Chapitre 4. Supports d’immobilisation innovants pour tests colorimétriques
1. Nouveaux supports pour test colorimétrique haut-débit
1.1. Comparaison de différentes architectures de surface
Dans le but d’optimiser l’effacité des tests d’hybridation colorimétriques, quatre matériaux ont été comparés pour l’immobilisation des oligonucléotides: (i) la membrane Multiscreen HTS-HA présentée précédemment, (ii) une surface de polystyrène brute classiquement utilisée pour les tests ELISA, (iii) une surface de polystyrène activée et (iv) un support adhésif double-face. Ces différents matériaux ont été assemblés en fond de puits d’une microplaque 96-puits afin de procéder à un test d’hybridation automatisé comme présenté au chapitre précédent. Dans chaque puits, une matrice de sondes spécifiques de trois polymorphismes a été immobilisée. Il s’agit des SNP associés aux antigènes érythrocytaires KEL1/KEL2, JK1/JK2 et FY1/FY2. La détection du signal a reposé une fois de plus sur un marquage des plots positifs par la phosphatase alcaline et l’utilisation d’une solution de substrat BCIP/NBT.
L’ensemble des résultats fournis par les tests colorimétriques, une étude des surfaces par microscopie électronique à balayage et une série d’expériences complémentaires de chimiluminescence, ont mis en evidence le fait que l’efficacité du test ne dépendait pas seulement de la densité de sondes sur la surface mais aussi de la capacité du matériau à retenir le précipité coloré.
L’ensemble de ces résultats sont présentés dans l’article 4 ci-après.
Article 4
Surface architectures for colorimetric microarrays
Gaelle C. Le Goff, Benjamin P. Corgier, Céline A. Mandon, Gabriel De Crozals, Carole
Chaix, Loïc J. Blum et Christophe A. Marquette
Le Goff G.C.
a, Corgier B.P.
a, Mandon C.A.
a, De Crozals G.
b, Chaix C.
b, Blum L.J.
aand Marquette C.A.
a*a
Laboratoire de Génie Enzymatique, Membranes Biomimétiques et Assemblages
Supramoléculaires, Institut de Chimie et Biochimie Moléculaire et Supramoléculaire, Université
Lyon1 ʹ CNRS 5246 ICBMS, Bât. CPE ʹ 43 Bd du 11 Nov., 69622 Villeurbanne, France.
b
UMR CNRS 5280, Université Claude Bernard Lyon 1, Bâtiment CPE
43, bd du 11 novembre 1918 - 69622 Villeurbanne, Cedex, France.
E-mail: christophe.marquette@univ-lyon1.fr; Fax: +334 72 44 79 70; Tel: +334 72 43 13 69
ABSTRACT
We report here an extensive comparison of support materials for colorimetric hybridization assays on
microarrays. Four surfaces with various chemistries and architectures (from flat to porous) were
evaluated: i) bare and ii) activated polystyrene surfaces classically used for ELISA; iii) a double-sided
adhesive support; and iv) a porous nitrocellulose/cellulose acetate membrane. Each substrate was
functionalized with a microarray of probes and subjected to an enzymatic colorimetric DNA hybridization
test for 3 SNPs genotyping. Tests were carried out in a 96-well assembly suitable for automated
high-throughput analysis. Colorimetry results, microscopy observations and a complementary
chemiluminescence study showed that the test efficiency not only depended on the surface probe density
but that the capacity of the material to retain the colored enzymatic product was also a critical parameter.
All parameters being considered, the adhesive coated surface appeared in the end to display the best
surface architecture for efficient colorimetric microarrays.
multiparametric diagnosis and prognosis testing. This fact is clearly evidenced by the many examples of
widely used routine point-of-care diagnostic tools (pregnancy tests, semen tests, virus and bacterium
presence tests: VIKIA® Rota-Adeno, HIV-1/2 (bioMérieux); Determine® HIV-1/2, HBsAg, Syphilis TP
(Inverness Medical)), designed as monoparametric lateral flow test immunoassays coupled with
colorimetric signal detection.
