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LTCC microreactor for urea determination in biological fluids
Sensors and Actuators B 141 (2009) 301–308
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Sensors and Actuators B: Chemical journal homepage: www.elsevier.com/locate/snb
LTCC microreactor for urea determination in biological fluids Karol Malecha a,∗ , Dorota G. Pijanowska b , Leszek J. Golonka a , Władysław Torbicz b a b
˙ Wyspia´ Faculty of Microsystem Electronics and Photonics, Wrocław University of Technology, Wybrzeze nskiego 27, 50-370 Wrocław, Poland Institute of Biocybernetics and Biomedical Engineering, Polish Academy of Sciences, Trojdena 4, 02-109 Warsaw, Poland
a r t i c l e
i n f o
Article history: Received 19 June 2008 Received in revised form 8 April 2009 Accepted 15 June 2009 Available online 24 June 2009 Keywords: LTCC Microreactor CFD Enzyme
a b s t r a c t This article presents design, fabrication and testing of an enzymatic microreactor with integrated heater and a temperature sensor. The microsystem was fabricated using low temperature co-fired ceramics (LTCCs) technology, which has a few advantages over silicon-based fabrication processes. Computational fluid dynamics (CFD) and finite element method (FEM) analysis was used to arrive at an optimum microreactor and heater design. The heater performance was evaluated using thermographic measurements, and the temperature resistance characteristics of the temperature sensor revealed good electrical properties and stability at higher temperatures (up to 140 ◦ C). As an enzyme carrier, the glass beads coated with polyacrylonitrile layer and porous glass beads were used. The microreactor was used to measure urea concentration with high output signal (ca. 2.5 pH units) and large scale. The maximal output signal enabled to apply presented microreactor for determination of urea in biological or environmental fluids. The properties of the LTCC-based enzymatic microreactor were comparable with the features of a similar one made in silicon. © 2009 Elsevier B.V. All rights reserved.
1. Introduction The low temperature co-fired ceramics (LTCC) is a well known technique for hybrid circuits and multilayer devices fabrication. It has been used in microelectronics since the end of 1980s [1]. A traditional LTCC structure consists of dielectric tapes, external and internal conductors, buried and surface passive components, thermal and electrical vias. The conductors and passive films are deposited by a standard screen-printing method. After that unfired ceramics layers are stocked together, laminated and co-fired at temperature of 850 ◦ C. Additional active and passive components can be added on the top and the bottom surface of fired structure [2–4]. The LTCC ceramics exhibits very good properties: low dielectric losses at high frequencies, hermeticity, biocompatibility, matching of coefficient of thermal expansion with silicon, high thermal dissipation, high temperature stability and relatively low cost [5,6]. The LTCC technology also enables of making three-dimensional (3D) structures what is especially desirable in case of microfluidics applications. Special micromachining methods are used to produce spatial structures inside LTCC modules: laser cutting, computer numerical control (CNC) machining, using of sacrificial volume materials, acetone etching, and low pressure lamination [7–9]. The most important features of the LTCC technology in respect to microdevices fabrication are the following: 3D structuring, integra-
∗ Corresponding author. Tel.: +48 071 355 4822; fax: +48 071 355 9718. E-mail address: [email protected] (K. Malecha). 0925-4005/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.snb.2009.06.026
tion of various components in one module, possibility of combining with various materials [10,11,25]. The advantages of LTCC structure, in comparison with silicon one, are as follows: lower price, shorter design and development time and possibility of integration of fluidic channels, heater, sensors and package in one LTCC module [12–15]. Microreactor technology has become a new and very promising field within a very short time in the fields of chemistry, process engineering and biotechnology [16]. The main benefits of microreactors with enzymes immobilized on beads are: easiness of arrangement in flow-through systems, simplicity of enzyme regeneration, very high surface-to-volume ratio in comparison with open-channel microfluidic systems and ability to efficient mixing of reagents under laminar flow regime. A considerable number of papers reporting successful application of enzymatic microreactors are reviewed in [17,18]. This miniaturized devices found application in many fields, especially in the analysis of proteins, nucleic acids and for kinetics studies of immobilized enzymes. Most of them are devices with enzymes immobilized on beads or microfluidic channel walls [19]. The report of using microfluidic system with enzyme immobilized on surface of porous beads was provided by Seong et al. [20], who fabricated PDMS/glass microreactor for determination of enzyme kinetics. A significant outcome of this study was the finding that the kinetics of enzyme was the same in a solution and after immobilization on the surface of beads. More recently, Alhadeff et al. [21] showed that sequential enzymatic double microreactor system with alcohol oxidase (AOD) and horseradish peroxidase (HRP) immobilized on aminopropyl glass beads could be used to quan-
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K. Malecha et al. / Sensors and Actuators B 141 (2009) 301–308 Table 1 Modeled fluid parameters. Fluid parameter −3
Density (kg m ) Dynamic viscosity (Pa s)
Value 1000 0.001
lower, up to 45 ◦ C) [22]. As a model enzymatic reaction, a hydrolysis of urea in the presence of urease was chosen. The obtained results were comparable with those for a similar system made in silicon [23]. 2. Numerical simulations
tifying ethanol from gasohol mixture. The sequential system was composed of two microreactors made of acrylic. Both were packed with the appropriate enzyme immobilized on surface of aminopropyl glass beads. AOD and HRP enzymes were used with phenol and 4-aminophenazone and the red-colored product was detected using a colorimetric analysis. The microreactor system was used to measure ethanol concentration with high sensitivity, high output signal and wide range. In this paper, a batch type flow-through enzymatic microreactor with integrated heater and a temperature sensor made in LTCC technology is presented. The LTCC microreactor consists of two chambers separated by the threshold. The batches in the form of glass or polymer coated glass beads with immobilized enzyme (urease) were loaded in one of two compartments of the microreactor. The vias, and the fluidic channels were patterned in DP951 A2 LTCC foils by the Nd-YAG laser (Aurel NAVS 30 laser trimming and cutting system). Finite element method (FEM) was applied to arrive at an optimum microreactor and heater design. The integrated heater was fabricated of conductive platinum ink by screen-printing method inside the LTCC module. The heater pattern was analytically calculated and designed to assure uniform distribution of the temperature in the area of the reaction chamber. The temperature distribution was verified by infrared thermographic measurements. In addition, the temperature sensor was embedded between heater and reaction chamber. The buried thermoresistor reveals good electrical properties and stability up to 140 ◦ C (a typical working temperature of the presented microreactor is much
