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Sensors and Actuators B 130 (2008) 436–448

Critical points in the fabrication of microfluidic devices on glass substrates

Mario Castaño-Álvarez a , Diego F. Pozo Ayuso b , Miguel Garcı́a Granda b ,
M. Teresa Fernández-Abedul a , Jose Rodrı́guez Garcı́a b , Agustı́n Costa-Garcı́a a,∗
a Departamento de Quı́mica Fı́sica y Analı́tica, Universidad de Oviedo, 33006 Oviedo, Asturias, Spain
b Departamento de Fı́sica, Universidad de Oviedo, 33007 Oviedo, Asturias, Spain

Available online 19 September 2007

Abstract
Miniaturized total analysis systems are becoming a powerful tool for analytical and bioanalytical applications. In this work, microfluidic channels
have been fabricated on glass substrates using photolithography and wet etching. Although these techniques are well-known and established, the
influence of different parameters on the fabrication process of microchannels is of great importance. Thus, practical aspects and critical points
of the procedure were considered and evaluated. Resulting glass chips with microfluidic channels were sealed with a cover plate to enclose the
channels. Different low and high temperature bonding procedures have been evaluated. Microfluidic chips have been used in combination with a
metal-wire end-channel amperometric detector for capillary electrophoresis (CE). The microfluidic channels and the amperometric detector have
been tested using p-aminophenol as model analyte demonstrating that these devices are useful for analytical applications.
© 2007 Elsevier B.V. All rights reserved.

Keywords: Glass microfluidic; Electrophoresis microchip; Photolithography; Wet etching; Thermal bonding; Electrochemical detection

1. Introduction such as Topas [14] or Zeonor [15,16] have also received

Micro-total analysis systems (␮TAS) have gained a
great interest, especially since capillary electrophoresis (CE)
microchips were proposed by Manz et al. [1,2]. These devices
have several characteristic features such as speed, versatility,
high performance, negligible consumption of reagent/sample
and waste generation as well as the possibility of integration of
various analytical steps, including sample preparation, mixing,
reaction, separation and detection.
Microfluidic fabrication clearly depends on the material
employed. These devices have been mainly made of sil-
icon and glass substrates using standard photolithographic
and etching techniques [3–5]. In the last years, polymer
materials are of increasing interest because their potentially
low manufacturing costs may allow them to be dispos-
able. Several polymers, including poly(methylmethacrylate)
(PMMA) [6,7], poly(dimethylsiloxane) (PDMS) [8,9], poly-
carbonate (PC) [10], polyester [11], polystyrene (PS)
[12] and poly(ethyleneterephthalate) (PET) [13], have been
employed. Recently, cyclic olefin polymers and copolymers
∗ Corresponding author. Tel.: +34 9 85103488; fax: +34 9 85103125.
E-mail address: costa@fq.uniovi.es (A. Costa-Garcı́a).
attention.
Fabrication processes on glass substrates are time-consuming
and clean-room facilities are required. Therefore, the result-
ing microchips are usually expensive for use as disposable
devices. However, characteristics such as good optical prop-
erties, efficiency in dissipating heat, well-understood surface
characteristics as well as high resistance to mechanical stress and
chemicals make them the first option. Moreover, their behavior
is quite similar to traditional fused silica electrophoresis capil-
laries [17]. Ideal geometries of resulting microfluidic channels
involve a high aspect ratio channel, which means deep channels
with parallel sides. Non-parallel walls occur in glass with wet-
etching procedures because this process occurs on the exposed
glass surface; hence, as the channel etches deeper, the walls are
also etched. The result is channels wider at the top than at the
base. An alternative to produce very deep channels with parallel
sides is the use of dry etching techniques, such as powder blast-
ing [18,19], plasma or deep reactive ion etching (DRIE) [20]
and laser ablation [21]. However, these techniques require high
cost instrumentation set-up and maintenance.
After channels microfabrication, the system has to be assem-
bled enclosing the channel networks or microstructures to allow
fluids to flow through the device. In most cases, a cover plate
made of the same material as the base is sealed to the device.

