Microfluidic immobilized enzyme

H. Mao, T. Yang, and P. S. Cremer, Design and characterization of immobilized enzymes in microfluidic... 

The second device was a multiplate-stacked microfluidic reactor, originally developed by Hessel, Lowe and co-workers for gas-phase transformations [lb] (Figure 3.2) and now adapted for biocatalytic processing with immobilized enzymes. 

Enzymatic bioreactor Immobilized enzyme biosensor Immobilized enzyme reactor Immobilized microfluidic enzyme reactor (IMER) Membrane reactor... 

A wide variety of applications for microfluidic bioreactors have been developed. One of the most common is immobilized enzyme reactors that are used for applications such as biosensors, medical diagnosis, and synthetic applications. Most of the... 

MicroChannel Enzyme Reactor. Enzymatic conversion is receiving attention because it is an environmentally friendly approach to synthesizing chemicals. Reactions can be carried out enzymatically in the solution phase or by immobilizing enzymes on microfluidic walls. The use of the latter type of reaction has the same mass and heat transfer advantages as the use of a microfluidic synthetic reactor. More information can be found in a published review article [6] and in the article microfluidic bioreactors in this encyclopedia. 

Mao, H., Yang, T, Cremer, P.S. (2002) Design and Characterization of Immobilized Enzymes in Microfluidic Systems. Anal. Chem. 74 379-385. 

Microfluidic bioreactors are a variety of devices that can be made by immobilizing a variety of biologically active substrates within a microfluidic device [1]. The ability to create a variety of biologically important devices is critical to enabling the true total analytical system. The variety of devices that can be made in this way ranges from immobilized enzyme reactors to enzymatic biosensors, immunoassys, and affinity chromatographic stationary phases. In order to form a microfluic bioreactor it is necessary to immobilize the active molecule within the device either directly onto the channel or onto a solid support within the channel such as a bead or a monolith. 

Microfluidic bioreactors have also been widely used to make immobilized enzyme biosensors. In this application the bioreactor is applied as a part of a detection device for a biologically important analyte. For instance, glucose can be detected by having a bioreactor that contains glucose oxidase immobilized in a microfluidic system [9]. The enzyme enables detection of glucose by electrochemical detection of the products of the bioreactor. 

To fulfill such requirements, attempts have been made in the past decade by researchers working on peptide mapping and proteomics through development of immobilized microfluidic enzymatic reactors. Microfluidic enzymatic microreactors are an alternative to in-solution method employing immobilization of proteases on microchaimels of chip-based reactors or surfaces of capillaries. The microreactors that enable proteolytic digestion by enzymes immobilized on solid supports are also referred to as immobilized enzyme reactors, IMERs. The great potential of IMERS for proteomic applications comprise rapid and enhance... 

Microfluidic Reactors with Immobilized Enzymes for Biocatalytic Transformations... 

Table 10.4 Enzymatic transformations in microfluidic reactors with immobilized enzymes.

Yakovleva, J., Davidsson, R., Lobanova, A., Bengtsson, M., Eremin, S., Laurell, T., Emneus, J., Microfluidic enzyme immunoassay using silicon microchip with immobilized antibodies and chemiluminescence detection. Anal. Chem. 2002, 74(13), 2994-3004. 

In order to increase the efficiency of biocatalytic transformations conducted under continuous flow conditions, Honda et al. (2006, 2007) reported an integrated microfluidic system, consisting of an immobilized enzymatic microreactor and an in-line liquid-liquid extraction device, capable of achieving the optical resolution of racemic amino acids under continuous flow whilst enabling efficient recycle of the enzyme. As Scheme 42 illustrates, the first step of the optical resolution was an enzyme-catalyzed enantioselective hydrolysis of a racemic mixture of acetyl-D,L-phenylalanine to afford L-phenylalanine 157 (99.2-99.9% ee) and unreacted acetyl-D-phenylalanine 158. Acidification of the reaction products, prior to the addition of EtOAc, enabled efficient continuous extraction of L-phenylalanine 157 into the aqueous stream, whilst acetyl-D-phenylalanine 158 remained in the organic fraction (84—92% efficiency). Employing the optimal reaction conditions of 0.5 gl min 1 for the enzymatic reaction and 2.0 gl min-1 for the liquid-liquid extraction, the authors were able to resolve 240 nmol h-1 of the racemate. 

An example of the immobilization of antibodies on channel surfaces was presented by Eteshola and Leckband [395]. A microfluidic sensor chip was developed to quantify a model analyte (sheep IgM) with sensitivities down to 17 nM. This was achieved by first immobilizing a layer of bovine serum albumine (BSA) onto the channel wall, followed by specific adsorption of protein A to which the primary antibody for IgM was coupled covalently. This antibody could capture IgM, which was detected with the secondary antibody, labeled with horseradish peroxidase (Scheme 4.91). This enzyme catalyzes the conversion of the fluorogenic substrate 3-(p-hydroxyphenyl)propioni c acid into a fluorophore, which was quantified off-chip with a spectrofluorometer. The measured fluorescence signal was proportional to the analyte concentration in the test sample. 

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