2.1. Computational fluid dynamics (CFD) analysis The CFD analysis was made to investigate the influence of the chambers geometry upon fluid flow conditions inside the structure. The first model of the microreactor consisted of two rectangular chambers separated by a threshold. The dimensions of the reaction chamber and chamber for reaction products were 6 mm (width) by 15 mm (length) by 280 m (height) and 6 mm by 7 mm by 280 m, respectively. The Navier–Stokes equation was solved with commercial software package ANSYS® /Flotran CFD. The modeling was made for two-dimensional steady, laminar, incompressible flow. The parameters of the modeled fluid are shown in Table 1. For all solutions, we applied a uniform flow rate Q at the inlet. This included specification of a zero velocity condition at the inlet in the direction normal to the inlet flow. Zero velocities in both directions were applied along the rigid walls, including where the walls intersected the inlet and outlet. We assumed incompressible fluid flow. In such cases, only the relative value of pressure is important, and zero relative pressure was applied at the outlet. The zero relative pressure value at the microreactor outlet means that a difference between pressure at the outlet and the ambient one was equal to zero. The numerical computations were carried out for several flow rate values Q (0.02–2 ml min−1 ). The resulting velocity field and fluid trajectories showed the recirculation regions that occurred in the corners of the microreactor. Recirculation regions were formed for all investigated flow rate values. The exemplary velocity field and fluid trajectories for rectangular reaction cham-
Fig. 1. Results of CFD simulations (in m s−1 ) for rectangular reaction chamber: (a) velocity field and (b) fluid trajectories (Q = 0.2 ml min−1 ).
Fig. 2. Results of CFD (in m s−1 ) simulations for rounded reaction chamber: (a) velocity field and (b) fluid trajectories (Q = 0.2 ml min−1 ).
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Fig. 3. Results of CFD (in m s−1 ) simulations for rounded reaction chamber partially filled with catalytic bed: (a) velocity field and (b) fluid trajectories (Q = 0.2 ml min−1 ).
Fig. 4. (a) Meander heating source and (b) optimized heater.
ber are presented in Fig. 1. Usually such areas tend to accumulate some impurities or air bubbles which make the effective pumping out of the entire reaction product from the microreactor impossible. On the basis of the CFD analysis, a new microreactor with rounded chambers was proposed. The new geometry of the chambers allowed to avoid fluid recirculation inside both microreactor chambers and then, an effective replacement of the fluid as well as beads with immobilized enzyme (e.g. for enzyme regeneration) was possible. Calculated velocity field and fluid trajectories for optimized reaction chamber without and with enzymatic catalytic bed are presented in Figs. 2 and 3, respectively. The catalytic bed was modeled as a porous media with hydraulic diameter of 100 m. Unfortunately, our version of the ANSYS(r) software precluded applying non-slip boundary condition on the surface of the porous media. In consequence, a straight lines for the fluid trajectories in case of the reaction chamber with catalytic bed were observed. As can be seen in Fig. 5 presence of catalytic bed inside reaction chamber provided to higher head loss and resulted in decreasing of mean fluid flow velocity. 2.2. Steady-state thermal analysis The enzyme (urease) activity strongly depends on temperature, that is why there was necessity to have stable heating source. Two different heater patterns were tested. The first one was a typical meander structure, as shown in Fig. 4a. This construction did not
give proper temperature distribution in the area of reaction chamber. Therefore, to obtain a uniform distribution of the temperature in the area of microreactor a special heater’s pattern was analytically calculated and designed [24,25]. First, a two-dimensional model of the heater was created. Next, shape of each of the heating meander line was modified to assure uniform distribution of the temperature in area of the microreactor. Then, based on the result for 2D model, three-dimensional one was created, as well. The new and optimized pattern of the heater is illustrated in Fig. 4b. In the model, a several simplifications were done to make the calculations much easier. Because of the relatively low temperature applied to the system, only convection and thermal conduction were considered during the analysis. Therefore, in our theoretical considerations, the properties of water and glass were used as a model fluid and catalytic bed, respectively. All the properties of the materials employed in the calculations are shown in Table 2. In the model, convection type heat exchange taking place at all external surfaces of the LTCC microreactor was taken into account. To reach desired temperature inside reaction chamber, various conditions of power supplied to the heater were investigated. Finally, the uniform temperature distribution was achieved. The FEM steady-state thermal analysis was applied to simulate and compare the temperature characteristics for both heating systems. The results of modeling are presented in Fig. 5. The difference between maximal and minimal temperature inside the microreactor did not exceed 6.5, 1.5 ◦ C for meander and the optimized heater,
Table 2 Material properties used in thermal analysis.
−3
Density (kg m ) Thermal conductivity (W m−1 K−1 ) Specific heat (J kg−1 K−1 ) Resistivity ( m)
LTCC
Platinum
Water
Catalytic bed (glass)
3100 3 450 1012
21,500 71.6 133 1.03 × 10−7
1000 0.61 4200 –
2900 0.9 729 –
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Fig. 5. The temperature distribution (◦ C) for: (a) meander, (b) optimized heating system and (c) optimized heating system with reaction chamber partially filled with catalytic bed.
respectively. According to the simulation results, the temperature was nearly uniformly distributed on the surface of the catalytic bed (Fig. 5c). 3. Technology Seven layers of DP 951 A2 Green TapeTM were used to make the microreactor. The thickness of the foil was equal to 137 m after firing. The vias, positioning orifices and the chambers were made in green tapes by Nd-YAG laser patterning. The heater and the temperature sensor were printed by standard screen-printing method through 325 mesh steel screen. All the patterned green tapes layers
are presented in Fig. 6. Layer (a) defines fluidic channel which connects the reaction chamber and chamber for reaction product. Layer (b) contains a threshold and cuts for the chambers of the microreactor. Layer (c) defines bottom of the microreactor. The optimized heater and the temperature sensor are screen-printed on layers (d) and (f), respectively. Layer (e) defines cuts for a temperature sensor terminals. Finally, layer (g) contains vias for a heater and a temperature sensor terminals. After screen-printing and laser cutting the all ceramic tapes were stacked into one module in a proper order, pressed in an isostatic press. In order to prevent chambers sagging, two-step lamination method was used [26]. First, all LTCC layers were initially pressed with low pressure of 20 atmospheres. Next,
Fig. 6. Design of the layers to construct the LTCC enzymatic microreactors.
