Anal. Chem. 1997, 69, 2877-2881
Fourier Transform Infrared Detection in
Miniaturized Total Analysis Systems for Sucrose
Analysis
B. Lendl, R. Schindler, J. Frank, and R. Kellner*
Institute for Analytical Chemistry, Vienna University of Technology, Getreidemarkt 9/151, A-1060 Wien, Austria
J. Drott and T. Laurell
Department of Electrical Measurements, Lund University, S-221 00 Lund, Sweden
Development of miniaturized total analysis systems (µ-TAS)
is the concern of intensive research activities due to the advantages envisioned by miniaturization, such as reduced sample and
reagent consumption as well as increased speed of analysis.1,2 For
optical detection in µ-TAS, fluorescence is usually used because
of the superior sensitivity provided by this technique.3-8 Besides
fluorescence, UV/visible spectroscopy for absorption measurements in miniaturized systems has also been reported.9-11
However, a common general drawback of using fluorescence or
UV/visible detection is that several reaction steps are frequently
required in order to derive a detectable reaction product. This
problem can be circumvented if Fourier transform infrared (FTIR) spectroscopic detection can be applied because nearly all
molecules exhibit characteristic absorbances in the infrared.12
In conventional flow injection analysis, it was already shown
that by multivariate evaluation of the mid-IR spectrum several
analytes can be determined directly in one sample if the matrix
is either constant or well characterized.13 In the case of nonconstant matrixes, FT-IR spectroscopy also enables simultaneous
determination if selective (enzymatic) reactions of the analytes
can be performed.14 FT-IR spectroscopic detection furthermore
allows one to shorten conventional reaction schemes, as in the
case of sucrose analysis, because sucrose hydrolysis alone
provides sufficient spectral information for quantitative analysis.15
Other methods recently proposed for sucrose determination in
flow systems employing spectroscopic detection usually require
three consecutive enzymatic reactions to obtain a detectable
reaction product.16-18
For UV/visible absorption measurements in µ-TAS, the optical
path lengths must be significantly reduced compared to the
conventionally sized flow systems. For FT-IR spectroscopic
detection this is not necessary, as in the conventional systems
short optical path lengths are already required. Therefore, it can
be expected that upon miniaturization FT-IR spectroscopy will gain
sensitivity compared to UV/visible spectroscopy. However, when
FT-IR spectroscopic detection in µ-TAS is applied, the diameter
of the IR beam must be reduced significantly at the place of
(1) Manz, A.; Grabner, N.; Widmer, H. M. Sens. Actuators B 1990, 1, 244248.
(2) van den Berg, A., Bergveld, P., Eds. Micro Total Analysis Systems, Proceedings of the µ-TAS ‘94 Workshop, Twente, NL, 1994.
(3) Harrison, D. J.; Manz, A.; Fan, Z.; Lüdi, H.; Widmer, H. M. Anal. Chem.
1992, 64, 1926-1932.
(4) Fan, Z. H.; Harrison, D. J. Anal. Chem. 1994, 66, 177-184.
(5) Raymond, D. E.; Manz, A.; Widmer, H. M. Anal. Chem. 1994, 66, 28582865.
(6) Effenhauser, C. S.; Manz, A.; Widmer, H. M. Anal. Chem. 1993, 65, 26372642.
(7) von Heeren, F.; Verpoorte, E.; Manz, A.; Thormann, W. Anal. Chem. 1996,
68, 2044-2053.
(8) Jacobson, S. C.; Hergenröder, R.; Kounty, L. B.; Ramsey, J. M. Anal. Chem.
1994, 66, 1114-1118.
(9) Moring, S. E.; Reel, R. T. Anal. Chem. 1993, 65, 3454-3459.
(10) Liang, Z.; Chiem, N.; Ocvirk, G.; Tang, T.; Fluri, K.; Harrison, J. D. Anal.
Chem. 1996, 68, 1040-1046.
(11) Verpoorte, E.; Manz, A.; Lüdi, H.; Bruno, A. E.; Maystre, F.; Krattinger, B.;
Widmer, H. M.; van der Schoot, B. H.; de Rooij, N. F. Sens. Actuators B
1992, 6, 66-70.
