Division of Electronics, Department of Technology, Uppsala University, P.O. Box 534, 751 21 Uppsala, Sweden.
(Received 25 May 1994; revised 4 October 1994; accepted 5 October 1994)
Abstract: Enzyme reactors were fabricated on silicon wafers using microstructur- ing technologies. The reactors were made of several parallel vertically-cut flow channels. The reactor structures occupied a wafer area of 3 * 15 mm. Reactors with two different channel densities were fabricated: 10 channels/ mm, 165 /xm deep; and 25 channels/mm, 235 /~m deep. Glucose oxidase was immobilised on the reactors and their corresponding enzyme activities were monitored by a colourimetric assay. It was shown that a reactor surface area increase of 3 times gave rise to a proportional enzyme activity increase in the reactor. The maximum glucose turnover rate for the reactor with 25 channels/ mm was approximately 35 nmol/minute and the corresponding apparent Km was approximately 17 raM. A wafer integrated enzyme reactor was also operated in a microdialysis-based system for continuous glucose monitoring, showing a linear response up to 4 mM glucose.
The development of microstructured chemical sensors is now generating increased interest. Some applications, e.g. solid-state pH-electrodes (Shindengen Co., Saitama, Japan) and H2 sensors (Sensitor AB, Link6ping, Sweden), have already
* Author to whom all correspondence should be addressed.
found commercial markets. Interest in micro- scaling and in implementat ion of existing macro- scale chemical instruments on silicon wafers is evidently growing rapidly (Arquint et al., 1994).
Several of the flow system components needed for the microstructuring of chemical analysis systems on silicon wafers are currently available. Micro-channels may easily be etched in silicon, and flow directing valves have been developed (Shoji et al., 1991), as well as wafer integrated micro-pumps (Bart et al., 1993) and precisely
defined cavities for sample injection (Jansson et al. , 1992).
For analysis of more complex molecules, enzyme reactors are required. These are an essential component of wafer integrated chemical analyzers for clinical applications, but have not yet received much attention. Suda et al. (1992) have described a silicon integrated enzyme reac- tor, incorporating a long v-groove channel as the enzyme coupling surface. Xie et al. (1992) described a wafer integrated reactor with 33 parallel v-grooves.
In this paper, the performance of a wafer integrated enzyme reactor with 30 parallel chan- nels, 165 tzm deep, and a channel density of 10/ mm (Laurell and Rosengren, 1994) is compared with that of a new reactor design, comprising 75 flow channels, 235 /xm deep, with a channel density of 25/mm, anisotropically etched into a < l l 0 > - o r i e n t e d silicon wafer. An untreated reactor was tested against a reactor submitted to a dedicated oxidation procedure, in order to establish whether the native silicon dioxide at the reactor surface would to serve as an adequate coupling site for enzyme immobilisation, or if an additional oxide layer would increase the amount of enzyme in the reactor.
2. M A T E R I A L S AND M E T H O D S
Microstructuring a flow-through cell in silicon, to create an enzyme reactor, aims to achieve a large surface area for enzyme immobilisation. The formation of micro-channels on a small wafer area has a surface enlarging effect. If equipped with flow connections, the structure can be used as a flow-through enzyme reactor. Either vertical channels or v-grooves can serve as micro-chan- nels; both are easily fabricated in silicon.
2.1. Reactor design
By using < l l 0 > - o r i e n t e d silicon, vertical walls can be fabricated in a wafer and thus a greater surface enlargement will be achieved than in a <100> wafer with a parallel v-grooved structure (Suda et a l . , 1993). Table 1 demonstrates the gain in geometrical properties with 250/xm deep vertical grooves, at a spacing equal to the channel width, over densely-spaced v-grooves, expressed in terms of the reactor sur face~wafer sur face ratio. As the channel frequency rises, the surface of
the vertical structure increases proportionally, while the surface of the v-groove structure remains unaffected.
