Microscale Bioreactor Studies 261
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*Author to whom all correspondence and reprint requests should be addressed.
Immobilized Enzyme Studies in a Microscale Bioreactor
F RANCIS  J ONES ,1 S COTT  F ORREST ,2 J IM  P ALMER ,2
Z ONGHUAN  L U ,2 J OHN  E LMORE ,2 AND  B ILL  B. E LMORE *,2
1
Chemical and Environmental Engineering,The University of Tennessee at Chattanooga,
615 MacCallie Avenue, Chattanooga, TN 37403-2598; and 2
Department of Chemical Engineering, Louisiana Tech University,
600 W. Arizona Avenue, Ruston, LA 71272,
E-mail: belmore@coes.latech.edu
Abstract
Novel microreactors with immobilized enzymes were fabricated using both silicon and polymer-based microfabrication techniques. The effectiveness of these reactors was examined along with their behavior over time. Urease enzyme was successfully incorporated into microchannels of a polymeric matrix of polydimethylsiloxane and through layer-bylayer self-assembly tech-niques onto silicon. The fabricated microchannels had cross-sectional dimen-sions ranging from tens to hundreds of micrometers in width and height. The experimental results for continuous-flow microreactors are reported for the conversion of urea to ammonia by urease enzyme. Urea conversions of >90%were observed.
Index Entries: Microscale bioreactor; polydimethylsiloxane microreactor;immobilized enzymes; urease enzyme; silicon wafer.
Introduction
The field of chemical process miniaturization is growing at a rapid pace with promising improvements i
n process control, product quality,and safety, (1,2). Microreactors typically have fluidic conduits or channels on the order of tens to hundreds of micrometers. With large surface area–to–volume ratios, rapid heat and mass transfer can be accomplished with accompanying improvements in yield and selectivity in reactive systems.Microscale devices are also being examined as a platform for traditional unit operations such as membrane reactors in which a rapid removal of reaction-inhibiting products can significantly boost product yields (3–6).
262Jones et al.
While microscale devices and systems are typically fabricated from silicon, alternative materials are being examined as suitable supports. Microsystem features as small as 1 µm may be fabricated precisely by using a variety of etching, molding, and milling techniques. Since enzymatic reactions typically occur at moderate operating conditions (moderate pres-sures and ambient temperatures), plastics may be considered an inexpen-sive alternative to silicon for use as microreactor fabrication material.
In this article, we report on the fabrication and performance of microreactors constructed of silicon and polydimethylsiloxane (PDMS). The resulting structures contain immobilized enzymes for converting bio-chemical substrates to useful products or for breaking down organics into waste streams.
Materials and Methods
Materials Used
Urease (EC 3.5.1.5 Type IX, Sigma-Aldrich: from Jack Beans) was used throughout the experiments. Before immobilizing urease onto the micro-reactor systems, the enzyme was evaluated for activity in the chosen buffer system (Tris[hydroxymethyl]aminomethane [THAM]). Free enzyme tests of the urease showed an approximate activity of 44,800 U/g of solid.reactive materials studies
Batch studies for evaluating immobilized enzyme activity and prop-erties of the “bioplastic” (urease entrapped in PDMS) material were con-ducted in 250-mL shake flasks in an environmentally controlled shaker/ incubator.
Continuous studies were performed in specially prepared micro-reactors molded from PDMS, designated PDMS (Sylgard™ 184 silicone elastomer; Dow Corning) poured onto silicon wafer molds. The micro-reactor molds were prepared using 4-in. silicon wafers of Type P, crystal orientation of <1-0-0>, resistivity of 1 to 2 Ω, and thickness of 457–575 µm from Silicon Quest (Santa Clara, CA). After preparation, mixtures of urease enzyme and PDMS (designated PDMS-E) were poured onto the micro-reactor mold and allowed to cure at ambient conditions.
A negative photoresist, SU-8 (Microchem), was used in the micro-reactor mold process for preparing the PDSM-E microreactors. When exposed to ultraviolet light, material may be removed via a wet etching process leaving high-definition features in micrometer dimensions. Additionally, a microreactor has been constructed in silicon onto which layer-bylayer self-assembled polyelectrolytes and enzymes are depos-ited. This system is being used for comparison with the PDMS-E system performance.
Scanning electron microscopy (SEM) images were taken with an AMRAY, 1800 series scanning electron microscope having a resolution of 0.2 µm. All objects in this work, except the silicon wafer microreactor, were first sputtered with a nickel layer a few nanometers thick in order to obtain an SEM image.