(12) Griffiths, P. R.; de Haseth, A. Fourier transform infrared spectrometry;
Chemical Analysis 83; John Wiley and Sons: New York, 1986.
(13) Guzman, M.; Ruzicka, J.; Christian, G. D. Vib. Spectrosc. 1991, 2, 1-14.
(14) Rosenberg, E.; Kellner, R. J. Mol. Struct. 1993, 294, 9-12.
(15) Lendl, B.; Kellner, R. Mikrochim. Acta 1995, 119, 73-79.
(16) Tzouwara-Karayanni, S.; Crouch, S. Food Chem. 1990, 35, 109-116.
(17) Olsson, B.; Stålbom, B.; Johansson, G. Anal. Chim. Acta 1986, 179, 203207.
(18) Garcia de Maria, C.; Townshend, A. Anal. Chim. Acta 1992, 261, 137143.
In this work, a flow system containing a micromachined
lamella-type porous silicon reactor and a novel mid-IR
fiber-optic flow cell were used for the enzymatic determination of sucrose in aqueous solution. The method relies
on the enzymatic hydrolysis of sucrose to fructose and
glucose catalyzed by β-fructosidase and on the acquisition
of FT-IR spectra before and after complete reaction.
β-Fructosidase was covalently bound to the porous silicon
surface of the channels in the microreactor. The porous
silicon was achieved by anodization of the silicon reactor
in a HF/ethanol mixture. For the measurement of small
amounts of aqueous solution, a miniaturized flow cell was
developed which consisted of two AgClxBr1-x fiber tips
(diameter, 0.75 mm) coaxially mounted in a PTFE block
at a distance of 23 µm. The flowing stream was directed
through the gap of the two fiber tips which served to define
the optical path length and to bring the focused mid-IR
radiation to the place of measurement. Using this construction, a probed volume of ∼10 nL was obtained. The
calibration curve was linear between 10 and 100 mmol/L
sucrose. Furthermore, the potential of this method was
demonstrated by the analysis of binary sucrose/glucose
mixtures showing no interference from glucose and by the
successful determination of sucrose in real samples.
S0003-2700(97)00017-6 CCC: $14.00
© 1997 American Chemical Society
Analytical Chemistry, Vol. 69, No. 15, August 1, 1997 2877
Figure 1. Cross-sectional SEM view of the porous microreactor.
measurement. One way to achieve this is the use of a conventional FT-IR microscope for focusing the IR radiation.19 Here we
present a novel approach based on mid-IR fiber optics, which
shows several advantages as compared to the use of an FT-IR
microscope. The dedicated optical design of the system is well
adapted to the flow-through cell, which allows for improved signalto-noise ratio as compared to the standard FT-IR microscope.
Further, additional flexibility is gained with this approach, as the
optical path length of the fiber cell can be accurately adjusted
from 1 to 500 µm, hence allowing the adaptation of the optical
path length to the lengths dictated by solvent (carrier) absorption.
Path lengths greater than 25 µm can be used in certain organic
organic solvents such as CCl4 or CHCl3, while aqueous solution
require path lengths of 25 µm or below. Furthermore, using midIR fiber optics, a local separation of several meters between a bulky
FT-IR spectrometer containing the interferometer and the highly
sensitive liquid nitrogen-cooled mercury cadmium telluride (MCT)
detector is possible in principle at present.20
A new technique to increase the surface area in silicon
micromachined enzyme reactors, based on electrochemical anodization of the silicon, yielding a spongy high surface area porous
silicon structure has recently been reported.21 This technique
enhances the available surface area as compared to reactors
produced by simple anisotropical etch where no extra surface area
more than what is created by the channel geometry is achieved.22-24
The microreactors were successfully used as a carrier matrix for
immobilized glucose oxidase in a system for amperometric
glucose monitoring. More recently it was shown that the enzyme
activity increased by a factor of 100 in such porous silicon
(19) Kellner, R.; Lendl, B. Anal. Methods Instrum. 1995, 2, 52-54.
(20) Jakusch, M. Diploma Thesis, Vienna University of Technology, 1996.
(21) Laurell, T.; Drott, J.; Rosengren, L.; Lundström, K. Sens. Actuators B 1996,
3, 161-166.
