Microreactors in Organic Chemistry and Catalysis, Second Edition (2013)

Masaya Miyazaki, Maria Portia Briones- Nagata, Takeshi Honda, and Hiroshi Yamaguchi

10.1. General Introduction

Bioorganic chemistry is a scientific discipline that represents the merger between biochemistry and organic chemistry. While biochemistry involves the study of biological processes using chemical methods, bioorganic chemistry attempts to expand organic-chemical studies, that is, synthesis of biomolecules, their structures, and kinetics of biochemical reactions toward biology. The broad area of bioorganic chemistry encompasses multifaceted researches on the use of biocatalysts for synthesis and screening of biomolecules. Biocatalysis has taken a strong position in the development of sustainable chemical transformations [1]. However, one of the drawbacks of biocatalytic processes is that biocatalysis is relatively slow to implement and this spells out the need for microscale processing techniques as a means to increase the speed at which bioconversion processes can be created [2].

Microreactors are microscale reactions vessels in which bioorganic and chemical reactions, for example, cell or enzyme-mediated reactions can be performed. Microreactor technology has been developed in many areas of science and technology. The advent of micro-fabrication technology allows for vast combinations of parameters to be simultaneously screened in short periods of time operating in small volumes of precious compounds. Microfluidics, a discipline, which involves processing fluids at microchips with microstructured geometries and microscale channel networks that precisely manage fluids and solutions, has been used to run chemical and biochemical assays. The microfluidic approach in microreactors has systematically been shown to outperform conventional macroscale reactor operation, in terms of the rate of substrate processing, which is attributed to the favorable mass and heat transfer characteristics of the microreactors. Mass and heat transportation are very efficient in micro- or nanoscale because of the relatively fast linear diffusion. In microfluidic systems, operations in micro- or nanoliter fluid volumes allow diffusion-limited reactions to occur significantly faster than the batch or large scale. An enhanced mass transfer allows for fast mixing of reagents and for an increase of reaction speed of chemical processes. In addition to mass transport, heat transport is considerably improved in microfluidic systems. An improved heat-exchanging efficiency allows for fast heating and cooling and a precise temperature control of chemical reactions. This is advantageous for certain sensitive reactions, as local high concentrations or temperatures can cause side reactions. Moreover, microreactors can take advantage of the microfluidic approach to achieve real-time biochemical analysis and detection with high efficiency, high-throughput, precision, and high sensitivity while requiring only microliter volume consumption of expensive compounds. The use of microfluidics that takes advantage of micro or nanoliter volumes ensures high efficiency and repeatability of biocatalytic processes [3]. In comparison to macroreactors, the small dimensions of microreactors allow usage of small amount of reagents under precisely controlled conditions ensuring an improved overall safety of the process [4]. Moreover, advantages of microsystems include shorter molecular diffusion distance, lightweight and compact system design, minimal amount of catalyst and waste products compared to equivalent macroscale reactors, laminar flow, effective mixing, better process control, and minimal energy consumption [5]. Additionally, the microchannel architecture could be designed to perform several processes or several assays combined in multiple steps of the analytical procedure in one integrated and automated platform. These integrations led to the concept of integrated microsystems, the so-called lab-on-chip (LOC) or micrototal analysis systems (μTAS).

Microreactor technology opens the gate to novel and improved analytical techniques while offering numerous advantages for different fields of applications in biomedicine, biochemical processing, and biotechnology. The developments in microreactor technology and micrototal analysis systems have their obvious impact on bioorganic chemistry. Significant advances in μTAS have led to rapid growth in enzyme microreaction technology [6, 7].

