**2. Background**

**1. Introduction**

158 Lab-on-a-Chip Fabrication and Application

cle carriers [1].

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 nanoparti‐

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 timeresolved; 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

**•** Microreactors with high surface-to-volume ratio (SVR) are able to absorb the heat evolv‐ ing 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

**•** 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 micro‐

**•** In biocatalytic applications, the efficiency of the microreactor can be further improved by immobilization of enzymes on nanoscale carriers accommodating in the reactor. Reusabil‐ ity of the biocatalyst makes the process economical and more environmentally friendly.

**•** To perform similar analyses in shorter timescale even in parallel is an anticipated objec‐ tive for screening and routine use in protein and enzyme research [3]. A desirable goal is the high throughput screening of enzymes and their substrates and inhibitors. The pro‐ spective fields of application of microreactors are quite wide and include biotechnology, as

**•** 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 [4]. Benefitting from system automation, this

**•** Small reagent volume is also a benefit of microreactors enabling economical and efficient

reactors, which overrides the achievable mixing efficiency of batch reactors [2].

well as combinatorial chemistry and enzyme-targeted drug discovery [4].

also eliminates errors associated with manual protocols.

screening of novel reaction paths and substrates.

development and enhanced safety due to smaller reactor volumes [2].

reactors, decreasing the product yield [2].

#### **2.1. Microreactors**

Analytical systems which comprise microreactors are characterized by outstanding repeata‐ bility and reproducibility, due to replacing batch iterative steps and discrete sample treat‐ ment by flow injection systems [4]. The possibility of performing similar analyses in parallel is an attractive feature for screening and routine use [3]. 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 [4].

Applications of microreactors can be divided into three classes [4]:


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 [5]. Losing the catalyst is a major drawback of this technique.

**Figure 1.** Lab-on-a-chip microreactors: (a) a monolith silica reactor [8], (b) a packed bed silica reactor [9], and (c) a packed bed MNP reactor [10]. Stability of immobilized enzymes: (d) after reuse of asparaginase in 10 cycles, 10 min each [7] and (e) during long-term operation of immobilized GOD [8].

*Filled reactors:* Microreactors utilizing immobilized catalysts (e.g., enzymes) have many advantages overthe 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 [6]. It was reported that immobilized asparaginase retained 95.7% of its activity after 10 cycles of use [7] (**Figure 1d**), while immo‐ bilized glucose oxidase enzyme (GOD) retained 97% of its original activity after cyclic regeneration and reuse [8]. Immobilization often extends the long-term stability and temper‐ ature 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 [7], and immobilized GOD retained the 95% of its activity [8] (**Figure 1e**) for 30 days.

ment by flow injection systems [4]. The possibility of performing similar analyses in parallel is an attractive feature for screening and routine use [3]. Microreactors have been integrated into automated analytical systems, as well as providing benefits from system automation, and

**•** Analytical use of biocatalysts to transform an analyte difficult to measure to an easy to

Microreactor systems can be further divided into classes based on the physical localization of

*Laminar flow reactors:* The majority of the commercial flow synthesis systems utilize laminar flow with soluble components and enzymes [5]. Losing the catalyst is a major drawback of this

**Figure 1.** Lab-on-a-chip microreactors: (a) a monolith silica reactor [8], (b) a packed bed silica reactor [9], and (c) a packed bed MNP reactor [10]. Stability of immobilized enzymes: (d) after reuse of asparaginase in 10 cycles, 10 min

*Filled reactors:* Microreactors utilizing immobilized catalysts (e.g., enzymes) have many advantages overthe traditional flow reactors. First of all, the catalyst (enzyme) can be recycled after usage; therefore, the process is more economical and environmental friendly. The

each [7] and (e) during long-term operation of immobilized GOD [8].

**•** Organic synthesis, when a target molecule is formed from components in flow

**•** Screening of substrates and enzymes examines their kinetic characteristics

this also eliminates errors associated with manual protocols [4]. Applications of microreactors can be divided into three classes [4]:

measure form

160 Lab-on-a-Chip Fabrication and Application

the catalyst:

technique.

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:


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 [15] was applied. Dependency of the kinetic parameters on the flow rate—and occasionally on further other parameters—was reported in many cases implying the limita‐ tions of the Michaelis–Menten model [8, 9, 14, 16].

In every on-chip study, *K*<sup>m</sup> and *k*cat as kinetic parameters were determined using various ways of product quantification such as capillary electrophoresis (CE) [7], amperometry [8], and fluorescent imaging [9, 16].



**Table 1.** Lab-on-a-chip microreactors with immobilized enzymes.

#### **2.2. Magnetic nanoparticles in microreactors**

The importance of MNPs as potential carriers of biomolecules is growing rapidly in biotech‐ nology and biomedicine. In LoC systems, nanosized magnetic particles provide quasihomogeneous 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 [24], biocatalytic [17], and bioanalytical [18] applications (**Table 1**).

Magnetite nanoparticles exhibit superparamagnetic or soft ferromagnetic behavior with high saturation magnetization resulting in high permeability values [19]. 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. [12] developed a microfluidic platform, where the magnetic field was concentrated between permalloy patterns (50 × 100) to produce a high magnetic field gradient overthe edges of them, thus being able to trap the magnetic beads. Li et al. [13] 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. [16] 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. [10] used an integrated electromagnet with active cooling for the entrapment of the magnetic particles in the reaction chamber (**Figure 1c**).

**References Enzyme Method Reusability Stability Particle [E] measurement**

Lilly–Hornby N/A N/A Silica

*K*m, *k*cat 600 nm

97% 30 days Monolith

reactor

95% silica Absorbance

Microbeads

80% N/A MNP Approximated

15 μm

Out of chip

In chip

Optical

Menten, EH plot

*K*m, *k*cat Amperometry, on-chip

*K*m, *k*cat Fluorescent imaging

Menten, LB plot

Seong et al. [9] Lilly–Hornby N/A N/A Microbeads Optical

The importance of MNPs as potential carriers of biomolecules is growing rapidly in biotech‐ nology and biomedicine. In LoC systems, nanosized magnetic particles provide quasihomogeneous 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 [24], biocatalytic [17], and

Magnetite nanoparticles exhibit superparamagnetic or soft ferromagnetic behavior with high saturation magnetization resulting in high permeability values [19]. 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. [12] developed a microfluidic platform, where the magnetic field was concentrated between permalloy patterns (50 × 100) to produce a high magnetic field gradient overthe edges of them, thus being able to trap the magnetic beads. Li et al. [13] 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. [16] 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

*K*m Fluorescent imaging

He et al. [8] GOD Michaelis–

phosphatase

**Table 1.** Lab-on-a-chip microreactors with immobilized enzymes.

**2.2. Magnetic nanoparticles in microreactors**

bioanalytical [18] applications (**Table 1**).

Slovakova et al. [16] Trypsin Michaelis–

Kerby et al. [14] Alkaline

162 Lab-on-a-Chip Fabrication and Application

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 severalreviews [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 [21].

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 ofthe enzyme. The immobilization of *Pc*PAL was carried outin liquid phase. For a detailed description, see [18]. After immobilization, negligible protein contents in the supernatants of the washing procedure were determined by the Bradford assay method [22]. The resulted enzyme-coated magnetic nanoparticles (*ec*MNPs) were used in two size varia‐ tions, with 250 and 600 nm diameters. Where otherwise not indicated the nominal *ec*MNP diameter is 250 nm.
