Anal Bioanal Chem (2009) 395:473478
DOI 10.1007/s00216-009-2980-9
ORIGINAL PAPER
Application of external micro-spectrophotometric detection
to improve sensitivity on microchips
Attila Gspr & Istvn Bcsi & Erika F. Garcia &
Mihly Braun & Frank A. Gomez
Received: 3 May 2009 / Revised: 28 June 2009 / Accepted: 15 July 2009 / Published online: 1 August 2009
# Springer-Verlag 2009
Abstract The goal of this work was to increase the
sensitivity of a UVVis spectrophotometer by decreasing
the background noise and lengthening the optical path. A
microphotometer has been modified to precisely select very
small parts of a microfluidic channel pattern of a chip and
to measure light absorbance on a magnified area of the
selected part of the channel. The viability of combining a
projection microscope and a spectrophotometer for external
absorbance measurements on disposable PDMS chips was
studied. Besides the external direct detection above a
microfluidic channel, the optical pathlength was lengthened
by detecting in the region of the perpendicular exit port.
Increasing the cross-sectional area of the zone of irradiation
improved the signal-to-noise ratio and the limits of
detection (LOD).
Keywords Chip . Poly(dimethylsiloxane) .
External UV detection . Micro-spectrophotometer .
Flow injection . Microfluidics/microfabrication . UV/Vis
A. Gspr (*) : M. Braun
Department of Inorganic and Analytical Chemistry,
University of Debrecen,
Egyetem tr 1,
Debrecen 4032, Hungary
e-mail: gaspara@tigris.unideb.hu
I. Bcsi
Cetox Ltd,
Egyetem tr 1,
Debrecen 4032, Hungary
E. F. Garcia : F. A. Gomez
Department of Chemistry and Biochemistry,
California State University, Los Angeles,
5151 State University Drive,
Los Angeles, CA 90032-8202, USA
Introduction
Microfluidic lab-on-a-chip technologies and devices repre-
sent a revolution in laboratory experimentation, bringing
the benefits of miniaturization, integration, and automation
to many research-based laboratories and industries. The use
of microfluidic chips has attracted great attention as they
offer several advantages over conventional analytical
techniques including small sample volume requirements,
portability, fast sampling times, ability to multiplex and
compatibility with other techniques [1]. The development
of novel and miniaturized detection systems employed in
microfluidic technologies will both simplify and hasten
analysis on chip platform. The most simple, universal, and
inexpensive detection mode is that based on UVvisible
spectrophotometry. Unfortunately, optical detection can be
problematic as microchips have small pathlengths greatly
reducing sensitivity and limits of detection (LOD). Hence,
few applications of spectrophotometric absorbance meas-
urements in microfluidic chip systems have been reported.
For example, a silicon flow cell was used to detect dyes
whereby the silicon was utilized as mirrored channel [2].
Haswell and coworkers used spectrophotometric detection
in chip for the determination of phosphate [3], nitrate [4],
and nitrite [5] integrating the optical fibers to the channel
ends. Numerous groups have used fiber optics in microchip
capillary electrophoresis [6, 7]. In this approach, alignment
of the fibers with the liquid channel is difficult. Of
particular concern is light, which circumvents the liquid
channel and reaches the detector. Others used optical
waveguides that are incorporated into the chip to enhance
the effective usage of the analytical light beam (effective-
ness of the interaction of the light with the analyte) [8, 9].
Numerous designs have been proposed to increase the
pathlength by passing the light along the length of the flow
channel rather than across it [2, 10, 11].
474
Poly(dimethylsiloxane) (PDMS) is a favorite material to
construct microfluidic chips from due to its low cost, ease
of fabrication and robustness [12]. There are a number of
studies that detail novel detection schemes for PDMS-based
microchips but they either require one, the compounds of
interest to be labeled (in the case of fluorescent detection)
or two, integration of detection components (for example,
conductivity and amperometry) into the chips [13]. The use
of label-free detection modes is growing and will increase
in usage in lab-on-a-chip devices. Similarly, incorporation
of sensor components into chips is quite developed [14].
On the other hand, it would be beneficial if neither labeling
nor chip modification were needed to detect molecules in
the ultraviolet and visible range using a PDMS-based
microchip. One advantage of PDMS is that it does not
absorb visible light and only slightly above 250 nm.
Herein, we describe the use of a microphotometer,
originally designed to measure photographic density on a
photographic plate, to measure light absorbance on a magni-
fied area of a microchannel. The instrument is a combination
of a projection microscope and a spectrophotometer. The
applicability of this instrument for external absorbance
measurements on disposable PDMS chips was studied.
Besides external direct detection above a microfluidic channel,
the optical pathlength was lengthened by detecting in the
