Lab-on-a-chip microreactors with immobilized enzymes.
In this chapter, a novel microfluidic device (MagneChip) is described which comprises microliter volume reaction chambers filled with magnetically fixed enzyme-coated magnetic nanoparticles (ecMNPs) and with an in-line UV detector. In the experiments, MNPs with phenylalanine ammonia-lyase (PAL)—an enzyme which catalyzes the deamination of l-phenylalanine (Phe) to (E)-cinnamate in many organisms—immobilized on the surface were applied as biocatalyst to study the characteristics of the MagneChip device. In the reaction chambers of this microfluidic device, the accurate in situ quantization of the entrapped MNPs was possible using a resonant coil magnetometer integrated below the chambers. Computational fluid dynamics (CFD) calculations were used to simulate the flow field in the chambers. The enzyme-catalyzed biotransformations could be performed in the chip with excellent reproducibility and of repeatability. The platform enabled fully automatic multiparameter measurements with a single biocatalyst loading of about 1 mg PAL-ecMNP in the chip. A study on the effect of particle size and arrangement on the catalytic activity revealed that the mass of ecMNPs fixed in the chamber is independent of the particle diameter. Decreasing the particle size resulted in increasing catalytic activity due to the increased area to volume ratio. A binary mixture of particles with two different particle sizes could increase the entrapped particle mass and further the catalytic activity compared to the best uniform packing. The platform enabled a study of biotransformation of l-phenylalanine and five unnatural substrates by consecutive reactions using same PAL-ecMNP loading. With the aid of the platform, we first demonstrated that PAL can catalyze the ammonia elimination from the noncyclic propargylglycine as substrate.
- Magnetic nanoparticles
- Magnetic chip reactor
- Microfluidic reactor
- Enzyme reaction
- Phenylalanine ammonia-lyase
Microreactors are usually defined as miniaturized reaction systems fabricated using methods of microtechnology and precision engineering. The term “microreactor” is the proposed name for a wide range of devices, having typically submillimeter channel dimensions which can be further divided into submicron sized components, for example, microparticle and nanoparticle carriers .
Before evolution of microreactor technology, the traditional way to conduct solution phase synthesis and analysis was the batch mode in stationary reactors with stirring or shaking to mix the reactants. Nowadays, microstructured devices offer greatly enhanced performance compared with conventional batch systems due to effects arising from the microscale domain:
Batch processes are space-resolved; therefore, the process must be readjusted in each demand for larger product quantities. In contrast, flow microreactor processes are time-resolved; therefore, the output of the reaction is determined by the flow rate and the operation time, and no further optimization is needed. This also leads to accelerated process development and enhanced safety due to smaller reactor volumes .
Microreactors with high surface-to-volume ratio (SVR) are able to absorb the heat evolving in an exothermic reaction more efficiently than any batch reactor. Therefore, the temperature distribution inside the microreactor is homogenous in the whole volume. In contrast, small SVR usually leads to uneven temperature distribution in large-scale batch reactors, decreasing the product yield .
Mixing quality is crucial for many reactions, where the molar ratio between the reactants needs to be controlled precisely. Short diffusion paths provide efficient mixing in microreactors, which overrides the achievable mixing efficiency of batch reactors .
In biocatalytic applications, the efficiency of the microreactor can be further improved by immobilization of enzymes on nanoscale carriers accommodating in the reactor. Reusability of the biocatalyst makes the process economical and more environmentally friendly.
To perform similar analyses in shorter timescale even in parallel is an anticipated objective for screening and routine use in protein and enzyme research . A desirable goal is the high throughput screening of enzymes and their substrates and inhibitors. The prospective fields of application of microreactors are quite wide and include biotechnology, as well as combinatorial chemistry and enzyme-targeted drug discovery .
Analytical systems which comprise microreactors are characterized by outstanding repeatability and reproducibility, due to replacing iterative steps in batch and discrete sample treatment by flow injection systems . Benefitting from system automation, this also eliminates errors associated with manual protocols.
