# Microstructured Reactors Designed by Stereolithography and Characterized by Fluorescent Probes

Authors: S. Corbel; F. Evenou; F. Baros; N. Martinet; M. Donner; M. C. Carré
Journal: International Journal of Photoenergy
 (2008)

---

## Abstract

The main objective of this research was to define a structured and functionalized support for future biomedical applications (model of “low-density bioarray”). The experiments were carried out by using stereolithography process with a special SU-8 photoresist and the reproducibility of the method was studied by analyzing the surface profile of the support. Finally, a matrix of regular controlled sized wells was fabricated. Chemical reactions leading to covalent grafting were run to demonstrate that the inner surface of the wells remains still reactive after polymerization. The grafting of fluorophores with carboxylic functions activated by N-hydroxysuccinimide was studied as function of time, in order to determine the best reactions, conditions. Then, the grafting of two distinct fluorescent probes was led simultaneously inside the wells, showing the possibility of spatial localization of diverse reactions on the same support. The covalent and localized bindings were confirmed by fluorescence spectroscopy and microscopy analyses.


---

## Body

## 1. Introduction

Probes
biomolecules   arrays are nowadays more and more used in research field to
detect, determine, or quantify complementary targeted biomolecules (DNA,
proteins, cells, tissues, etc.). An important point is the probe biomolecule
immobilisation on the support which must be well located, stable (a covalent
binding with the support is suitable) while keeping binding properties of
biomolecules. Among the different technology processes for reaching these
structured biosensor supports, thin polymer film-based substrates are currently
used to afford robust patterns [1–5]. So, the shape of the polymeric structure
was devised to fit the “tools” used in Life Sciences Laboratories: multichannel
micropipets for multiple simultaneous depositions, microplates readers for
analyses.In this paper,
we demonstrate that photolithography remains a simple and reproducible process
for polymer patterning that generates surface-confined amino groups which can
be subsequently used for immobilisation of compounds. Through the use of
stereolithography process, we have fabricated a polymeric matrix including millimetre-sized
wells on the surface of glass slides.Thanks
to the hydrophobic nature of the polymer, the ministructures so made can also
provide ideal 3D aqueous environment  which might be very convenient for the
capture of biomolecules.The work can
be resumed in three steps:(1)
the stereolithography
process to get the polymeric matrix,(2)
the covalent grafting
of fluorescent probes to demonstrate that the surface remains reactive in the
wells and finally,(3)
the fluorescence
to validate the grafting.


## 2. Localization of Reactive Sites on a Microscope Slide by Stereolithography

### 2.1. The Supports and Products

The supports,
glass microscope slides silanised with an amino linker, 3-aminopropyltriethoxysilane
(APTS), chemicals, and solvents were  purchased from Sigma-Aldrich  (Chimie Tech Services, Anthony, France). After having tested
different monomers, SU-8 photoresist (MicroChem Corp., Newton, MA USA)  was selected.  Its formulation is based of EPON SU-8 resin
(glycidyl-ether-bisphenol-A novolac) dissolved inγ-butyrolactone (52 wt%), and a triaryl
sulfonium salt as the photoinitiator. The main interesting physical characteristics
are the low density (d=1.164) which allows an easy recoating and the
high glass temperature (Tg> 200°C) suitable for thermal treatment.Stability of
the cured resin was tested by immersing coated
slide in different solvents at room temperature. The polymer appeared very
stable in solvents such as methylene chloride, absolute ethanol, and acetone (2 days
for each of them) in propylene glycol methyl ether acetate (PGMEA), sodium
bicarbonate buffer pH 8.5 (5 days). In pure DMF, the film was completely and
rapidly stripped from the surface keeping its patterned structure.After the
demonstration that polymer issued from SU-8 was sufficiently resistant, we investigated
the step of   shaping by polymerization.


