Understanding the dynamics of gene expression and regulation forms the foundation of synthetic biology. Upon completion of the construction of a synthetic biological component, the first step is functional assessment of gene expression. It is desirable to analyze the variation in gene expression with respect to different environmental stimuli in order to precisely identify the functions of synthetic parts/systems [18]. Current methods for the assessment of gene expression involve the use of fluorescent protein expression in microplate readers and flow cytometers. However, these assessment tools are still insufficient for screening the rapid response of a cellular system to different environmental stimuli, and the detection limit restricts the analysis to proteins that are highly expressed. Such limitations of current technologies should be resolved, and better methods are required for the development of synthetic biology. However, in microfluidic devices, cells can be confined to a very small space and, hence, the signal from even a small concentration of a protein (in particular, regulatory proteins) is amplified several fold, thus allowing real-time monitoring of the activity of the protein within a cell [19]. Without exploiting the advantage of the concentrator offered by microfluidics, it is almost impossible to determine the effect of regulatory proteins as their expression level is below the detection range of a macroscale device. There are several microfluidic devices for better understanding gene expression and regulation, which are highlighted in the following section. Miniaturized methods to monitor and control gene expression and regulation of synthetic biological parts on a chip can be largely categorized as follows: droplet-based methods for single-cell analysis and array-based method for the analysis of the effect of environmental changes on gene expression.

Droplet-based, quantitative detection of gene expression has been achieved even at the single-cell level [20] and many review and research papers have already highlighted the unique advantages of droplet-based microfluidics for monitoring gene expression [1,21,22]. For example, Huebner et al. encapsulated single cells into aqueous microdroplets and then detected the expression of a fluorescent protein individually [15]. Due to the capability for high-throughput analysis (>10⁷ sample throughput per day), droplet-based gene expression analysis can be applied to many biological studies. Also, as shown in Figure 1(a), Shim et al. demonstrated the compartmentalization of single bacterial cell within a droplet of picoliter volume on a chip [23]. The chip not only facilitated the study of the dynamics of protein expression but also measured enzymatic activity in individual cells. This can be a powerful tool for investigating the heterogeneity of cells in identical culture environments. However, some of the bottleneck issues related to droplet-based microfluidics include droplet shrinkage, size variations, encapsulation of cells based on poisson distribution and intra-group variations. In addition to droplet-based methods, microfluidic-array-based high-throughput devices have been developed [24–27]. In particular, Thompson et al. reported a microfluidic array device for high-throughput analysis of gene expression profiles using the phenomenon of diffusive mixing in a cell culture chamber [25]. King et al. developed a similar array-based high-throughput microfluidic device capable of analyzing gene expression in living cells and revealed a distinct dynamics in gene expression (Figure 1(b)) [24].

Regulation of gene expression in response to both intracellular and extracellular stimuli can be analyzed using microfluidic devices. Methods to introduce intracellular stimuli require highly elaborate microdevices or functional nanoparticles to access the insides of the cell. Recently, nanoparticles have been widely developed as a novel means for applying intracellular stimuli to regulate gene expression, but this is beyond the scope of our review [28]. Instead, extracellular methods are used, which typically involve the application of mechanical or chemical stimuli to regulate gene expression in the cells [29]. Microfabricated and mechanically confined microenvironments have been used to investigate the persistence of antibiotic resistance in E. coli by allowing cells to grow and divide in straight microchannels [30]. Using these mechanical confinement interfaces, Balaban et al. revealed the phenotypic switching that occurs between cells in the absence of antibiotics (with normal growth rates) and that in the presence of antibiotics (with reduced growth rates), and thus was able to relate the inherent heterogeneity in a bacterial cell population to persistence. In addition, microfluidic confinement of single cells is used to study the behavior of quorum sensing on growth, as microfluidic confinement allows us to control and monitor gene expression in a single cell [31]. Temperature gradients in a microfluidic device have been developed using a typical Y-shaped channel [32] and time-specific switching of temperature is used to investigate the patterning of cells [33]. Temperature gradient generator devices can be considered useful for controlling and understanding the effects of extracellular stimuli on gene expression and regulation. Furthermore, chemical stimuli with spatiotemporal gradients have been very widely used to actively regulate gene expression. Many microfluidic devices with biochemical interfaces have been developed that use gradient flow to regulate gene induction or inhibition [34–36]. Charvin et al. reported that a microfluidic device can control gene expression temporally and monitored the long-term fluorescence response [37]. Lastly, since oxygen plays a crucial role in regulating cells, a microfluidic oxygen gradient generator device has been developed by using arrays of electrodes transducing current into oxygen via electrolysis [38].