Small reagent volume is also a benefit of microreactors enabling economical and efficient screening of novel reaction paths and substrates.
Microreactors have a high potential in industry, as developments by microreactors can be faster transferred into production at lower costs than batch processes.
Despite of the rapid development of enzymatic microreactors in the recent decade, important design questions still need to be answered.
Reaction kinetics is a key parameter of device design. Widely used kinetic parameters are deduced from the Michaelis–Menten model, which is valid only in batch reactions. In flow systems, the flow effects should be also considered. Immobilization of the enzymes causes further complication in modeling. Immobilization may affect the intrinsic kinetic parameters and may influence the availability of the enzyme. The kinetic model should also consider that the liquid phase containing the substrate and product is moving compared to the solid phase containing the immobilized enzyme.
When supported catalyst-filled microreactors are used, reproducible filling of the supported catalyst into the reactor space is not always straightforward. Even more challenging is the quantification of the actual load of the carriers.
Long-term stability of the reactor and the reproducibility of the measurements may be affected—among other factors—by the flow rate, the substrate concentration, and the morphology of the immobilized biocatalyst.
This chapter presents results carried out by a microfluidic microreactor system, the so called MagneChip platform including four serial reaction chambers with individually removable permanent magnets. The results were achieved by experiments using phenylalanine ammonia-lyase (PAL) from Petroselinum crispum immobilized on the surface of magnetic nanoparticles (MNPs) and filled into one or more chamber of the MagneChip.
Biotransformations with PAL under different conditions were performed mostly using the natural substrate
the reproducibility of biotransformation in the microreactor system,
the effects of the long-term use and cyclic reuse of the biocatalyst on the biocatalytic activity,
the effects of the particle size on the biocatalytic activity,
the optimal substrate concentration and flow rate of the in-chip biotransformation,
the effect of immobilization and the use of flow microreactor on the kinetic constants, and
the biotransformations of further substrates with PAL.
Analytical systems which comprise microreactors are characterized by outstanding repeatability and reproducibility, due to replacing batch iterative steps and discrete sample treatment by flow injection systems . The possibility of performing similar analyses in parallel is an attractive feature for screening and routine use . Microreactors have been integrated into automated analytical systems, as well as providing benefits from system automation, and this also eliminates errors associated with manual protocols .
Applications of microreactors can be divided into three classes :
Organic synthesis, when a target molecule is formed from components in flow
Analytical use of biocatalysts to transform an analyte difficult to measure to an easy to measure form
Screening of substrates and enzymes examines their kinetic characteristics
Microreactor systems can be further divided into classes based on the physical localization of the catalyst:
Laminar flow reactors: The majority of the commercial flow synthesis systems utilize laminar flow with soluble components and enzymes . Losing the catalyst is a major drawback of this technique.
Filled reactors: Microreactors utilizing immobilized catalysts (e.g., enzymes) have many advantages over the traditional flow reactors. First of all, the catalyst (enzyme) can be recycled after usage; therefore, the process is more economical and environmental friendly. The reactions are highly reproducible as the catalyst (enzyme) concentration is fixed in the system. Immobilization of enzymes often causes decrement in biocatalytic activity and choosing the appropriate immobilization technique is challenging . It was reported that immobilized asparaginase retained 95.7% of its activity after 10 cycles of use  (Figure 1d), while immobilized glucose oxidase enzyme (GOD) retained 97% of its original activity after cyclic regeneration and reuse . Immobilization often extends the long-term stability and temperature resistance of the enzymes, and in several cases, even the catalytic activity is increased compared with the soluble form. Immobilized asparaginase retained the 72.6% of its original activity for 10 weeks , and immobilized GOD retained the 95% of its activity  (Figure 1e) for 30 days.