0925-4005/$ – see front matter © 2007 Elsevier B.V. All rights reserved.
doi:10.1016/j.snb.2007.09.043
 M. Castaño-Álvarez et al. / Sensors and Actuators B 130 (2008) 436–448
The procedure for glass-to-glass bonding is not always straight-
forward as is shown in bibliography. Thus, various bonding
methods such as anodic bonding, silicon fusion bonding and
thermal bonding have been developed [4,5,22–25]. The thermal
bonding process is used often in quartz and glass microchannels,
although channel deformations are possible due to the high tem-
perature required. Alternatively, low temperature process based
on intermediate layers [26–32] has been used for glass-to-glass
bonding.
Glass microfluidic devices can be employed for cap-
illary electrophoresis (CE). These capillary electrophoresis
microchips require a sensitive and miniaturized detection sys-
tem. Electrochemical detection (EC), especially amperometric
detection, has been successfully employed in CE microchips
due to advantages such as inherent miniaturization, sensitiv-
ity, low cost, portability and compatibility with microfabrication
technology [33].
Electrochemical detection requires an appropriate design
of the detector in order to ensure electrical isolation from
the high separation voltage. Three approaches have been
reported for coupling EC detection to CE: end-channel, in-
channel and off-channel [34]. Although in-channel [35,36]
and off-channel [37] formats are supposed to be more sen-
sitive, the end-channel configuration has been more widely
employed due to the simplicity and variety of detector designs
[9,11,14,38–40].
In this work, a fast, low cost and reliable process for
the fabrication of microfluidic devices on different glass sub-
strates has been developed. The requirement for clean-room
conditions was less rigorous than those reported in the bib-
liography. A rigorous study on the critical points affecting
the fabrication procedure has been performed. Considerable
attention has been directed toward simplifying and improv-
ing the reliability of the different manufacturing steps. Finally,
a simple procedure for glass-to-glass bonding has been also
described. The developed microchip has been applied to
capillary electrophoresis demonstrating its analytical applica-
tion.
2. Experimental
2.1. Reagents and materials
p-Aminophenol (pAP), tris(hydroxymethyl) aminomethane
(Tris), glycine (Gly) and sodium silicate solution (SiO2 ·Na2 O,
35%) were purchased from Sigma–Aldrich (St. Louis,
MO 63103, USA). Potassium chloride, sodium hydroxide,
ammonium hydroxide (25%), hydrogen peroxide (30%),
acetone, isopropyl alcohol, nitric (65%), acetic (99%), sulfuric
(95–97%) and hydrochloric (37%) acid were obtained from
Merck (D-6100 Darmstadt, Germany). Hydrofluoric acid
(48%), ammonium fluoride solution (40%) were supplied by
Fluka (9471 Buchs, Switzerland).
p-Aminophenol solution was prepared daily in the running
buffer and protected from light. All solutions were filtered
through nylon syringe filters (Cameo 30N, 0.1 ␮m, 30 mm)
obtained from Osmonics (Minnetonka, MN 55343, USA).
437
Water was purified employing a Milli-Q plus 185 equip from
Millipore (Bedford, MA 01730). All other reagents were of
analytical reagent grade.
Micropipettes, 0.250 and 1.0 mL tips, and 1.5 mL tubes
obtained from Eppendorf (22331 Hamburg, Germany) were also
employed.
The rest of volumetric material (flasks, pipettes, vessels. . .)
was of analytical reagent grade.
2.2. Microchip fabrication
Microfluidic channels have been fabricated in different glass
substrates using photolithography and wet-etching technique.
Fabrication process has been performed in a clean room and
involved various steps (Fig. 1):
2.2.1. Substrate preparation
Different glass substrates have been evaluated: com-
mercially available soda-lime microscope glass slides
(76 mm × 25 mm × 1 mm, Menzel-Glässer, Germany), pol-
ished borofloat, and soda-lime glass (75 mm × 25 mm × 1 mm,
Vitrotec S.A., Spain). Pyrex wafers (10 cm diameter, Vitrotec
S.A., Spain), which were cut in small pieces, have been used
too.
Glass surfaces have to be adequately cleaned before pho-
toresist deposition. Different washing procedures have been
evaluated.
The first one (C1) is described as follows:
(1) Substrates were left soaking in deionized water with com-
mercial detergent overnight.
(2) They were sonicated in deionized water with commercial
detergent at 40 ◦ C for 30 min.
(3) Substrates were rinsed several times with deionized water
in an ultrasonic bath.
(4) They were sonicated in isopropyl alcohol for 30 min in order
to eliminate the water.
(5) Finally, they were blown dry with nitrogen gas.
The second washing (C2) consisted of:
(1) Substrates were left soaking in deionized water with com-
mercial detergent overnight.
(2) Substrates were rinsed with deionized water and acetone in
order to eliminate organic material.
(3) They were cleaned in piranha solution (H2 SO4 :H2 O2 , 3:1)
for 15 min in an ultrasonic bath.
(4) They were rinsed with deionized water in an ultrasonic
bath.
(5) They were sonicated in isopropyl alcohol for 30 min in order
to eliminate the water.
(6) Finally, they were blown dry with nitrogen gas.
The last washing tested (C3) was based on:
(1) Substrates were left soaking in deionized water with com-
mercial detergent overnight.
438 M. Castaño-Álvarez et al. / Sensors and Actuators B 130 (2008) 436–448