The miniaturization of chemical reactors presents many fundamental and practical advantages for the synthetic organic chemists. The small dimensions of microreactors allow for the use of minimal amounts of reagents under precisely controlled conditions. The reaction time with a substrate is directly proportional to the length of the microchannel. In addition, control of reaction variables such as thermal or concentration gradients within the microreactor allows new methods for efficient chemical transformations with high space-time yield. The benefit of microreactors result from their physical properties such as enhanced mass and heat transfer as well as regular flow profiles leading to improved yields with increased selectivities. These microdevices also make it possible to rapidly screen reaction conditions and improve the overall safety of the process since the generation of hazardous intermediates in situ is safe as only very small amounts are generated and directly reacted in a closed system. The possibility of scale-up of synthetic procedures is also one of the benefits of using microreactors. The first part of this chapter deals with the recent progress and application of microreactors in chemical synthetic processes. The chapter also focuses on the conceptual implications of microreactor technology for synthetic chemists. Various organic reactions performed in microreactors are presented. Moreover, studies that report on biomolecular synthesis in microfluidic reactors are also covered in this chapter.

The emergence of microfluidic reactors has significantly enhanced the possibilities for chemical synthesis, particularly the enzyme-catalyzed transformations at different stages of biocatalytic process development [8–10]. Enzymatic microreactors can be classified based on two approaches; heterogenous and homogenous biocatalysis, as represented using immobilized enzymes and enzymes in solution, respectively. Immobilized enzyme microreactors (IMERs), which are actually reactors with enzymes immobilized on solid supports, have several advantages over conventional solution-based systems. A low expenditure of enzyme is one of the advantages of immobilization. An additional advantage is the ease in separation of immobilized enzymes from the reaction mixture thus, decreasing or eliminating protein contamination of the product. The immobilized enzyme microreaction systems afford control of reaction space by separating the catalyst from the reaction solution, therefore reducing contamination or inhibition, improvement of catalyst stability, and durability, in addition to reusability.

IMERs are commonly used in medical diagnostics and therapy [11], particularly in DNA digestion and analysis [12], and for DNA-based diagnostics, and genotyping. The analysis of genetic materials typically requires for amplification of DNA, which is performed by polymerase chain reaction (PCR). By utilizing repeated thermal cycling (heating and cooling), enzyme-mediated exponential replication of DNA takes place thus rendering the many copies of DNA readily detectable. Direct gene analyses of biological samples are of high impact in clinical diagnostic field particularly in research for infectious and genetic diseases. Miniaturizing PCR systems offers the advantage of a much faster process brought about by then much lower thermal mass of the reaction chamber, which consequently allows for rapid heating and cooling. Moreover, reduction of dimension, size, and weight of the diagnostic instruments enables production of easy-to-handle microdevices that may provide for faster, automated analyses of minute amount of samples. Small thermal mass due to microscale enables a significant reduction in thermal cycling times from several hours to minutes. PCR in microreactors ensures a large number of parallel amplification analysis and can lead to more accurate information and a better understanding of the analyzed processes [13]. Although typical DNA amplification in microreactors involved injection of substrates for PCR reaction and polymerase enzyme into the reaction zone, followed by thermal cycling, recently, there has been an attempt to use immobilized DNA polymerase in recording the DNA synthesis process in order to increase sensitivity detection during DNA strand extension.

Microreactor technologies also open new possibilities in immunoanalytical applications. The conventional enzyme-linked immunosorbent assay (ELISA) approach has a number of limitations for its application in resource-limited settings due to its requirement for long assay times, large amount of expensive reagents and equipment required, and cumbersome liquid handling. ELISA, however, can be readily transferred to microfluidic format. Microfluidics-based immunoassays are potentially beneficial in terms of maximizing sensitivity, production of fast and accurate results, and minimizing sample volume and reagent requirements [14–16]. Recent developments in microfluidic technology have also been focused on the applications of microreactors in enzymatic diagnostics.

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.

An important analytical application of enzymatic microreactors is their use in biocatalysis, in order to transform a difficult-to-measure analyte into an easily measurable form. This could be represented by microreactors designed for digestion of proteins to convert them to more readily measured peptides. Applications of microreactors in continuous-flow chemistry have expanded rapidly over the past two decades, with numerous reports of higher conversions and yields compared to conventional batch equipment. In this chapter, a comprehensive discussion on the most recent trends in the development of enzymatic microreactors and their current applications are covered.