The reactors filled with immobilized (bio)catalyst can be further divided into two groups according to the type of the supporting material of the (bio)catalyst:
Monolith reactors: The reactor is defined as monolith reactor where the supporting material is fixed in the reactor volume, and the (bio)catalyst is nonremovable; therefore, the chip is single use. Nanostructured materials are used to further increase the SVR. Examples include silica monolith reactors  (Figure 1a) or most recently reactors incorporating nanofibrous material made by electrospinning .
Packed bed reactors: The reactor is defined as packed bed reactor where the supporting materials are beads, even on microscale or nanoscale. The (bio)catalyst is immobilized onto their surfaces. Nanotechnology enables functional modifications of the beads, for example, making them magnetic. The reactor can be loaded with the suspension of the beads (Figure 1b) and viscous  or magnetic [12, 13] forces are utilized to keep the particles fixed in the reaction chamber.
Kinetic studies could be carried out with ease in microreactors by changing the attributes of the reaction, for example, the inflow substrate concentration. Because the most often used Michaelis–Menten model cannot be applied to flow reactors; in several cases [9, 14], the Lilly–Hornby model  was applied. Dependency of the kinetic parameters on the flow rate—and occasionally on further other parameters—was reported in many cases implying the limitations of the Michaelis–Menten model [8, 9, 14, 16].
In every on-chip study, Km and kcat as kinetic parameters were determined using various ways of product quantification such as capillary electrophoresis (CE) , amperometry , and fluorescent imaging [9, 16].
|Mu et al. ||Asparaginase||Michaelis–|
|MNP||Out of chip|
CE out of chip
|He et al. ||GOD||Michaelis–|
|Out of chip|
|Kerby et al. ||Alkaline|
|K m, kcat|
|Slovakova et al. ||Trypsin||Michaelis–|
|K m, kcat||600 nm|
|Seong et al. ||Lilly–Hornby||N/A||N/A||Microbeads||Optical|
2.2. Magnetic nanoparticles in microreactors
The importance of MNPs as potential carriers of biomolecules is growing rapidly in biotechnology and biomedicine. In LoC systems, nanosized magnetic particles provide quasi-homogeneous systems, high dispersion, high reactivity, low diffusion limits, and possibility of magnetic separation. The MNPs are usually collected in microsized reaction chambers. The collection and separation from the fluid stream are accomplished by external magnetic field. Such microreactors were found to be highly effective in biodetection , biocatalytic , and bioanalytical  applications (Table 1).
Magnetite nanoparticles exhibit superparamagnetic or soft ferromagnetic behavior with high saturation magnetization resulting in high permeability values . To date, magnetic manipulation of magnetic beads utilizing a magnetic bead separator array seems to be one of the most promising technique of precise handling of biocatalysts in chip. Do et al.  developed a microfluidic platform, where the magnetic field was concentrated between permalloy patterns (50 × 100) to produce a high magnetic field gradient over the edges of them, thus being able to trap the magnetic beads. Li et al.  used external hard magnet to develop a concentrated magnetic field perpendicular to the channel at a certain position of the chip. The particles accumulated at the designated place. Slovakova et al.  used a pair of hard neodymium magnets positioned in a given angle to develop a magnetic field parallel to the channel structure. It was reported that in this case, the particles are arranged parallel with the channel axis, and also, the reaction efficiency was reasonably higher than in orthogonal configurations. Lien et al.  used an integrated electromagnet with active cooling for the entrapment of the magnetic particles in the reaction chamber (Figure 1c).
Because of the widespread applications of MNPs in biotechnology, biomedical, and material science, more and more synthesis techniques have been developed to obtain different kinds of MNPs. Exhaustive discussions on the available synthesis techniques (e.g., coprecipitation, microemulsion, thermal decomposition, solvothermal, sonochemical, microwave assisted, chemical vapor deposition, combustion synthesis) can be found in several reviews [20, 21]. The synthetic methods will determine the shape, the size distribution, size, the surface chemistry of the particles, and consequently their magnetic properties. Various optimization methods could be used to obtain proper MNPs suitable for the desired research and commercial applications .
