A new generation of scanners for DNA chips

Francois Perraut a,*, Alexandre Lagrange c, Patrick Pouteau a, O. Peyssonneaux c,Pierre Puget a, G. McGall d, Lionel Menou b, Richard Gonzalez b, Pierre Labeye c,

Frederic Ginot a

a Equipe bioMerieux-LETI, CEA/LETI/DSYS, 17, rue des Martyrs, 38054 Grenoble Cedex, Franceb bioMerieux, Parc Club du Moulin a Vent, 33 av. Docteur Levy, 69693 Venissieux Cedex, France

c CEA/LETI/DSYS, 17, rue des Martyrs, 38054 Grenoble Cedex, Franced Affymetrix, 3380 Central Expressway, Santa Clara, CA 95051, USA

Received 17 May 2001; received in revised form 7 March 2002; accepted 15 May 2002

Abstract

Today, most of the DNA chips are used with fluorescent markers. Associated with fluorescence confocal scanners, this technology

achieves remarkable performances in terms of sensitivity and accuracy. The main technical issues related to these scanners have

already been reviewed. However, these scanners are costly, especially when high density chips are used. In this case, a mechanical

precision of 1 mm or less is required to achieve the measurement precision required. This cost level prevents the spread of this

technology in the diagnostic market. We will present a new concept for scanners with equivalent or superior performances, with a

cost cut of 5�/10. This concept is inspired from the field of optical disk and reader. Basically, an optical format is added to the chip,

before DNA deposition. This format contains tracks which are superimposed to the DNA features. These tracks define the path that

an optical head of a CD player must follow in order to scan the surface of the DNA chip. Such a head is a very cheap component,

and has a precision of less than 100 nm thanks to real-time focus and tracking. These functions are fulfilled by electromagnetic

actuators mounted on the support of the frontal lens. We show here that it is possible to use such a head to build a fluorescence

confocal scanner with equivalent or even better performances than conventional scanners. # 2002 Elsevier Science B.V. All rights

reserved.

Keywords: Confocal scanner; Biochip format; Microarrays

1. Introduction

DNA chip technology is an enabling technology

which is now used in various fields of research in

biology. Indeed, this is a powerful analytical tool of

genetic material. Associated with fluorescence confocal

scanners, this technology achieves remarkable perfor-

mances in terms of sensitivity and accuracy (Ramsay,

1998; Basarsky et al., 2000). However, these scanners are

costly, specially when high density chips are used, like

those manufactured by Affymetrix, a Californian com-

pany who are a leader in this field.

The cost of the existing scanners is affordable for

some markets, like that of the research in the pharma-

ceutical industry. However, in the routine diagnostic

market, in which molecular diagnostic is still in its

infancy, it represents an extra cost without replacing the

conventional tests; this cost level is not acceptable, and

prevents the spread of the DNA chip technology.

We have addressed the cost issue of the current

scanners for high density chips. To reach the required

performances, the sampling of fluorescence acquisition

must be sufficient to get robust data: numerous readings

per feature, hence high quality mechanics and electro-

nics, and use of a small excitation spot. Also, confocal

reading is usually used to increase the sensitivity by

rejecting the light coming from other plans than that of

the biological signals. The more confocal the system, the

better is the signal to noise ratio but the higher is the

need for accuracy for the focus and tilt adjustments of

* Corresponding author. Tel.: �/�/33-4-3878-5544; fax: �/33-4-

3878-5787

E-mail address: francois.perraut@cea.fr (F. Perraut).

Biosensors and Bioelectronics 17 (2002) 803�/813

www.elsevier.com/locate/bios

0956-5663/02/$ - see front matter # 2002 Elsevier Science B.V. All rights reserved.

PII: S 0 9 5 6 - 5 6 6 3 ( 0 2 ) 0 0 0 7 3 - 8

the DNA chip. Indeed, the relative position of the

scanner and the biochip is defined by 5 degrees of

freedom (three translations and two rotations), and

good and fast data acquisition for fluorescent features of10 or 30 mm wide necessitates a very precise control of

these five coordinates (less than 1 mm for X and Y

movements). That is not easy to reach over large

surfaces (1 cm or more), and is expensive when

addressed by conventional robotics and electronics

used in the existing scanners. That is why some users

prefer a non confocal reader (Cheung et al., 1999), and

also why so few scanners do exist for biochips withfeatures smaller than 80 mm (Singh-Gasson et al., 1999).