Microscale Bioreactor Studies263 Preparation of Biomaterial and Layer-by-Layer Self-Assembly The combination of PDMS and urease enzyme to form a microreactor from the resulting “bioplastic” material (PDMS-E) has been reported pre-viously (7). When enzyme concentrations were maintained at 2.5% (w/w) or less, the resulting microreactor cured with good structural integrity and high definition (e.g., well-formed microchannels and >90% retention of triangular transverse packing features in the microchannels).
For enzyme attachment to the silicon microreactor tested, a layer-by-layer technique was employed to build a multilayer system of polyions and enzyme. Deposition of multilayers was accomplished by alternating posi-tively and negatively charged layers of polydimethyldiallyl ammonium chloride (PDDA) and polystyrene sulfonate (PSS), respectively, to which was attached urease enzyme. After depositing in succession three layers of PDDA, PSS, and PDDA, three layers of urease enzyme were alternately deposited with three layers of PDDA. The resulting architecture is described as follows:
PDDA/PSS/PDDA + (UR/PDDA)3
Batch Studies for Evaluating Enzyme Immobilization
and Pdms-E Characteristics
To assess the enzymatic activity of the PDMS-E complex, “nonstruc-tural” PDMS preparations with various enzyme fractions were prepared and cured in glass petri dishes. On curing, these preparations were removed and cut into cubes nominally 3 mm on a side. Equal weights of these cubes (~10 g/reactor) were placed in 250-mL shake flasks. A 150-mL preparation of 0.1 mol/L THAM buffer solution containing 0.1 mol/L of urea was placed in each of three flasks. The pH of the buffer/urea medium was adjusted to 7.5 by the addition of HCl. Shake flasks were placed on a shaker incubator at
25°C and 200 rpm. Sample volumes of 2 mL were removed periodically for ammonia analysis. Additionally, swelling studies were conducted by periodically removing PDMS-E cubes from the urea solution, removing surface water, and weighing to assess the degree of water adsorption by the biopolymer (8).
Microreactor Fabrication
PDMS-E microreactor
The devices employed in this work were fabricated using silicon wa-fers as either microreactors or molds for the PDMS-E. The silicon wafers were treated by standard photolithographic techniques to form the desired features. Process steps to fabricate micromolds have been presented else-where (8). Figure 1 depicts a PDMS-E microreactor after curing on a silicon wafer mold. F igure 2 shows an SEM micrograph of a portion of the microchannel with triangular features fixed within the channel. PDMS-E microreactors were fabricated in 50-, 500-, and 1000-mm lengths to study
264Jones et al.
performance characteristics. Table 1 gives the various dimensions and operating conditions under study. Each microreactor enzyme loading was tested at various flow rates. Mean residence times were calculated taking
into account the volume occupied by the triangular mixing features.
Fig. 1. PDMS-E microreactor containing 1.7 wt% urease.
Fig. 2. SEM micrograph of portion of the microchannel with triangular features.
Microscale Bioreactor Studies
265
T a b l e  1D i m e n s i o n s  a n d  O p e r a t i n g  C o n d i t i o n s  o f  P D M S -E  M i c r o r e a c t o r
U r e a  F e e d  S o l u t i o n
C h a n n e l  w i d t h P e r  c h a n n e l  v o l u m e E n z y m e  “l o a d i n g ”F l o w  r a t e M e a n  r e s i d e n c e R e a c t o r
D e s c r i p t i o n
(µm )/l e n g t h  (m m )
(m m 3)/s u r f a c e  a r e a  (m m 2)g “E ”/g  P D M S (m L /m i n )t i m e  (m i n )
1a S i x  c h a n n e l s 500/502.67/48.30.250.0600.27(w i t h  t r i a n g u l a r 0.500.0062.67 t r a n s v e r s e  f e a t u r e s )0.75
0.001
16.01
2a S i x  c h a n n e l s 500/50027.7/484.8
0.250.0602.77(w i t h  t r i a n g u l a r 0.250.0237.28 t r a n s v e r s e  f e a t u r e s )
0.500.00627.650.50
0.001
165.56
3a S i x  c h a n n e l s 500/100055.4/970
0.250.0486.92(w i t h  t r i a n g u l a r 0.500.02314.56 t r a n s v e r s e  f e a t u r e s )
0.75
0.006
27.65

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