### 2.2. The Coating and Photopolymerization

The process comprised the coating, curing, and baking
operations. Polymerization occurred on one side of the slide, except on the frosted end. Prior to use,
silanized glass slides were rinsed with acetone and dried. SU-8 photoresist
(2 mL) was dropped on the centre of each slide and spun on its surface using a
spin coater (Karl Süss, RC
Spin Coater CT62 V08, Saint Jeoire, France) at a spin speed of 600 rpm during 30 seconds. Solvent
was evaporated by a baking on a hot plate at 95°C
during 5 minutes. Higher baking times were found to lead to air bubbles
formation inside SU-8 film.Stereolithography
process was used to polymerize photoresist at the surface of the slide (Figure1). This technology enables the fabrication of many complex shapes and has been
presented in previous papers [6]. The Spectra-Physics Model 2020 argon ion
laser was used for irradiations in the near ultraviolet
(wavelength 363.8 nm) with an exposure of 300 mJ/cm2. Matrix of
regular polymeric wells was obtained by successive duplications of an empty
disc pattern inside a full polymer square, in the four directions (right, left,
up, down). The shape is composed of a basic element like a hollow cylinder
surrounded by a filled surface around it (Figure 2).Figure 1
Structuration of the
support by stereolithography.Figure 2
Shape of the
polymeric structure obtained on the support (model of “low-density bioarray”).A program has been written in C++
for tracing the pattern with galvanometric mirrors directly on the basis of
geometric parameters, without going through the usual steps of
CAD, slicing, and manufacturing. To get compact
cross-linking, we performed the hatch spacing up to a value less than the laser
beam diameter, in the two orthogonal directions(x,y). The spot laser diameter
was 0.2 mm in width, while the hatch space was set at 0.1 mm. When drawing the
shape, the software uses two kinds of vectors. The first ones are
used for tracing circles and the others are used for filling the outside
surface of the circle with horizontal (x) and vertical (y) lines called
hatching.The improvement consisted of taking into account each point of coordinates
within or outside the circle. Furthermore, there was a
possibility to store hatch information in a file in order to verify the good
accuracy in particular to determine whether there is an intersection between
the hatching line (x or y) and contour lines as circles. Therefore, there was a
double exposition at the intersection of circles and hatching lines as also at
the intersections of horizontal (x) and vertical (y) lines of the hatching step.In order to
achieve the cross-linking of the exposed parts, a postexposure bake was carried
out on a hot plate at 95°C during 2 minutes and then the slide was immersed in
SU-8 developer under agitation for about 5 minutes, then had been dried.
Finally, a strong adhesion of the polymer on the surface of the slide was
obtained by a postcuring under UV lamps for 15 minutes and a baking on a hot
plate at 95°C for 30 minutes.