In contrast to genomics and proteomics, metabolomics helps explain actual cellular behavior and hence is gaining increased importance in biological studies [39]. By investigating the metabolite, a biologist can analyze an organism’s phenotype faster. For the synthetic biologist, metabolomics is especially crucial in predicting the product yield of an engineered metabolic network. However, decoding the metabolome is very difficult because, with the macro-scale devices, there are no tools to amplify the signal from metabolites that are at submicromolar concentration. The cell extracts usually contain various metabolites in diverse ratios. For that reason, high-resolution separation and sensitive detection are required for metabolite analysis [40]. Tools for metabolite analysis include nuclear magnetic resonance (NMR) spectroscopy, gas chromatography coupled to mass spectrometry (GC-MS), and liquid chromatography coupled to MS (LC-MS) [41]. These instruments favored the development of a database of metabolites and enable label-free, multiple-compound detection. However, the cost, handling, and maintenance of the instruments limit their applications [9] and a time consuming sample preparation step is always needed to guarantee the sensitivity of the techniques. Microfluidics makes new ways for the study of metabolites. Microfluidics can offer a platform for faster sample preparation, better separation, and robust analysis. However, faster analysis does not normally promise a better detection [40]. Extensive microfluidics-based metabolite detection methods have been reviewed elsewhere [40]. In this section, the possibility of monitoring a metabolite (both intracellular and extracellular) on a microfluidic chip is discussed.

Another main emphasis of synthetic biology is to understand symbiosis in microbial communities, which works efficiently in multi-cellular environments to perform complex tasks like cellulose degradation, methanogenesis, nitrogen fixation, and degradation of toxic compounds. It is important to understand natural cell communities before developing an artificial cellular community (e.g., quorum sensing systems). Microfluidics not only offers a way for co-culture of several species of bacteria but also provides a platform for single-cell culture. Although bacteria live in symbiosis with other microbes in nature, co-culture of microbes in the laboratory had always been a difficult task due to the competition and dominance between different groups of microbes. Microscale spatial separation of different species of microbes provided with chemical communication helped in the co-culture of different microbial species [54]. Controlled co-culture of a microbial community may help understand and harness beneficial natural microbial communities to create a synthetic community with novel function.

The uncultivable microbial species area major challenge in microbiology. Despite the presence of a large pool of microorganisms that grow in the laboratory, a vast majority of the microorganisms are uncultivable even with a rich medium. The group of uncultivable microbes is of particular interest to the synthetic biologist as they may provide evidence for evolution and are the main source of novel genes. Isolation of a pure culture of uncultivable microbes is impossible without isolation chip (Ichip)-based microfluidics. An Ichip offers a miniaturized diffusion chamber that helped isolate a significant and novel group of microorganisms from environmental samples. The microbial species presented in the Ichip were different from those obtained with a rich medium in a Petri dish [55].