Fig. 1. Schematic representation of microfabrication process on glass substrates.
(2) Substrates were rinsed with deionized water and acetone in
order to eliminate organic material.
(3) Substrates were cleaned with NaOH 0.1 M for 15 min in an
ultrasonic bath.
(4) They were rinsed with deionized water in an ultrasonic bath.
(5) They were sonicated in isopropyl alcohol for 30 min in order
to eliminate the water.
(6) Finally, they were blown dry with nitrogen gas.
After washing, substrates were dried in an oven at 90 ◦ C for
15 min.
2.2.2. Photolithography
Glass substrates, depending of photoresist to be used, were
spin-coated with Microposit Primer (Shipley, UK) in order
to improve the photoresist adherence. The primer-treated or
un-treated substrates were coated with a positive photoresist.
Two different photoresist, S1813 (Shipley, UK) and ma-P1275
(Microresist Technology, Germany), were used in this study.
Conditions of the spin-coating step (speed, acceleration and
time) were optimized. After photoresist deposition, the sub-
strates were soft-baked in an oven at 90 ◦ C for 30 min. The UV
lithography was processed using chromium photomasks. These
masks have been fabricated using a low cost laser photomask
system developed in our laboratory [41]. This makes possible
an easy modification of the designed pattern, as shown in Fig. 2,
obtaining high-resolution mask (<5 ␮m).
The glass substrates, coated with photoresist, were exposed
to UV radiation (λ = 365 nm), through the fabricated mask,
placed by simple contact on top of the photoresist. Thus, a
mask aligner is not necessary to use. The exposure dose was
calculated considering the power of the lamp, which has been
previously characterized. The density of energy used was 150
and 252 mJ cm−2 for S1813 and ma-P1275, respectively.
Development of the photoresist was accomplished in 90 s
by immersing the exposed substrate into the developer, MF-311
(Shipley, UK) for S1813 and ma-D331 (Microresist Technology,
Germany) for ma-P1275. After rinsing with deionized water, the
temperature and time of the photoresist hard baking were studied
for each photoresist.
2.2.3. Wet chemical etching
Finally, glass substrates were immersed in the etching solu-
tion. Different HF-based solutions and etching times as well as
several types of stirring have been evaluated.
After etching, substrates were rinsed with deionized water
and the photoresist was removed by dipping in acetone.
2.2.4. Glass bonding
After microchannel fabrication, the system has to be assem-
bled, enclosing the channel networks or microstructures to allow
fluids to flow through the device. The device assembly has been
made using different bonding processes. Before bonding, it is
necessary to make connection holes to the flow channel (for sup-
plying buffer and sample solutions). Those were manufactured
by drilling the microslide with diamond drills (2 mm diameter)
at the end of the microfluidic channels.
Since glass bonding depends on the chemical surface state of
the wafers to be sealed, the glass has to be adequately clean and
flat. Different techniques based on intermediate layer as well
 M. Castaño-Álvarez et al. / Sensors and Actuators B 130 (2008) 436–448
Fig. 2. Photomask pattern with (I) twin T and (II) Π designs.
as thermal bonding have been evaluated. The micromachined
substrate and cover plate were treated as follows:
(1) Cleaning: sonicate in deionized water for 30 min and fur-
therly in acetone for 15 min.
(2) Hydrophilic treatment: the substrates to be bonded were
treated with NH4 OH:H2 O2 (3:1) solution for 30 min to
give them hydrophilic properties. Next, the substrates were
rinsed with deionized water, isopropyl alcohol and blown
dry with nitrogen gas.
(3) Intermediate layer: the cover plate was treated with several
intermediate layers. A two-component epoxy glue (Araldit),
a thin adhesive tape and a solution of sodium silicate
(SiO2 ·Na2 O) were chosen for sealing the glass plates. Dif-
ferent procedures for deposition of the intermediate layers
have been evaluated.
(4) Bonding: the treated cover plate surface was immediately
brought in contact with the glass substrate. Different bond-
ing temperatures have been evaluated for annealing the
device.
2.3. Electrochemical detector
The amperometric detector was situated in the waste reser-
voir (B) with a three-electrode configuration. The reference and
counter electrodes were coupled in a 250 ␮L micropipette tip.
The reference electrode consisted of a 1 mm diameter silver
wire anodized in saturated KCl, introduced in a tip through a
syringe rubber piston. The tip is filled with saturated KCl solu-
tion and contains a low resistance liquid junction. A platinum
439
wire (0.3 mm diameter) that acted as auxiliary electrode was
externally fixed with insulating tape.
The working electrode was manually aligned at the outlet
of the separation channel (as previously reported [14]) and it
consisted of a 250 ␮m diameter gold wire (Alfa Aesar, Ger-
many). The wire was introduced in the reservoir with the aid of
a micropipette tip, then, it was adhered to the microchip with
Araldit (Vantico AG, Basel, Switzerland). Finally, 1 cm long
piece of a 250 ␮L micropipette tip is adhered to the chip hole
fixing the wire.
2.4. Apparatus
In order to fabricate the photomasks, a chromium layer must
be deposited on top of a glass substrate. This layer was deposited