In our study, the surface of MNPs was chemically modified by sol–gel method, which resulted in the formation of a core–shell silica-MNP carrier. Then, the surface was functionalized by epoxy groups, which were able to form stable, covalent binding with the amino, thiol, or hydroxide groups of the enzyme. The immobilization of PcPAL was carried out in liquid phase. For a detailed description, see . After immobilization, negligible protein contents in the supernatants of the washing procedure were determined by the Bradford assay method . The resulted enzyme-coated magnetic nanoparticles (ecMNPs) were used in two size variations, with 250 and 600 nm diameters. Where otherwise not indicated the nominal ecMNP diameter is 250 nm.
3. The MagneChip platform: construction and operation
MagneChip is a microfluidic platform centered on a chip consisting of several reaction chambers enabling accumulation (and release) of MNPs. This magnetic microreactor chip can utilize the benefit of excellent separation ability of MNPs in magnetic field. In various applications, the MNPs covered by biologically active molecules (e.g., bioreceptors) are immobilized on their surfaces may be used. Magnetic techniques enable anchoring the particles inside certain compartment of microreactors, where the accumulated magnetic particles can form a dense layer. After filling (in a consecutive step), reagents can flow through the chip, while bioreaction occurs inside the microchambers and the resulted product flows through the chip. The outflow can be collected and/or quantified outside the chip, for instance by absorbance method. Because the enzyme to be immobilized on the MNP surfaces can be chosen freely, a wide variety of applications are possible (Figure 2). Taking the advantage of the continuous-flow operation, product formation can be monitored for a long time under various conditions over the same anchored ecMNP layer. MagneChip can be reinitialized periodically which enables multiparameter experiments, and therefore, reaction kinetics can be characterized in a fully automated way. Because the flow control system of the platform allows changing the actual substrate over the ecMNP layer, reactions can be screened even with unexplored substrates (Scheme 1). This feature renders MagneChip as a tool for substrate discovery as well.
3.1. Basic aims and principles
A microfluidic test bench was developed for carrying out microreactor experiments with MagneChip (Figure 3). The test bench consisted of two syringe pumps for dispensing reagents, a thermostable chip holder and a zoom microscope for the optical inspection of the chip. The chip holder had four magnet drawers enabling to push permanent magnets under reaction chambers of the chip and also pull them out as the magnetic field is no longer required.
MagneChip reaction chambers (volume of ~1 μl) were designed to accomplish the following requirements:
Because (bio)chemical reaction occurred inside the reaction chambers operating under continuous-flow conditions, a relative homogenous flow velocity distribution was required. This condition could be fulfilled because laminar flow was developed in the chamber.
MNPs were accumulated in the chambers, and their drifting was prevented by an external magnetic field. The critical flow rate and the amount of accumulated ecMNPs could be increased using prolated channels providing sufficient amount of (bio)catalysts to reach reasonable conversion of the desired reaction.
A resonant coil magnetometer was installed under the reaction chambers. Using the magnetometer, the accumulated amount of ecMNPs could be measured with high accuracy.
A four reaction chamber MagneChip layout is presented in Figure 4a. CFD simulations revealed that the flow velocity distribution inside of the chambers varied in a scale of two (Figure 4b). Depending on the typical flow rates used in MagneChip, reaction residence time in the chambers may vary from 1 to 10 s (Figure 4c).
3.2. Construction method of MagneChip
Figure 4a depicts the arrangement of a four-chamber chip used for testing the enzymatic reactions.
The chip was constructed by PDMS molding technology. SU-8 photoresist structures were prepared as a molding master, resulting in a channel height of 110 μm. PDMS was poured on the master and was kept on room temperature for 1 day. After cross-linking, the PDMS replica was released and the PDMS channel bodies were bonded to standard microscope glasses after oxygen plasma treatment. Some of the chips were equipped with a resonant coil magnetometer placed under the chambers for MNP quantity measurement . The coil was embedded in an intermediate PDMS layer. For further construction details, see .