### 2.3. Dimension Characteristics of  the Polymeric Shapes and Accuracy

Surface
profiles measurements have been recorded with the optical microfocus measurement
system (UBM) and actual heights of the SU-8 wells measured by scanning at the
surface of the polymeric structure build on the support.First, we
defined the elementary shape of a cross-section, which is a circle of 13 mm diameter centred in a square of 17 mm side. By duplicating this pattern once,
we obtained two identical wells as represented on Figure3. Thus, to define the diameter of circle equal to 13 mm, we have to take into account
the diameter of the beam (200 μm).(a) Top view of the geometrical shape of two large wells. (b) ExperimentalAB line profile (dth = 13 mm).
(a)(b)The wells
so defined and fabricated have been analyzed by the optical profilometer along the
directionAB as  shown on Figure 3.The line profile represents the
thickness of the polymer along the lineAB crossing the
centres of the 2 wells. The origin of the vertical axis z which defines the height
corresponds to the level of the horizontal plane of the glass support (z=0).We can observe
that the patterns are clean and correctly reproduced. The polymeric layer seems
uniform with a relatively constant thickness, except on the edge of the square
where the polymer is thicker.These small
peaks due to an over polymerization may be explained, as mentioned before, by
the scanning of the pattern and therefore by a supplementary curing on the
borders of the shape. It can be also mentioned that the surface of the polymer
is not perfectly plane. Nevertheless, the thickness of the polymer remained
more or less constant (between 31 and 38μm) with a variation which can be estimated
to 18%.We have
searched in a second step to manufacture smaller wells, by decreasing the
diameter regularly as far as reaching a minimum value. Although the chosen
resin allows the manufacturing of micro structures, the process is limited by
the focus diameter of the laser beam. We kept the basic
pattern which was a circle centred inside a square. The shape was duplicated by
successive scanning according to one direction; we imposed a decreasing
diameter from 6 to 1 mm, while maintaining constant the interval between the
circle and the outside square (Figure4). The
analysis of the profile, concerning a surface measuring 45 × 12 mm, is represented
on Figure 4. Whatever
the size of the well, the shape was correctly reproduced.(a) Top view of the
theoretical shape of linear matrix with decreasing diameter; (b) line profile of 6 polymer wells with decreasing diameter from 6 to 1 mm.
(a)(b)First of
all, we notice that it is possible to manufacture wells until 1.8 mm of theoretical diameter
presenting satisfactory lateral profiles. Indeed, it was difficult to clean the
inside of the smallest wells (around 1 mm). Between the wells, the polymer walls
have a height of 27μm on average. As it has been observed previously, the
polymer was thicker on the sides with a value varying between 36 and 42 μm. We
also found the small peaks of over polymerization affecting the outside limit
of each well corresponding to the intersections of the hatching lines.The above
results imply the feasibility of more complex patterns. For example, Figure5
shows the fabrication of a 2D matrix with a constant diameter of the wells and
the resulting profile. The fabrication was performed by duplicating several
times the same elementary shape according to x and y directions. The surface analysis was
made along lines L1 and L2 crossing the centres of the wells as shown on Figure 5.(a) Top view of the theoretical matrix surface with the directionsL1 and L2; (b) surface profile along L1 and L2.
(a)(b)One recovers
the peaks of over polymerization already mentioned. Far from the edges, the polymer
surface remains constant according to one axis. However, the polymer is
globally thicker inL2 than in L1 due to the angle of the laser beam with the
vertical axis in the scanning system and the consequent different irradiation
intensity.In order to
determine the accuracy we have scanned a grid in polymer (45× 18 mm) in the two
orthogonal directions with hatch spacing of 1 mm. The corresponding line
profile is presented on Figure 6. At first sight, we can observe that the
polymer thickness is practically constant and equal to 30 μm on the whole
length. These results are otherwise very encouraging since we could define
small square wells of 1 mm side delimited by thin walls in polymer.Figure 6
Line profile of a polymeric matrix with a 1 mm hatch spacing.


## 2.1. The Supports and Products

The supports,
glass microscope slides silanised with an amino linker, 3-aminopropyltriethoxysilane
(APTS), chemicals, and solvents were  purchased from Sigma-Aldrich  (Chimie Tech Services, Anthony, France). After having tested
different monomers, SU-8 photoresist (MicroChem Corp., Newton, MA USA)  was selected.  Its formulation is based of EPON SU-8 resin
(glycidyl-ether-bisphenol-A novolac) dissolved inγ-butyrolactone (52 wt%), and a triaryl
sulfonium salt as the photoinitiator. The main interesting physical characteristics
are the low density (d=1.164) which allows an easy recoating and the
high glass temperature (Tg> 200°C) suitable for thermal treatment.Stability of
the cured resin was tested by immersing coated
slide in different solvents at room temperature. The polymer appeared very
stable in solvents such as methylene chloride, absolute ethanol, and acetone (2 days
for each of them) in propylene glycol methyl ether acetate (PGMEA), sodium
bicarbonate buffer pH 8.5 (5 days). In pure DMF, the film was completely and
rapidly stripped from the surface keeping its patterned structure.After the
demonstration that polymer issued from SU-8 was sufficiently resistant, we investigated
the step of   shaping by polymerization.