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Fig. 7. Fragment of the chamber (6 mm width) obtained by standard LTCC process (a) and with use of fugitive phase (b).
Fig. 8. The LTCC-based microreactor.
spatial structures were filled with by fugitive phase and laminated second time with standard pressure of 200 atmospheres. After the lamination, the LTCC module was co-fired in air at a modified temperature profile with slower ramp rate up to 450 ◦ C and a maximum at 875 ◦ C. The two-step lamination considerably decreased sagging and contraction of microreactors’ chambers during lamination and firing process. The comparison between chambers obtained from standard LTCC process and those obtained from the two-step lamination method is shown in Fig. 7. Finally, surface of the microreactor was covered by glaze layer (ITME, Warsaw) and joined to a transparent polymer polydimethylsiloxane (PDMS) [27]. The ready to use microreactor structure is presented in Fig. 8.
ature of 37 ◦ C and cooling time to ambient temperature were equal to 50 and 30 s, respectively. Shorter heating and cooling times are related to much higher thermal conductivity of the silicon material. 4.2. Temperature sensor The embedded temperature sensor was located between the heating system and the reaction chamber. The sensor was made of DP 3630 resistive ink, which is inexpensive in respect to typically used Pt ink [28]. Resistance changes of the DP 3630-based sensor versus temperature were investigated experimentally. The resistance values of thermoresistor were measured twenty two times in
4. Results 4.1. Heating system The integrated heater located inside the LTCC module was fabricated of a conductive Pt ink by screen-printing method. The heater pattern was analytically calculated and designed to ensure a uniform distribution of the temperature inside the microreactor. The temperature distribution on the surface of the microreactor reaction chamber was measured by an infrared (IR) thermo-scanner. In order to investigate the temperature field using IR thermography system, the LTCC microreactor was painted black to keep the uniform emissivity over the surface. The exemplary temperature distribution in the area of reaction chamber filled with catalytic bed is presented in Fig. 9. For the temperature inside microreactor 37 ◦ C, the obtained heating power was 90 mW. According to the transient measurements, heating time necessary to reach stable temperature of 37 ◦ C and cooling time to reach 25 ◦ C were equal to 200 and 150 s, respectively. In case of a similar microreactor made in silicon heating time required to obtained stable temper-
Fig. 9. The temperature field inside reaction chamber filled with catalytic bed, measured by an IR thermo-scanner. Temperature inside the microreactor T = 37 ◦ C, heating power P = 90 mW.
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Fig. 10. Resistance of the embedded DP 3630 temperature sensor versus temperature.
a range from 30 to 130 ◦ C at 5 ◦ C intervals, while the temperature was recorded using a Pt-100 PTC resistor. The measurements were made by Agilent 34970A data acquisition switch unit using twowire method. The temperature coefficient of resistance (TCR) values were calculated based on the resistances at 30 and 130 ◦ C. The average value of the TCR was equal to ca. 3500 ppm ◦ C−1 . The electrical parameters of the resistors were similar to the properties of thickfilm platinum thermoresistors. The buried sensor thermoresistor reveal good electrical properties and stability up to 140 ◦ C. However, the typical working temperature of the presented microreactor is much lower, i.e. up to 45 ◦ C. For the fabricated thermoresistor, the resistance change per temperature degree was around 0.16 ◦ C−1 . The characteristics of DP 3630 embedded LTCC temperature sensor is presented in Fig. 10. 4.3. Measurements As a batch for the microreactors, two types of the supports with immobilized enzyme (urease) was prepared. In the first case, the enzyme was immobilized onto porous glass beads (Sigma, PG 100120), while in the second case, the glass beads (Sigma, PG 100-120) were coated with polymer layer of polyacrylonitrile (PAN). Then, the enzyme molecules were adsorbed on the beads surface [23]. To remove the non-properly attached enzyme molecules, prior to the measurements the LTCC microreactors were washed with phosphate buffer at flow rate of 0.2 ml min−1 for 30 min. After initial washing, the beads with immobilized enzyme were loaded into the microreactors. The measurements of microreactors were performed in a closed loop flow-through system, where sample was circulated through the microreactor. A schematic diagram and photograph of the experimental setup is presented in Fig. 11. The pH changes resulted from hydrolysis of urea catalyzed by urease was measured by the micro pH sensor placed in the sample reservoir. The pH changes in the sample were recorded by the pH combined glass microelectrode PHC 3359-08 Radiometer. In 10 min time intervals the concentration of urea in sample was changed by a standard addition method. The measurements were performed in 5 mM phosphate buffer containing 0.1 M sodium chloride of initial pH 6.1. The effectiveness of the LTCC-based microreactors was tested in terms of the long-term stability, amount of the urease immobilized onto the carrier as well as enzyme carrier material. To compare influence of the enzyme carrier material (glass or polymeric beads) on urease activity, the microreactors were loaded with equal amount of beads. A single load of the glass/polymeric beads with immobilized enzyme was 3 mg per microreactor. Resulting calibration curves for both microreactors are shown in Fig. 12.
Fig. 11. Schematic diagram of the experimental setup (not to scale).