The new concept we present in this paper is inspired

from the field of optical disk and reader. Basically, an

optical format is added to the chip, before DNA

addition. This format is composed of tracks which are

superimposed onto the DNA features. These tracks

define the path that an optical head of a CD player must

follow in order to scan the entire surface of the DNAchip. Such a head is a very cheap component, and

reaches a submicronic spatial precision thanks to con-

tinuous dynamic focus and tracking. These functions are

fulfilled by electromagnetic actuators mounted on the

support of the frontal lens. The actuators allow for a

dynamic focus and tilt correction, and the format allows

the DNA chip itself to synchronise the fluorescent

measurement. Our scanner uses the same kind ofphoto-sensor (photomultiplier tube or avalanche photo-

diode) and laser as the conventional readers because

these components are imposed by the fluorescent dye

and by the expected reader performances (sensitivity,

speed). However, we estimate that the cost of our

scanner is divided by 5�/10 compared to others. In this

paper, we demonstrate the feasibility of such a low cost/

high performance system composed of a scanner andoptically formatted DNA chips.

2. Scanner principles

2.1. Optical format of the biochip

A chip with an optical format (Fig. 1) is constituted of

a glass substrate supporting guiding tracks, synchroni-sation marks and a reflective thin layer (not shown in

Fig. 1).

The guiding tracks are materialised by microscopic

grooves etched at the surface of the wafer. A small part

of the excitation light is reflected by the chip surface like

with any optical interface. This reflected light contains

the diffracting orders generated by the network formed

by the guiding tracks, and is projected to a four cellphotosensor. This sensor generates an information

named Tracking Error which is used to dynamically

control the position of the spot in order to hold it on the

current following track via a feedback loop (see Section

2.2).

Synchronisation marks are interruptions of the tracks.

These marks are positioned at precise positions relative

to the DNA chip pattern. On a given track, and for

every DNA feature crossed by the track, one mark is

placed just after the beginning of the feature, and

another is placed just before its end. During the scan

of the tracks, these marks generate a signal used to

synchronise the fluorescent measurements. Since the

synchronisation is done by the chip itself without

external encoders, the fluorescent measurements are

correct even if the linear stage has defaults (clearance,

delay). These marks also allow a dynamic data proces-

sing to perform during the scan of the chip and avoid

the use of high level image processing after the

fluorescence image acquisition.

A reflective thin layer is placed just under the surface

of the chip. Refractive index and thickness of this layer

are chosen in such a way that the reflectivity of the DNA

chip surface for the incident light is fixed and relatively

independent of the refractive index of the biological

buffer present at the surface of the DNA chip during the

reading. Since this interface between the chip and the

surrounding buffer is clearly detectable over the whole

surface, it is possible to perform a dynamic control of

the focus (see Section 2.2). This dynamic focus function

absorbs positioning defaults of the chip within its

cartridge and of the cartridge in the scanner holder, as

well as mechanical defaults of the scanning stages,

without any expensive component. Also, the thin layer

is designed to minimise the reflectivity of this interface at

the fluorescence wavelength to avoid fluorescent losses.To assure the chemical compatibility between this

optical layer and the chemical steps necessary for DNA

Fig. 1. Optical format of the chip: this chip supports guiding tracks

(gray pattern) which guide the spot laser (dark circle) during the scan,

and synchronisation marks to synchronise the fluorescent measure-

ment dynamically. The optical format is precisely superposed to the

DNA pattern of the DNA chip.

F. Perraut et al. / Biosensors and Bioelectronics 17 (2002) 803�/813804

loading of the chips, a silica layer is added on the top of

the index matching layer.

2.2. System overview

To read the kind of chip previously described, we

have built a breadboard which supports fluorescence

confocal detection and control position devices (Fig. 2).

The scanning of the chip area is achieved by twolinear stages: the first one translates the actuator and a

458 mirror for the fast X axis; the second one moves the

chip along the slow Y axis. This linear stages provide the

macroscopic translations.