Two versions of the enzyme reactor (named "50 tzm reactor" and "20 txm reactor") were manufactured, offering different enzyme immo- bilisation surface areas. The 50 tzm reactor photo mask comprised 30 lines to form the channels, 50 /xm wide, 13 mm long and at intervals of 50 /xm. The 20/zm reactor photo mask incorporated 75 parallel lines to form the channels, 20 /xm wide, 13 mm long and with 20 tzm spacing. Figure 1 illustrates the surface enlarging effect as the channel frequency is increased and the channels are etched deeper.
From the rear side of the wafer a flow inlet and a flow outlet were etched into the entrance and exit basins of the enzyme reactor. Figure 2 gives a schematic top view of the reactor structure. The grey areas represent the wafer surface; the white areas represent the flow channels and the entrance and exit basins; the flow inlet and outlet, etched through the wafer from the rear, are shown in black.
2.2. Reactor fabrication
Anisotropic wet etching of silicon in K O H features a strongly direction-dependent etch rate. The {111} planes of the crystal yield the lowest etch rate, while the {110} planes display a considerably higher rate. This rate difference can be a factor of over 400, depending on the etch conditions (Kendall, 1975).
In a < l l 0 > - o r i e n t e d silicon wafer, the {111} planes run perpendicular to the wafer surface. A wafer with an etch mask aligned to these planes will, when exposed to an anisotropic wet etchant, produce vertical channels (c.f. Fig. 8).
An oxidised <110> wafer was used to fabricate the reactor structure, where the silicon dioxide was used as the masking layer during etching. The flow inlet and outlet were etched from the rear side, while the flow channels and the entrance and exit basins were simultaneously etched from the front side. The etching was performed in K O H solution (70 g/100 ml H20 ) at 80°C, giving an etch rate of 1.7/xm/min in the <110> direction.
Initially an anisotropic etch step was required to determine the precise orientation of the { 111 } planes, to enable the alignment of the photo masks during reactor structuring. If the masks
TABLE 1 Comparison of the reactor surface~wafer surface ratio for v-groove and vertical channel structures. The vertical channels are assumed to be 250 txm deep.
Fig. 1. Cross-section view of the two reactor designs, illustrating the gain in reactor surface through increasing
the etch depth and lamella frequency.
II
were poorly aligned to the {111} planes, sideways etching could occur, interfering with the forma- tion of a parallel lamella structure and causing a loss of valuable reactor surface.
The reactor was sealed with a Pyrex glass lid, completing the flow-through cell. The Pyrex glass was bonded anodically to the wafer surface (Gustavsson, 1988) (Fig. 3). A glass/silicon bond was formed when the structure was heated to approximately 450°C, and 1000 V applied across the glass/silicon interface. The choice of glass as the sealing material enabled visual inspection of the reactor status during operation.
Two versions of the 20 /xm reactor were fabricated: one batch was made according to the procedure above; the other also underwent a thermal oxidation process, growing a 280 .~ thick oxide layer, before bonding the glass lid.
An adapter for connection of the enzyme reactors to the flow-through system was fabricated in PVDF (Fig. 4). A reactor was placed in the adapter so that the two O-rings covered the flow inlet and outlet. Two different adapters with connecting tubing were used: one during the
II
Fig. 2. Schematic top view of the reactor structure. The grey areas represent the wafer surface, the white areas represent the channels, and the flow inlet and outlet are shown in black.
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1 ~l~l'~Xz 7
Fig. 3. Anodic bonding o f the glass lid to the reactor surface. The heater was set to 450°C. A t a voltage o f approximately +1000 V across the silicon structure, the
two surfaces were forced into a glass/Si bond.
immobilisation procedure and one during reactor testing, to ensure that only enzyme bonded to the silicon structure was studied. An opening was cut in the top cover of the adapter to enable visual inspection of the reactor during operation.