In both cases, for microreactors loaded with enzyme immobilized onto bear glass beads and polymer coated beads, the output signal obtained for maximal concentration of urea (Curea = 345 mM) was very high ca. 2.5 pH units. Experiments indicate an upper detection limit of ca. 145 and 250 mM for urease immobilized onto glass beads (ROC2) and glass beads coated with PAN (ROC1), respectively, which is higher than the maximum tolerable concentration of urea in blood for patients with kidney deficiency (the range 30–80 mM). The performance of microreactors at elevated temperature was investigated. Measurements were performed at two temperatures 25 and 37 ◦ C. As can be seen in Fig. 13, the calibration curves are sigmoid-like, however, this shape is better expressed for the microreactor measured at the elevated temperature 37 ◦ C. In this case, the response approached the saturation at lower concentration of urea (29.2 mM, i.e. log Curea = −1.53). In the case of the microreactor measured at 23 ◦ C, the saturation was approached at higher concentration of urea around 144 mM (log Curea = −0.88). This means that enzymatic reaction rate of the urea hydrolysis was higher for higher temperature of the microreactor operation. In addition, slopes of the calibration curves for urea concentration in range of log Curea from −2 to −1.5 for the two temperatures 37 and 23 ◦ C are different and equal to 2.5 and 2.2 pH unit/dec, respectively.
Fig. 12. Calibration curves for microreactors with different batches: urease immobilized onto glass beads (ROC2) and glass beads coated with PAN (ROC1) in their 1st day of operation.
K. Malecha et al. / Sensors and Actuators B 141 (2009) 301–308
Fig. 13. Calibration curves for microreactors loaded with 3 mg of urease immobilized onto glass beads (ROC2) investigated at the two temperatures 37 and 25 ◦ C.
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The shape of the microreactor chambers significantly influence gas bubbles formation and their removal during flow of the fluid through microreactor. The LTCC with rounded chambers are much in favor the rectangular ones. Analytically calculated and optimized heater pattern ensured uniform distribution of temperature inside the microreactor. The temperature distribution has been verified by the infrared thermographic measurements. The temperature sensor was made of inexpensive DP 3630 ink. It was embedded between the heating system and the reaction chamber of the microreactor. The buried sensor thermoresistor reveal good electrical properties and stability up to 140 ◦ C. For both microreactors loaded with enzyme immobilized onto bear glass beads and polymer coated beads, the output signal was very high ca. 2.5 pH units. The properties of the LTCC-based enzymatic microreactor were comparable with the features of a similar one made in silicon. The main features of the presented batch type enzymatic microreactor are the following: variety of materials for an enzyme support, easiness of upload, easy control of enzyme activity and very good long-term stability. Presented LTCC-based microreactor can be used as a standalone device or can be applied as an integrated part of much more sophisticated micro-total analytical system (TAS) for ex vivo measurements of the urea concentration in biological fluids as well as for pharmaceutical industry for evaluation of effect of different drugs on enzymes activity. Current research is focused on design of a device consisting of efficient mixing channels, heating system, temperature sensor, enzymatic microreactor and a detection system. Acknowledgement The authors wish to thank Ministry of Science and Higher Education for the financial support (Grant No. N N515 410534). References
Fig. 14. Responses for the microreactor loaded with 3 mg of urease immobilized onto glass beads (ROC2) on the 1st, 2nd and the 18th day of its operation.
The second important microreactor parameter, besides the analytical signal level, it is long-term stability. The repetition of the measurements of the microreactor loaded with 1.5 mg porous glass beads was done after 1 and 17 day(s) of its permanent washing with the phosphate buffer solution. The responses of the microreactor contained half the weight of carrier with respect to the ROC2 on the 1st, 2nd and 18th day of its operation is presented in Fig. 14. The responses shown as characteristics were very similar, the difference between the maximum analytical signals on the 2nd and 18th day of microreactor operation were ca. 0.1 and 0.2 pH unit, respectively. Results obtained for the silicon type microreactor [23] and LTCC microreactors shown that for both analytical signals were comparable. An advantage of microreactors use is a very good long-term stability and easy manipulation of the measured concentration range that can be achieved through control of the amount of the batch with immobilized enzyme loaded in a microreactor. 5. Conclusions A simple and inexpensive LTCC batch type enzymatic microreactor with integrated heater and temperature sensor was designed, manufactured and positively tested.
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Biographies Karol Malecha was born in Poland in 1981. He received his MSc degree in material science engineering from Wroclaw University of Technology, Poland in 2005. Currently he is a PhD student at the Faculty of Electronics Microsystem and Photonics, Wroclaw University of Technology, where he is involved in modeling and manufacturing of microfluidic systems made in LTCC technology. He is IMAPS member. Dorota Pijanowska received the MSc in biomedical engineering from the Faculty of Precision Mechanic in the Warsaw University of Technology. She completed PhD thesis on analysis of factors determining parameters of ion sensitive field effect transistors as the sensors of biochemical quantities in 1996. Next in 2006, she received her Doctor of Science degree Her research interests include fabrication and characterization of (bio)chemical sensors and analytical microsystems, and their applications in biomedical diagnosis and environmental monitoring. Leszek Golonka was born in Poland in 1946. He received his MSc and PhD degrees in electronics from the Wroclaw University of Technology, Poland in 1969 and 1976, respectively. In 1991, he received the DSc degree. Since 1996, he has been a professor at the Wroclaw University of Technology. His current research activity includes thick-film and low temperature co-fired ceramics (LTCC) devices, sensors and microsystems. He is IMAPS, IEEE and PTTS (Polish society for sensor technology) member. Władysław Torbicz obtained his MSc (1958) in electrical engineering from the Warsaw University of Technology and PhD (1965) in electronic engineering and DSc (1989) in biomedical engineering from the Polish Academy of Sciences. Since 1991 he is a professor in the Institute of Biocybernetics and Biomedical Engineering of the Polish Academy of Science. His research work was devoted to electronic components of control systems, memory devices and biomedical devices. Since early 1980 his field of research work are chemically sensitive semiconductor devices, mainly of the ISFET type and their applications in biomedical diagnosis and environmental.