(22) Laurell, T.; Rosengren, L.; Drott, J. Biosens. Bioelectron. 1995, 10, 289299.
(23) Murakami, Y.; Toshifumi, T.; Yokoyama, K.; Tamiya, E.; Karube, I.; Suda,
M. Anal. Chem. 1993, 65, 2731-2735.
(24) Strike, D. J.; Thiébaud, P.; van der Sluis, A. C.; Koudelka-Hep, M.; de Rooij,
N. F. Microsyst. Technol. 1994, 1, 48-50.
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Analytical Chemistry, Vol. 69, No. 15, August 1, 1997
microreactors when they were compared to the original nonporous
structures.25
EXPERIMENTAL SECTION
Reagents. Sucrose and glucose standard, 0.2 mol/L each,
were prepared by dissolving 34.23 g of sucrose and 18.02 g of
glucose (both analytical grade) in 500 mL of distilled water. A
sodium acetate buffer solution (pH 4.7) was prepared by dissolving
27.2 g of sodium acetate in 1 L of distilled water and adjusting
the pH with 1 N HCl. This buffer system exhibits little absorbance
between 950 and 1200 cm-1.
Enzyme Reactor Fabrication. The microreactor was fabricated in (110) silicon, p-type (20-70 W cm) using anisotropic wet
etching. The micromachined reactor comprised a structure of
32 parallel channels, 50 µm wide, 11 mm long, and 240 µm deep.
The total length of the reactor structure, including the flow inlet
and outlet, was 13.1 mm and the total width 3.15 mm (Figure 1).
A surface-enlarging porous silicon layer on the channel
structure was achieved by anodizing the microreactors in a
solution of HF (48%) and ethanol (96%), mixing ratio 1:1. The
anodization was performed at a constant current density of 50 mA/
cm2 for 5 min. Illumination with a standard Hg light source was
supplied from the anodic side (rear side) of the reactor during
anodization. After the anodization the reactor was thoroughly
rinsed in distilled water.
Enzyme Immobilization. The procedure to immobilize
β-fructosidase to the porous silicon reactor consisted of three
steps: silanization, glutaraldehyde activation, and enzyme coupling, carried out in beaker solutions and under gentle agitation.
The microreactor was silanized with (3-aminopropyl)triethoxysilane (APTES; Sigma Chemicals Co., St. Louis, MO). A 1 g
sample of APTES was dissolved in 9 mL of water and the pH
adjusted to 3.5 with 6 mM HCl. Silanization was accomplished
in a beaker placed in a water bath at 75 °C for 2 h. After the
silanization the reactor was thoroughly rinsed in distilled water.
(25) Drott, J.; Lundström, K.; Rosengren, L.; Laurell, T. Submitted to J. Micromech.
Microeng.
Figure 2. Sketch of the developed miniaturized fiber-optic flow cell
allowing for the acquisition of an FT-IR spectrum of 10 nL. The
diameter of the AgCxlBr1-x was 750 µm.
The silanization was followed by glutaraldehyde (GA) activation:
2 mL of 2.5% GA (grade II, Sigma Chemicals Co.) in 18 mL of 0.1
M phosphate buffer solution (PBS; pH 7) for 2 h at ambient
temperature. The GA activation was followed by rinsing the
reactor in PBS overnight. A solution of β-fructosidase (Boehringer
Mannheim GmbH), 10.6 mg in 2 mL of PBS, was used for enzyme
coupling. Immobilization was performed for 7.5 h at ambient
temperature. After immobilization, the microreactors were rinsed
and stored at 8 °C in PBS.
Apparatus. A Bruker IFS 88 FT-IR spectrometer equipped
with a narrow-band MCT detector was used for acquisition of FTIR spectra. The flow system was set up with a Cavro XP 3000
syringe pump (syringe size 500 µL) a Valco ten-port selection
valve, and poly(tetrafluoroethane) (PTFE) (i.d., 0.5 and 0.3 mm)
and poly(ether ether ketone) (PEEK) (i.d., 0.17 mm) tubings as
well as fittings from Global FIA. A HP 8452A diode array
spectrometer equipped with a 1 cm cuvette was used for UV/
visible measurements performed within the reference method.