2.3. Enzyme immobilisation
Standard methods for immobilising an enzyme on silica gel use silicon dioxide as the coupling site on the surface of the support matrix (Weetall, 1976). Similarly, the surface layer of silicon dioxide in the fabricated wafer integrated reactors
Biosensors & Bioelectronics
may be used to couple an enzyme (Laurell et al., 1994). Heating during anodic bonding inevitably creates a thin dioxide layer on the silicon surface. In order to verify that this native silicon dioxide was sufficient for enzyme immobilisation, another reactor was submitted, for comparison, to a dedicated oxidation pro- cedure to form a thick oxide layer for the enzyme coupling.
Each reactor was silanised with 3-aminopropyl- triethoxysilane (Sigma Chemical Co., St Louis, USA) and 1 g APTES in 9 ml of water; pH was adjusted to 3.5 with 6 M HC1. The APTES was pumped through the reactors (flow rate 36 /zl/ min) for 3 hours, followed by thorough washing with water (flow rate 100 /zl/min) for 30 min. Silanisation was performed in a water bath at 75°C. A 2.5% glutaraldehyde (GA) solution (grade II; Sigma Chemical Co., St Louis, USA) in 0.1 M phosphate buffer (PBS) was subsequently pumped through the reactors for 4 hours at a flow rate of 36 p~l/min. GA activation was followed by washing with PBS for over one hour (flow rate 36 /xl/min). Finally, glucose oxidase (GOD) coupling, using 10 mg glucose oxidase (EC 1.1.3.4, type X-S, Aspergi l lus niger) in 2 ml PBS, was performed for two hours, (flow rate 7 /.d/min). Reflection light microscope inspection of the reactor after enzyme immobilisation showed that the silicon surface had a weak orange colour, indicating that protein had coupled to the surface; c.f. the brick red colour observed when enzyme is immobilised on controlled pore glass (Danielsson, 1979).
Side view Silicon wafer
i Entrance and exit of f low
Top view
Hole for visual inspection of the reactor.
Fig. 4. A PVDF adapter provided f low connections to the reactor. An opening was cut in the top cover to enable visual inspection o f the reactor during operation.
The enzyme activity in the reactors was investi- gated using a spectrophotometrical assay, the Trinder reagent, in line with an HPLC absorbance detector (Waters 486 Tunable Absorbance Detec- tor; Millipore Corp., Milford, MA, USA). The Trinder reagent (von Gallati, 1977) yields an absorbance change proportional to the change in H202 concentration (Fig. 5). GOD-catalysed conversion of glucose produces H202 in pro- portion to the glucose concentration. The Trinder reagent could thus be used to determine indirectly the glucose oxidase turnover rate, provided that the peroxidase activity in the reagent solution exceeded the GOD activity.
Trinder reagent solutions were prepared: 25 mM phenol (pro analysi; Merck, Darmstadt, Germany) and 2 mM 4-Amino-2,3-dimethyl- 1-phenyl-3-pyrazolin-5-on (pro analysi; Merck, Darmstadt, Germany). Glucose was added to the reagent solutions to give preparations with glucose concentrations of 2, 5, 10 and 50 mM. Approximately 100 units of peroxidase (EC 1.11.1.7, type VI, from horseradish; Sigma Chemical Co., St Louis, USA) was subsequently added to a reagent volume of 5 ml for each glucose concentration.
The reagent solution was pumped either through the reactor, measuring the absorbance of the developed colour compound at 492 nm,
or past the reactor to provide the zero absorbance level, as shown in Fig. 6.
2.5. Microdialysis system
The reactor performance was also tested in a microdialysis-based glucose monitoring system (Laurell, 1992). A microdialysis probe, perfused by a stepping motor-controlled syringe pump, continuously sampled glucose for the enzyme reactor (Fig. 7). The reactor converted glucose to gluconic acid and H202 according to the following equation.
GOD Glucose + 02 < = = > Gluconic acid + n202
(a)
Consequently, any change in glucose concen- tration produced a corresponding change in the dissolved oxygen concentration, which was monitored by an oxygen electrode. The electrode signal was amplified by a Keithley 428 picoamperemeter and sampled by a computer.