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Sensors and Actuators B: Chemical journal homepage: www.elsevier.com/locate/snb
LTCC microreactor for urea determination in biological fluids Karol Malecha a,∗ , Dorota G. Pijanowska b , Leszek J. Golonka a , Władysław Torbicz b a b
˙ Wyspia´ Faculty of Microsystem Electronics and Photonics, Wrocław University of Technology, Wybrzeze nskiego 27, 50-370 Wrocław, Poland Institute of Biocybernetics and Biomedical Engineering, Polish Academy of Sciences, Trojdena 4, 02-109 Warsaw, Poland
a r t i c l e
i n f o
Article history: Received 19 June 2008 Received in revised form 8 April 2009 Accepted 15 June 2009 Available online 24 June 2009 Keywords: LTCC Microreactor CFD Enzyme
a b s t r a c t This article presents design, fabrication and testing of an enzymatic microreactor with integrated heater and a temperature sensor. The microsystem was fabricated using low temperature co-fired ceramics (LTCCs) technology, which has a few advantages over silicon-based fabrication processes. Computational fluid dynamics (CFD) and finite element method (FEM) analysis was used to arrive at an optimum microreactor and heater design. The heater performance was evaluated using thermographic measurements, and the temperature resistance characteristics of the temperature sensor revealed good electrical properties and stability at higher temperatures (up to 140 ◦ C). As an enzyme carrier, the glass beads coated with polyacrylonitrile layer and porous glass beads were used. The microreactor was used to measure urea concentration with high output signal (ca. 2.5 pH units) and large scale. The maximal output signal enabled to apply presented microreactor for determination of urea in biological or environmental fluids. The properties of the LTCC-based enzymatic microreactor were comparable with the features of a similar one made in silicon. © 2009 Elsevier B.V. All rights reserved.
1. Introduction The low temperature co-fired ceramics (LTCC) is a well known technique for hybrid circuits and multilayer devices fabrication. It has been used in microelectronics since the end of 1980s [1]. A traditional LTCC structure consists of dielectric tapes, external and internal conductors, buried and surface passive components, thermal and electrical vias. The conductors and passive films are deposited by a standard screen-printing method. After that unfired ceramics layers are stocked together, laminated and co-fired at temperature of 850 ◦ C. Additional active and passive components can be added on the top and the bottom surface of fired structure [2–4]. The LTCC ceramics exhibits very good properties: low dielectric losses at high frequencies, hermeticity, biocompatibility, matching of coefficient of thermal expansion with silicon, high thermal dissipation, high temperature stability and relatively low cost [5,6]. The LTCC technology also enables of making three-dimensional (3D) structures what is especially desirable in case of microfluidics applications. Special micromachining methods are used to produce spatial structures inside LTCC modules: laser cutting, computer numerical control (CNC) machining, using of sacrificial volume materials, acetone etching, and low pressure lamination [7–9]. The most important features of the LTCC technology in respect to microdevices fabrication are the following: 3D structuring, integra-
∗ Corresponding author. Tel.: +48 071 355 4822; fax: +48 071 355 9718. E-mail address: [email protected] (K. Malecha). 0925-4005/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.snb.2009.06.026
tion of various components in one module, possibility of combining with various materials [10,11,25]. The advantages of LTCC structure, in comparison with silicon one, are as follows: lower price, shorter design and development time and possibility of integration of fluidic channels, heater, sensors and package in one LTCC module [12–15]. Microreactor technology has become a new and very promising field within a very short time in the fields of chemistry, process engineering and biotechnology [16]. The main benefits of microreactors with enzymes immobilized on beads are: easiness of arrangement in flow-through systems, simplicity of enzyme regeneration, very high surface-to-volume ratio in comparison with open-channel microfluidic systems and ability to efficient mixing of reagents under laminar flow regime. A considerable number of papers reporting successful application of enzymatic microreactors are reviewed in [17,18]. This miniaturized devices found application in many fields, especially in the analysis of proteins, nucleic acids and for kinetics studies of immobilized enzymes. Most of them are devices with enzymes immobilized on beads or microfluidic channel walls [19]. The report of using microfluidic system with enzyme immobilized on surface of porous beads was provided by Seong et al. [20], who fabricated PDMS/glass microreactor for determination of enzyme kinetics. A significant outcome of this study was the finding that the kinetics of enzyme was the same in a solution and after immobilization on the surface of beads. More recently, Alhadeff et al. [21] showed that sequential enzymatic double microreactor system with alcohol oxidase (AOD) and horseradish peroxidase (HRP) immobilized on aminopropyl glass beads could be used to quan-
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K. Malecha et al. / Sensors and Actuators B 141 (2009) 301–308 Table 1 Modeled fluid parameters. Fluid parameter −3
Density (kg m ) Dynamic viscosity (Pa s)
Value 1000 0.001
lower, up to 45 ◦ C) [22]. As a model enzymatic reaction, a hydrolysis of urea in the presence of urease was chosen. The obtained results were comparable with those for a similar system made in silicon [23]. 2. Numerical simulations
tifying ethanol from gasohol mixture. The sequential system was composed of two microreactors made of acrylic. Both were packed with the appropriate enzyme immobilized on surface of aminopropyl glass beads. AOD and HRP enzymes were used with phenol and 4-aminophenazone and the red-colored product was detected using a colorimetric analysis. The microreactor system was used to measure ethanol concentration with high sensitivity, high output signal and wide range. In this paper, a batch type flow-through enzymatic microreactor with integrated heater and a temperature sensor made in LTCC technology is presented. The LTCC microreactor consists of two chambers separated by the threshold. The batches in the form of glass or polymer coated glass beads with immobilized enzyme (urease) were loaded in one of two compartments of the microreactor. The vias, and the fluidic channels were patterned in DP951 A2 LTCC foils by the Nd-YAG laser (Aurel NAVS 30 laser trimming and cutting system). Finite element method (FEM) was applied to arrive at an optimum microreactor and heater design. The integrated heater was fabricated of conductive platinum ink by screen-printing method inside the LTCC module. The heater pattern was analytically calculated and designed to assure uniform distribution of the temperature in the area of the reaction chamber. The temperature distribution was verified by infrared thermographic measurements. In addition, the temperature sensor was embedded between heater and reaction chamber. The buried thermoresistor reveals good electrical properties and stability up to 140 ◦ C (a typical working temperature of the presented microreactor is much
