Microfluidic enzymatic microreactors

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... 

Enzymatic microreactors (7.5 nL) have been fabricated in the microfluidic chip to prepare the tryptic digest of equine (horse) myoglobin (14.2 pmol/p.L)... 

Peterson, D.S., Rohr, T., Svec, F., Frechet, J. M.J., Enzymatic microreactor-on-a-chip Protein mapping using trypsin immobilized on porous polymer monoliths molded in channels of microfluidic devices. Anal. Chem. 2002, 74(16), 4081M088. 

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. 

Fig. 11 Top SU-8 based microfluidic system, which includes an enzymatic microreactor, a chromatographic device, and an integrated ionization emitter tip. Bottom SEM photograph of a section of a monolithic phase prepared from LMA/EDMA. Reproduced from [133]...

In addition to serving as the stationary phases, monolithic materials are also finding numerous other applications in the microfluidic world. Their use in on-chip solid-phase extraction and preconcentration,as supports for immobilization of enzymes to form enzymatic microreactors for protein mapping,static mixers,and valves represent just a few examples of modules in the microfluidic toolbox and further growth is inevitable. 

Another application of microfluidic enzymatic reactors is in the enormous and diverse challenges of proteomic investigations. Enzymatic microreactors present proteomics with a valuable analytical tool for protein analysis. Most of applications of IMERs are currently directed at protein analysis by protein digestion and peptide mapping. 

Most recently, a modular microfluidic reactor and in-line filtration system for the rapid and small-scale evaluation of biocatalytic reactions have been demonstrated by O Sullivan and others [153]. The system combined a substrate with a biocatalyst in free solution. The PMMA enzymatic microreactor worked by co-flowing the enzyme and substrate through a T-channel and mixing was achieved by staggered herringbone micromixer (SHM). The filtration unit composed of gaskets made of PDMS... 

Many advantages have been reported with the use of microfluidic reactors in biocatalytic reactions. These advantages can potentially enable the rapid evaluation of different reaction conditions, overcoming the time constraints associated with biocatalytic process development. Biocatalysis by enzymatic microreactors have been widely reported. Enzymatic microreactors are classified based on their applications such as microreactors for enzymatic diagnosis and genetic analysis, for enzyme-linked immunoassays, and for analysis of proteins. Microreactors for genetic analysis have been widely exploited, while integration of microfluidics... 

Electrophoretic microfluidic chips feature a number of microreactor characteristics and have been used for conducting chemical and biochemical reactions in channels and microfabricated chambers, mixing reagents, microextraction and microdialysis, post- and preseparation derivatizations, etc. The most recent achievements are reviewed in Ref. 63 and other similar publications. These integrated microdevices perform PCR amplification, cell sorting, enzymatic assays, protein digestion, affinity-based assays, etc. In this section we describe such integrated microsystems and the most recent advances in this field. 

Enzymatic syntheses within the microfluidic platform were also reported. The construction and novel compound synthesis from a synthetic metabolic pathway consisting of a type III poly-ketide synthase (PKS) known as 1,3,6,8-tetrahydroxynaphthalene synthase (THNS) from Streptomyces coelicolor and soybean peroxidase (SBP) in a microreactor were performed (Fig. 5) [11]. THNS immobilized to Ni-NTA agarose... 

Enzymatic syntheses within the microfluidic platform were also reported. The construction and novel compound synthesis from a synthetic metabolic pathway consisting of a type HI polyketide synthase (PKS) known as 1,3,6,8-tetrahydroxynaphthalene synthase (THNS) from Streptomyces coelicolor and soybean peroxidase (SBP) in a microreactor were performed (Fig. 5) [11]. THNS immobilized to Ni-NTA agarose beads was prepacked into a microfluidic channel, while SBP was covalently attached to the walls of a second microfluidic channel precoated with a reactive poly(maleic anhydride) derivative. The result was a tandem, two-step hiochip that enabled synthesis of novel polyketide derivatives. The first microchan-nel, consisting of THNS, resulted in the conversion of malonyl-CoA to flaviolin in yields of up to 40% with a residence time of 6 min. This conversion is similar to that obtained in several-milliliter batch reactions after 2 h. Linking this microchannel to the SBP microchannel results in biflaviolin synthesis. During the course of this work, we discovered that the substrate specificity of THNS could be manipulated by simply changing the reaction pH. As a result, the starter acyl-CoA specificity can be broadened to yield a series of truncated pyrone products. When combined with variations in the ratio of acyl-CoA and... 

Microreactors are ideal for directing complex enzymatic synthesis, such as multienzyme catalysis and cascade reactions. There have been a grovdng number of studies [173-178] that represent the implementation of microreactions for multistep enzymatic catalysis and a list is presented in Table 10.5. Microfluidic biocatalytic... 

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