During all operations when reagent solutions, immobilising agents, or microdialysis samples were pumped through the reactor, a micropore filter (Gelman Acrodisc LC 13 0.45 /xm) was placed at the pump flow exit, to prevent particles from catching in the reactor and obstructing the liquid flow. The filter was replaced after each step in the enzyme immobilising procedure.
4-Amino-antipyrin Phenol
2H202 + ? +
HO
C H ~ ~ N H 2
Peroxidase
Chinonimin colour compound
~ N + 4H20
c%_r z C H ~ ~ N +
O
Fig. 5. The Trinder reagent produces a coloured compound in proportion to the concentration of H202. Here the reagent is used to monitor H202 produced by the GOD-catalysed conversion of glucose.
Fig. 6. By pumping the Trinder reagent past or through the reactor, the enzyme activity could be estimated using a Waters 486 Tunable Absorbance Detector.
3. RESULTS
3.1. Reactor fabrication
The achieved geometrical properties of the enzyme reactors, (Table 2) did not fully agree with the dimensions predicted by the layout of the computer-generated photo mask. This was caused by a non-uniform transfer of the mask pattern throughout the mask fabrication and the wafer patterning processes. Also, in the definition of the lamella width, a slight mask misalignment gave rise to a loss in lamella width. This was especially pronounced in the case of the 20 txm reactor (Fig. 8). Furthermore, the channel length was shortened slightly during etching due to the fact that the end sides of the lamellae exposed crystal planes which display a high etch rate (Fig. 9).
3.2. Enzyme activity determination
The enzyme activities in the reactors were determined by pumping through glucose contain- ing Trinder reagent at various flow rates, produc- ing the absorbance recordings shown in Fig.
10. The absorbance change, AA, found when switching the reagent flow through and past the reactor structure, gave a measurement of the concentration, AC, of the produced coloured compound, according to the Lambert/Beer Law,
AA A C - lc (2)
where z~A is absorbance change, AC is the concentration change [mM], l is optical path [cm], (1 cm) and e denotes the molar absorption coefficient [cm2/txmol]: e for the chinonimin colour compound is 9.0 [cm2//~mol] at 492 nm.
The actual glucose turnover rate, Ve, was determined by multiplying each calculated AC by the flow rate (eq. (3)) thus giving the colour compound production in mmol/min, which in turn yielded the H202 production from the GOD- catalysed glucose conversion. The factor of two accounts for the fact that two molecules of H202 are required to produce one molecule of colour compound:
Fig. 7. The reactor was operated in a microdialysis-based system for continuous glucose monitoring.
T A B L E 2 Achieved geometr ical propert ies of the two reactor structures. Dead volume estimates were made for channel widths of 33 Ixm in the 20 Ixm
reactor and 50 p,m in the 50 p,m reactor.
50 ~m 20 p,m reactor reactor
Entrance + exit basin area 14 mm 2 17 mm 2 Channel depth 165 p,m 235 o,m Channel width 49-50 p,m 32-33 ism Lamella width 48-52 ~m 3-11 o,m Lamella length 12.7 mm 12.4 mm Lamella structure area 164 mm 2 498 mm 2 Total reactor area 178 mm e 515 mm 2 Dead volume in reactor 4.3 mm 3 9.0 mm 3 Loss of lamella length 0.3 mm 0.6 mm
ra t e [ l /mini a n d AC is c o n c e n t r a t i o n c h a n g e
[mM] . T o e n s u r e that t h e T r i n d e r r e a c t i o n was a l l o w e d
to d e v e l o p fu l ly , b e f o r e e n t e r i n g t h e f l o w - t h r o u g h
c u v e t t e , a d e l a y l ine was i n t r o d u c e d b e t w e e n t h e
# !
) 8
Fig. 8. Anisotropic etching of <110> silicon can prod- uce deep channels separated by standing walls, forming a surface enlarging structure. The SEM image gives a lengthwise view of the lamella structure of the 20 ton reactor. The varying lamella width was caused by a non-uniform photo mask and a slight misalignment of
the mask to the {111} planes.