## 2.2. The Coating and Photopolymerization

The process comprised the coating, curing, and baking
operations. Polymerization occurred on one side of the slide, except on the frosted end. Prior to use,
silanized glass slides were rinsed with acetone and dried. SU-8 photoresist
(2 mL) was dropped on the centre of each slide and spun on its surface using a
spin coater (Karl Süss, RC
Spin Coater CT62 V08, Saint Jeoire, France) at a spin speed of 600 rpm during 30 seconds. Solvent
was evaporated by a baking on a hot plate at 95°C
during 5 minutes. Higher baking times were found to lead to air bubbles
formation inside SU-8 film.Stereolithography
process was used to polymerize photoresist at the surface of the slide (Figure1). This technology enables the fabrication of many complex shapes and has been
presented in previous papers [6]. The Spectra-Physics Model 2020 argon ion
laser was used for irradiations in the near ultraviolet
(wavelength 363.8 nm) with an exposure of 300 mJ/cm2. Matrix of
regular polymeric wells was obtained by successive duplications of an empty
disc pattern inside a full polymer square, in the four directions (right, left,
up, down). The shape is composed of a basic element like a hollow cylinder
surrounded by a filled surface around it (Figure 2).Figure 1
Structuration of the
support by stereolithography.Figure 2
Shape of the
polymeric structure obtained on the support (model of “low-density bioarray”).A program has been written in C++
for tracing the pattern with galvanometric mirrors directly on the basis of
geometric parameters, without going through the usual steps of
CAD, slicing, and manufacturing. To get compact
cross-linking, we performed the hatch spacing up to a value less than the laser
beam diameter, in the two orthogonal directions(x,y). The spot laser diameter
was 0.2 mm in width, while the hatch space was set at 0.1 mm. When drawing the
shape, the software uses two kinds of vectors. The first ones are
used for tracing circles and the others are used for filling the outside
surface of the circle with horizontal (x) and vertical (y) lines called
hatching.The improvement consisted of taking into account each point of coordinates
within or outside the circle. Furthermore, there was a
possibility to store hatch information in a file in order to verify the good
accuracy in particular to determine whether there is an intersection between
the hatching line (x or y) and contour lines as circles. Therefore, there was a
double exposition at the intersection of circles and hatching lines as also at
the intersections of horizontal (x) and vertical (y) lines of the hatching step.In order to
achieve the cross-linking of the exposed parts, a postexposure bake was carried
out on a hot plate at 95°C during 2 minutes and then the slide was immersed in
SU-8 developer under agitation for about 5 minutes, then had been dried.
Finally, a strong adhesion of the polymer on the surface of the slide was
obtained by a postcuring under UV lamps for 15 minutes and a baking on a hot
plate at 95°C for 30 minutes.