by evaporation using an Emitech K950X (UK). The substrates
were placed at the top of a high vacuum chamber. This chamber
is pumped by a Varian DS-102 rotary pump assisted by a Turbo-
molecular pump (Boc Edwards, UK) reaching a pressure of less
than 10−5 bar. In the bottom of the chamber, the chromium is sit-
uated inside a tungsten basket. After high vacuum was reached,
a current of 10 A was applied through the basket, increasing
its temperature and thus evaporating the metal, which is spread
all over the chamber walls and substrates. A thin layer of the
metal is obtained on the glass substrates and the layer thickness
(100 nm) is controlled by a K150X Film Thickness Monitor
(Emitech, UK).
Photoresist deposition was made by spin-coating using a sin-
gle wafer spin processor model WS-400A-6NPP from Laurell
Technologies Corporation (North Wales, USA). The process
440 M. Castaño-Álvarez et al. / Sensors and Actuators B 130 (2008) 436–448
involves depositing a small puddle of photoresist onto the cen-
tre of the glass substrate and then spinning the substrate at high
speed fixed previously. Centripetal acceleration will cause the
photoresist to spread to, and eventually off, the edge of the
substrate leaving a thin film of photoresist on the surface [42].
A 473 nm Nd:YAG laser (CrystaLaser, USA) was employed
for the direct photoresist exposure and the fabrication of pho-
tomasks. The glass substrates with the photoresist were moved
during the laser beam exposure using two high-accuracy trans-
lation stages M-605.2DD (Physik Instrumente, Germany) of
50 mm (Y-axis) and 25 mm (X-axis). A shutter SH05 (ThorLabs,
USA) was also employed for controlling the laser emission.
Translation stages and shutter were interfaced to Pentium 3,
500 MHz, 128 MB RAM computer system and controlled by
custom software based on LabView.
The final exposure of the samples has been made employing
a 365 nm UV lamp (LTF Labortechnik, Germany).
Thermal bonding was made in a muffle furnace from
Nabertherm (Germany).
An ultrasonic bath, a magnetic stirrer Agimatic-E and a
digitally controlled oven from JP Selecta (Spain) were also
employed.
Characterization of microfluidic channels were performed
using a confocal microscope Leica TCS SP2/AOBS (Leica
Microsystems, Germany). The images were processed employ-
ing Leica confocal software.
Amperometric detection was performed with an Autolab
PGSTAT 10 (ECO Chemie, Netherlands) potentiostat interfaced
to a Pentium Celeron, 333 MHz, 64 MB RAM computer sys-
tem and controlled by Autolab GPES software version 4.9 for
Windows 98.
2.5. Electrophoresis procedure
The separations in CE microchips were electrokinetically
driven using two high-voltage power supplies (HVPS, MJ series)
with a maximum voltage of +5000 V from Glassman High Volt-
age (High Bridge, NJ 08829-0317, USA). They were interfaced
to a Pentium Celeron, 333 MHz, 64 MB RAM computer sys-
tem and monitored by a DT300 Series Board, DT Measure
Foundry 4.0.6 software for Windows 98. Positive connections
were located in A and C reservoirs and grounds where situated in
B and D reservoirs (Fig. 2). In this way, a high voltage between
A and B (separation channel) and between C and D (injection
channel) can be applied. High-voltage electrodes consisted of
0.3 mm diameter, 1 cm long platinum wires (Goodfellow) were
inserted into each of the reservoirs and connected by means of
crocodile clips to the HVPS.
Prior to electrophoresis, microchips were initially rinsed with
Milli-Q water for 15 min and then with the running buffer for
10 min. Washing was made with the aid of a simple vacuum
system and reservoirs were filled with the running buffer solu-
tion. The microchip was fixed in its holder and a Faraday cage
was used for housing it in order to minimize electrical interfer-
ences. After baseline stabilization, reservoir C is filled with the
sample solution and injections were performed by applying the
desired voltage between sample (C) and sample waste (D) reser-
voir. Since no voltage was applied to the other two reservoirs,
an “unpinched” injection was performed. Separation was carried
out by applying the corresponding voltage to the running buffer
reservoir (A) with the detection reservoir (B) grounded. Then,
the detection potential is applied and the electropherogram is
recorded. All experiments were performed at room temperature.
2.5.1. Safety considerations
High-voltage power supplies should be handled with extreme
care to avoid electrical shock.
3. Results and discussion
3.1. Fabrication parameters
Glass is an isotropic material that is wet etched with HF-based
solutions in a non-directional manner, resulting in hemispheri-
cal channel structures. Ideal geometries involve high aspect ratio
channels, that means, deep channels with parallel sides. Differ-
ent parameters that affect the aspect ratio of the channel have
been evaluated and optimized.
Pretreatment of the glass substrates has been the first param-
eter studied. Substrates surface has to be adequately cleaned in
order to get a good adherence of the photoresist (PR). Thus,
neutral (C1, deionized water), acid (C2, piranha) and alkaline
(C3, NaOH) solutions have been tested. Similar results in the
final microchannel have been shown with the different wash-
ings. However, C2 washing was employed in the next part of the
work due to this washing allowed improving the initial condi-