The fluorescence path of our scanner is constituted of

a laser for the excitation, a dichroıc beam splitter and a

frontal lens to focus the light on the probe area. The

fluorescence light is collected by the frontal lens,reflected by the dichroıc filter and focused by a lens to

the confocal pinhole. A photomultiplier tube is used to

measure the fluorescence level. Excitation light is

stopped by a set of filters.

To measure the position errors (tracking error and

focus error), a part of the excitation light is reflected by

the reflective thin layer placed at the surface of the chip.

This light is split in two parts: the first one for the focus

error and the second one for the tracking error

measurement (see Fig. 2).

The focus error measurement is done by the use of an

astigmatic lens which projects a circular dot of the laser

spot on a four cell sensor when the focus is good. When

the chip is no longer at focus, the dot is distorted

towards an ellipse. The direction and length of the long

axis of the ellipse, which are measured by the four cell

sensor, are representative of the direction and the value

of the defocus (Laurent, 1997; Lagrange, 1998).

In the same way, The Tracking Error is measured by

the projection of the diffraction pattern of the guiding

tracks on an other four cell sensor. When the frontal

lens is no longer centred on the current track, the

diffraction pattern is modified on the four cell sensors.

This modification is quantified by the system (Laurent,

1997; Lagrange, 1998).

The frontal lens is mounted on an electromagnetic

actuator which has two degrees of freedom: the first one

along the optical axis to correct a defocus, and the

second one along an axis perpendicular to the scan

direction (see Fig. 3) to hold the spot on the current

track. This actuator is controlled by an electronic board.

Two feedback loops from the position sensors to the

actuator achieve the controls of Focus and Tracking.

Indeed, the actuator provides microscopic translations

Fig. 2. Reader breadboard diagram.

F. Perraut et al. / Biosensors and Bioelectronics 17 (2002) 803�/813 805

to continuously correct the mechanical defaults of low

cost linear stages and chip packaging.

The synchronisation marks are detected by the

Tracking Error sensor with a different combination of

the four cells to calculate a ‘Longitudinal Error’ instead

of the ‘Lateral Error’ used for the tracking function. The

signals generated by these marks are used to start andstop the fluorescence measurements corresponding to

the DNA features. In the present work, this operation

has been done after completion of the scan. In the

future, this signal will be used as a trigger for the

photocounting electronic board.

3. Materials and methods

3.1. Reader breadboard set up

Our reader breadboard is schematised in Fig. 2. Theexcitation path is composed of: (a) a 635 nm/15 mW

diode laser (Blue Sky Research, PS024-00) adjusted to

get 4.4 mW on the chip; (b) a dichroıc beam-splitter for

Cy5 Dye (Omega 650 DRLP). The excitation light is

focused through the glass of the chip by an aspheric lens

mounted onto a CD actuator (kind gift of A. Fargeix).

The size of the excitation spot on the chip area is

controlled by a diaphragm. This diaphragm reduces theincident beam diameter, hence the numerical aperture of

the lens for the excitation light. This was necessary to get

a large spot on the chip, as the DNA features of the

DNA chip used here are 29 mm wide. In this work, the

size of the spot is set to 5 mm, diameter measured at 1/e2,

and corresponding to a 0.15 excitation numerical

aperture. The laser is powered by a power supply

(AMS Electronique PS028-00) whose current is modu-lated at 2 MHz to prevent the instability created by

reflected light coming back into the laser. A l /4 slide is

placed just after the laser (Melles Griot 02WRQ007-

635.9). A 6359/2 nm band pass filter (Omega

EX635BP10) is placed after the laser to stop any

fluorescence light coming from the cavity.

The fluorescence detection path is composed of: (a)

the aspheric lens with a total numerical aperture of 0.6;(b) two identical filters to stop excitation light (Schott

RG 665); (c) a 44.5 mm focal length achromat with anti-

reflexion coating (Melles Griot LPX 045); (d) a 50 mm

pinhole (CVI); and (e) a photocounting photomultiplier

(Hamamatsu H7155-21). The output of the photomul-

tiplier is connected to a 12 bit, 500 Ksamples/s counting

board (National Instrument AT-MIO16E2).The macroscopic X, Y scanning movements are

achieved by two linear stages, respectively for fast and

low scan axis (Etel TLMB03-50-EP) controlled by an

axis controller board (Delta Tau, PMAC Lite). In the

present work, scan speed is varied between 20 and 40

mm/s along the fast axis. The biological results were

obtained at the 20 mm/s speed. The scan range of the

both axis is 8.3 mm.A holder allows Affymetrix cartridges or microscopic

slides to be mounted.