In the particular field of microarrays, the necessity to image the chip surface to distinguish the specific
response of each one of the different spots has led to an intensive utilization of fluorescence imagers.
Despite the fact that this approach was giving very good results when sensitivity was required, the
technology restrictions impacted negatively on the democratization of the tool. Indeed, none of the health
insurance authorities, even in developed countries, have budget to reimburse expensive tests, even real
breakthrough technologies in pathology early diagnosis. The unique economically viable solution seems
then to be the adaptation of the pre-existing colorimetric single parameter assays to automated
high-throughput multiparameter tools. So far, such approaches have been based either on colloidal gold
nanoparticles for DNA labeling [1-3], on molecular beacon-functionalized gold nanoparticles [4, 5] or on
colored particles such as dyed polystyrene microspheres used for the generation of the colorimetric signal
[6]. Enzyme labels were also used (mainly horseradish peroxidase (HRP) and alkaline phosphatase (AP))
and appeared of great interest when signal amplification was required. Practical applications using these
labels on microarrays were reported, for instance for the detection of PCR products on nitrocellulose [7] or
agarose films [8], or used in combination with multiplex asymmetrical PCR [9, 10]. In those different
studies, colorimetric strategies were shown to drastically lower the costs of the high-throughput and
mass-scale testing tools.
Our group worked for the last three years on the development of a cost-efficient high-throughput
analysis platform (HiFi-assay) for multiparametric nucleic acid assays based on such an enzyme catalyzed
support was shown to critically impact the final test quality.
We are reporting here a comparison study between the different surface architectures which were
foreseen for this system. Indeed, different surface materials and coating strategies were tested and
appeared to have different levels of impact on both the immobilization of the probe microarray and the
quality of the colorimetric staining of positive spots. Flat polystyrene surface, polymeric adhesive coated
surface (polyacrylic based) and porous nitrocellulose based membranes were thus tested and
characterized.
2- MATERIALS AND METHODS
2.1- Materials
Synthetic oligonucleotide probes and primers were supplied by Eurogentec (Belgium). The sequence
and length of the six couples of probes and primers (2 per SNP) were previously optimized [11] (the 3
associated SNPs are described in Electronic Supplementary Material Table 1). For intellectual property reasons, probes and primers sequences will not be described here. Alkaline phosphatase-labeled
streptavidin and BCIP/NBT ready-to-use solution were purchased from Sigma-Aldrich (Lyon, France).
The bottomless 96-well plates were purchased from Greiner bio-one SAS (Courtaboeuf, France). The
porous surface was an original membrane-bottomed 96-well plates (Multiscreen HTS-HA) purchased
from Millipore (France). The white polystyrene surface was PolyShrinkTM purchased from Luckysquirrel
(Belen, US). The white double sided adhesive 7966WDL, medical grade, was obtained from 3M (France).
2.2- Spotting procedure
)RUROLJRQXFOHRWLGHPLFURDUUD\VSUHSDUDWLRQWKH¶-amino modified probe sequences were prepared in acetate buffer 0.1 mol.L-1, KCl 0.1 mol.L-1, bromophenol blue 0.25 mg.mL-1, pH 5.5 to reach a final
substrate was air-dried at room temperature and was then ready to be used.
The polystyrene surface and the double sided adhesive were, when mentioned, activated for one hour
with 1% (v/v) glutaraldehyde in 0.1 mol.L-1 acetate buffer, pH 5.5.
A double sided tape bearing 6 mm diameter holes was used to assemble the polystyrene support with a
bottomless 96-well plate whereas the spotted adhesive support was directly assembled with the bottomless
plate thanks to its adhesive property.