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Fig. 9. Side view of the flow entrance basin in the 20 izrn reactor, showing the ends of 235 Izrn deep flow channels. The loss of lamella length is indicated by the triangular remains at the bottom of the reactor, in front
of each lamella.
1,0- 62.5 ~l/min 37.5 ~tl/min
• "~ 0,6-
0,4- A
0,2-
0'0 I ' I I I ' I I l I . . . . I . . . . I . . . . l . . . . I . . . . I ' r ' '
0 5 10 15 20 25 30 35 40 Time [min]
Fig. 10. Absorbance recording for 2 mM glucose in Trinder reagent pumped successively through and past the 20 ixrn reactor at flow rates of 62.5 and 37.5 p.l/ rain. When the flow bypassed the reactor, the volume in the reactor developed a deeper colour owing to a further progressed enzymatic glucose breakdown: an absorbance peak therefore appeared each time the flow
was resumed through the reactor.
reactor outlet and the absorbance detector inlet. No change in AA was found and so the shorter connection line was used thereafter.
Next, Trinder reagent preparations with differ- ent glucose concentrations were pumped through each reactor. The Ve values were calculated and plotted versus glucose concentration. Ve vs.
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glucose concentration for the 20/xm, oxidised 20 /xm and 50/xm reactors is shown in Fig. 11. The equation of the curve matches the following theoretical expression, for an enzyme activity recording (Cantor & Schimmel, 1980). The coef- ficients of the curve fit gave the apparent Km and Vmax values for each reactor structure:
Vm x Ve - 1 + K m (4)
So
where Ve is turnover rate of the enzyme re- actor [mmol/min], So is substrate concentration [mM], Vmax is maximum enzyme turnover rate [mmol/min] and Km is substrate concentration at Vmax/2 [mM].
Linear regression of a reciprocal plot of glucose turnover rate versus glucose concentration, a Lineweaver Burk plot, also gave the Vm~, and the apparent Km parameters, where 1/Vma~ was the y-axis intercept and - 1~Kin the x-axis intercept (Fig. 12). If these two Vma~ and apparent Km estimates from Figs. 11 and 12 did not differ significantly, they could be considered a fair estimate of the reactor enzyme activities (Cantor & Schimmel, 1980).
The apparent Km and Vm,~ values given by the two plots are presented in Table 3. The V,,a~ estimation from the Ve VS. So plot (Fig. 11) gave a maximum turnover rate 3.3 times higher in the 20 /xm reactor than that in the 50 /xm reactor (3.8 times according to the Lineweaver Burk plot, Fig. 12); comparing the maximum turnover
3 0 , 0 , , , , I , , , , I , , i , I . . . . I , , L , I , , ~ ,
20.0- 15,0- ~ Y =32'7/(1+17"4/x)
> 10,0- A
. . . . . . . . . . . . . ~ - . . . . 5,0-
y = 10,1/(l+ll.7/x) A"
0 , 0 . . . . I . . . . I . . . . I . . . . I . . . . I . . . .
0 10 20 30 40 50 60 Glucose conc. [mM]
Fig. 11. Curve fit of the GOD turnover rate, Ve, versus glucose concentration for 20 p~m (-+-), oxidised 20 ~ (-o-) and 50 jxrn (-A-) reactors. The Vmax and apparent Km coefficients were estimated from the curve for each
t l l l l l l l l l l l t l l l l l l l l , i l l J J l l
A, js't~
y = 0,0976 + 1,18x R= 0,998
! 1 . "1"
• . I y = 0,0279 + 0,561x R= 0,9996
~ = 0,9977
0 ' 0 . . . . I . . . . I ' l ' t . . . . I . . . . I g ' i '
0 0,1 0,2 0,3 0,4 0,5 0,6
Glucoseconc -1 [mM "1 ]
Fig. 12. A reciprocal plot of glucose turnover rate versus glucose concentration also yielded the Vmax and apparent Km parameters. The performance of the 20 txm (-+-), oxidised 20 txm (-o-) and 50 txm (-zl-)
reactors are plotted.
