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.; Lu ¨ di, 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.; Hergenro¨der, 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.; Lu ¨ di, 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
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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.; Lundstro¨m, 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.; Thie´baud, P.; van der Sluis, A. C.; Koudelka-Hep, M.; de Rooij, N. F. Microsyst. Technol. 1994, 1, 48-50.
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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.; Lundstro¨m, 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.
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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.
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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 O ¨ CH. Received for review January 3, 1997. Accepted April 23, 1997.X AC9700179 X
Abstract published in Advance ACS Abstracts, June 15, 1997.
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