of known molecular probes cur-rently single strand DNA sequences, oligonucleo-

.tides or gene fragments . These probes are able torecognise selectively its complementary strands toform a duplex. When DNA chips is put in contactwith solution containing different unknown targets,the hybridisation occurs only on the areas where thecomplementary strands are present. The detection ofthese areas on the solid support allows to identify thetargets. Currently the detection of hybridisationevents on the support is performed by using markers

Ž .previously fixed on all the targets Fig. 2 .In the literature, the ‘‘Gene chips’’ or ‘‘DNA

chips’’ terminology is employed in a wide way andincludes macroarrays and microarrays. Standard defi-nitions are not yet clearly exposed. Generally, thedifference between macro and microarray concernsthe number of active areas and their size. Macroar-rays correspond to devices containing some tensspots of 500 mm or larger in diameter and made byhand or by using standard robots. Microarrays gen-rally concern devices containing thousands spots ofsize less than 500 mm. Their fabrication requiresspecial robotic equipment.

DNA chips mean device containing high densityŽ .of active areas several hundreds of thousands spots

( )E. Souteyrand et al.rApplied Surface Science 164 2000 246–251248

Fig. 2. Principle of DNA chips: on solid support, patchwork of active areas are arrayed. Each area contains one type of oligonucleotidesŽ . Žsingle DNA strands as probes. DNA chips are dipped into a solution containing targets to be detected various single DNA strands

.carrying markers . When targets are complementaries with probes immobilised on surface, hybridisation process occurs. By scanning thesurface, thanks to the markers, hybridised units can be located.

of tens of micrometer in diameter. Taking into ac-count the small size of each active area to be formedand the high number of spots to be made, DNA chipsfabrication needs the use of tools issued from mi-crotechnology.

4. Elaboration process

There are several steps in the implementation ofDNA chips:

Ø choice of the substrate: glass, silicon substrate,etc.

Ø chemical surface preparationØ choice of probes and designØ chip fabrication requiring to address reagents at

precise location on the supportØ hybridisation processØ readout processØ software: data processing, image processing,

bioinformatics

Many options for each step and many possiblecombinations can be considered. Characteristics of

ŽDNA chips size, number of active area, probes,.design, etc. are depending on the targeted applica-

tions and will be determining factors in the choice ofthe technology ways used for implementation.

Whatever the size of DNA chips, the most cur-rently used support is glass slide because of its lowcost and its transparency. This last point is importantfor the detection of hybridisation on the active facein contact with solution. The detection by fluores-cence spectroscopy coupled with confocal micro-scope can be performed in closed cell through rearface. Numerous fluorescent markers as fluoresceine,rhodamine Cys3, Cys5 can be directly fixed on DNAsingle strand targets. A double labelling techniquehas also been developed: DNA targets carry onlybiotin molecules whose small size does not perturbthe hybridisation process. After hybridisation oc-curred, the formed duplexes were fluorescent la-belled with streptavidinphycoerythrin conjugatesthanks the strong affinity between streptavidin and

w xbiotin 1 . Hybridisation detection by fluorescence issensitive technique with high spatial resolution, semiquantitative but requires to avoid the quenching andblenching phenomena. The detection by using ra-dioactive labels as 125I, 32 P or 33 P is a more quantita-tive method but the weak spatial resolution, thesafety and waste disposal inherent problems haveturned off studies towards alternatives solutions.Several detection techniques based on mass spec-

( )E. Souteyrand et al.rApplied Surface Science 164 2000 246–251 249

troscopy, optical principle or electrochemical reac-tions have been studied. For instance, electrochemi-cal techniques as cyclic voltammetry and chrono-coulometry using redox active molecules were widelyemployed in the case of DNA sensors made of

Ž . w xcarbon substrate or metallic electrodes Au, Pt. 2 .The application of the electrochemical detection

to DNA chips constituted with several hundredsspots implies a complex multiplexing device, thecontrol of electrical insulating between electrodes ata micrometer scale and the guarantee of any electro-chemical interference. This device has not yet beendeveloped.