Miniaturized Fiber-Optic Flow-Through Cell. A detailed
construction drawing of the home-made fiber optic flow through
cell is depicted in Figure 2. Two AgClxBr1-x fiber pieces (diameter,
750 µm) with plane-parallel faces were assembled coaxially to each
other. The closeness of the measuring chamber and rigid
positioning of the fiber tips were achieved by means of PTFE
ferrules clamped by aluminum screws, allowing precise axial
guidance. The distance between the two fiber end faces was
adjusted using a measuring microscope. The geometric design
of the inner part of the cell, made of PTFE, guarantees parallelism
of all optical surfaces and minimizes the dead volume. This PTFE
body was inserted in a aluminum housing with internal screw
threads for conventional FIA fittings enabling connection to the
flow cell to the miniaturized system. The assembled cell was
mounted on a home-made positioning device which allowed
positioning to maximize light throughput. The positioning table
as well as two KBr lenses (diameter, 38 mm; focal length, 35 mm)
mounted on an optical bench were set on a platform which was
placed in the sample compartment of the FT-IR spectrometer. The
IR radiation was focused onto the fiber tip by use of a KBr lens
and then, after passing through the flow cell, collected by the
second KBr lens and directed to the MCT detector. The faces of
the two AgClxBr1-x fibers used were cut and smoothed using a
microtome equipped with a freshly broken glass knife to minimize
losses induced by coupling the IR radiation to the fibers.
Manifold and Procedure. The silicon microreactor containing the immobilized β-fructosidase was placed in a Plexiglas body
Figure 3. Manifold used: syringe size, 500 µL; (holding coil) i.d.
0.5 mm, l ) 250 cm; (c1) i.d. 0.17 mm, l ) 10 cm; (c2) i.d. 0.3 mm, l
) 10 cm; (c3) i.d. 0.3 mm, l ) 25 cm, (c4) i.d. 0.3 mm, l ) 25 cm. The
inset shows a detailed arrangement of the silicon microreactor placed
in the Plexiglas body. Sealing was achieved with a 100 µm thick latex
layer.
and connected to the flow system by means of conventional FIA
fittings. To avoid clogging of the microreactor, a filter disk (pore
size, 10 µm, purchased from Upchurch Scientific) was placed in
the Plexiglas body so that the flowing stream was filtered before
reaching the microreactor. A detailed sketch of the arrangement
together with the complete experimental setup used is depicted
in Figure 3. In the first step, the part of the manifold located
downstream from the selection valve was flushed with buffer
solution by first aspirating buffer into the holding coil, and second,
directing it through the bypass (c3) andsafter switching the
selection valvesalso through the enzyme reactor via the flow cell
to waste. Then a background spectrum was recorded. In the
second step, sample was aspirated into the holding coil. One part
of the sample was pumped directly to the detector via the bypass
coil (c3) and coil 4 whereas the second part of the aspirated sample
was directed through the enzyme reactor and consecutively
transported to the detector. From the different (3, 6, 8.5, and 11
µL) sample volumes investigated, 8.5 µL was chosen as a
compromise between a small total sample volume and sensitivity
(dispersion factor, 1.5). The same amount of unreacted sample
was brought via the bypass (c3) to coil 4. In order to obtain
complete sucrose hydrolysis the “stopped-flow technique”26 was
applied when the sample had reached the silicon microreactor.
After restarting the flow (50 µL/min) two spectra were recorded.
The first spectrum corresponded to the sample that was located
in coil 4 during the stopped-flow period. The second spectrum
was then recorded from the sample that was located in the
microreactor during the stopped flow period.
Reference Method. As a reference method for the analysis
of the real samples, a test kit from Boehringer Mannheim for the
analysis of glucose and sucrose in liquid samples was used.
RESULTS AND DISCUSION
Data Acquisition and Calibration Curve. As a consequence
of the limited sensitivity of FT-IR spectroscopic measurements in
aqueous streams, the signals produced by the enzymatic reaction
must be maximized. In the method for sucrose analysis presented
here, the analytical readout is taken from the difference spectrum
obtained by subtracting the mid-IR spectrum of the sample after
(26) Olson, S.; Ruzicka, J.; Hansen, E. H. Anal. Chim. Acta 1982, 136, 101112.
