Supplementary Data

Appendix A: Model mechanism structure selection

The experimental data used for the initial model development step was obtained from µBR experiments performed in the commercial silicon and glass microfluidic platfrom presented in Figure 1. The oxygen measurements were performed using four optical fibres (placed in positions 1, 2, 4 and 5) connected to a four-channel optical oxygen meter (FireStingO2 from PyroScience, Aachen, Germany). The lifetime luminescent measurement was performed through Pyro Oxygen Logger software provided by PyroScience.

A solution of equal activities of glucose oxidase and catalase (20 U/mL) in PBS buffer (pH 7.2) was introduced through inlet 1, while different concentrations of glucose (between 5 and 100 mM) were introduced through inlet 2 at different flowrates (0.1 to 1 µL/s) in order to generate oxygen consumption data corresponding to different holdup times. Different amounts of catalase were added (20, 60 and 120 U/mL) through an inlet between sensors 3 and 4 (see Figure 1). The flowrate used to introduce extra catalase solution in the inlet 3 was equal to the flowrate values in inlet 1 and inlet 2.

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Table S1 – Kinetic parameters for mechanism AMechanism A

Kinetic Constant Initial Value Reference

k I [M−1 s−1] 13997 [20]

k II [M−1 s−1] 2.1×106 [20]

kV [s−1 ] 1152 [20]

Figure S1 – Mechanism A. Comparison of the concentration profiles for glucose, gluconic acid and oxygen inside the microreactor: simulation results (blue solid line) vs. experimental data (red dashed line).

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Table S2 – Kinetic parameters for mechanism B when catalase is assumed to be consumed during the reaction

Mechanism B*Kinetic

ConstantInitial Value

Optimized/Estimated Value

k I [M−1 s−1] 13997 1.2×1011

k II [M−1 s−1] 2.1×106 6.65×1011

k IV [s−1] 1150 1.59×105

kVI [M−1 s−1] 4.0×108 3.98×108

Figure S2 - Mechanism B*. Comparison of the simulation results (blue solid line) and concentration profiles (no addition of extra catalase) inside the microreactor (red dashed line).

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Figure S3 – Mechanism B*. Comparison of the concentration profiles for glucose, gluconic acid and oxygen inside the microreactor before and after addition of catalase: simulation results (blue solid line – before catalase; red dashed line - after) vs. experimental data (green dashed line).

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Table S3 – Kinetic parameters for mechanism B when catalase acts as a classic catalyst

Mechanism BKinetic

ConstantOptimized/

Estimated Valuek I [M

−1 s−1] 1.63×1011

k II [M−1 s−1] 4.68×1011

k IV [s−1] 1.59×105

kVI [M−1 s−1] 2.8×108

Figure S4 – Mechanism B. Comparison of the simulation results obtained for the concentration profiles inside the microreactor before (blue solid line) and after addition of catalase (red dashed line).

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Figure S5 – Mechanism B. Comparison of the concentration profiles for glucose, gluconic acid and oxygen inside the microreactor before and after addition of catalase: simulation results (blue solid line – before catalase; red dashed line - after) vs. experimental data (green dashed line).

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Table S4 – Kinetic parameters for mechanism CMechanism C

Kinetic Constant

Optimized/Estimated Value

k I [M−1 s−1] 8.2×10−6

k III [M−1 s−1] 7.0×10−4

Figure S6 – Mechanism C. Comparison of concentration profiles for glucose, gluconic acid and oxygen inside the microreactor: simulation results (solid line) vs. experimental data (dashed line).

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Table S5 – Kinetic parameters for mechanism DMechanism D

Kinetic Constant

Optimized/Estimated Value Comment

k I [M−1 s−1] 4.76×104 before

extra catalase

k III [M−1 s−1] 4.87

kVI [s−1] 4.2×108

k I [M−1 s−1] 5.63×104 after

extra catalase

k III [M−1 s−1] 2.61

kVI [s−1] 6.93×108

Figure S7 - Mechanism D. Comparison of the simulation results obtained for the concentration profiles inside the microreactor before (blue solid line) and after addition of catalase (red dashed line).

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Appendix B: Enzyme activity measurement in mL- and µL- scale reactors

Figure S8 – The absorbance curves recorded in the presence of catalase for 5 mM (A), 10 mM (B) and 20 mM (C) of hydrogen peroxide.

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Figure S9 - Dynamic response of the oxygen consumption rates, expressed as the number of moles of oxygen dissolved in a stock glucose solution: 2.5 mM (A), 10 mM (B) and 100 mM (C) of glucose.

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Figure S10- Dynamic response of the oxygen consumption rates, expressed as the number of moles of oxygen dissolved in the glucose/catalase solution. Experiments where the activity of catalase is equal to the activity of the glucose oxidase (A), 10 times higher than glucose oxidase (B) and 100 times higher than glucose oxidase (C), respectively, were compared for different glucose/glucose oxidase reaction velocities (0.402 U/mL, 0.304 U/mL and 0.245 U/mL).

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Figure S11 – The stable response of the oxygen consumption rates, expressed as the number of moles of dissolved oxygen measured by each sensor spot. The oxygen detection was studied for the catalase/glucose oxidase reaction in the presence of glucose (A). The addition of extra catalase (B) and hydrogen peroxide (C) solutions was performed inside the microchannel between sensor 3 and sensor 4. The numbers on the plots correspond to the sensor spot positions inside the microreactor shown in Figure 1.

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