The breadboard is controlled by a custom software

written in graphic language (National Instrument Lab-

View) which runs under Windows NT operating system

installed on an Intel Microcomputer.

The focus control feedback loop has a 1500 Hz band

pass, and the tracking control feedback loop has a 900Hz band pass. The sampling rate for the acquisitions of

the fluorescence signals and of the synchronisation

signals is 20 kHz.

3.2. Optical format manufacturing

The realisation of the optical format has been done in

LETI facilities, using well-known procedures of micro-electronics only slightly modified for silica. To produce

optically formatted wafers compatible with Affymetrix

production equipment, round wafers 200 mm in dia-

meter were used. This format is common in microelec-

tronics industry. Fused silica was used to be compliant

in terms of contamination with microelectronics facil-

ities. After completion of wafer formatting, these round

wafers were cut at the Affymetrix square format.The manufacture of the substrates consisted of the

realisation of the guiding tracks and synchronisation

marks, as well as the realisation of the alignment marks

for the Affymetrix technology (DNA synthesis, wafer

sawing, chip packaging), the marks required for the

proper operation of the reader currently used with

Affymetrix chips (the GeneArray Scanner†, built by

Agilent), and finally the coatings, first by the refractiveindex layer necessary for an efficient continuous focus

and tracking of our reader, and second by the silica layer

necessary for DNA chemistry.

The diagram of the different steps of fabrication is

shown in Fig. 4.

The photolithography steps have been achieved on a

stepper (ASM, ASM 90). This equipment exposes the

optical format chip by chip. The equipment used has aresolution of motive of 0.35 mm and a precision of

positioning of 0.12 mm. Indeed, this precision was not

required for our application (1 mm is enough).

Fig. 3. Frontal lens mounted on the electromagnetic actuators.

F. Perraut et al. / Biosensors and Bioelectronics 17 (2002) 803�/813806

Etching of the tracks in the substrate was performed

by reactivate ionic etching (RIE) with CHF3 and O2

gases (Margail, 1999). The depth of 850 A had been

calculated for proper function of tracking. The homo-

geneity of depth of etching is 9/3%. The synchronisation

marks are two 2 mm interruptions of the guiding tracks

separated by 2 mm (see Fig. 6).

The marks required by the Affymetrix technology and

the reference reader were made with an amorphous

silicon layer. The reflectivity obtained after completion

of the process is around 30%.

The index matching layer was made of silicon nitride

(Si3N4) with a refractive index of 2. This material is used

routinely in microelectronics. This deposition of 820 A

has been achieved by low pressure chemical vapor

deposition (LPCVD) (Applied Materials, Applied

5000; see step 9). The silica layer for chemical compat-

ibility is 5000 A thick, and was also deposited by

LPCVD. The exact thickness of the silica layer does

not have to be very precise. Therefore, it also constitutes

a protection layer for the silicon nitride layer during the

chemical treatments used for surface derivatisation

(Pease et al., 1994).

Each chip contains 1692 guiding tracks of 2.4 mm,

with a pitch of 4.8 mm. Each track supports 283

synchronisation marks, spaced at a pitch of 28.8 mm.

One oligonucleotide cell will be superimposed to the

optical format in such a way that it covers six guiding

tracks and is included between two synchronisation

marks.

3.3. Oligonucleotide synthesis and chip packaging

Oligonucleotide synthesis was performed by Affyme-

trix as described elsewhere (Pease et al., 1994). Thesubstrates prepared as described above were processed

as done in routine at Affymetrix, and packaged chips

were produced, compatible with the GeneChip† line of

equipment, namely the Fluidics station (Affymetrix)

which automates the hybridisation and washing steps,

and the GeneArray scanner (Agilent, Palo Alto, CA)

which reads the fluorescence of the chips at a resolution

of 3 mm.The DNA chips synthesised are custom chips dedi-

cated to an antiretroviral genotypic resistance assay. It

consists of 280�/280 oligonucleotide cells of 28.8�/28.8

mm, covering numerous variants of the HIV-1 genome.