3.3. Method of MNP quantification in the reaction chambers
The magnetic behavior of MNPs initiated the development of an inductive method to quantify the nanoparticles. The measurement is based on the resonance frequency shift of a passive electrical resonant circuit, where a flat inductor coil integrated in a silicone elastomer film acts as a sensor. From the suspension of MNPs flowing into the chip, MNPs were anchored within the reaction chambers by external permanent magnets. The ecMNP amount inside the chamber affected the inductance; therefore, the resonance frequency was changed. The method also enabled on-line monitoring of the actual ecMNP quantity in the chamber. This test arrangement enabled to study the effect of particle size and arrangement on the chamber filling MNP mass and also on the catalytic activity of the PAL bound to the ecMNPs .
3.4. Operation methods of MagneChip
3.4.1. Fluid handling steps
The experiments in MagneChip (Figure 3) involved four steps: (1) filling up the chip with MNPs, (2) absorbance calibration, (3) experiment cycles, and (4) chip cleaning.
Chip filling. In the chip-filling step, an MNP suspension was driven through the chip by applying a slight air pressure (0.2–0.3 bar) to the vial containing the MNP suspension and connected to the inlet of MagneChip via a PTFE tube (Figure 3) at 25°C. During the filling process, the MNPs were accumulated in the reaction chambers due to the permanent magnets placed in moveable drawers enabling “on/off” switching of the magnetic field. Once the chamber most distant from the inlet (Figure 3, Chamber 4) was saturated, the permanent magnet of the chamber at preceding position was turned on (Figure 3, Chamber 3). The same procedure was repeated (Figure 3: Chambers 2 and 1) until all chambers were filled up. Each chamber of the MagneChip device could capture ca. 250 μg of ecMNP biocatalyst .
Calibration and experiment cycles: During the forthcoming steps, the valve at the inlet of the MagneChip (Figure 3) was switched to the substrate (reagent) circuit. The flow controller performed the dosage of the substrate and other chemicals as dictated by the programmed sequence.
Chip cleaning: At the final, chip-cleaning step, the magnetic drawers of the MagneChip (Figure 3) were drown out and a washing solution was driven through the chip to remove ecMNPs.
The individual steps in a series of experiments, called experiment cycle (Figure 3, Experiment cycles), involved a Reaction step and a Re-initialization step. A series of experiments could consist of several experiment cycles depending on the number of parameters to be changed.
Reaction step. In a Reaction step, a substrate-containing solution was flowing through the chip at a constant flow rate, and the specific absorbance of the product was continuously monitored in the outflow of the chip.
Re-initialization step. In the Re-initialization step, the feed of the substrate-containing solution was stopped, and the chip was flushed with a washing buffer, while the magnetic particles were retained by the permanent magnets.
3.4.2. Reaction step variants
The substrate feed (with continuous monitoring of the absorbance in the outflow at a previously selected wavelength) was accomplished according to one of the following variants. The cycle ended when the predesigned step time had been passed or when the reaction reached saturation.
Repeatibility test. The feed of the substrate started (1st cycle) or continued at unchanged flow rate.
Flow rate test. The feed of the substrate started (1st cycle) or continued, while the flow rate changed cycle by cycle.
Substrate concentration test. The substrate-containing solution and the washing buffer were feed in parallel at a predesigned ratio resulted in a predefined dilution of the substrate at the chip inlet. The dilution ratio could be different cycle by cycle.
Substrate screening. The actual substrate was loaded into the substrate syringe through a bypass valve from the actual container of the substrate stock (A-F, in Figure 3), and the feed of the substrate began at a predefined flow rate. To change to the next substrate, a Re-initialization step was performed, followed by loading the next substrate into the substrate syringe from the substrate stock (A-F).
3.5. Quality assessment of the operations in MagneChip
A series of subsequent measurements performed by the system were considered as reliable if all the following conditions were met :
independent measurements were reproducible using the same type of ecMNP biocatalyst,
the product of the enzyme reaction could be measured selectively in the UV–Vis range,
product and substrate could be completely removed through the washing steps,
the enzymatic activity of the ecMNP biocatalyst remained unchanged during the measurement
and last but not least, the ecMNP layer in the magnetic reactors remained unharmed during the measurement cycles.