tions of the substrate for the final bonding procedure. Next, the
substrates were dried in an oven since this process can reduce
the thermal stress between the glass substrate and the photore-
sist, subsequently added, resulting in a longer survival time of
the photoresist during the wet etching.
After washing, the photoresist has to be deposited on the
glass substrates by spin-coating technique. The surface of glass
materials oxidize very easily. The surface oxide forms long range
hydrogen bonds with water adsorbed from the air. When the PR
is spun onto surface, it adheres to the water vapour rather than
to the surface, and poor adhesion results. Water adsorbed can
be eliminated by soft-baking in an oven. Other two procedures,
based on the modification of the interface surface, have been
evaluated for improving the adherence of the PR (S1813). First,
100 nm thickness chromium layer was evaporated onto the glass.
The Cr layer did not improve the PR adherence and the survival
time of the PR in the wet etching decreased.
In the second procedure, a hexamethyldisilizane (HMDS)
based primer solution was deposited on the glass substrate during
20 s and then, it was spin-coated at 4000 rpm for 25 s. The primed
surface improved the PR adherence; thus, peeling of the PR
was not observed for a long time. Therefore, in all next studies
that involved the use of the S1813 PR the primer solution was
employed.
The performance of the S1813 PR was compared with the ma-
P1275 PR. Both of them have a very different viscosity, which
affects the thickness of the coating layer. Final film thickness and
other properties will depend, therefore, on the nature of the resin
 M. Castaño-Álvarez et al. / Sensors and Actuators B 130 (2008) 436–448
Fig. 3. Profile comparative obtained by confocal microscopy for (A) S1813 and (B) ma-P1275 photoresist with an etching time of 30 and 40 min, respectively.
(viscosity, drying rate, percent solids, surface tension, etc.) and
the parameters chosen for the spin process. Factors such as final
rotational speed and acceleration contribute to the properties of
coated films.
The S1813 PR was spun onto the glass substrate using speeds
ranging from 3000 to 6000 rpm and times between 20 and 40 s.
In all cases, a thickness layer around 1 ␮m was obtained. A
speed of 5500 rpm and a time of 30 s have been used in the next
steps of the work, due to the higher homogeneity of the PR layer
deposited throughout the entire glass surface.
PR ma-P1275 was deposited using two speed ramps due to
its high viscosity. Thus, the PR has to be initially extended on
the substrate by spin coating with a speed of 250 rpm for 10 s.
Finally, it was spun on at 3000 rpm for 20 s. In these conditions,
a layer around 7 ␮m thickness was obtained. An increase in
the thickness layer allowed a long survival time during the wet
chemical etching, reaching deeper channels. Moreover, this PR
has shown a better adherence than S1813 PR, even without using
the primer solution. An example of this is shown in Fig. 3, where
two samples, made with these two different photoresists are com-
pared. These images were taken using a confocal microscope,
which gives a three-dimensional image of the sample (Fig. 3,
right), as well as its depth profile in a selected region (Fig. 3,
left). In some depth profiles, an unreal noise was observed in the
base of channels, which was due to residual reflection during the
acquisition of the confocal images. Using these depth profiles,
the geometric factor (channel width divided by channel depth)
of each sample can be calculated, reaching the conclusion that
for the same fabrication parameters, ma-P1275 PR allows to get
441
a better value of this parameter. The best geometric factor should
present a value proximal to 1.
After PR deposition, the channel pattern has to be trans-
ferred to the resist. Thus, the PR was exposed by two different
irradiation methods: direct writing and masked exposure.
For direct writing method, a laser writing system has been
designed and implemented. The basic principle of this system is
the focalization of a low cost Nd:YAG laser light (λ = 473 nm)
on the photoresist, creating a minimum light spot, which moves
along the surfaces describing the desired motive (Fig. 2). By
controlling the power of light and the velocity of the movements,
it is possible to create on the photoresist a pattern of irradiated
lines with the desires width.
This method resulted very time-consuming and was not able
to easily reproduce the same pattern many times. However, it
was successfully used to make photomasks, which have been
employed in the second method.
In masked exposure method, a UV lamp was used to transfer
the pattern from the photomask to the PR layer on substrates
as can be seen in Section 2. Thus, the use of a mask-aligner
is not required. The UV light modified the chemical structure
of the PR, making it soluble on the developer solution. The
development time was optimized to 90 s. With smaller times,
the complete development of the PR was not obtained. Higher
times could raise part of the non-exposed PR.
Once PR has been developed, a post-develop bake or hard-
bake was used to improve resist’s wet-etching resistance by
hardening it. This makes the PR more difficult to remove in
aggressive etches with HF-based solutions. Thus, temperatures
442 M. Castaño-Álvarez et al. / Sensors and Actuators B 130 (2008) 436–448