This chip is used for the development of a diagnostic test

within bioMerieux (unpublished data).

Optically formatted chips were used for the experi-

ments with our system. Standard custom HIV-1 chipswere used for the reference experiments, for wavelength

compatibility, since the reference system uses fluores-

ceine while our system uses Cy5†, a red fluorochrome.

3.4. Photometric controls

To investigate the influence of the optical format on

the light-directed DNA synthesis and on the fluores-

cence level, analyses were done by epi-fluorescencemicroscopy. The microscope was composed of a Nikon

Labophot 2A with epi-fluorescence tube and CCD

attachment, a cooled CCD Camera (LE2IM HiSiS24),

a Nikon PlanApo x2/0.1 objective or a Leitz EF x10/

0.25 objective. Two successive filters for the Cy5 dye

were used: a standard cube (Nikon 41008 HQ Cy5) and

an additional band pass filter (Omega 670DF 40 EM

XF45).Images were pre-processed by dark subtraction and

flat field correction. By using these materials and this

method, very precise photometric measurements are

obtained. The precision is only limited by the photon

shot noise.

3.5. Biological material and hybridisations

A human plasma positive for HIV-1 was used for

biological validation of the system. Viral RNA was

extracted from the plasma.

An enzymatic amplification was performed for Pro-

tease and reverse Transcriptase gene fragments. A single

pool of amplifications was used for all the experiments.

The amplicons were then fragmented and labelled by a

proprietary process, as described elsewhere (Monnot etal., 2001). Part of the pool was labelled with fluorescein

for the reference experiments, the other part with the

Cy5 dye (Amersham Pharmacia Biotech) for the valida-

Fig. 4. Optical format manufacturing. Step 1: glass wafer. Step 2: low

pressure chemical vapour deposition (LPCVD) of amorphous silicon.

Step 3: photolithographic step (alignment marks for photosynthesis

process), photoresist coating�/exposure through a mask. Step 4: resist

development. Step 5: reactive ionic etching (RIE) of silicon layer and

stripping resist. Step 6: photolithographic step (tracks), photoresist

coating�/exposure through a mask. Step 7: resist development. Step 8:

RIE etching of glass and stripping resist. Step 9: LPCV deposit of

index matching layer Si3N4 and silica.

F. Perraut et al. / Biosensors and Bioelectronics 17 (2002) 803�/813 807

tion of our system. Labelled amplicons were purified

and hybridised to the DNA array.

Oligonucleotides respectively labelled with fluorescein

and Cy5 were added to the hybridisation mix, to light upcontrol checkerboards located at the corners of the

chips.

Hybridisation and washing were performed using a

protocol derived from the one described else-

where (Troesch et al., 1999), using the Affymetrix

GeneChip† Fluidics Station 400.

3.6. Biological data analysis

Reference experiments were imaged by the Affymetrix

GeneArrayTM scanner, and analysed by the GeneChip†

software, followed by Excel† Macros for amino acid

calls.

Today, the data produced by our system contains two

sets of data, one containing the fluorescence signals, the

other containing the synchronisation signals. Fluores-cence images were reconstituted using these two sets of

data, simply by using the synchronisation marks to

realign the successive lines of the fluorescence image.

The resulting fluorescence images were then formatted

to become compatible with the Genechip† software.

Analysis was then done as above. No normalisation of

the fluorescence signals based on the time elapsed

between two synchronisation marks was done at thisstage of the work.

4. Technical results

4.1. Depth of section

The depth of section of the confocal scanner has been

measured at the full width at half maximum (FWHM) in

coherent mode to 25 mm (see Fig. 5). We have applied a

procedure described by Wilson (1990). Briefly, a plane

mirror is scanned axially through focus, and the

intensity of the reflected light is recorded through the

confocal path (without any filter). This measurement is

well matched by the theoretical result (24.86 mm) given

by the relation (1) (Tiziani et al., 1996):

FMWH�0:443�l=(1�cos u) (1)

where l is the wavelength and u refer to the numerical

aperture sin u . For this measurement, the wavelength is

635 nm and the numerical aperture for the excitation is

equal to 0.15.