In order to test the fulfillment of the first group of conditions, a control measurement was performed after each series of experiments; that is, the first step of the sequence was repeated in the last step under the same conditions, and the specific activity of the immobilized biocatalyst (UB) at saturation concentrations of
3.5.1. Reproducibility of the individual measurements
Reproducibility of the chip-filling process: The first single chamber of the MagneChip was filled with MNP suspension. Biotransformation of
The filling–refilling results indicated that neither that the homogeneity of the MNP suspension nor the filling procedure of the chambers had remarkable effect on the reproducibility of the measurements. The significant difference between the UB values of MNP biocatalyst in shake vials and in MagneChip indicated increased effectivity of the biocatalysts in MagneChip device .
Reproducibility of independent measurements: Biotransformation of
3.5.2. Optical inspection of the reaction chambers
During the experiments, the chip was optically inspected by a zooming microscope and a monochrome hi-speed smart camera. Before evaluating the measurement sequence, the plan view of the chip was stored as a reference (∏ref). At the end of the step i of the measurement sequence, the plan view of the chip was sampled again (∏seq,i) and it was compared to the reference as follows :
where (j,k) are the pixel coordinates of the plan view image; therefore, the changes in accordance to the reference image are indicated by white pixels. The total number of white pixels is defined as chamber difference score (SC) used as a marker for describing the changes of the MNP layer arrangement. Therefore, the changes compared with the image of the first cycle (reference) were indicated by white areas during the consecutive cycles of the measurement.
In practice, SC values under 2000 reflected to negligible changes. However, SC > 3000 indicated serious structural change of the ecMNP layer, for instance, the complete breakthrough of a bubble (Figure 5b) . Air bubbles usually did not split at the channel entrance, rather passed at one side along the chamber wall. Numerical simulations revealed (Figure 5c–f) that the velocity profile became asymmetric due to the bubble passage and the overall mass flow rate through the porous MNP layer significantly decreased (28.6–20.7 μL min−1, roughly 72% of its original value), while the remaining fluid passed through the developed tunnel. The passing bubble could drift away particles which decreased the total mass of the biocatalyst in the reaction chamber. Therefore, the biocatalytic activity of the damaged chamber decreased and the consequent measurements were no longer reliable.
Reliability assessment of the measurements was based mostly on the following parameters:
Chamber difference score (SC)—Over SC > 4000 (average), the measurement was declined.
Control measurement—Over 5% of error, the measurement was declined.
Each of the experiments carried out by the platform was justified based on the above criterion.
3.5.3. Reproducibility of cyclic reactions
A crucial feature is the reproducibility of cyclic reactions performed by the system. To check the reproducibility of the test reactions, the MagneChip was filled with MNP biocatalyst, and biotransformation of
The product quantity in cycle by cycle—calculated by taking the integral of the absorbance plot—clearly indicated that the chip was successfully re-initialized in every cycle throughout the experiment, and the reaction was repeated reproducibly seven times (average product quantity of P = 0.12 ± 1.5% μmol) . The moderate mean value of the chamber difference score SC = 1322 (1609 max) reflected negligible changes in the MNP layer.
4. The MagneChip platform: application examples
4.1. Characterization of the PAL reaction with
l-phenylalanine ( l-1a) in MagneChip
4.1.1. Influence of the substrate flow rate on the biotransformation
MagneChip was filled with ecMNP biocatalyst, and biotransformations of
The reaction velocity was calculated for each cycles. By increasing the flow rate, the calculated reaction velocity increased until reaching saturation at about 25 μL min−1 (Figure 7).