perpendicular exit port.
Experimental
Chemicals and reagents
Stock solutions of Brilliant Blue FCF food dye (FD&C
blue#1 or E133 from Szilas Ltd, Hungary) and 0.1 mM
phosphate complexed in molybdenum blue compound (the
phosphate was mixed with a reagent containing ammonium
molybdate, potassium antimonyl tartarate and sulfuric acid
followed by ascorbic acid according to Ref. [15]) were
Fig. 1 The construction of the
microphotometer (Schnellpho-
tometer, Carl Zeiss, Jena) used
for spectrophotometric detection
on chip. The optical fiber of a
miniaturized spectrophotometer
(Avaspec-2048-2, Avantes, The
Netherlands) was positioned
behind the projection screen.
The magnified picture about the
projection screen is shown in
the right side
A. Gspr et al.
prepared in water. All solutions (methanol, water) were
degassed and filtered through a 0.45-m syringe filter.
Microphotometer
The analytes injected into the chip were detected by a
modified microphotometer (Schnellphotometer-II, VEB
Carl Zeiss, Jena; Fig. 1) equipped with a fiber optic
miniaturized spectrophotometer. For many years, micro-
photometers (microdensitometers) have been used for
spectrographs to evaluate photographic plates (photoem-
ulsions placed on glass plates). This device directs a steady,
narrow beam of light through the photographic plate to a
light sensitive detector. As the plate was stepped along the
dark and transparent part of the plate, the detector measured
and recorded the amount of light.
The microphotometer consists of an xy translational
stage to move and accurately control the position of the
microfluidic chip where the absorbance measurement is
intended to be carried out. The chip can be fixed onto a
movable stand and positioned manually by means of
adjustable screws with 0.0025 mm accuracy. The part of
the chip to be investigated should be positioned above and
below the lighting and projective objectives, respectively.
After focusing, approximately a 1-mm2 area of the chip can
be projected onto the screen to obtain an enlarged picture of
the spot (Fig. 1, right side). The screen provides a rapid
visual survey of the section of the channel under examina-
tion and in fine detail. Approximately 100 magnification
of the section of the chip under study can be obtained on
the screen.
On the spot of the chip projected onto the screen, a
rectangle opening with variable sides can be specified by
adjusting slits in both horizontal and vertical directions
(Fig. 1, right side). Through the specified rectangle, the
light of the projected picture can pass to the detector, thus
the absorbance measurement can be limited only to the
specified area of the chip (inside a given length of the
projection screen
adjustable slit
magnified
picture of the chip
focus setting
projecting objective
chip on movable stand
lighting objective
Application of external micro-spectrophotometric detection
microfluidic channel) resulting in good utilization of light
for the spectrophotometric detection of the analyte. The
horizontal and vertical apertures of the slits are 05 and 0
20 mm, respectively.
In our experiments, a tungsten lamp was used as the light
source. The optical fiber of a miniaturized spectrophotom-
eter (Avaspec-2048-2, Avantes, The Netherlands) was
positioned behind the projection screen. The light reached
the optical fiber after passing through the slit of the adjusted
area and collected by lens. The spectrophotometric meas-
urements were controlled and recorded by Avasoft
(Avantes, Eerbeek, The Netherlands). Since the detection
was performed externally, any point along the chip could be
detected. The obtained absorption data were exported to a
Microsoft Excel program for further analysis. The standard
PDMS chip with glass slide could be used for the
spectrophotometric detection in the visible range.
Chip fabrication
The PDMS chips were prepared by using a mold created by
soft photolithography [10]. The pattern consisting of
standard T-type and cross T-type channel of 50 m wide
was designed using AutoCAD software (San Rafael, CA)
and printed as a high resolution (20,000 dpi) photomask
(CAD/Art Services, Inc., Bandon, OR). Negative type
photoresist (SU-8 2025, Microchem, Newton, MA) was
spin-coated onto a 3 silicon wafer at 2,600 rpm for 30 s to
a thickness of 30 m. The photoresist-coated wafer was
baked for 15 min at 95 C. The pattern on the mask was
transferred to the wafer through UV exposure for 2 min.
The exposed wafer was baked at 95 C for 5 min and
unexposed areas were removed by rinsing with SU-
Fig. 2 Schematic representation
of the standard T-type channel
chip made from PDMS from the a,
side (a) and from above (b). The
top of the E exit port is covered
with a thin layer of PDMS
including a channel to lead the
Fluid channel
C S
waste liquid out from the chip.
The arrows indicate the
possible detection sites of
the external detection with
the microphotometer
b,
C
475
8 developer (Microchem, Newton, MA). The PDMS chip
was fabricated by cast molding of a 10:1 mixture of PDMS
oligomer and cross-linking agent (Sylgard 184, Dow
Corning, Midland, MI). The PDMS mixture was degassed