Table 1
Etching rates for different glass substrates and HF-based solutions
HF-based solutions Soda-lime microscope glass slides Soda-lime glass Borofloat glass Pyrex

Etching rate (␮m/min)a

5% (HF:NH4 F 1:7):9.25% HCl 0.25 ± 0.07 – – –
HF:H2 O 1:10 0.5 ± 0.1 – – 0.6 ± 0.1
HF:NH4 F:H2 O 1:5:5 (BOE) 1.3 ± 0.2 0.8 ± 0.1 0.4 ± 0.1 –
HF:NH4 F 1:6 (BHF) 0.7 ± 0.1 0.6 ± 0.1 0.15 ± 0.05 0.10 ± 0.02
HF:HNO3 :AcOH:H2 O 1:2:1:4 (HNA) 1.3 ± 0.3 0.9 ± 0.2 0.10 ± 0.02 –
a Etching rate expressed as mean ± S.D., where “S.D.”: standard deviation of at least three samples.

comprised between 100 and 145 ◦ C as well as times ranging
from 30 to 45 min have been tested. A temperature of 130 ◦ C for
30 min (S1813 PR) and 110 ◦ C for 30 min (ma-P1275 PR) was
chosen taking into account the PR layer survival during the wet
etching.
Last step, and the most critical in the microfabrication pro-
cess, was the wet etching of the glass substrates. Glass is a
complex mixture based mainly on SiO2 and other compounds
such as Na2 O, CaO, MgO, etc. HF is used to dissolve the glass
and other chemical compounds, which are difficult to dissolve in
water. Some authors [4,5] suggested, for glass substrates, a wet
etching using HF-based solution with HCl, which is supposed
to dissolve the formed precipitates. Moreover, we demonstrated
experimentally that different stirring forms affect directly the
good performance of wet etching. Thus, ultrasonic-vigorously
and magnetic stirring were tested. The best results were obtained
with magnetic stirring (3000 rpm), while the other ones caused
the peeling and rising of the PR.
In this work different etching solutions, based on hydroflu-
oric acid, with and without HCl, have been tested. In Table 1
the different etching rates obtained for each solution, acting on
different kinds of glass substrates are shown. The higher etching
rate (for soda-lime microscope glass slides) was obtained using
the buffered oxide etch (BOE-HF:NH4 F:H2 O 1:5:5). As can
be seen, the NH4 F adjust the etching performance increasing
the etching rates when the BOE and the buffered HF (BHF-
HF:NH4 F 1:6) solutions are compared with HF:H2 O (1:10)
solution. Adding nitric and acetic acid to the etching solu-
tion (HNA-HF:HNO3 :AcOH:H2 O 1:2:1:4) also improved the
etching ratio, nevertheless, this solution was very aggressive
decreasing the survival time of the photoresist (<15 min). When
the soda-lime microscope glass substrates are compared with
the other soda-lime glass substrate a decrease in the etching rate
is observed. This difference can be attributed to the fabrication
process of the glass, so that, the two glasses can present differ-
ent SiO2 composition. Similar results are shown between the
common glass (soda-lime) and borosilicate glass (borofloat and
Pyrex). The borofloat and Pyrex glass present a higher propor-
tion of SiO2 than soda-lime and then, they are more difficult to
etch, decreasing the etching rate. The only advantage of using
borosilicate glass was that no white precipitates were found
on the surface while precipitates whether were observed with
common glass.
In Table 2 the geometric factor (channel width divided by
channel depth) is shown for soda-lime microscope glass-slides,
etched with each solution. Fig. 4 presents a comparative between
the aspects of the channels obtained in each case. In this case, a
lower geometric factor meant a better aspect ratio (narrow and
deep channels). Thus, from these results, it can be seen that the
best geometric factor (for soda-lime substrates) corresponds to
BHF solution. Due to its better geometric factor, good etching
rate and longer survival time of the photoresist (approximately
60 min), the BHF solution was used in the next studies.
As the literature proposes the use of HCl in order to dis-
solve the formed precipitates and increase the etching rate, this
compound was added to the BHF solution in different concentra-
tions. Thus, when HCl was added to the BHF solution between
0 and 15%, the geometric factor was increased from 5 to 12
for 0 and 15% HCl, respectively. Therefore, the performance of
wet-etching with HCl was worse resulting channels with a bad
aspect ratio, it means, very wide and less deep channels. Thus,
although the HCl can dissolve the formed precipitates, it can
also raise the photoresist making wider channels.
In order to compare the shape and quality of each kind of
etching process, Fig. 5 depicts microscope images and depth
profiles of the obtained microchannels. As can be seen on the
depth profiles (Fig. 5, left), when the HCl concentration was
increased, the channel wide was also increased from 100 ␮m
(0% HCl) to 300 ␮m (15% HCl). However, the depth was not
increased with the same ratio, it even decreased. Therefore, the
best etching solutions for soda-lime glass have resulted to be the
BOE solution and especially the BHF solution without using
HCl.
The optimized wet-etching process makes unnecessary the
use of more expensive methods such as RIE, DRIE, powder
blasting or laser ablation [18–21].
3.2. Bonding process
The system has to be assembled in order to close the
microchannels. Thus, different bonding processes have been
Table 2
Geometric factors for different HF-based etching solutions
HF-based solutions Geometric factor
Soda-lime microscope glass slides
5% (HF:NH4 F 1:7):9.25% HCl 63
HF:H2 O 1:10 72
HF:NH4 F:H2 O 1:5:5 (BOE) 7
HF:NH4 F 1:6 (BHF) 5
HF:HNO3 :AcOH:H2 O 1:2:1:4 (HNA) 9
 M. Castaño-Álvarez et al. / Sensors and Actuators B 130 (2008) 436–448 443

Fig. 4. Profile comparative obtained by confocal microscopy for different HF-based etching solutions (A) HF:NH4 F 1:6 (BHF), (B) HF:NH4 F:H2 O 1:5:5 (BOE),
(C) HF:HNO3 :AcOH:H2 O 1:2:1:4 (HNA), (D) HF:H2 O 1:10 and (E) 5% (HF:NH4 F 1:7):9.25% HCl.
444 M. Castaño-Álvarez et al. / Sensors and Actuators B 130 (2008) 436–448

Fig. 5. Profile comparative for wet etching with BHF solution with increasing levels of HCl, (A) 0%, (B) 1%, (C) 5% and (D) 15%.

tested. Although currently the procedure used to seal the
microchannels is the thermal bonding, low temperature proce-
dures based on intermediate layers have also been investigated.
Commercial adhesive (Araldit) and adhesive tape were
rejected. The first procedure was discarded because of a non-
homogeneous glue layer was obtained. Therefore, the cover
plate was not adequately sealed along the channel and thus,
the fluid cannot flow through the microchannel. Similar results
were obtained with the adhesive tape; moreover, the film had not
resistance enough, which caused an ease rupture.
The next procedure evaluated was based on an intermediate
layer of SiO2 ·Na2 O (water glass). This procedure had been pre-
 M. Castaño-Álvarez et al. / Sensors and Actuators B 130 (2008) 436–448 445

Table 3
Optimal conditions for microfluidic fabrication on soda-lime glass substrates
Step Optimal conditions

Pre-treatment Commercial detergent, deionized water, acetone,

piranha solution (H2 SO4 :H2 O2 , 3:1), isopropyl
alcohol and blown dry with N2 (approximately 1 h)
Lithography
Spin-coating Primer (HMDS) 4000 rpm, 25 s
Photoresist S1813 5500 rpm, 30 s
Soft-baked 90 ◦ C, 30 min
UV exposure 115 s (150 mJ cm−2 )
Developed MF-311 developer → 90 s
Hard-baked 130 ◦ C, 30 min
Wet etching HF:NH4 F 1:6, 30 min
Thermal bonding 650–675 ◦ C, 5 h