4.2. Focus

The accuracy of the dynamic autofocus was measured

by the use of the confocal channel without any

fluorescence filters. Like for the measurement of thedepth of section above, the (reflected) light intensity

variation observed in this channel when the chip is

moved around its focus position quantifies the defocus

distance. However, in order to increase the amplitude of

the light intensity variation upon chip defocus, the

confocal pinhole was defocused and set at a position

where the chip defocus sensitivity is the highest. (When

the pinhole is at focus of its lens, the light intensity goingthrough the pinhole is at its maximum. This light

intensity is therefore less sensitive to a defocus of the

chip.)

A calibration curve linking chip defocus and light

intensity was then done, as above for the measurement

of the depth of section.

Then, the chip was voluntarily tilted so that it was no

longer perpendicular to the optical axis. The scan waslaunched and light intensity was recorded during the

scan. With a tilt of 3.78 along a 4-mm scan, the accuracy

of the focus was estimated to be 9/1 mm.

The focus control is very robust and it is this

characteristic of our scanner which allows very thin

optical sectioning and a spatial quantification of the

fluorescence to be done even when the positioning of the

chip is not correct.

4.3. Tracking

To evaluate the tracking accuracy, we also use the

confocal channel. However, the confocal assembly(pinhole�/PM, see Fig. 2) was replaced by a CCD

camera (Photometrics Image Point) with a relay lens

constituted of the frontal aspheric lens of the CD

actuator and a 300 mm achromat. This set up had a

magnification of 75, and projected the image of the

tracks on the CCD sensor. The chip with tracks is

mounted on a manual stage able to move perpendicu-

larly to the tracks. In this way we can simulate andcontrol a tracking movement. A dark field illumination

of the tracks is provided by an external light source and

an optical fibre illuminator.

Fig. 5. Measured coherent depth of section: Experimental result: the

variation in detected signal as a plane mirror is scanned axially through

focus. The curve was obtained using 635 nm laser diode with 5 mm spot

diameter, 3.5 mm/0.15 NA CD lens, 40.5 mm focus lens, 50 mm

diameter confocal pinhole. Z step�/5 mm.

F. Perraut et al. / Biosensors and Bioelectronics 17 (2002) 803�/813808

In open loop mode, that is without tracking control, a

translation of the chip along the Y axis (perpendicular

to the tracks) induces a proportional translation of its

image formed on the CCD. On the contrary, when the

tracking control is at work, the image is locked and does

not moved perceptively, because the CD actuator holds

the spot on the current track. In practice, however, there

is a slight remaining error that can be estimated by

subtracting two images obtained for displacement of �/

100 mm and �/100 mm, a range corresponding to the

correction range of the tracking. We measure 0.66 mm

between these images.

The performances of the tracking control are thus

excellent and permit an accuracy of 9/0.33 mm for total

correction range of 9/100 mm. This range allows to scan

a 5 mm width biochip with a tilt of 28 relative to the fast

axis direction (X axis in Fig. 2) with a positionning

accuracy of 9/0.33 mm.

4.4. Synchronisation signal

Fig. 6 illustrates the signal over noise ratio of the

detection of the synchronisation marks. The amplitude

of peaks and the shape of the curve allows detection of

the marks without ambiguity and with high accuracy.

This accuracy gives a fluorescence image with a high

spatial resolution. In its present status, our system does

not use these marks to trigger fluorescence acquisition.

The marks are simply used to realign the lines of the

fluorescence image.

4.5. Compatibility between the optical format and the

biochip manufacture and fluorescence

4.5.1. Alignment between chip format and chip

oligonucleotides

The aligned superposition of the guiding tracks and

synchronisation marks and of the DNA features was

checked by epifluorescence microscopy. Fig. 7 shows thecorrect superposition of the different pattern. A small

shift of 2.5 mm was observed, which is within the

uncertainty expected, and lower than the security

margins put on the format.

4.5.2. Fluorescence level

A fluorescence quantification was done with for-

matted chips and standard silica chips to check if

differences in fluorescence behaviour were detectable.