and baked at 80 C for 30 min. The PDMS replicas were
peeled off from the mold.
Holes of 0.3 mm diameter for the liquid connections to C
(carrier) and S (sample) ports, and hole of 0.5 mm diameter
for the perpendicular detection to the E (exit) port (Fig. 2.)
were punched (Schmidt Technology GmbH (St. Georgen,
Germany) through the PDMS chip. The inner diameter of
the E port can be decreased to 50400 m if a short piece
of fused-silica capillary is tightly inserted into the port.
Then, the chip was irreversibly sealed onto a standard glass
slide of 1 mm thickness (VWR, USA) using air plasma
oxidation (PDC-32G, Harrick Plasma, Ithaca, NY, USA).
Figure 3 shows the pieces of fused-silica capillaries of
50 m and 400 m ID (360 m and 600 m OD,
respectively) inserted into the port of PDMS chip.
In order to remove the liquid coming from the exit port
and to keep clear and bubble-free the top of the E port for
the spectrophotometric detection, the E port is covered
(sealed by air plasma oxidation) with a thin (about 1 mm)
layer of PDMS including a 0.2-mm-wide, 50-m-deep, and
5-mm-long channel. One end of this channel is positioned
to the E port, and a 0.3-mm diameter for the liquid
connections to the W (waste) port (Fig. 2.) was punched
through the PDMS layer at the other end of the channel.
Injection of sample plugs
Sample plugs were injected into the chip by pressure or
high voltage. Upon detection of the sample plugs in the
Detection site
Detection site through the exit port
through the channel
W
E
PDMS layers
Glass/quartz slide
S
W
E
476
200 m
Fig. 3 Microscopic pictures around the pieces of fused-silica
capillaries of 50 (a) and 400 m (b) ID (360 and 600 m OD,
respectively) inserted into the port of the PDMS chip
microfluidic channel, a subnanoliter volume of dye sample
(length in channel less than 200 m) was used for the
detection. Electrokinetic injection was used in the cross T-
type channel. Here, 400 V was applied to produce constant
flow in the sample channel and when the junction of the
chip filled with the sample, 800 V was applied to the ends
of the separation channel. For the electrokinetic injections
two miniaturized high-voltage (0.22 kV) power supplies
were used (Microply-01, Cetox Ltd., Hungary). The dye
sample migrated toward the cathode (electrolyte, 25 mM
phosphate, 50 mM SDS; pH, 3).
For detection of the sample plugs through the E port,
only a single-channel peristaltic pump (Ismatec IPC,
Switzerland) was used. The sample (0.12 L) was injected
into the peristaltic pump tubing (ID, 0.3 mm), previously
filled with water, and pumped into the S port while the C
port was closed.
Results and discussion
One of the most well-known relationships in analytical
chemistry is the LambertBeer law. This law states that
absorption is proportional to the light pathlength and the
concentration of the absorbing species. Since the obtained
absorbance does not depend on the cross-sectional area of
the irradiated sample (cuvette), the absorbance should be
A. Gspr et al.
the same even if the cross-sectional area of the irradiation is
changed. This is advantageous for detection on chips,
because it means that we get the same absorbance value
even if we measure a very small area of the sample plug
(e.g., 104 mm2) in the microfluidic channel (its width is
typically less than 100 m) as compared to the conven-
tional measurement through a cuvette of six orders of
magnitude larger irradiated area (a 1-cm-side cuvette).
Using the microphotometer and selecting a section of the
microfluidic channel on the magnifying projection screen,
we can precisely select very small parts of the channel
pattern of the chip for measuring absorbance. On the
projected picture of the chip, an area of rectangular
dimension inside the microfluidic channel (in the analyte
plug) can be specified by adjusting the slits horizontally
and/or vertically (Fig. 1, right side). Thus, a rectangle with
sides of 250 m and 2200 m can be specified for the
absorbance measurements. Using the microphotometer, the
absorbance measurement is limited only to the small area of
the chip inside the microfluidic channel; therefore, very
good utilization of light for the analyte detection can be
achieved. In Fig. 4, the absorbance signals collected for
60 s were compared using slit areas of 20, 40, 200, and
400 m2 (104 times larger on the projected, magnified
screen). Although the obtained absorbance values are not
altered (as expected due to the LambertBeer law),
considerable differences can be found in the signal noise.
Increasing the cross-sectional area of the irradiation
decreases the noise.
The LOD of a measurement depends on the signal-to-
noise (standard deviation) (LOD 3s=S, where and S
50 mAU
a,
b,
c,
d,
0 20 40 60
Time (s)
Fig. 4 The absorbance signals using different slit areas on the
projected picture a 2, b 4, c 20, and d 40 mm2 (the projected areas on
chip were 104 times smaller; sample: blue dye (FD&C blue#1), =
640 nm) The standard deviations were calculated to 0.015 (a), 0.0049
(b), 0.0015 (c), and 0.0014 (d)
Application of external micro-spectrophotometric detection 477