Analytical Chemistry, Vol. 69, No. 15, August 1, 1997
2879
Table 1. Difference in Absorption at 998-1034 cm-1
Taken from the Difference Spectrum during the
Analysis of a 100 mmol/L Sucrose Standard as a
Function of the Stopped-Flow Time
stopped-flow time (s)
∆ mAU (998-1034
cm-1)
0
30
60
120
180
240
300
2.2
4.9
13.8
29.1
33.5
32.3
33.7
complete hydrolysis from that measured before reaction. Therefore, considering the enzyme substrate determination as reported
here, the “end point” method is of advantage, compared to the
kinetic one, since in this approach maximum intensities in the
corresponding difference spectra are obtained. For a complete
conversion of the analyte in a short time, a high enzyme activity
is necessary. This demand is linked to the available surface area
of the enzyme reactor used.
When the sample was continuously pumped through the
microreactor at a flow rate of 50 µL/min, just partial reaction of a
100 mmol/L sucrose standard occurred. Therefore, the flow was
stopped as soon as the sample filled the microreactor in order to
increase the contact time between the immobilized β-fructosidase
and the analyte so that complete reaction could be achieved. By
varying the stopped-flow time, it was found that a period of at
least 3 min was required to achieve complete hydrolysis of a 100
mmol/L sucrose standard (Table 1). However, a stopped-flow
time of 5 min was adopted for further measurements to assure
sufficient reaction time. The two parts of the sample (the
unreacted as well as the reacted one) exhibited different dispersion
as a consequence of different flow lines. To correct for that, the
recorded absorption of the isosbestic point at 1116 wavenumbers
was taken as an internal reference. The spectrum of the reacted
sample was multiplied by a constant factor to achieve the same
absorption at 1116 wavenumbers as in the spectrum of the
nonreacted sample. In Figure 4a, the mid-IR spectra recorded
during the analysis of a 100 mM sucrose standard as well as the
corresponding difference spectrum are shown. The bands appearing in the region displayed (1250-900 cm-1) are due to C-C
and C-O stretching modes.27 To establish a calibration curve in
aqueous solution, triplicate analyses of five standards covering
the range from 10 to 100 mmol/L sucrose was performed (Table
2).
Analysis of Sucrose Standards and Binary Sucrose/
Glucose Mixtures. To investigate the influence of glucose on
the developed method, increasing amounts of glucose (25, 50, 75,
and 100 mmol/L) were added to a 100 mmol/L sucrose standard
and analyzed. The results obtained are listed in Table 3 and show
clearly that the developed method is not subjected to interference
from glucose.
Analysis of Real Samples. The sucrose content of five
different soft drinks was determined by the developed method.
For this purpose, the soft drinks were diluted 1:1 with distilled
water and degassed by sonification prior to analysis. Standard
addition was performed with three real samples adding 25, 50,
and 100 mmol/L sucrose to the diluted samples. Every soprepared solution was subjected to triplicate analysis. As can be
seen from the results shown in Table 4, the slopes of the
calibration curves obtained from standard addition agree well with
(27) Hineo, M. Carbohydr. Res. 1977, 56, 219-227.
2880 Analytical Chemistry, Vol. 69, No. 15, August 1, 1997
Figure 4. (a) Spectra recorded during the analysis of a 100 mM
sucrose standard. Spectrum i corresponds to the standard before
reaction and spectrum ii to the one after reaction. Spectrum iii is the
calculated difference spectrum from which the analytical readout was
taken. (b) Spectra recorded during the analysis of a real sample.
Spectrum iv, before reaction; spectrum v, after reaction; and spectrum
vi, calculated difference spectrum.