2.3- Oligonucleotide assay procedure
Blood samples were prepared according to reference [11]. Oligonucleotide assays were carried out on
an EVO75 robot (TECAN, Switzerland) equipped with a heater. For all experiments, the protocol
involved the following steps. i) Wells were washed with phosphate buffer saline (PBS, 0.1 mol.L-1, pH
7.4) and saturated with PBS mixed with LowCross Buffer 1:5 (v/v) (Candor Bioscience, Wangen,
Germany). ii) Samples were diluted 2.5 times in the saturation buffer and transferred to the wells and
incubated for 30 min at 55°C. iii) The wells were then loaded with the alkaline phosphatase-labeled
streptavidin solution (2 μg.mL-1) and incubated for 30 min at 55°C). iv) Finally, 200 μL of BCIP/NBT
substrate solution were added in each well for signal generation. The microtiter plate bottom was imaged
using a flatbed scanner (HP Scanjet 3770, Hewlett-Packard) in greyscale (from 0 to 65535 arbitrary units
(a.u.)) with a 2400 dpi resolution. Image analysis (signal quantification) was performed using GenePix Pro
5.0 software (Axon Instrument).The signal intensity per spot was calculated as the median intensity for all
pixels included in a circular feature defining the spot and corrected using a local background evaluation.
Wells were washed with PBS after hybridization, labeling and signal generation steps. For the porous
surface, washing steps were performed through filtration under vacuum condition. For all other surfaces,
measurements were realized using SuperSignalTM solution (Pierce, France) as horseradish peroxidase
substrate in order to achieve bright chemiluminescent signals. The emitted light was collected and
integrated for 180 seconds using a charged coupled device (CCD) cooled camera light measurement
system (EM-CCD, Hamamatsu).
3- RESULTS
The present study aims to demonstrate the impact of the surface architecture of a high-throughput
colorimetric assay on the test results. For that purpose we chose to compare the hybridization efficiency of
real sample targets in a multiplexed assay useful for blood group genotyping [11]. Here, target molecules
were 100-250 bp long PCR products obtained through multiplex PCR of whole blood extracted DNA. The
target sequences were specific to 3 single nucleotide polymorphisms (SNPs) associated with clinically
important blood group antigens (Kell, Kidd and Duffy systems) and the selectivity toward detection of
SNPs in the target sequences was achieved by carrying out all hybridization at 55°C. The principle of the
assay is presented in figure 1-a. Whole blood samples undergo genomic DNA extraction, followed by amplification of target sequences using multiplex PCR and biotinylated primers. The resulting amplicons
are mixed with streptavidin-phosphatase alkaline conjugates and contacted with the immobilized probes
for hybridization. Finally, the alkaline phosphatase ± streptavidin system enables a colorimetric detection RI K\EULGL]DWLRQ WKH LPPRELOL]HG HQ]\PH¶V reaction with its substrate (BCIP/NBT) generates a purple precipitate on hybridized spots. The stained microarrays were finally imaged using an ordinary flatbed
scanner and the grayscale image analyzed for genotype determination.
A schematic view of the different surface architectures tested within this study is presented in figure
1-b. From left to right the surface complexity increases from a flat classical polystyrene substrate (bare or
glutaraldehyde activated), to a pressure sensitive adhesive (PSA) acrylic based coated 2D polymer and
conditions of incubation and pipetting.
Figure 1 (a): Principle of the blood group genotyping colorimetric assay, from [11]. (b): Schematic view of the three different surface architectures tested within the study for oligonucleotide probe immobilization.
Figure 2 draws a global comparison between the different assay efficiencies: the blood group
genotyping results of two patients are presented together with the microarray composition. The first
remarkable observation is the signal weakness onto bare polystyrene compared to other surfaces. In
particular, SNP specific probes immobilized onto bare polystyrene did not even generate measurable
signal from their interaction with PCR products. Moreover, neither bare polystyrene nor glutaraldehyde
Figure 2 Blood group genotyping results for two patients using the four different tested surfaces.