25
<=
15- l"
¢J
10- 2.4 (10)
5- 4.8 (20) tVV~
9.6 (40) 19.2 }80) 24 (100)
0 50 100 150 Time [min]
Fig. 13. The 20 gm reactor was operated in a system for microdialysis-based glucose sampling. The oxygen electrode current was monitored as the dialysis probe was exposed to glucose solutions of 5, 1 0 , 2 0 , 40, 80, 100, 2 and 4 rnM. The oscillations in the curve were
caused by pump oscillations.
TABLE 3 Apparent Km and Vm~x for the three enzyme reactors investigated (20 wm oxidised 20 v,m and 50 txm), estimated from a Ve vs. So plot and a Lineweaver Burk plot.
Reactor apparent K m Vma x
Ve VS. So Lineweaver Burk Ve vs. So Lineweaver Burk
rates of the oxidised 20 /xm reactor and the 20 /,~m reactor, the ratio was 1.0 (0.9 by estimates based on the Lineweaver Burk plot).
3.3. Microdialys is -based glucose moni tor ing
The reactor was connected to a microdialysis- based glucose sampling system. A Clark-type oxygen electrode was used to measure the changes in dissolved oxygen in the flow system as the glucose concentration around the microdialysis probe was varied. Figure 13 illustrates the system response as the probe was immersed sequentially in glucose solutions of 5, 10, 20, 40, 80, 100, 2 and 4 mM (note that dialysis has a diluting effect on the sample). The numbers in parenthesis in the figure indicate the bulk g lucose concentrat ion around the microdialysis fibre. The corresponding calibration plot for the measurement system is shown in Fig. 14. At a flow rate of 25 txl/min
20 , , , , I . . . . i , , , , i . . . . i L i i t
o
o
o 1 0 -
-~ ',
"o.
" - - " . . . . . . . . . . o - . . . . . . . o
0 , , , , t . . . . , , . . . . . . f , , , ,
0 5 ll0 115 20 25
Glucose concentration [mM]
Fig. 14. Calibration plot (oxygen electrode current vs. glucose concentration in the reactor), for the reactor response during microdialysis-based glucose monitor- ing. At approximately 10 mM glucose, the oxygen electrode reaches its minimum level as all oxygen is
consumed by GOD-catalysed glucose conversion.
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T. Laurell et al. Biosensors & Bioelectronics
and with a dialysis probe length of 30 mm, the glucose dialysis factor is 24% (Laurell, 1992). Therefore, the glucose concentrations given on the x-axis of Fig. 14 are the concentrations in the sample flow following the microdialysis step.
4. DISCUSSION
Increasing the lamella density from 10/mm to 25/ mm, and accounting for the increased etch depth of the 20 /xm reactor, a total area enlargement of 2.9 times was achieved compared to the 50 tzm reactor. The area enlargement should yield a corresponding enzyme activity increase. The curve fits in Figs. 11 and 12 suggest a rise in maximum substrate turnover rate of between 3.2 and 3.8 times, which indicates that the surface enlarging strategy is successful.
No significant difference in Vmax was found between the 20 ~tm and oxidised 20/xm reactors, indicating that the native silicon dioxide produced during anodic bonding was sufficient for the enzyme coupling.
It should be noted that the increase in estimated enzyme activity exceeded that expected from a direct reactor area comparison. This can be partially explained by the hypothesis that the enzyme layer at the lamella surface was better utilised than the layer in the entrance and exit basins, owing to differences in flow distribution. If the enzyme activity contribution from the basins were excluded from the calculations, the area comparison between the 20/xm and the 50 p~m reactors would concern the lamella region only, which is 500 mm 2 vs. 164 mmZ: a surface 3.0 times larger in the 20 /.~m reactor. This corresponds better to the empirical value of the enzyme activity increase, i.e. a factor of 3.2-3.8.