2.1. Computational fluid dynamics (CFD) analysis The CFD analysis was made to investigate the influence of the chambers geometry upon fluid flow conditions inside the structure. The first model of the microreactor consisted of two rectangular chambers separated by a threshold. The dimensions of the reaction chamber and chamber for reaction products were 6 mm (width) by 15 mm (length) by 280 m (height) and 6 mm by 7 mm by 280 m, respectively. The Navier–Stokes equation was solved with commercial software package ANSYS® /Flotran CFD. The modeling was made for two-dimensional steady, laminar, incompressible flow. The parameters of the modeled fluid are shown in Table 1. For all solutions, we applied a uniform flow rate Q at the inlet. This included specification of a zero velocity condition at the inlet in the direction normal to the inlet flow. Zero velocities in both directions were applied along the rigid walls, including where the walls intersected the inlet and outlet. We assumed incompressible fluid flow. In such cases, only the relative value of pressure is important, and zero relative pressure was applied at the outlet. The zero relative pressure value at the microreactor outlet means that a difference between pressure at the outlet and the ambient one was equal to zero. The numerical computations were carried out for several flow rate values Q (0.02–2 ml min−1 ). The resulting velocity field and fluid trajectories showed the recirculation regions that occurred in the corners of the microreactor. Recirculation regions were formed for all investigated flow rate values. The exemplary velocity field and fluid trajectories for rectangular reaction cham-
Fig. 1. Results of CFD simulations (in m s−1 ) for rectangular reaction chamber: (a) velocity field and (b) fluid trajectories (Q = 0.2 ml min−1 ).
Fig. 2. Results of CFD (in m s−1 ) simulations for rounded reaction chamber: (a) velocity field and (b) fluid trajectories (Q = 0.2 ml min−1 ).
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Fig. 3. Results of CFD (in m s−1 ) simulations for rounded reaction chamber partially filled with catalytic bed: (a) velocity field and (b) fluid trajectories (Q = 0.2 ml min−1 ).
Fig. 4. (a) Meander heating source and (b) optimized heater.
ber are presented in Fig. 1. Usually such areas tend to accumulate some impurities or air bubbles which make the effective pumping out of the entire reaction product from the microreactor impossible. On the basis of the CFD analysis, a new microreactor with rounded chambers was proposed. The new geometry of the chambers allowed to avoid fluid recirculation inside both microreactor chambers and then, an effective replacement of the fluid as well as beads with immobilized enzyme (e.g. for enzyme regeneration) was possible. Calculated velocity field and fluid trajectories for optimized reaction chamber without and with enzymatic catalytic bed are presented in Figs. 2 and 3, respectively. The catalytic bed was modeled as a porous media with hydraulic diameter of 100 m. Unfortunately, our version of the ANSYS(r) software precluded applying non-slip boundary condition on the surface of the porous media. In consequence, a straight lines for the fluid trajectories in case of the reaction chamber with catalytic bed were observed. As can be seen in Fig. 5 presence of catalytic bed inside reaction chamber provided to higher head loss and resulted in decreasing of mean fluid flow velocity. 2.2. Steady-state thermal analysis The enzyme (urease) activity strongly depends on temperature, that is why there was necessity to have stable heating source. Two different heater patterns were tested. The first one was a typical meander structure, as shown in Fig. 4a. This construction did not
give proper temperature distribution in the area of reaction chamber. Therefore, to obtain a uniform distribution of the temperature in the area of microreactor a special heater’s pattern was analytically calculated and designed [24,25]. First, a two-dimensional model of the heater was created. Next, shape of each of the heating meander line was modified to assure uniform distribution of the temperature in area of the microreactor. Then, based on the result for 2D model, three-dimensional one was created, as well. The new and optimized pattern of the heater is illustrated in Fig. 4b. In the model, a several simplifications were done to make the calculations much easier. Because of the relatively low temperature applied to the system, only convection and thermal conduction were considered during the analysis. Therefore, in our theoretical considerations, the properties of water and glass were used as a model fluid and catalytic bed, respectively. All the properties of the materials employed in the calculations are shown in Table 2. In the model, convection type heat exchange taking place at all external surfaces of the LTCC microreactor was taken into account. To reach desired temperature inside reaction chamber, various conditions of power supplied to the heater were investigated. Finally, the uniform temperature distribution was achieved. The FEM steady-state thermal analysis was applied to simulate and compare the temperature characteristics for both heating systems. The results of modeling are presented in Fig. 5. The difference between maximal and minimal temperature inside the microreactor did not exceed 6.5, 1.5 ◦ C for meander and the optimized heater,
Table 2 Material properties used in thermal analysis.
−3
Density (kg m ) Thermal conductivity (W m−1 K−1 ) Specific heat (J kg−1 K−1 ) Resistivity ( m)
LTCC
Platinum
Water
Catalytic bed (glass)
3100 3 450 1012
21,500 71.6 133 1.03 × 10−7
1000 0.61 4200 –
2900 0.9 729 –
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Fig. 5. The temperature distribution (◦ C) for: (a) meander, (b) optimized heating system and (c) optimized heating system with reaction chamber partially filled with catalytic bed.
respectively. According to the simulation results, the temperature was nearly uniformly distributed on the surface of the catalytic bed (Fig. 5c). 3. Technology Seven layers of DP 951 A2 Green TapeTM were used to make the microreactor. The thickness of the foil was equal to 137 m after firing. The vias, positioning orifices and the chambers were made in green tapes by Nd-YAG laser patterning. The heater and the temperature sensor were printed by standard screen-printing method through 325 mesh steel screen. All the patterned green tapes layers
are presented in Fig. 6. Layer (a) defines fluidic channel which connects the reaction chamber and chamber for reaction product. Layer (b) contains a threshold and cuts for the chambers of the microreactor. Layer (c) defines bottom of the microreactor. The optimized heater and the temperature sensor are screen-printed on layers (d) and (f), respectively. Layer (e) defines cuts for a temperature sensor terminals. Finally, layer (g) contains vias for a heater and a temperature sensor terminals. After screen-printing and laser cutting the all ceramic tapes were stacked into one module in a proper order, pressed in an isostatic press. In order to prevent chambers sagging, two-step lamination method was used [26]. First, all LTCC layers were initially pressed with low pressure of 20 atmospheres. Next,
Fig. 6. Design of the layers to construct the LTCC enzymatic microreactors.