Table 1 Limit of detection values of phosphate (in molybdenum blue

0,60 complex) using on-channel and in-exit-port detections with fiber optic
spectrophotometry for chips, and with conventional spectrophotome-
try including 1-cm-length cuvette (=640 nm)
Absorbance

0,40 LOD (M) Volume of sample used

for the detection (nL)

On-channel detectiona
0,20
20 m2 50 0.6
40 m2 38 1.2
0,00 200 m2 5.6 6.0
0 20 40
Time (s)
60 80
400 m2 5.3 12
In-exit-port detectionb 0.78 120
Fig. 5 Absorption signals of sample plugs injected by voltage into the Conventional spectrometryc 0.16 1,000,000
chip (sample: blue dye (FD&C blue#1), sample volume, 0.15 nL, =
640 nm) a
Cross-sectional areas of the irradiation on the chip were ranged
between 20400 m2
b
are the standard deviation of the response and slope of the The height of the exit port was 2.5 mm
c
calibration curve, respectively). Increasing the cross- Supporting that at least 1 cm height of sample should be loaded into
sectional area of the irradiation from 20 to 400 m2 the cuvette
decreases the standard deviations from 0.015 to 0.0017
(resulting in similar improvement in LOD values), respec- be increased due to higher Joule-heat formation during
tively. Further increasing the area did not improve the electrophoretic applications, attenuated electroosmosis, less
standard deviation. These data explain that a larger achievable resolution for separations, etc.). Transverse
irradiation area leads to a large number of parallel detection detection through a given length of the channel provides
in the sensor at the same time and the standard deviation of for more sensitive detection, but is not amenable for
the large number of parallel detections should be smaller. detection of small plugs of sample (which are typical in
The use of electrokinetic injection allows for the microfluidic applications). Furthermore, transverse detec-
injection of subnanoliter volumes (plugs of about 100 m tion frequently requires complicated adjustment of the
length in the channel of 50 m width and 30 m height) optical fibers. The most simple solution to increasing the
into the channel of a cross T-type chip and with high height of the liquid layer is when the detection is performed
reproducibility (Fig. 5). The use of the microphotometer in the exit port perpendicular to the end of the microfluidic
allows for the absorbance measurement within the spot of channel. Using a microphotometer, a 50-m-wide hole of
the flowing subnanoliter plug. the exit port can be specified for the detection (Fig. 3).
The height of the flowing liquid layer in the channels of Since microscopic bubbles are often trapped at the bottom
microchips is extremely small (3050 m) and should not of the capillary thereby complicating spectrophotometric