Table 2. Calibration Curve for Sucrose Analysis
sucrose standard (mmol/L)
∆ AU (998 -1034 cm-1) (mAU)
rel std dev, rsd (%), (n ) 3)
10
25
50
75
100
4.4
9.8
10.9
3.6
17.5
5.8
25.0
1.5
33.1
3.4
slope, b (mAU mmol/L)
intercept, a (mAU)
resid std dev, sy (mAU)
std dev of the method, sx0 (mmol/L)
regression coeff r
0.301
0.206
0.109
2.93
0.997
Table 3. Influence of the Glucose Content on the
Sucrose Determinationa
glucose added (mmol/L)
∆ mAU (998-1034 cm-1)
0
25
50
75
100
33.3
32.4
31.9
32.3
33.2
a Different amounts of glucose added to a 100 mmol/L sucrose
standard.
the one obtained from the analysis of aqueous standards (Table
2). Therefore, it can be concluded that spectral interferences
related to the matrix were successfully eliminated by calculation
of the difference spectra. The spectra recorded during the
analysis of a real sample (sample C) are depicted in Figure 4b.
The spectral features of sucrose are clearly overlapped by matrix
absorption (spectra iv and v). However, by calculation of the
Table 4. Results Obtained from the Triplicate Analysis
of Real Samplesa
sucrose contentb (mmol/L)
soft drink
sample
IR method
ref method
A
B
C
D
E
218 (2.4)
1 (-)
53 (6.7)
306 (4.0)
204 (2.8)
215 (2.8)
1 (-)
57 (1.7)
305 (2.1)
208 (2.0)
slope
(mAU mmol/L)
0.328
0.293
0.338
a For the samples with which standard addition was performed the
slope of the corresponding calibration curve is shown as well. b Values
in parentheses are rsd’s (%).
difference spectrum, spectral features related to sucrose hydrolysis
are obtained. Furthermore, the results obtained by IR analysis
are confirmed by the reference method.
Performance of the Fiber-Optic Flow-Through Cell. To
verify the path length adjusted using the light microscope, a midIR spectrum of the empty flow cell was recorded. From the
recorded interference fringes which resulted from multiple reflections of the IR beam at the fiber tip/air interfaces, the path length
of the empty flow cell was calculated to be 23 µm, resulting in a
probed volume of ∼10 nL. The noise level of the FT-IR spectra
when measuring in aqueous solution was determined by recording
a 100% line. Two single-beam spectra were measured one after
the other using Blackman-Harris three-term apodization and
averaging 128 coadded scans at a spectral resolution of eight
wavenumbers. The time required for all operation steps such as
spectrum acquisition, Fourier transformation, and storage of the
spectrum on the hard disk was 10 s. Rationing of the two singlebeam spectra and calculation of the decadic logarithm gives the
100% line expressed in absorption units. In the spectral region
of interest (950-1200 cm-1) the rms value (standard deviation of
the noise) was calculated to be 9.2 × 10-5 AU. Therefore, it can
be extrapolated that spectral features on the order of 2.8 × 10-4
AU can be detected by this experimental setup, assuming the
spectral features to be at least 3 times the noise level. A 100 mM
sucrose solution exhibits absorption of 0,145 AU at 1070 wavenumbers with this experimental set-up. Calculation of the amount
of sucrose present between the two fiber tips reveals that 1 ng/
10 nL sucrose is sufficient for IR detection under this experimental
conditions.
CONCLUSIONS
The introduction of Fourier transform infrared spectroscopy
as a novel detection scheme in miniaturized total analysis systems
represents a significant step toward the development of techniques
capable of obtaining molecular-specific information in miniaturized
flowing streams. The great amount of chemical information
contained in FT-IR spectra was exploited to reduce the number
of enzymatic reactions needed for the determination of sucrose
in aqueous standards as well as in real samples such as soft drinks.
Both the development of a new molecular specific detector based
on a miniaturized IR fiber-optic flow cell and the application of
silicon microreactors with in situ fabricated porous silicon layers
as a surface-enlarging matrix for immobilized enzymes can be
considered to be the cornerstones in the novel µ-FT-IR analysis
system presented herein.
ACKNOWLEDGMENT
Acknowledgment is given to the Austrian Science Foundation
for the financial support provided by the Project P11338 ÖCH.
Received for review January 3, 1997. Accepted April 23,
1997.X
AC9700179
X
Abstract published in Advance ACS Abstracts, June 15, 1997.
Analytical Chemistry, Vol. 69, No. 15, August 1, 1997
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