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Fig. 7. Fragment of the chamber (6 mm width) obtained by standard LTCC process (a) and with use of fugitive phase (b).
Fig. 8. The LTCC-based microreactor.
spatial structures were filled with by fugitive phase and laminated second time with standard pressure of 200 atmospheres. After the lamination, the LTCC module was co-fired in air at a modified temperature profile with slower ramp rate up to 450 ◦ C and a maximum at 875 ◦ C. The two-step lamination considerably decreased sagging and contraction of microreactors’ chambers during lamination and firing process. The comparison between chambers obtained from standard LTCC process and those obtained from the two-step lamination method is shown in Fig. 7. Finally, surface of the microreactor was covered by glaze layer (ITME, Warsaw) and joined to a transparent polymer polydimethylsiloxane (PDMS) [27]. The ready to use microreactor structure is presented in Fig. 8.
ature of 37 ◦ C and cooling time to ambient temperature were equal to 50 and 30 s, respectively. Shorter heating and cooling times are related to much higher thermal conductivity of the silicon material. 4.2. Temperature sensor The embedded temperature sensor was located between the heating system and the reaction chamber. The sensor was made of DP 3630 resistive ink, which is inexpensive in respect to typically used Pt ink [28]. Resistance changes of the DP 3630-based sensor versus temperature were investigated experimentally. The resistance values of thermoresistor were measured twenty two times in
4. Results 4.1. Heating system The integrated heater located inside the LTCC module was fabricated of a conductive Pt ink by screen-printing method. The heater pattern was analytically calculated and designed to ensure a uniform distribution of the temperature inside the microreactor. The temperature distribution on the surface of the microreactor reaction chamber was measured by an infrared (IR) thermo-scanner. In order to investigate the temperature field using IR thermography system, the LTCC microreactor was painted black to keep the uniform emissivity over the surface. The exemplary temperature distribution in the area of reaction chamber filled with catalytic bed is presented in Fig. 9. For the temperature inside microreactor 37 ◦ C, the obtained heating power was 90 mW. According to the transient measurements, heating time necessary to reach stable temperature of 37 ◦ C and cooling time to reach 25 ◦ C were equal to 200 and 150 s, respectively. In case of a similar microreactor made in silicon heating time required to obtained stable temper-
Fig. 9. The temperature field inside reaction chamber filled with catalytic bed, measured by an IR thermo-scanner. Temperature inside the microreactor T = 37 ◦ C, heating power P = 90 mW.
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Fig. 10. Resistance of the embedded DP 3630 temperature sensor versus temperature.
a range from 30 to 130 ◦ C at 5 ◦ C intervals, while the temperature was recorded using a Pt-100 PTC resistor. The measurements were made by Agilent 34970A data acquisition switch unit using twowire method. The temperature coefficient of resistance (TCR) values were calculated based on the resistances at 30 and 130 ◦ C. The average value of the TCR was equal to ca. 3500 ppm ◦ C−1 . The electrical parameters of the resistors were similar to the properties of thickfilm platinum thermoresistors. The buried sensor thermoresistor reveal good electrical properties and stability up to 140 ◦ C. However, the typical working temperature of the presented microreactor is much lower, i.e. up to 45 ◦ C. For the fabricated thermoresistor, the resistance change per temperature degree was around 0.16 ◦ C−1 . The characteristics of DP 3630 embedded LTCC temperature sensor is presented in Fig. 10. 4.3. Measurements As a batch for the microreactors, two types of the supports with immobilized enzyme (urease) was prepared. In the first case, the enzyme was immobilized onto porous glass beads (Sigma, PG 100120), while in the second case, the glass beads (Sigma, PG 100-120) were coated with polymer layer of polyacrylonitrile (PAN). Then, the enzyme molecules were adsorbed on the beads surface [23]. To remove the non-properly attached enzyme molecules, prior to the measurements the LTCC microreactors were washed with phosphate buffer at flow rate of 0.2 ml min−1 for 30 min. After initial washing, the beads with immobilized enzyme were loaded into the microreactors. The measurements of microreactors were performed in a closed loop flow-through system, where sample was circulated through the microreactor. A schematic diagram and photograph of the experimental setup is presented in Fig. 11. The pH changes resulted from hydrolysis of urea catalyzed by urease was measured by the micro pH sensor placed in the sample reservoir. The pH changes in the sample were recorded by the pH combined glass microelectrode PHC 3359-08 Radiometer. In 10 min time intervals the concentration of urea in sample was changed by a standard addition method. The measurements were performed in 5 mM phosphate buffer containing 0.1 M sodium chloride of initial pH 6.1. The effectiveness of the LTCC-based microreactors was tested in terms of the long-term stability, amount of the urease immobilized onto the carrier as well as enzyme carrier material. To compare influence of the enzyme carrier material (glass or polymeric beads) on urease activity, the microreactors were loaded with equal amount of beads. A single load of the glass/polymeric beads with immobilized enzyme was 3 mg per microreactor. Resulting calibration curves for both microreactors are shown in Fig. 12.
Fig. 11. Schematic diagram of the experimental setup (not to scale).
