128 C.A. Marquette, L.J. Blum / Analytica Chimica Acta 506 (2004) 127–132
(imidodiacetic acid) Sepharose Fast Flow were obtained then washed with 25 ml of equilibrating buffer and stored at
from Pharmacia. PDMS precursor and curing agent (Sylgard 4 ◦ C.
184) were supplied by Dow Corning. All buffers and aque-
ous solutions were made with distilled demineralized water. 2.3. Biochips preparation
2.2. Bioactive beads preparation The biochips were prepared by arraying spotting solutions
as 0.3 l drops. The spotting solutions were prepared by
Glucose oxidase supporting beads were prepared by in- mixing 45 l of an aqueous solution composed of glycerol
teraction of Ni-charged IDA-Sepharose with Gox modiﬁed 2.5%, 0.05 M NaCl with either 5 l of wet beads bearing
with histidine. The histidine grafting on the Gox carbo- d(A)22 , 5 l of human IgG bearing beads, or 5 l of glu-
hydrate moiety was performed using a method previously cose oxidase bearing beads added of 5 l of luminol bearing
described for horseradish peroxidase modiﬁcation . beads.
Brieﬂy, 5 mg of Gox dissolved in 200 l of distilled water To design the low density array, 0.3 l drops were de-
and 80 l of a 0.1 M NaIO4 solution were added and mixed posited on a ﬂat poly(vinyl chloride) (PVC) substrate ev-
under stirring for 20 min. Then, 40 l of a 0.2 M histidine ery 3 mm and dried under a tungsten lamp during 30 min.
solution in 0.1 M carbonate buffer, pH 9, and 480 l of The presence of glycerol in the spotting solution enables the
0.1 M carbonate buffer, pH 9, were added to the activated achievement of homogeneous and smooth dry beads spots.
glucose oxidase and incubated 2 h under stirring. At that The dry bead spots were then transferred to the PDMS in-
time, the reaction was complete and 20 l of freshly pre- terface by pouring a mixture of precursor and curing agent
pared 0.1 M NaBH4 solution in water were added and mixed onto the PVC substrate and cured for 90 min under a tung-
for 15 min in order to stabilize the bounding of histidine on sten lamp. The biochip preparation was then terminated by
the activated Gox. peeling off the PDMS polymer (Fig. 1).
The Gox-His was then separated from non-reacted species In a similar way, the active ﬂuidic biochip was prepared
by desalting on a Sephadex G25-M column. Afterwards, by depositing 0.3 l drops of the different spotting solu-
500 l of IDA-Sepharose beads were equilibrated with tions (see above) on a PVC master composed of 12 channels
30 mM Veronal Buffer, 30 mM KCl, pH 8.5, and perfused (w:2.5 mm, h:1 mm, l:15 mm), prepared by sealing differ-
with 100 mM NiCl2 solution. On the Ni-IDA-Separose ent PVC parts together with epoxy glue, and following the
column thus obtained, 5 mg of the Gox-His desalted as de- same protocol as presented above. Peeling off the PDMS
scribed above were deposited. The column was then washed substrate and covering it with a ﬂat PVC surface leads to
with 10 ml of equilibrating buffer and stored at 4 ◦ C. the achievement of 12 active macro channels (Fig. 3).
Luminol was immobilized on DEAE-Sepharose beads
by electrostatic interactions. Five hundred microliters of 2.4. Nucleic acid assay
DEAE-Sepharose were packed from ethanol slurry into a
plastic column, equilibrated with 10 ml of 30 mM Veronal As a model oligonucleotide hybridization process, a
buffer, 30 mM KCl, pH 8.5, and perfused with 1 ml of 10 mM d(T)22 –d(A)22 couple was used. The low density arrays
luminol solution in equilibrating buffer. The column was and the active macro-ﬂuidic systems were assayed with
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 ﬂat
PVC 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 with
biotin labelled d(T)22 at a concentration of 0.5 nM.
C.A. Marquette, L.J. Blum / Analytica Chimica Acta 506 (2004) 127–132 129
the same general protocol. Brieﬂy, the biochip or a partic- solidiﬁed PDMS, bead emergence appeared on the surface
ular channel were saturated during 30 min by a solution of the polymer corresponding to the spots deposited (Fig. 1).
of buffer A (30 mM Veronal (diethylbarbiturate), pH 8.5 No beads residue were observed on the PVC master after the
containing 30 mM KCl, 0.2 M NaCl, 0.1% tween and 1% removal of the PDMS layer, demonstrating the high transfer
BSA), then incubated for 1 h with biotinylated d(T)22 at efﬁciency of the beads to the PDMS interface. Those bead
a particular concentration in buffer A. The biochip or a islands were then able to anchor active bio-molecules at the
particular channel were then washed 10 min with buffer interface, through the Sepharose beads (Fig. 2).