Measurements were done with the epifluorescence

microscope described in Section 3. The results reportedin Table 1 indicate that optical format does not change

significantly the fluorescence intensity.

Fig. 6. Synchronisation signal generated by the system: the zero transitions marked by arrows correspond to the synchronisation marks.

Fig. 7. Alignment between chip format and DNA features.

F. Perraut et al. / Biosensors and Bioelectronics 17 (2002) 803�/813 809

5. Biological validation

The biological validation of the system was conducted

by the team responsible for HIV-1 genotyping within

bioMerieux. A viral RNA was amplified, fragmented,

labelled, and hybridised to HIV-1 chips according to the

procedures described in Section 2. The HIV-1 chip is a

custom chip manufactured by Affymetrix under bio-

Merieux specifications. It contains 280�/280 oligonu-

cleotide cells of 28.8�/28.8 mm. To render more

informative the comparison between the reference

system and our new system, a sub-optimal amplification

protocol was chosen for the test, which produces a low

concentration of amplicons and some non specific

background. An optimal amplification protocol was

also used as control.

The chips were hybridised in the same conditions,

with the same buffers, and with the same amplifications

pool for both the reference and our system. The only

difference is the final labelling step, as the reference

system requires fluorescein and our system works with a

Cy5 label, chosen to decrease the cost of the laser source

of the scanner. The determination of 33 amino acids

within the protease gene relevant for antiviral therapy

monitoring was achieved, and the results are shown in

Table 2.

These data clearly indicate the negative impact of a

sub-optimal amplification in HIV genotyping as correct

amino acid calls decrease drastically from 100 to 50%.

Even worse for medical diagnostic, the rate of errors is

very high, up to 12%. On the other hand, the new system

rates a correct call at 89% and an error rate of 3%.

Similar results were obtained with the RT gene over 42

interrogated codons.

In any case, we show here that our system can

decrease the constraints put on the amplification step,

which is usually the most tricky step in molecular

diagnostic because of interference between some sam-ples (patient blood is not under control) and the

amplification chemistry or enzymology.

A detailed analysis of fluorescence intensities in

relation to amino acid calls show that errors occur

exclusively for faint signals, both for the reference

system and for our new system.

To better understand these good results with our

system, we have conducted statistical and signal analysison both readers as explained below. The analysis was

performed in five steps:

1) calculation of the number of photoelectrons emitted

by the PMT photocathode by computing the gain

between photo-counts and photoelectrons (Klobu-

char et al., 1971),

2) comparison of the number of photoelectrons be-

tween the two scanners,

3) measurements of the ratio between specific signal

and offset in both cases,4) calculation of the fluorescent energy coming from

the space out of focus and not stopped by the

confocal pinhole (Tiziani et al., 1996; Wilson, 1990),

5) analysis of the distribution of variance versus mean

intensity.

The results are:

1) For the maximum specific signals, the energy

expressed in photo-electrons per pixel (same size

of pixels for both systems) after the photo-cathode

of photomultiplier is 1.4 times greater as thereference scanner despite the use of Cy5 on our

system (Cy5 is well known to emit more light than

fluorescein). We attribute the relatively low intensity

obtained with our system to a relatively low

labelling yield with Cy5. Thus, these data indicate

that the good results obtained with our system are

not due to a stronger light intensity coming from

Cy5.2) The ratio between specific signal and offset is 10

times larger on our system than on the reference

scanner. This increase of ratio leads to an improve-

Table 1

Fluorescence level on a standard and a formatted chip: fluorescence

levels are given in relative fluorescence unit

Fluorescence signal Standard chip Formatted chip

Specific max 1950933 2200944

Specific min 350920 500918

Signal was measured with an epifluorescence microscope and a

cooled CCD camera. Chips were analysed after hybridisation with a

complementary target labelled with Cy5. Arbitrary units.

Table 2

Comparison between the reference system and our system

Optimal amplification protocol Sub-optimal amplification protocol Sub-optimal amplification protocol

Reference system Reference system Formatted chip and new reader

No. replicates 2 4

% correct codon call 100 50 89

% error 0 12 3

% uncalled 0 38 8

The protease gene of HIV virus was analysed at 33 codons important for antiviral therapy monitoring.