## 2.3. Dimension Characteristics of  the Polymeric Shapes and Accuracy

Surface
profiles measurements have been recorded with the optical microfocus measurement
system (UBM) and actual heights of the SU-8 wells measured by scanning at the
surface of the polymeric structure build on the support.First, we
defined the elementary shape of a cross-section, which is a circle of 13 mm diameter centred in a square of 17 mm side. By duplicating this pattern once,
we obtained two identical wells as represented on Figure3. Thus, to define the diameter of circle equal to 13 mm, we have to take into account
the diameter of the beam (200 μm).(a) Top view of the geometrical shape of two large wells. (b) ExperimentalAB line profile (dth = 13 mm).
(a)(b)The wells
so defined and fabricated have been analyzed by the optical profilometer along the
directionAB as  shown on Figure 3.The line profile represents the
thickness of the polymer along the lineAB crossing the
centres of the 2 wells. The origin of the vertical axis z which defines the height
corresponds to the level of the horizontal plane of the glass support (z=0).We can observe
that the patterns are clean and correctly reproduced. The polymeric layer seems
uniform with a relatively constant thickness, except on the edge of the square
where the polymer is thicker.These small
peaks due to an over polymerization may be explained, as mentioned before, by
the scanning of the pattern and therefore by a supplementary curing on the
borders of the shape. It can be also mentioned that the surface of the polymer
is not perfectly plane. Nevertheless, the thickness of the polymer remained
more or less constant (between 31 and 38μm) with a variation which can be estimated
to 18%.We have
searched in a second step to manufacture smaller wells, by decreasing the
diameter regularly as far as reaching a minimum value. Although the chosen
resin allows the manufacturing of micro structures, the process is limited by
the focus diameter of the laser beam. We kept the basic
pattern which was a circle centred inside a square. The shape was duplicated by
successive scanning according to one direction; we imposed a decreasing
diameter from 6 to 1 mm, while maintaining constant the interval between the
circle and the outside square (Figure4). The
analysis of the profile, concerning a surface measuring 45 × 12 mm, is represented
on Figure 4. Whatever
the size of the well, the shape was correctly reproduced.(a) Top view of the
theoretical shape of linear matrix with decreasing diameter; (b) line profile of 6 polymer wells with decreasing diameter from 6 to 1 mm.
(a)(b)First of
all, we notice that it is possible to manufacture wells until 1.8 mm of theoretical diameter
presenting satisfactory lateral profiles. Indeed, it was difficult to clean the
inside of the smallest wells (around 1 mm). Between the wells, the polymer walls
have a height of 27μm on average. As it has been observed previously, the
polymer was thicker on the sides with a value varying between 36 and 42 μm. We
also found the small peaks of over polymerization affecting the outside limit
of each well corresponding to the intersections of the hatching lines.The above
results imply the feasibility of more complex patterns. For example, Figure5
shows the fabrication of a 2D matrix with a constant diameter of the wells and
the resulting profile. The fabrication was performed by duplicating several
times the same elementary shape according to x and y directions. The surface analysis was
made along lines L1 and L2 crossing the centres of the wells as shown on Figure 5.(a) Top view of the theoretical matrix surface with the directionsL1 and L2; (b) surface profile along L1 and L2.
(a)(b)One recovers
the peaks of over polymerization already mentioned. Far from the edges, the polymer
surface remains constant according to one axis. However, the polymer is
globally thicker inL2 than in L1 due to the angle of the laser beam with the
vertical axis in the scanning system and the consequent different irradiation
intensity.In order to
determine the accuracy we have scanned a grid in polymer (45× 18 mm) in the two
orthogonal directions with hatch spacing of 1 mm. The corresponding line
profile is presented on Figure 6. At first sight, we can observe that the
polymer thickness is practically constant and equal to 30 μm on the whole
length. These results are otherwise very encouraging since we could define
small square wells of 1 mm side delimited by thin walls in polymer.Figure 6
Line profile of a polymeric matrix with a 1 mm hatch spacing.


## 3. Fluorophores Covalent Grafting and Characterization

Glass
surfaces are well adapted   for microarray applications because they present good mechanical
properties, with low intrinsic fluorescence. They also possess a relatively
homogeneous chemical surface which can be easily modified and furthermore, they
are not very expensive. For our study, we have selected commercially available
microscope slides already silanized by a hetero linker bearing an amino
terminal function.Furthermore,
for a future sensor application, the spots need to be uniform, accurate without
spreading the analytes solution to the adjacent areas of the macroarrays. We
have thought that these requirements could be achieved through the creation of
wells embedded in a polymer covering the surface.


### 3.1. Fluorophores Covalent Grafting

Chemical
reactions leading to covalent grafting were run to demonstrate that the inner
surface of the wells remains active after the polymerization.We have
chosen as models two distinct wavelength fluorescent probes already studied in
our laboratory [7, 8]. These fluorophores are “molecular rotors”
easily synthezised: the first compound
2-cyano-5-(4-(N,N-dimethylamino)phenyl)-2,4-pentadienoic acid (CiCOOH) is a cinnamilydene
derivative [7]; the second one, 7-(N,N-diethylamino)coumarin-3-carboxylic acid
(CouCOOH), has a coumarin structure [9] (Figure 7). The two fluorescent probes
comprise in their structure a carboxylic acid function well suited for a covalent
robust amide bond with the amino of the surface.Structure formulas of the two fluorophores CiCOOH and CouCOOH.
(a)(b)As previously
described about covalent graftings of other acidic compounds on solid
substrates [10], the acidic function has been activated with
N-hydroxysuccinimide.Grafting of
fluorophore active esters to amino solid support through amide bond, occurs at room
temperature, safe from light, in sodium bicarbonate buffer, pH 8.5. After
grafting, the slides were carefully washed by successive immersions under
magnetic stirring, in deionised water, absolute ethanol, and dichloromethane.
Then, they were air dried and stored until fluorescence analysis. The covalent
bindings on the slides were confirmed by fluorescence spectroscopy and
microscopy analyses.