B (30 mM Veronal, pH 8.5 containing 30 mM KCl, 0.2 M The low density array format was used to design pro-
NaCl), afterwards saturated with buffer A during 20 min tein and oligonucleotide biochips based on Sepharose
and then incubated with peroxidase-labelled streptavidin beads bearing human IgG or d(A)22 homo oligonucleotide.
at a 1 g/ml concentration during 30 min. The array or a Fig. 1 presents the immobilisation process and a mi-
particular channel were then washed 20 min with buffer B crograph of a 100 d(A)22 spots array revealed with a
and was then ready to be measured. streptavidine–peroxidase conjugate after an hybridization
of the entire biochip with a biotin labelled d(T)22 . A 8%
2.5. Protein biochip assay standard deviation of the chemiluminescent signal was
found within the array. Similar results were obtained when
In a way similar to that used for the nucleic acid biochip, incubating the protein array, composed of immobilized hu-
the protein biochip was saturated during 30 min by a solu- man IgG, with biotin labelled anti-human IgG antibodies.
tion of buffer A, then incubated for 1 h with biotinylated The present biochip preparation procedure appeared then
anti-IgG antibody at a particular concentration in buffer A. to enable the achievement of homogenous array of the two
The biochip was then washed 10 min with buffer B, saturated most studied types of bio-molecules. Moreover, changing
with buffer A during 20 min and then incubated with perox- the properties of the beads used could lead to a larger scale
idase labelled streptavidin at a 1 g/ml concentration during of immobilized bio-molecule. Thus, oligosaccharide  or
30 min. The array was then washed 20 min with buffer B kinase  substrate could be easily immobilized and used
and was then ready to be measured. as analytical tools.
The non-speciﬁc signal generated by the non-speciﬁc ad-
2.6. Biochips measurement sorption on either the PDMS or the Sepharose material were
evaluated by preparing a biochip with unmodiﬁed beads
The ready-to-measure nucleic acid and protein biochips (not bearing any bioactive compound) and running the pro-
were bring into contact with a buffered solution (30 mM tein or nucleic assay in the presence of a maximum amount
Veronal, 30 mM KCl, pH 8.5) containing in addition of labelled antibody or labelled nucleic acid (100 g and
200 M luminol, 500 M hydrogen peroxide and 20 M 7 pmol, respectively). An extraordinary low non-speciﬁc sig-
p-iodophenol. At that time, the arrays were introduced in nal, close to the limit of the instrumentation sensitivity, was
the charge coupled device (CCD) light measurement system found.
(Intelligent Dark Box II, Fuji Film) and the emitted light The PDMS assisted biochip demonstrates here its great
integrated during 3 min. potential capabilities in array technology, since a low stan-
The enzyme substrate biochip was used in a similar way; dard deviation and an almost non-existing background were
a buffered solution (30 mM Veronal, 30 mM KCl, pH 8.5) observed.
containing a particular concentration of glucose and 200 M
of the triggering agent (1,3-dichloro-5,5-dimethylhydantoin) 3.2. Quantitative measurement with the active ﬂuidic
was injected in each channel just before the introduction biochip
of the biochip in the measurement system, and the signal
integrated during 3 min. The macro-ﬂuidic biochips were prepared by spotting
The biochip pictures obtained were quantiﬁed with a Fuji the bioactive bead solution on a three-dimensional master
ﬁlm image analysis program (Image Gauge 3.12). leading to the achievement of 2.5/1/15 mm (w/h/l) channels
(Fig. 3). The different solutions used in each assay formats
were manually introduced in the channels under vacuum
3. Results pressure, and stand to react during a stop ﬂow duration (as
described in the Section 2).
3.1. Low density arrays A ﬁrst study was performed to evaluate the ability of
the biochip to quantitatively detect nucleic acid. Samples
Low density arrays were prepared by spotting aqueous so- with different biotin labelled d(T)22 concentrations were in-
lution composed of glycerol 2.5%, NaCl 0.05 M containing jected in each particular channel, hybridized and revealed.