F. Perraut et al. / Biosensors and Bioelectronics 17 (2002) 803�/813810

Fig. 8. Distribution of intensity variance vs. mean intensity for DNA features: our system (A) and Agilent scanner (B). Each curve is given in

photoelectrons and the scale on variance axis is the same in both figure. We observe that the variance is very spread in the low intensity zone in B.

This phenomenon is not observed with our system (A).

F. Perraut et al. / Biosensors and Bioelectronics 17 (2002) 803�/813 811

ment of the lowest detection limit. One part of this

increase is perhaps due to the Cy5 (less non specific

adsorption than fluorescein) or to a better electronic

stage (lower noise in our reader) but the major partof this increase is due to a better confocal filtering:

the sharper depth of section reachable with the

dynamic auto-focus reduces the offset level coming

from the space out of focus by a factor 4 compared

to the reference reader. This improvement decreases

the detection limit by a factor of 2. Then, faint

signals are better detected with our breadboard.

3) The spatial resolution is also a very important issuewhich must be considered. To investigate this

further, we have plotted the variance of pixel

intensities against its average for each DNA feature

for both systems (see Fig. 8). For an ideal reader,

such a plot gives a straight line with a slope of 1

because the only noise is the photon shot noise. In

Fig. 8, due to the Y axis scale, this slope is hardly

visible. Interestingly, we observe a very high var-iance for faint intensities in the reference system

whereas it is almost normal in our system. The high

noise observed on the reference scanner appears

when a faint feature is close to a bright one: despite

image processing, the value of a faint feature is

perturbed when it is closed to a bright feature. The

better result with our reader is due to a better spatial

localisation of fluorescence (better correspondencebetween pixels and features). Thus, the number of

uncalled and errors are decreased (see Table 2).

6. Discussion

We have defined a new concept to realize low cost

DNA chip scanners without sacrificing neither sensitiv-

ity or spatial resolution, and have shown its feasibility.The potential of this new technology is enormous, in

addition that it is economically advantageous.

The quality of the focus allows a decrease in the depth

of section of the scanner, collection of the maximum of

fluorescence, and avoids fluctuations of fluorescence

collection over the chip due to variable focus. All these

factors contribute to increase the specific signal, to

increase the signal to noise ratio, and to increase thereliability of the comparison of fluorescence levels

coming from different areas of the chip.

On the other hand, the tracking function increases the

spatial resolution of the scanner, while using low quality

stages and associated electronics. This increases the

spatial signal to noise ratio, also contributing to increase

the reliability of the comparison of fluorescence levels

from different area of the chip. This improvement couldalso be used to read DNA features smaller than those

used today, hence enhancing chip information or

reducing its size. Singh-Gasson et al. have already

pointed out the lack of scanners able to read 16 mm

features (Singh-Gasson et al., 1999).

In the present work, synchronisation marks were used

only to realign the successive lines of the fluorescencescan. However, they should also enable normalisation of

the fluorescence signals by the time elapsed between the

marks defining a DNA feature. This will still increase

the precision of the fluorescence quantification.

Moreover, the controlled superposition of the optical

format and DNA feature has side advantages, like

enabling reading only part of the chips, for example to

scan again at a lower speed the faintest signals.Finally, all these advantages can be used either to

increase the precision of the biological information, or

to decrease the constraints put on the quality of the

sample and sample preparation.

Acknowledgements

A lot of people have contributed to this work inaddition to the authors. M. Belleville and E. Desgrange

designed the lithographic masks for optical format

manufacture. D. Renaud managed the adaptation of

the silicon equipment line to silica wafers. P. Brincard

and his team achieved the manufacture of the formatted

wafers. R. Campagnolo and his team supported reg-

ularly electronics and mechanics of the reader bread-

board. A. Fargeix gave us the actuators. M. Ida helpedthe initiation of this work. G. Vernet provided the

biological materials and helped discussing biological

results. M. Mittmann, J. Fidanza L. Kajisa and H.

Quang gave helpful technical assistance within Affyme-

trix. M. Bergeon accompanied the whole work to ensure

industrial interest. We also thank the R&D directions of

bioMerieux, CEA/LETI, and Affymetrix which have

constantly supported this work.

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