Enzymatic microfluidic

Apart from immunoassays, enzyme assays can also be used to detect certain substrates in a clinical diagnostic setting. The benefits of performing enzymatic assays on microchips are the analytical power and minimal reagent use in microfluidic systems combined with the selectivity and amplification factors that come with biocatalysis. 

D.C. Duffy, H.L. Gillis, J. Lin, N.F. Sheppard Jr, and G.J. Kellogg, Microfabricated centrifugal microfluidic systems characterization and multiple enzymatic assays. Anal. Chem. 71, 4669-4678... 

H. Irth A microfluidic-based enzymatic assay for bioactivity screening combined with capillary liquid chromatography and mass spectrometry. Lab Chip 2005, 5,... 

In the electronics indnstry, a Pentinm compnter chip has hundreds of millions of transistors and only a hundred pins in and out. If each transistor had to be addressed individually, it would be impossible to have such a chip. Microfluidic systems are similarly easy to control n fluid lines can be controlled by 21og n control hues. Additionally, the pressure to actuate a valve depends on the width of the control line, so by choosing our pressure carefully, one thinner fluid line can be closed while the wider fluid lines remain open due to insufficient pressure. This idea has been used to develop microfluidic systems that can screen enzymatic libraries and perform in vitro transcription translation of DNA to protein in approximately 30 minutes. 

Research on microchip protein analysis has been very active for cellular protein functional assay, clinical diagnostics, and proteomics studies. Once again, the microfluidic technology plays an important role in protein assays. Immunoassay, protein separation, and enzymatic assay will be described in detail in subsequent sections. 

FIGURE 10.20 Schematic representation of the microfluidic device used for the two-step enzymatic detection of glucose [1043]. Reprinted with permission from the American Chemical Society. 

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)... 

Richter, T., Shultz-Lockyear, L.L., Oleschuk, R.D., Bilitewski, U., Harrison, D.J., Bi-enzymatic and capillary electrophoretic analysis of non-fluorescent compounds in microfluidic devices Determination of xanthine. Sensors Actuators B 2002, 81, 369-376. 

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. 

Xu, Z.R., Fang, Z.L., Composite poly(dimethylsiloxane)/glass microfluidic system with an immobilized enzymatic particle-bed reactor and sequential sample injection for chemiluminescence determinations. Anal. Chim. Acta 2004, 507,129-135. 

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. 

In some cases, substrates and enzymes are not soluble in the same solvent. To achieve efficient substrate conversion, a large interface between the immiscible fluids has to be established, by the formation of microemulsions or multiple-phase flow that can be conveniently obtained in microfluidic devices. Until now only a couple of examples are published in which a two-phase flow is used for biocatalysis. Goto and coworkers [431] were first to study an enzymatic reaction in a two-phase flow in a microfluidic device, in which the oxidation ofp-chlorophenol by the enzyme laccase (lignin peroxidase) was analyzed (Scheme 4.106). The surface-active enzyme was solubilized in a succinic acid aqueous buffer and the substrate (p-chlorophenol) was dissolved in isooctane. The transformation ofp-chlorophenol occurred mainly at... 

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. 

Whereas fluorescence is typically measured during non-separation, mix-and-read protocols offering high readout throughput, several approaches exist today that involve separation steps in front of the actual detection. In the Caliper Labchip technology, a multi-parallel microfluidic separation system coupled with fluorescence detection allows the monitoring of enzymatic reactions. Quantification of substrate and product of the enzymatic reaction, after separation from each other as well as from the test compound, minimises artefacts and offers ratiometric results, although with comparatively lower... 

Kr enkova, J. and Foret, F. (2004). Immobilized microfluidic enzymatic reactors. Electrophoresis 25 3550-3563. 

Herrmann, M., Veres, T., and Tabrizian, M. (2006) Enzymatically generated fluorescent detection in micro channels with internal magnetic mixing for the development of parallel microfluidic ELISA. Lab on a Chip, 6, 555 560. 

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