Whatever the used techniques for reading out ofthe hybridisation, note that a limiting effect for thesensitivity and the quantification is due to the possi-ble adsorption of no complementary targets on thesurface without any hybridisation process. Carefuland strong washes are required to improve stronglythe signal-to-noise ratio.

This means that another key point is the surfacepreparation aiming either to bind robustly the singleDNA strand as probes on the support or to active thesurface for starting in situ DNA synthesis. Chemicaltreatment must be chosen as a function of the usedmaterials. For instance, on gold electrodes, oligo-nucleotides were previously modified with thiol ter-minated linkers and then deposited on electrodes for

Ž24 h. Deposition of silane polymer APTS, APMS,.GPTS, etc. with adequate terminal reactive function

is generally performed. Drawbacks lie in the weakmonitoring of polymerisation reaction. This leads toa no reproducible immobilisation of oligonucleotidesin terms of quantity yielding impossible a precisequantification of biological phenomena. Moreover,poor quality of polymer grafting forgets the reuse ofthe DNA chips.

5. Two main strategies for the fabrication of DNAchips

For low and medium number of spots, challengeconsists in robust and reproducible immobilisation ofalready synthesised oligonucleotides combined withprecise addressing device. This method presents ad-vantages to employ purified and well-defined oligo-nucleotides. For macroarray, the located deposition

of biological materials can be made by micropipetteor by commercial robot spotting. For microarray, thenumber of spots must be highly increased and areaof every active spot must be diminished. To implantsuch device in fair time, commercial but more oftenhomemade high-speed robots are used. These robots

Ž .dispense nanovolumes of reagents 100 nl–35 pland are based on various technologies such as ondrop touch system, automatic nano-pipette, injector,micro-valves or piezoelectric injecting device.

For high-density chips, in situ synthesis of oligo-nucleotides directly on the support is necessary toavoid the use of thousands bottles of reagents.

Elaboration of high-density DNA chips performedw xby Affymetrix 3 is based on an original idea com-

bining the photolithography process with the DNAsynthesis cycles. In this case, similar as above de-scribed DNA synthesis is employed but DMT pro-tecting group is replaced by photosensitive protect-ing function. The deprotection step is effective onlyunder lighted area. The use of photolithographicprocess with adequate masks allows for each photo-chemical synthesis cycle to precisely define areas tobe reactive for next coupling step. Thus, all possiblecombinations to build a given length DNA sequenceare from the four primary bases by this process witha set of appropriate masks. For instance, they are48 s 65 536 different possibilities of 8-mer se-quences. All these oligonucleotides can be realiseddirectly on glass support owing to 4=8s32 cyclesof synthesis with only 4 masks. The first high-den-sity gene chips have been performed on 1.28=1.28cm2 substrates: each active area corresponds to a50-mm size.

Direct in situ oligonucleotide synthesis on supportw xis also developed by Southern’s group 4 , by a

w xFrench team at Ecole Centrale de Lyon 5 and byw xProtogen 6 . This last one has adapted ‘‘ink jet’’de-

vices coupled with homemade synthesiser. The mainproblem is to well monitor the addressing of reagentsat located sites, to avoid the spreading of reagentdrop and contamination and to slow down the sol-vent evaporation for giving time for chemical reac-

Ž .tions coupling step to be occurred. To confine thereagent drops, Protogen designs active areas by usualphotolitographic process, and applied a chemicaltreatment to provide a hydrophilic character. Thesurrounding inactive areas were previously yielded

( )E. Souteyrand et al.rApplied Surface Science 164 2000 246–251250

hydrophobic. Drop ejection of reagents by piezoelec-tric devices allows a large versatility to make simul-taneously various oligonucleotides at different sur-face areas.