### 3.2. Fluorescence Spectroscopy Study of the Grafted Probes

The covalent
binding of each fluorophore was first conducted on a separate slide to define
their respective fluorescence properties. For this study, the slide was
immerged in the sodium bicarbonate buffer, pH 8.5, containing the fluorophore,
under slight agitation during 4 hours. After the general work-up described
above, the fluorescence has been measured at different sites all along the
slides. The fluorescence spectra were recorded on a Horiba Jobin-Yvon
Fluorolog3 spectrofluorimeter and were corrected from lamp variations. Experiments
were carried out at room temperature, the glass slide being in front-face position
in order to avoid light reflection. The excitation/emission fluorescence fields
of the two fluorophores were well separated as expected considering previous
studies. The conditions of excitation-emission were 470–565 nm and 410–467 nm for
Fluorophores CiCONHX and CouCONHX, respectively.We have  gathered (Figures8 and 9) the emission spectra obtained with each fluorophore. For the
cinnamylidene derivative (CiCOOH grafting), the fluorescence signal Ai was recorded at six places on the slide (Figure 8). The signal B (background) of
the native slide has been also reported; it has been subtracted from signal A1 to exhibit the grafted cinnamilydene fluorescence (A1−B); so the maximum
fluorescence is well characterized at 565 nm for a 470 nm excitation. Two
remarks might here be done: on the one hand, the signal is not uniformall over the slide, with a 28%
variation observed between two extreme measures: this fact may be imputed on
the inhomogeneity of the native slide, but on the other hand the fluorophore
response is distinct from the support one at this wavelength excitation.Figure 8
Emission spectra obtained with the cinnamylidene derivative (CiCOOH grafting);Ai:
recorded at 6 different positions on the slide; B: background signal of the
original slide; A1−B: signal corrected from background.Figure 9
Emission spectra obtained with the coumarine derivative (CouCOOH grafting).For
the coumarine derivative (CouCOOH grafting) the fluorescence signal was also recorded
at six places of the slide (Figure9). We have not reported the signal of the
native slide which was negligible at this wavelength excitation. In this case,
we can see a low deviation of the fluorescence signal; indeed, a difference of 11%
between the two extreme spectra has been observed. Another interesting feature
is the very important observed fluorescent signal.These
two fluorophores show specific fluorescent characteristics which have been useful
for the characterization of localized graftings.


### 3.3. Fluorescence Microscopy Study for the Localization of the Grafted Probes

In
order to determine the optimal reactions’ conditions on the structured support,
the grafting of these two fluorophores was studied in function of time (Figure10).
Grafting reaction was carried out by successive deposition of the same volume
of known fluorophore concentration solution into the macro wells in a saturated
sodium bicarbonate buffer atmosphere. The reaction occurs with time, the first
deposition corresponding to the longest-time reaction and the last one to the
shortest-time reaction.Time evolution in covalent grafting of CiCOONHS ((a) 6 hours, 9 hours, 24 hours, 48 hours, and 80 hours) and CouCOONHS ((b)
3 hours, 6 hours, 9 hours, 24 hours, 48 hours). The best conditions were reached after 24 hours of reaction.
(a)(b)The
fluorescence intensities in  macrowells were measured with a Zeiss Axiotech
epifluorescence microscope, coupled  to a Hamamatsu IP28 photomultiplier. Two
filter sets were used in order to differentiate the two fluorescent probes
CiCOOH and CouCOOH. All measurements were repeated five times, at different
places of the well, the diameter of the excitation spot being fixed to 200μm. As we can see on
Figure 10, the best conditions of grafting the two fluorophores were obtained
after 24 hours of reaction.Then, the
grafting of the two distinct fluorescent probes (CiCOOH) and (CouCOOH) was led
simultaneously inside the wells. For this, we have
deposited the two probes alternatively in a matrix of 10 wells to confirm that
the inner area was still reactive after the polymerization step (Figure11).
The aim of this experiment was to prove that many different reactions may be conducted
on a same support which is a condition of future developments in the industry
for diagnostics test.Figure 11
Matrix of 2× 5
wells (diameter = 5 mm; distance between wells = 3 mm) with CiCOONHS and CouCOONHS
grafted alternatively by deposition of 10 μL≈60 nmol of each fluorophore solution.The fluorescence of each probe was recorded in
five selected places in the same well, by fluorescence microscopy. Dispersion
of measurements is shown on Figure12 using the filters set relative to CouCOOH
probe. Even if homogeneity in a well seems not as good as those obtained on a
bare slide, due to the smaller size of the observation spot, the two probes are
clearly differentiated, in spite of a weak overlap between the two filter sets.Figure 12
CiCOONHS and CouCOONHS alternatively grafted in the 10 wells of the matrix analyzed by fluorescence microscopy (filters Zeiss:λexc [395–440]; λem> 470 nm; magnification ×5; gauge 4).