viously tested with good results [30,43]. Thus, different solution

concentrations (1–35%), spin-coating speeds (3000–8000 rpm)
and times (5–30 s) as well as several temperatures (20–700 ◦ C)
have been evaluated.
When high concentrations of silicate sodium and low spin-
coating speed are used, the intermediate layer is too thick and
therefore a water layer is formed avoiding the good sealing of the
glass. On the other hand, if low concentrations and high spin-
coating speed are employed, a thin layer is obtained causing
short hardened time and unsuccessful adhesion of the cover plate
occurs. The optimal conditions were obtained using a solution
concentration of 3%, with a spin speed of 5000 rpm during 30 s.
Initially, low temperatures (RT, 100 ◦ C) were used to seal the
system, but a reversible bonding was obtained and the solutions
did not flow properly through the channels. Then, the substrate
was heated in a muffle furnace at 650–675 ◦ C for 5 h obtaining
a successfully sealing of the system. Usually, high tempera-
ture procedures do not require an intermediate layer; hence,
the thermal process was successfully made without using the
intermediate layer. When the glass temperature was proximal
to the melting point a homogenous external mechanical pres-
sure throughout the glass was necessary for the accomplishment
of the thermal bonding. This pressure was applied by a home-
made refractory steel device, compound by an external chassis
in which two sample holders can be inserted. The glass cover
plate and the micromachined substrate were placed on these
sample holders, and then they are covered by two steel plates
and compressed by two screws. Fig. 6 shows the scheme of
the device employed for thermal bonding. This thermal bonding
method avoids the use of much more expensive equipments such
as those used in anodic bonding [44].
In Table 3 the optimal conditions of each step to fabricate a
microfluidic device on soda-lime glass substrates with channels
of 50 ␮m width and 20 ␮m depth approximately are shown.
3.3. Microchip performance
The resulting microfluidic system with a 4.5 cm long sep-
aration channel (between running buffer reservoir, A, and
waste/detection reservoir, B) and 1.5 cm long injection chan-
Fig. 6. Schematic representation and picture of the thermal bonding device.
nel (between sample reservoir, C, and sample waste reservoir,
D) was employed for capillary electrophoresis (CE) with amper-
ometric detection.
The performance of CE-microchip and the electrochemical
response of the gold wire working electrode were studied using
p-aminophenol (pAP) as model analyte. A 50 mM Tris–Gly
pH 9.0 was employed as running buffer. At this pH, pAP
(pKa = 10.46) is a neutral mark, so that, it can be used for charac-
terizing the electroosmotic flow (EOF). Measuring the migration
time of pAP and knowing the effective length of the channel
(distance from injection to detection point: Leff = 4 cm), it is
possible to calculate the electroosmotic velocity (cm s−1 ). The
electroosmotic mobility (cm2 V−1 s−1 ) can be obtained dividing
the electroosmotic velocity by the electric field applied (V cm−1 )
in the channel.
A 100 ␮M solution of pAP was employed in the evaluation
of the glass microchip using an injection voltage of +1500 V
applied for 10 s. Since pAP presents an oxidation process, a
detection potential of +0.75 V was used [14,39]. Parameters such
as peak current (ip ), migration time (tm ) and half-peak width
(w1/2 ) are shown in Table 4 as well as the theoretical plate num-
ber (N), electroosmotic velocity (veo ) and mobility (μeo ) for pAP
using different electric fields (E).
When the separation voltage was varied from +1000 to
+1500 V, an increase in the peak current and a decrease in the
446 M. Castaño-Álvarez et al. / Sensors and Actuators B 130 (2008) 436–448
Table 4
Analytical characteristics for pAP (100 ␮M) using different separation voltages in the glass-microchip with an end-channel gold wire detector
Voltage (V) E (V/cm) ip (nA) tm (s)
+1000 222 6.6 77.2
+1500 333 18 39.6
+2000 444 11 23.3
Conditions, injection: 10 s at +1500 V; Ed = +0.75 V (vs. Ag/AgCl), running buffer: 50 mM Tris–Gly pH 9.0.
Fig. 7. Electropherogram for 100 ␮M pAP using glass-microchip with an
end-channel gold wire detector. Conditions: Vsep = +1500 V; injection: 10 s at
+1500 V; Ed = +0.75 V (vs. Ag/AgCl), running buffer: 50 mM Tris–Gly pH 9.0.
half-peak width and migration time was observed. If the separa-
tion voltage was increased to +2000 V a decrease in the half-peak
width and migration time was also observed, but a lower peak
current was measured. Moreover, in this case, a higher baseline
noise level as well as bubbles formation due to excessive Joule
heating happened.
The best performance of the glass CE-microchip in combina-
tion with the end-channel gold wire detector was obtained using
a separation voltage of +1500 V. Fig. 7 depicts an electrophero-
gram for a 100 ␮M pAP solution using a separation voltage of
+1500 V.
Analytical characteristics of the new glass microchip with the
amperometric detector were quite similar to those shown pre-
viously on commercial microchips [14,39] demonstrating, thus,
its possible analytical and bioanalytical applications. Further-
more, the glass microdevices have demonstrated a longer life
time than polymer commercial microchips.
4. Conclusions
Microfluidic channels have been successfully fabricated
in different glass substrates using photolithography and wet-
etching technique. Practical aspects and critical points that affect
to all of the different parameters on the fabrication process of
glass microfluidic devices have been rigorously evaluated and
optimized. An accurate, fast and low cost methodology has been
developed, permitting the fabrication of a butch of complete
microchips in less than 8 h.
The importance of the substrate pretreatment and the effect
of photoresist thickness as well as the different methodologies
for transferring the pattern design to the PR have been discussed.
w1/2 (s) N (m−1 ) EOF (veo , cm/s) μeo (×10−4 cm2 /V s)