6. Interest of silicon as support for DNA chips

Silicon is also employed as solid support for DNAchips implementation for various reasons. Microtech-nology on this materials is well developed and offersthe opportunity to design multiplexing device fromsilicon. The semiconducting properties of this mate-rials can also be exploited for the development ofnew methods for detecting hybridisation.

Thus, electrochemical deposition of oligonu-clotides via pyrole groups has been developed by

w xLivache et al. 7 . Presynthesised oligonucleotidesŽ .ODN were previously linked covalently to pyrole.The electropolymerisation of pyrole mixed with py-role-ODN leads in one step to the formation of anadherent conductive polymer layer on the polarisedsurface. These methods of immobilisation require

Ž .conductive surface Au, Pt, etc. and a selectingswitching of the electrodes to polarise individuallyeach electrode. In this case, silicon microtechnologyserves for making multiplexing silicon support onwhich array of gold electrodes can be drawn. Under-ground electrical connections allows to apply locatedpolarisation for electropolymerisation. As describedin literature, this method allows obtaining microar-

Žrays actually 128 electrodes are performed on the.substrate but seems fastidious because of functional-

isation of each electrode needs individual elec-tropolymerisation with appropriate ODN-pyrole solu-tion.

w xNanogen 8 uses also multiplexing device insilicon to make active DNA chips. Active areas aredelimited by gold dots and can be selectively po-larised with positive sign. As DNA strand targetscarry negative charge via phosphate groups, they arefastly driven to the positively charged sites. Polarisa-tion allows the release of the diffusion phenomenalimiting the kinetics of the hybridisation process byimproving strongly the concentration of DNA targetsclose to the surface. As a consequence, hybridisationprocess occurs in a few seconds when usually, hy-bridisation process is performed in half an hour.

After hybridisation, a light positive polarisation canremove from the surface the non-hybridisated strandsin order to increase the signalrnoise ratio of hybridi-sation reading out.

In the same way, the negative charges present inDNA strands allow a direct detection of hybridisa-tion without the use of labelled targets. This tech-nique of detection consists to follow the field effectsinduced by hybridisation process inside a blockingelectrode. This effect is due to the arrival on thesurface of new charge carried by complementarystrands. The GENFET is a field effect Transistormade of Silicon covered with thin dioxide siliconlayer on which polymeric membrane is deposited.DNA single strands are then immobilised. WhenGenfet is put in contact with a solution containingcomplementary DNA strands, a signal is obtained.No signal has been recorded when the solution does

w xnot contain the non-complementary strands 9 .Another property of semiconductor structure can

also be exploited for direct detection of hybridisa-tion. Surface potential can be locally measured bylight excitation. This surface photopotential dependson the surface charges and so, it is sensitive to the

w xhybridisation 10 . Adapted to DNA chips, this tech-nique presents advantages to map all the surface ofthe structure by only scanning a light beam. Thisdirect detection of hybridisation avoids labelling ofDNA targets. A unique simple structure made ofSirSiO interface with ohmic contact on rear face is2

required whereas active face is composed of poly-meric membrane on which oligonucleotides spots arearrayed.

Silicon microtechnology can also be used to solvethe problem of drop confinement. Planar substratescan be advantageously replaced by 3D-structuredsubstrates. Chemical or electrochemical etching canbe easily performed on silicon substrates.

7. Conclusions

DNA chips are considered as new tools, allowingimportant advances in genomics thanks the opportu-nity to record thousands information in parallel. Wideperspectives as well in research as for numerousapplications are connected with the development ofDNA chips. These tools are highly perfectible in

( )E. Souteyrand et al.rApplied Surface Science 164 2000 246–251 251

Žterms of quantification improving signalrnoise ra-. Žtio , reproducibility, robustness in order to reuse the

.chips . A part of their development will depend onthe adaptation of microtechnology to their peculiarproblems where surface chemistry must perfectlymonitor at a micro scale.

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