4.1.2. Calculation of kinetic parameters
MagneChip was filled with ecMNP biocatalyst, and biotransformations of
The linear fitting method proposed by Lilly et al.  was applied for the calculation of the kinetic constants of the biotransformation of
|Kinetic parameter||MagneChip||Shake vial|
|K m (mM)||2.5||9.1|
|k cat (s−1)||2.8 × 10−2||3.2 × 10−2|
|kcat/Km (s−1 M−1)||11.3||3.5|
It was found that the apparent Km value was reasonably smaller in MagneChip (2.5 mM) than in shake vial (9.1 mM). Turnover number (kcat) and specificity constant (kcat/Km) were determined also for both reaction modes. While in the shake vial, the turnover number was somewhat higher (3.2·× 10−2 s−1) than the in chip (2.8·×·10−2 s−1), the specificity constant turned out to be significantly higher in chip (11.3 s−1 M−1) as compared with the shake vial (3.5 s−1 M−1). This may be attributed to the smaller Km value in the MagneChip indicating significant contribution of diffusion effects to the higher apparent Km value in shake vial.
4.2. Effect of particle size on the enzyme activity
The accumulated quantity of ecMNPs in the reaction chambers was determined by the embedded resonant magnetometer of the MagneChip device . The measurements revealed that the total mass of the accumulated particles was approximately the same for two different particle sizes (m = 241.6 μg, ecMNP600, d = 600 nm and m = 248.3 μg, ecMNP250, d = 250 nm) . The total particle mass could be only increased using a binary mixture (m = 283.6 μg, MNP250:600) of the particles. This experiment resulted in a significantly higher MNP mass (17%) captured in the magnetic chamber as compared with the chamber capacity filled with MNPs of uniform particle sizes .
MagneChip was filled with different sized MNP biocatalysts, and biotransformations of
Results of the measurement using variously sized ecMNPs as biocatalysts are summarized in Figure 9. In fact, the ecMNP600-filled chambers yielded the lowest final concentration of product as indicated by the lowest specific absorbance (AU = 1.07, at 295 nm) at the chip outlet. Filling the chip by ecMNP250 resulted in an increase of the measured absorbance by 46% (AU = 1.56, at 295 nm). Because the chambers contained the same filling mass (m = 241.6 μg for ecMNP600 and m = 248.3 μg for ecMNP250) and therefore the same enzyme amount, the difference between the MNP250- and the MNP600-filled reactors can only be attributed to other factors, for example, to the differences in total surface area .
The major difference can also stem from the remarkably smaller average microchannel diameters between the particles within the ecMNP250-filled chamber as compared to the ecMNP600-filled one. This can result in shortened diffusion path and therefore better mass transport . An additional 40% increment was achieved using the 1:1 particle mixture, which was obviously resulted as a synergy of the higher enzyme content (17%) due to the higher chamber capacity and enhanced transport phenomena due to the small average microchannel diameter .
4.3. Testing multiple substrates in MagneChip
Substrate screening experiments were performed with a single ecMNP-loading in the chip passing the solutions of the different substrates (Scheme 1:
Noteworthy, all the four unnatural substrates (rac-1b,c,e,f) which were transformed by the MNP biocatalyst with higher specific biocatalytic activity (UB) than that of
4.4. Characterization of an enzyme reaction with a novel substrate
By a reaction performed in the MagneChip device, it was first demonstrated that PAL can catalyze the ammonia elimination from the acyclic
MagneChip, filled by PAL-ecMNPs, was used for the microscale biotransformation of
Our results proved that the MagneChip microfluidic device is a reliable, reproducible, and efficient tool which was capable of fast, reliable, and fully automated screening and kinetic characterization of PcPAL substrates using minimal solvent (~500 μl) and biocatalyst (~1 mg MNP) amounts for a test compound. Compared with shake vial, the volumetric productivity of the MNP biocatalyst in the chip exceeded the one of the shake vial by more than three orders of magnitude. The platform was also capable of studying enzymatic reactions with undiscovered substrates of PcPAL. The above results suggest that the MagneChip platform would be successfully utilized as a novel and flexible tool for enzyme-catalyzed biotransformations.