## 3.1. Fluorophores Covalent Grafting

Chemical
reactions leading to covalent grafting were run to demonstrate that the inner
surface of the wells remains active after the polymerization.We have
chosen as models two distinct wavelength fluorescent probes already studied in
our laboratory [7, 8]. These fluorophores are “molecular rotors”
easily synthezised: the first compound
2-cyano-5-(4-(N,N-dimethylamino)phenyl)-2,4-pentadienoic acid (CiCOOH) is a cinnamilydene
derivative [7]; the second one, 7-(N,N-diethylamino)coumarin-3-carboxylic acid
(CouCOOH), has a coumarin structure [9] (Figure 7). The two fluorescent probes
comprise in their structure a carboxylic acid function well suited for a covalent
robust amide bond with the amino of the surface.Structure formulas of the two fluorophores CiCOOH and CouCOOH.
(a)(b)As previously
described about covalent graftings of other acidic compounds on solid
substrates [10], the acidic function has been activated with
N-hydroxysuccinimide.Grafting of
fluorophore active esters to amino solid support through amide bond, occurs at room
temperature, safe from light, in sodium bicarbonate buffer, pH 8.5. After
grafting, the slides were carefully washed by successive immersions under
magnetic stirring, in deionised water, absolute ethanol, and dichloromethane.
Then, they were air dried and stored until fluorescence analysis. The covalent
bindings on the slides were confirmed by fluorescence spectroscopy and
microscopy analyses.


## 3.2. Fluorescence Spectroscopy Study of the Grafted Probes

The covalent
binding of each fluorophore was first conducted on a separate slide to define
their respective fluorescence properties. For this study, the slide was
immerged in the sodium bicarbonate buffer, pH 8.5, containing the fluorophore,
under slight agitation during 4 hours. After the general work-up described
above, the fluorescence has been measured at different sites all along the
slides. The fluorescence spectra were recorded on a Horiba Jobin-Yvon
Fluorolog3 spectrofluorimeter and were corrected from lamp variations. Experiments
were carried out at room temperature, the glass slide being in front-face position
in order to avoid light reflection. The excitation/emission fluorescence fields
of the two fluorophores were well separated as expected considering previous
studies. The conditions of excitation-emission were 470–565 nm and 410–467 nm for
Fluorophores CiCONHX and CouCONHX, respectively.We have  gathered (Figures8 and 9) the emission spectra obtained with each fluorophore. For the
cinnamylidene derivative (CiCOOH grafting), the fluorescence signal Ai was recorded at six places on the slide (Figure 8). The signal B (background) of
the native slide has been also reported; it has been subtracted from signal A1 to exhibit the grafted cinnamilydene fluorescence (A1−B); so the maximum
fluorescence is well characterized at 565 nm for a 470 nm excitation. Two
remarks might here be done: on the one hand, the signal is not uniformall over the slide, with a 28%
variation observed between two extreme measures: this fact may be imputed on
the inhomogeneity of the native slide, but on the other hand the fluorophore
response is distinct from the support one at this wavelength excitation.Figure 8
Emission spectra obtained with the cinnamylidene derivative (CiCOOH grafting);Ai:
recorded at 6 different positions on the slide; B: background signal of the
original slide; A1−B: signal corrected from background.Figure 9
Emission spectra obtained with the coumarine derivative (CouCOOH grafting).For
the coumarine derivative (CouCOOH grafting) the fluorescence signal was also recorded
at six places of the slide (Figure9). We have not reported the signal of the
native slide which was negligible at this wavelength excitation. In this case,
we can see a low deviation of the fluorescence signal; indeed, a difference of 11%
between the two extreme spectra has been observed. Another interesting feature
is the very important observed fluorescent signal.These
two fluorophores show specific fluorescent characteristics which have been useful
for the characterization of localized graftings.