22.2 1700 0.05 2.3
10.2 2100 0.10 3.0
7.4 1400 0.17 3.9
The most critical step of microfabrication was the wet etching
of the glass substrates. Different etching solutions, based on
hydrofluoric acid, with and without HCl, as well as several types
of stirring, were evaluated. The best microfluidic channels, with
better geometric factor, were attained using BHF (HF:NH4 F
1:6) solution with a magnetic stirred. The resulting channels
were worse when HCl was added to the etching solution since
it increased the geometric factor.
The device assembly has been made using different bonding
processes. Different techniques based on intermediate layer were
initially tested and discarded. The best sealing of channels was
obtained by thermal bonding at 650–675 ◦ C for 5 h.
Resulting microfluidic devices have been successfully used
for capillary electrophoresis with electrochemical detection. An
end-channel gold wire detector has been easily integrated on
the glass microchip for amperometric detection. Thus, the elec-
troosmotic flow (EOF) of the new glass microchip has been
characterized using pAP as neutral mark. This advances the pos-
sible analytical and bioanalytical applications of the new glass
microfluidic device.
Acknowledgements
This work has been supported by the FICYT under projects
IB05-151C1 and IB05-151C2. Mario Castaño Álvarez thanks
FICYT-Principado de Asturias for the award of a Ph.D. grant.
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Biographies
Mario Castaño Álvarez obtained his BSc degree in chemistry, focus in analyt-
ical chemistry, in 2002 (University of Oviedo) and his MSc degree in analytical
chemistry in June 2005 (title: Amperometric detectors for capillary electrophore-
sis microchips). He is currently a PhD candidate at University of Oviedo and
his work is focused on fabrication and characterization of microfluidic devices
with electrochemical detection.
Diego Francisco Pozo Ayuso received the degree in physics from the Uni-
versity of Oviedo, Spain. Since 2003, he is working in optical waveguide
fabrication and characterization in the Physics Department of the University
of Oviedo. In 2005, he received a grant to doctoral courses from this University.
Moreover, this year he received the pedagogic aptitude for teachers of Sec-
ondary Education, obtained a master in management of the environment from
Asturias Business School and worked in an environmental control company. He
is currently working as researcher for a coordinated project between the inmuno-
electroanalisis group and the integrated optic group of the University of Oviedo.
His research interests are fabrication and characterization of optical waveguides,
capillary electrophoresis microchips (MCE) and biosensors microdevices based
on Mach–Zehnder interferometer.
Miguel Garcı́a Granda received his degree in physics in 2003 from the Uni-
versity of Oviedo (Spain). During 2003 he joined the Hanh-Meitner Institut
448 M. Castaño-Álvarez et al. / Sensors and Actuators B 130 (2008) 436–448
in Berlin (Germany) where he worked in chalcopyrite solar cells fabrication
techniques and structural haracterization. In 2004, he received a doctoral grant
from the Spanish Ministry of Education and Science. Currently, he is pursu-
ing his PhD which is directed towards the study of optical waveguides design,
fabrication and characterization, as well as Mach–Zehnder optical modulators,
within a cooperation framework between the Universities of Oviedo (Spain) and
Paderborn (Germany).
Marı́a Teresa Fernández Abedul obtained her BSc degree in chemistry, focus
in analytical chemistry, in 1989 (University of Oviedo) and the PhD in chem-
istry in 1995 (University of Oviedo). Since September 1994, she is senior
lecture in analytical chemistry (University of Oviedo). She works in the Immu-
noelectroanalysis Research Group of the University of Oviedo and has been
supervisor of several research projects developed at the Electrochemistry labora-
tories of the Department of Physical and Analytical Chemistry of the University
of Oviedo. Nowadays his research is focused on the development of elec-
trochemical immunosensors and genosensors employing both enzymatic and
non-enzymatic labels as well as fabrication and characterization of microfluidic
devices with electrochemical detection.
José Rodrı́guez Garcı́a is a professor at the University of Oviedo (Spain). He
received the teaching degree from the University of Oviedo, and the BSc and
Extraordinary PhD degrees in physics from the University of Santander (Spain).
From 1982 to 1988, he worked in electromagnetic field analysis on dielectric
waveguides in the Electronics Department of the University of Santander. As
member of the COST-216 European Project, he has worked at the ETH (Zurich)
as well as for the CTNE and NESTLE Companies. Since 1988, he has been a pro-
fessor at the University of Oviedo and since 1993, supervisor and coordinator of
the Secondary School. He is a member of the Electromagnetism Academy (USA)
and member of the SPIE society. He was selected as Man of the Year 1997 by
the ABI and he was included in: Who’s Who in electromagnetics, Who’s Who in
the world and Who’s Who in contemporary achievements. He conducts research
in the following areas: electromagnetic field theory, modelling, characterization
and evaluation of integrated optical waveguides and devices.
Agustı́n Costa Garcı́a obtained his BSc degree in chemistry, focus in analytical
chemistry, in 1974 (University of Oviedo) and the PhD in chemistry in 1977 (Uni-
versity of Oviedo). Since February 2000, he is professor in analytical chemistry
(University of Oviedo). He leads the Immunoelectrochemical Research Group
of the University of Oviedo and has been supervisor of several research projects
developed at the Electrochemistry laboratories of the Department of Physical
and Analytical Chemistry of the University of Oviedo. Nowadays his research is
focused on the development of electrochemical immunosensors and genosensors
employing both enzymatic and non-enzymatic labels.