In order to quantitatively compare the four architectures, the colorimetric signals of the dT16 positive
control spots were quantified (figure 3-a). A clear hierarchy of the different surfaces was obtained: from
bare polystyrene (giving the smallest signals and the largest errors) to the porous membrane surface
(giving the highest intensities and the smallest errors). A first interpretation of this trend could rely on the
difference in the immobilization efficiency of the probes onto the different surfaces. Indeed, as can be
seen on SEM images in figure 4, the hierarchy of the surface complexity follows the hierarchy observed in
figure 3-a. This might suggest that the surface density of the immobilized probes, which increases with the
specific surface of the support, is causing the signal difference between the different architectures.
However, it is worth of note that spot intensities depend also on the colorimetric staining efficiency,
which could be itself conditioned by the surface structure. Indeed, different surface architectures are not
the positive control spots using chemiluminescent signals. Error bars are standard deviations, n=4.
To clarify the situation, a chemiluminescent detection (based on horseradish peroxidase label) of the
positive control spots was also performed. This light emitting system is solely dependent upon the amount
of label enzyme present at spot location and then upon the surface density of probes, avoiding thus
product accumulation effect encountered for the colorimetric assay. The quantified results are presented in
figure 3-b (all the different surfaces being white and imaged the same way). As expected, the porous
surface generated once again the highest signal and even if glutaraldehyde activation of polystyrene
increases the surface density of the immobilized probes (higher chemiluminescent signal), it remains far
lower than on the porous mesh. The major difference with the colorimetric profile was the low signal
obtained on the adhesive surface: the spot intensities, almost identical to those obtained onto polystyrene
surfaces, are the proof of low probe density.
Chemiluminescence results confirm that the hierarchy obtained using the colorimetric detection (figure
3-a) was then not only related to different surface densities of probes but also to the capacity of the surface
to efficiently retained the precipitating enzyme colored product. Here, adhesive surface was the most
adhesion of the colorimetric staining.
To complete the comparison of the different surface architectures, the size of the stained spots was also
compared using figure 2 images. Spots diameters of SNPs specific spots were found to be 350 μm, 200
μm and 300 μm for porous surface, adhesive coated surface and activated polystyrene, respectively
(because of signal weakness and low reproducibility no reliable measure can be reported for bare
polystyrene). These spot size differences are due to the surface hydrophobicity of the different substrate
which impact on the spreading of the drops just after spotting. In the case of the porous surface, the
spotted volume is completely absorbed within the
material thanks to capillary forces, leading to a
larger spot size. For the three other surfaces, the hierarchy of the spot size can be related to the measuredcontact angles: 88° and 73° for adhesive coated surface and activated polystyrene, respectively.
Figure 4 Scanning electron microscopy images of the different surfaces tested. (a): porous surface, (b): adhesive coated surface and (c): activated polystyrene.
microarray appeared easier. Indeed, the classical ELISA testing surface (polystyrene or activated
polystyrene), even if capable of immobilizing sufficient amounts of probes are not optimum for
colorimetric signal generation, because of their flatness and their low capacity to retain precipitating
product. On the other side, porous surfaces such as nitrocellulose/cellulose acetate membrane are able to
immobilize large amounts of accessible probes and also to generate intense colorimetric signal. The only
drawback of these porous surfaces is the large size of the obtained spots which reduces the spot density
capability of the system. Finally, adhesive coated surface appeared to be the best compromise between
immobilized probe density, spot density and colorimetric signal intensity. Moreover, an additional
interesting feature of this adhesive material for probe immobilization is its possible integration in complex
system thanks to fast and easy assembly through its adhesive property.
ACKNOLEDGMENTS
The authors thank the Etablissement Français du Sang Rhône-Alpes, especially Jean-Charles Brès and
Dominique Rigal, for providing genotyped multiplex PCR products and the set of probe sequences used