100 M

0.75 a, 0.79
b,

75 M

0.59
0.5
Absorbance

Absorbance

50 M

0.39

0.25
25 M
0.19
c,
10 M
0 5 M
0 25 50 75 100
Concentration (M) -0.01

Fig. 6 Calibration graphs obtained by conventional spectrophotomet- Fig. 7 Signals of standard solutions of phosphate in concentration
ric measurement with 1-cm-length cuvette (a), in-exit-port of a chip range of 5100 M (sample: phosphate (in molybdenum blue
(b) and on-channel of a chip (c) detection complex), sample volume, 0.2 L =640 nm)
478
detection, capillaries of 250400 m ID are typically used.
The height of the detection cell (E exit port) was 2.5 mm
(optical path length in the chip), comparable to classical
cuvette-based spectrophotometric techniques, but the vol-
ume of the cell is only 120 nL (for 250 m ID capillary),
which is in the range of the volume of solution found in
microchannels in a chip. This exit port detection is not
suitable for detection of small sample plugs (e.g. in case of
electrophoretic separations in chip), but it can be applied in
flow injection analysis (FIA) using sample plugs of
submicroliter volumes. The dual use of microfluidics
integrating FIA and separation systems has recently been
proposed [16]. Figure 6 shows the measurement of standard
solutions of phosphate (in molybdenum blue complex) in
the concentration range of 5100 M. The 0.2 L volumes
of sample solutions were transferred by use of a peristaltic
pump (ID of tubing was 250 m) and injected into the chip
via the S sample port. As can be seen, even low-micromolar
concentrations of a phosphate sample can be reproducibly
detected. LOD values of phosphate using on-channel and
in-exit-port detections with fiber optic spectrophotometry
for chips, and with the conventional spectrophotometry was
summarized in Table 1. While the attainable sensitivity less
than five times worse using in-exit-port detection, the
needed sample volume almost 10,000 times smaller than in
conventional spectrophotometry. The LOD values are far
from the desired level when on-channel detection is
applied.
In Fig. 7, the calibration graphs obtained by on-channel
and exit port detection are compared. The latter one is more
than 50 times more sensitive due to the longer optical path
length. In this figure, the calibration graph obtained by
conventional spectrophotometric measurement with cuvette
(1 cm side) is also compared.
Conclusions
We have described the use of a microphotometer coupled to
a spectrometer for detection of analytes in a microchip
format. Although the size of the microphotometer does not
fit well to the microfluidic scale (which is a general
problem for numerous other detection techniques, including
LIF or MS detections, used for microfluidics), the applied
microphotometer makes universal detection mode possible
(not the case for LIF). Improved LODs were achieved in
microchannels. To enhance the effective usage of the
A. Gspr et al.
analytical light beam, detection, both inside the 50400-m-
wide hole of the exit port and in the flowing subnanoliter
volume of sample plug, are needed. In the proposed
experimental setup, the interaction of the light with the
analyte is better than in the case of fiber optics in a
spectrometer arranged to the channel without the micropho-
tometer. On the other hand, the use of adjustable slits on the
projection screen of the microphotometer maximizes the
irradiated area (e.g., the whole area of a subnanoliter sample
plug), vis-a-vis, all the light which reaches the detector
passes through the liquid in the channel and not outside the
channel. The sensitivity of the spectrophotometric measure-
ments can be improved by detecting in the exit port having a
small inner diameter.
Acknowledgments The authors gratefully acknowledge financial
support for this research by grants from the National Scientific
Research Fund, Hungary K75286), the National Office for Research
and Technology (Baross-2-2007-0028) and the National Science
Foundation (DMR-0351848, CHE-0515363, and OISE-0754138).
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