Continuous Flow Reactors from Microfluidic Compartmentalization of Enzymes within Inorganic Microparticles

Compartmentalization and selective transport of molecular species are key aspects of chemical transformations inside the cell. In an artificial setting, the immobilization of a wide range of enzymes onto surfaces is commonly used for controlling their functionality but such approaches can restrict their efficacy and expose them to degrading environmental conditions, thus reducing their activity. Here, we employ an approach based on droplet microfluidics to generate enzyme-containing microparticles that feature an inorganic silica shell that forms a semipermeable barrier. We show that this porous shell permits selective diffusion of the substrate and product while protecting the enzymes from degradation by proteinases and maintaining their functionality over multiple reaction cycles. We illustrate the power of this approach by synthesizing microparticles that can be employed to detect glucose levels through simultaneous encapsulation of two distinct enzymes that form a controlled reaction cascade. These results demonstrate a robust, accessible, and modular approach for the formation of microparticles containing active but protected enzymes for molecular sensing applications and potential novel diagnostic platforms.


Materials
Fluorescence recovery after photobleaching (FRAP) of Cy5-R5 peptide in silica microparticle Figure S3: FRAP experiment. Fluorescence recovery after photobleaching (FRAP) experiment was conducted for silica microparticle formed using 1080 mM silicic acid and 1 mg/ml Cy5 labelled R5 peptide. Before the FRAP R5 fluorescence is even throughout the microparticle (i).
After photobleaching an area from the microparticle (ii) dark spot appeared. The black spot was evident even 3 min after the photobleaching (iii), confirming that the centre of microparticle has also a solid matrix. Figure S4: Encapsulation efficiency (EE%). EE% of BSA-FITC and FITC into silica microparticles.

Encapsulation efficiency (EE%) of FITC-BSA and FITC
Encapsulation efficiency for all used silicic acid conditions were measured ( Fig S4) Figure S5: Operation of microfluidic reactor.

Operation of microfluidic reactor
Step required for filling and operation of the microfluidic reactor with silica microparticles.

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Microfluidic reactor (Fig. S1) with substrate inlet, microparticle inlet and product outlet were designed with AutoCAD so that microparticles could be immobilised and tightly packed. This was possible by adding 25 µm filters, which allow liquid flow through the reactor while keeping the 100 µm sized microparticles inside. The dimensions of the reactor are: 2 mm width and 5.9 mm length.
Microparticles were packed while in oil and the de-emulsification was done inside the device. First the device was filled with oil keeping all the inlets and outlet open. Next, the substrate inlet was closed placing a steel pin inside the inlet followed by insertion of microparticles (in oil). After the whole reactor volume was full of microparticles, the microparticle inlet was closed and 10 % PFO solution was flown through the device from substrate outlet approximately for 5 min. Now, the oil could be replaced by flushing with water approximately 20 min or until no oil was evident. Finally, the substrate flow could be started and experiment started.

Reaction time calculations of microfluidic reactor for one enzyme β-gal and two enzyme GOx/HRP systems
Microfluidic reactor with volume of 1.40E −09 m 3 was used to pack enzyme silica micro particles into a tight array. In order to obtain kinetic information (Km) of the encapsulated enzymes, calculations of reaction times were conducted. This reaction time can be then used calculate to reaction rate v (µM/min) knowing the amount of product (resorufin) at the detection region of the device. This was done transforming the measured product fluorescence (resorufin in both systems) to a concentration (µM) by using a calibration curve measured with free resorufin (Fig. S7).
The substrate has time to be transformed to the product in the reactor depending on the used flow.
The calculated reaction times (s) are summarised in Table S1. For quantifying the Michelis-Menten kinetics of our device 500 µL/h flow rate was chosen for β -gal and 31 µL/h for GOx/HRP system.
These flow rates were chosen from yield data ( Fig. S6b and 6e), because both values lie in the region where the increase of yield seems to be linear against the flow rate/time. Bulk reaction rate of two enzyme GOx/HRP system in bulk Figure S6: Michaelis-Menten kinetics of GOx/HRP system free and encapsulated into microparticles.

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Encapsulating GOx/HRP system into the microparticles has less negative effect in the bulk effect on enzyme kinetics compared to the single enzyme, β-gal, system. This is due to the fact that it is beneficial to have the enzymes involved in the cascade on close proximity. However, the substrate has to still travel from bulk into the microparticle, which contributes to the slower reaction rate.

Microfluidic reactor fluorescence intensity
The product fluorescence in function of substrate concentration shows linear response in both systems examined (Fig. 4). The intensity was monitored from the end of reactor device at the detection area (see article Fig. 5). Background intensity with buffer was measured for each device and subtracted.