In comparison with the 20/.~m reactor, a plain flow-through cell, 235 /zm deep, without the lamella structure, would offer an area of 49 mm 2, which is only about 10% of the 20 /zm reactor area. On the other hand, an enzyme reactor 235 /xm deep, 3 mm wide and 15 mm long, with a total reactor volume of 10.6 /.d and based on a CPG glass gel (CPG density 3.16 ml/g, CPG coupling area 51.4 m2/g) would offer an area for enzyme immobilisation of approximately 172 000 mm 2, which is 330 times that of the 20 /zm reactor. It should, however, be pointed out that not all of the surface in the glass gel can be efficiently used, owing to substrate transport
298
limiting effects in the microporous glass grains, and thus an enzyme activity ratio below 330 should be expected.
The wafer integrated reactors fabricated so far hold much less enzyme than comparable reactors filled with enzyme-carrying CPG. In spite of the lower enzyme activity, the reduced dimensions of the wafer integrated reactor require smaller amounts of enzyme, owing to the low sample volumes and low flow rates. This implies that wafer integrated enzyme reactors have the poten- tial for application in chemical analysis systems based on microsampling techniques, such as microdialysis or flow injection analysis systems.
Since GOD activity is dependent on the presence of oxygen, the maximum glucose con- centration in the reactor is limited to the concen- tration of dissolved oxygen, if complete glucose conversion is to be achieved. For monitoring clinical glucose levels (0-20 mM), dilution of the sample before measurement would therefore be desirable. In room temperature physiological saline solution, the dissolved oxygen concen- tration is 0.26 mM (8.2 mg/1) (Hitchman, 1978). Consequently, for a glucose concentration in the reactor of 0.26 mM and a flow rate of 10 /xl/ min, the enzyme reactor must be able to convert 2.6 nmol glucose per minute. In the present state, the 20 /xm reactors have an estimated turnover rate of approximately 0.5 nmol/minute at a glucose concentration of 0.26 mM, according to Figs. 11 and 12. This indicates that the enzyme activity in the reactors requires an approximately five-fold increase to achieve complete glucose conversion. It seems reasonable to expect that future versions of the reactor will meet and exceed this goal.
In the microdialysis-based system for continu- ous glucose withdrawal (Figs. 13 and 14) the reactors responded linearly up to 4 mM glucose. The linear response was limited by the amount of dissolved oxygen, and an increased linearity could be achieved by diluting the sample before introduction to the reactor. It should, however, be noted that since the reactor does not hold an excess of enzyme, it may display a long-term drift, caused by the gradual loss in enzyme activity through the denaturing effect of enzymatically produced H202. This effect is typical of enzyme- based systems.
We suggest that <110> silicon and anisotropic etching, providing vertically-cut flow channels, is used when surface enlarging structures are required in wafer integrated enzyme reactor applications.
Further increasing the reactor surface area, through a reduced lamella width and deeper etching, may yield a structure with an enzyme activity that exceeds the requirements for com- plete glucose conversion. The reactor will then display a stable response to glucose and will be unaffected by enzyme activity loss during operation.
Microdialysis provides a continuous sample flow at very low flow rates, in the/zl/min-range, and is therefore well suited to miniaturised wafer integrated analysis systems.
Microstructuring technology promises to con- tribute significantly to the ongoing efforts to miniaturise chemical analysis components. Most reports so far have focused on micro-scaling of the sensor elements, and several chemical micro- sensors, such as pH electrodes and oxygen electrodes, are now commercially available. The next task is to integrate flow channels, valves, pumps, enzyme reactors, sample inlets etc. together with the sensing elements in micro- scale, to achieve a totally integrated analysis system. Some reasons for the drive towards the micro-scaled systems are:
* large-volume batch fabrication, offering cheap and replaceable components;
* low operational cost due to low consumption of chemicals and analytes;
* short signal lag and rise times; * small units with low power consumption.
The development of silicon integrated enzyme reactors is a further step towards a complete wafer integrated chemical multi-analysis system. Our future efforts will focus on improving the enzyme activity, to open the way to reduced reactor size and prolonged, stable reactor oper- ation.
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