1:10 (v/v) of wet modiﬁed Sepharose beads at the surface Fig. 4A presents the evolution of the chemiluminescent sig-
of a PVC substrate, drying the spots under a tungsten lamp nal obtained as a function of the oligonucleotide amount in-
and pouring a PDMS solution on it. After the removal of the troduced in each channel. As a matter of fact, the nucleic
130 C.A. Marquette, L.J. Blum / Analytica Chimica Acta 506 (2004) 127–132
Fig. 2. The different possibilities of the active PDMS biochips. (A) A Sepharose bead bearing oligonucleotide emerges from the PDMS surface. A
biotinylated tracer is hybridized to the immobilized nucleic acid and revealed with a peroxidase labelled streptavidin. (B) A protein modiﬁed Sepharose
bead is included in the PDMS polymer. A biotin labelled antibody reacts with the immobilized antigen and is revealed with a peroxidase labelled
streptavidin. (C) An IDA-Sepharose bead bearing glucose oxidase (Gox) and a luminol-charged bead emerge from the PDMS surface. The Gox oxidizes
the glucose present in the reaction medium, generating H2 O2 which in turn, reacts with the immobilized luminol to produce light.
acid biochip enables the quantitative detection of biotiny- format, a 3 l sample volume could be used, lowering the
lated oligonucleotide amounts in the range 58 pM–23 nM. number of nucleic acid molecules detected to 108 , which is
Compared to the detection limits obtained with ﬂuorescence close to the best results obtained in previous works [3,8,11].
[8,11] or radiolabeled-based  systems, involving gener- In a next study, the performance of the protein array to
ally highly complex micro array designs, the present method detect free antigen in a competition format was evaluated.
exhibits a slightly lower sensitivity but a larger detection Samples containing human IgG at different concentrations
range. The present detection limit of 58 pM corresponds to were incubated in a particular channel, together with biotin
an amount of 1010 molecules in a 300 l assay volume. labelled anti-IgG antibodies. The chemiluminescent reading
Nevertheless, when using the biochip in a low density array of the biochip leads to the obtaining of the competition curve,
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 PVC
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 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.
C.A. Marquette, L.J. Blum / Analytica Chimica Acta 506 (2004) 127–132 131
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 macro
ﬂuidic biochip, an enzyme-based system was designed
(Fig. 1), in which chelating beads bearing glucose oxidase
 were co-entrapped at the PDMS interface with luminol
charged beads. In the reaction sequence, the glucose oxi-
dase catalyses the oxidation of glucose and the production
of hydrogen peroxide which reacts in the presence of a trig-
gering agent  with the immobilized luminol to generate
light. It has been previously shown that this conﬁguration,
with a different immobilisation system , enabled the re-
alization of sensitive biosensor array without contamination
problem between the different spots. Samples containing
glucose at different concentrations were injected in the
biochip and the light intensity produced was measured con-
comitantly on the entire biochip. The obtained calibration
curve is presented in Fig. 4C. Again, the performances of
the PDMS biochip appeared to be competitive with regard
to the sensitivity and the linear range presented in previous
reports dealing with chemiluminescent detection of enzyme
substrate [16,18–21]. Moreover, we demonstrated in a pre-
vious study that six different oxidase enzymes could be used
at the same time under the same experimental conditions,
allowing to obtain a six-parameter biosensor. The present
technology could then be transferred in a close future to the
design of PDMS based enzyme substrate biochip for the
detection of multiple parameters.
PDMS as an immobilisation matrix has been successfully
used to design multi-purpose biochips i.e. for either nu-
cleic acids, proteins or enzymes. It is shown that the ability
of 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 a
Fig. 4. Performances of the PDMS-based biochip. Dose–response and generic biochip production. Indeed, the present spotting pro-
micrograph of (A) the nucleic acid biochip (B) the protein biochip and (C) cedure enables the use of a large scale of immobilization
the enzyme biochip. The biochips were measured during 3 min and the
image quantiﬁed with a Fuji ﬁlm quantiﬁcation program. B0 represents
procedures and chemistries, and of chemical synthesis on
the light intensity obtained in the absence of free IgG and B the light beads, prior to the spotting.
intensity obtained in the presence of a particular IgG concentration. Moreover, the use of PDMS material allows to consider
the design of micro-ﬂuidic systems integrating some sens-
ing elements directly trapped at the macro-channel/ﬂowing
which a linearization by the “logit” function is presented on
solution interface. A ﬁrst step to those systems has been
passed with the presented macro-ﬂuidic systems integrating
B nucleic acids, proteins or enzymes. The performances of the
logit = ln present analytical systems are competitive with previously
B0 − B
described methods [3,8,11,16,18–21].
where B is the signal obtained at a ﬁxed free antigen con-
centration and B0 the signal obtained in the absence of free
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