In both cases, for microreactors loaded with enzyme immobilized onto bear glass beads and polymer coated beads, the output signal obtained for maximal concentration of urea (Curea = 345 mM) was very high ca. 2.5 pH units. Experiments indicate an upper detection limit of ca. 145 and 250 mM for urease immobilized onto glass beads (ROC2) and glass beads coated with PAN (ROC1), respectively, which is higher than the maximum tolerable concentration of urea in blood for patients with kidney deficiency (the range 30–80 mM). The performance of microreactors at elevated temperature was investigated. Measurements were performed at two temperatures 25 and 37 ◦ C. As can be seen in Fig. 13, the calibration curves are sigmoid-like, however, this shape is better expressed for the microreactor measured at the elevated temperature 37 ◦ C. In this case, the response approached the saturation at lower concentration of urea (29.2 mM, i.e. log Curea = −1.53). In the case of the microreactor measured at 23 ◦ C, the saturation was approached at higher concentration of urea around 144 mM (log Curea = −0.88). This means that enzymatic reaction rate of the urea hydrolysis was higher for higher temperature of the microreactor operation. In addition, slopes of the calibration curves for urea concentration in range of log Curea from −2 to −1.5 for the two temperatures 37 and 23 ◦ C are different and equal to 2.5 and 2.2 pH unit/dec, respectively.
Fig. 12. Calibration curves for microreactors with different batches: urease immobilized onto glass beads (ROC2) and glass beads coated with PAN (ROC1) in their 1st day of operation.
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Fig. 13. Calibration curves for microreactors loaded with 3 mg of urease immobilized onto glass beads (ROC2) investigated at the two temperatures 37 and 25 ◦ C.
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The shape of the microreactor chambers significantly influence gas bubbles formation and their removal during flow of the fluid through microreactor. The LTCC with rounded chambers are much in favor the rectangular ones. Analytically calculated and optimized heater pattern ensured uniform distribution of temperature inside the microreactor. The temperature distribution has been verified by the infrared thermographic measurements. The temperature sensor was made of inexpensive DP 3630 ink. It was embedded between the heating system and the reaction chamber of the microreactor. The buried sensor thermoresistor reveal good electrical properties and stability up to 140 ◦ C. For both microreactors loaded with enzyme immobilized onto bear glass beads and polymer coated beads, the output signal was very high ca. 2.5 pH units. The properties of the LTCC-based enzymatic microreactor were comparable with the features of a similar one made in silicon. The main features of the presented batch type enzymatic microreactor are the following: variety of materials for an enzyme support, easiness of upload, easy control of enzyme activity and very good long-term stability. Presented LTCC-based microreactor can be used as a standalone device or can be applied as an integrated part of much more sophisticated micro-total analytical system (TAS) for ex vivo measurements of the urea concentration in biological fluids as well as for pharmaceutical industry for evaluation of effect of different drugs on enzymes activity. Current research is focused on design of a device consisting of efficient mixing channels, heating system, temperature sensor, enzymatic microreactor and a detection system. Acknowledgement The authors wish to thank Ministry of Science and Higher Education for the financial support (Grant No. N N515 410534). References
Fig. 14. Responses for the microreactor loaded with 3 mg of urease immobilized onto glass beads (ROC2) on the 1st, 2nd and the 18th day of its operation.
The second important microreactor parameter, besides the analytical signal level, it is long-term stability. The repetition of the measurements of the microreactor loaded with 1.5 mg porous glass beads was done after 1 and 17 day(s) of its permanent washing with the phosphate buffer solution. The responses of the microreactor contained half the weight of carrier with respect to the ROC2 on the 1st, 2nd and 18th day of its operation is presented in Fig. 14. The responses shown as characteristics were very similar, the difference between the maximum analytical signals on the 2nd and 18th day of microreactor operation were ca. 0.1 and 0.2 pH unit, respectively. Results obtained for the silicon type microreactor [23] and LTCC microreactors shown that for both analytical signals were comparable. An advantage of microreactors use is a very good long-term stability and easy manipulation of the measured concentration range that can be achieved through control of the amount of the batch with immobilized enzyme loaded in a microreactor. 5. Conclusions A simple and inexpensive LTCC batch type enzymatic microreactor with integrated heater and temperature sensor was designed, manufactured and positively tested.
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Biographies Karol Malecha was born in Poland in 1981. He received his MSc degree in material science engineering from Wroclaw University of Technology, Poland in 2005. Currently he is a PhD student at the Faculty of Electronics Microsystem and Photonics, Wroclaw University of Technology, where he is involved in modeling and manufacturing of microfluidic systems made in LTCC technology. He is IMAPS member. Dorota Pijanowska received the MSc in biomedical engineering from the Faculty of Precision Mechanic in the Warsaw University of Technology. She completed PhD thesis on analysis of factors determining parameters of ion sensitive field effect transistors as the sensors of biochemical quantities in 1996. Next in 2006, she received her Doctor of Science degree Her research interests include fabrication and characterization of (bio)chemical sensors and analytical microsystems, and their applications in biomedical diagnosis and environmental monitoring. Leszek Golonka was born in Poland in 1946. He received his MSc and PhD degrees in electronics from the Wroclaw University of Technology, Poland in 1969 and 1976, respectively. In 1991, he received the DSc degree. Since 1996, he has been a professor at the Wroclaw University of Technology. His current research activity includes thick-film and low temperature co-fired ceramics (LTCC) devices, sensors and microsystems. He is IMAPS, IEEE and PTTS (Polish society for sensor technology) member. Władysław Torbicz obtained his MSc (1958) in electrical engineering from the Warsaw University of Technology and PhD (1965) in electronic engineering and DSc (1989) in biomedical engineering from the Polish Academy of Sciences. Since 1991 he is a professor in the Institute of Biocybernetics and Biomedical Engineering of the Polish Academy of Science. His research work was devoted to electronic components of control systems, memory devices and biomedical devices. Since early 1980 his field of research work are chemically sensitive semiconductor devices, mainly of the ISFET type and their applications in biomedical diagnosis and environmental.