## 3.3. Fluorescence Microscopy Study for the Localization of the Grafted Probes

In
order to determine the optimal reactions’ conditions on the structured support,
the grafting of these two fluorophores was studied in function of time (Figure10).
Grafting reaction was carried out by successive deposition of the same volume
of known fluorophore concentration solution into the macro wells in a saturated
sodium bicarbonate buffer atmosphere. The reaction occurs with time, the first
deposition corresponding to the longest-time reaction and the last one to the
shortest-time reaction.Time evolution in covalent grafting of CiCOONHS ((a) 6 hours, 9 hours, 24 hours, 48 hours, and 80 hours) and CouCOONHS ((b)
3 hours, 6 hours, 9 hours, 24 hours, 48 hours). The best conditions were reached after 24 hours of reaction.
(a)(b)The
fluorescence intensities in  macrowells were measured with a Zeiss Axiotech
epifluorescence microscope, coupled  to a Hamamatsu IP28 photomultiplier. Two
filter sets were used in order to differentiate the two fluorescent probes
CiCOOH and CouCOOH. All measurements were repeated five times, at different
places of the well, the diameter of the excitation spot being fixed to 200μm. As we can see on
Figure 10, the best conditions of grafting the two fluorophores were obtained
after 24 hours of reaction.Then, the
grafting of the two distinct fluorescent probes (CiCOOH) and (CouCOOH) was led
simultaneously inside the wells. For this, we have
deposited the two probes alternatively in a matrix of 10 wells to confirm that
the inner area was still reactive after the polymerization step (Figure11).
The aim of this experiment was to prove that many different reactions may be conducted
on a same support which is a condition of future developments in the industry
for diagnostics test.Figure 11
Matrix of 2× 5
wells (diameter = 5 mm; distance between wells = 3 mm) with CiCOONHS and CouCOONHS
grafted alternatively by deposition of 10 μL≈60 nmol of each fluorophore solution.The fluorescence of each probe was recorded in
five selected places in the same well, by fluorescence microscopy. Dispersion
of measurements is shown on Figure12 using the filters set relative to CouCOOH
probe. Even if homogeneity in a well seems not as good as those obtained on a
bare slide, due to the smaller size of the observation spot, the two probes are
clearly differentiated, in spite of a weak overlap between the two filter sets.Figure 12
CiCOONHS and CouCOONHS alternatively grafted in the 10 wells of the matrix analyzed by fluorescence microscopy (filters Zeiss:λexc [395–440]; λem> 470 nm; magnification ×5; gauge 4).


## 4. Conclusions and Future Works

This
preliminary work validates the fabrication process for generating polymeric
matrix of wells, the size, the geometry, the number, the localization of which
being well controlled. We have demonstrated the possibility to localise
reactive sites in a polymer matrix shaped on a commercial glass slide. In this
way, three main points have been developed. The local area on functionalized
substrates delimited by polymer wells remained reactive as demonstrated by the
covalent  graftings of suited fluorophores followed by spectrofluorimetry or
fluorescence microscopy  analyses. We have cured the resin SU-8 with a closely
spaced hatch pattern until 1mm resolution. An over polymerization at the
crossing points of the plotting lines has been demonstrated. This problem will
be resolved by developing a higher-density microarrays synthesized by
photolithographic methods analogous to the techniques used in the semiconductor
industry. This original method is based on dynamic photolithographic masks
provided by  Texas Instrument (Tex, USA) with digital light processor (DLP), often called  digital micromirror device (DMD) [11, 12]. Future biomedical implementations include
antibody array where different antibodies could be grafted into the wells to
realize micro ELISA tests; an immunohistochemistry robot could be used to treat
the glass slides.



---

*Source: 757510-2008-07-31.xml*