Separation and screening of living cells is an essential preparatory step in not only cell-based biological and physiological studies, but also practical applications such as cell engineering, clinical immunoassay, and drug tests. Whole-cell assay is a pre-requisite in toxicogenomics to study the biological impact of toxic compounds. However, conventional macroscale separation methods in biology, such as sieve filtration and gradient centrifugation, have a high risk of causing damage to the cells due to the strong mechanical stress caused by the ultra-fast rotating speed or the viscous forces generated by micropores on the membranes, and the separation favors collection of cells of similar size rather than individual cells. Also, more elaborate methods have been developed, such as fluorescence-activated cell sorting (FACS) and magnetically activated cell sorting (MACS), that require a large volume of samples and are also labor and time intensive [56]. For miniaturized FACS, Y-shaped junctions are widely utilized for positioning individual cells at the center of a laminar flow controlled by optical tweezers. After detecting a fluorescence signal [57], an EOF [58] can be implemented to switch the position of the cell from the center to one of the edges based on the fluorescence signal. The main advantage of the microfluidic FACS, compared with the conventional FACS, is the ability to sort cells at a faster rate (~100 cells/s) [57]. Also, microfluidic approaches are free from contamination with cells of the previous run as these microchips are disposable, being fabricated from cost-effective materials. In a similar manner, for miniaturized MACS, sample cells are labeled using magnetic beads with an antibody acting as an anchor between the magnetic beads and the cells [59–61]. Then, magnetic fields are induced to control the position of the cells for continuous separation and sorting. Compared with a fluorescence signal, magnetic fields can extend over longer distances and manipulate cells simultaneously, resulting in higher throughput (1011 cells in 30 min) [62].

Active sorting mechanisms rely on external forces such as an optical, magnetic, dielectrophoretic, or acoustic force. However, another class of efficient and continuous cell sorting devices was developed by using passive sorting mechanisms. These mechanisms were combined with several mechanical and physical properties of cells, such as size, density, shape, deformability, and polarizability. The cell separation using the micropillar structure [63,64] and obstacle structures [65,66] are fabricated in microfluidic channels, depend on size and deformability of cells. This approach does not require an additional driving force besides a hydrodynamic flow. Hence, it offers a simple and continuous way to separate any particle or cell on the basis of size [67]. Due to superior abilities of microfluidic technology in the control of the environment surrounding a cell, it is possible to cultivate cells in controllable conditions such as chemical concentration, ambient temperature, and external forces for a longer duration. Controlling chemical concentration gradient and other environmental conditions provides a high possibility for cell separation based on the native and robust chemotactic [17,68], chemostatic [69], and quorum sensing responses of the cell [31]. This approach inherently has no physical or mechanical stress that can cause cell death or mutation. Also, this technique can be utilized to find a unique cell type that has optimized resistance and survival abilities in a certain environment.

It is evident from the above discussion that the main bottleneck in the progress of synthetic biology is the need to completely understand biological systems. Despite the many advances in the technology to assess a biological system, it is very difficult to understand their behavior because of the asynchronous nature exhibited even in a homogenous population of cells. Recently, microfluidics technology has been proving its potential to offer a means to detect biological response at the single-cell level by automation and multiplexing. However, microfluidics technology should be made simpler so that even non-experts can work with it. Microfluidic devices should form a part of the laboratory, like other devices in the lab (for example, PCR machine). Microfluidics offers individual platforms for different fields of biology, such as DNA synthesis, sequencing, DNA amplification, cell free-protein expression and functional genomics, proteomics, and metabolomics. A combination of all these devices would be a robust and time-saving platform for diagnosis of various diseases. Functional screening of drugs with better efficacy would be made simpler with the aid of microfluidics.

The field of synthetic biology has been progressing rapidly with the success of rebooting life from a chemically synthesized genome [71] and multiplex automated genome engineering (MAGE) for large-scale programmed evolution of cells in a week’s time for an improved phenotype [72]. With the robust and advanced analysis methods contributed by microfluidics technology, it will be possible to better understand and analyze biological systems and hence engineer them efficiently. Microfluidic devices can help in screening population of millions of organisms to find the most efficient producer of target enzyme or a fuel. Microfluidics technology will provide the hope that the synthetic biologists dream of constructing and understanding the machinery of life (like engineers control mechanical devices) is not too far from reality.

Recent progress in genetic engineering and molecular biology has opened a new era of science called synthetic biology. The study intends to build artificial organisms by applying the engineering approach to biology [73]. Synthetic biology is considered to have a huge impact in various industrial fields by providing cheap and readily accessible drugs, developing new anti-cancer drugs, and producing biofuels. One excellent example is artificial artemisinin, an antimalarial drug produced by engineered E. coli [2]. The synthetic biologists deal with biological parts (modules, circuits, and systems), similar to modern electronic engineers in many ways. In electronics, engineers design circuits using quantitative knowledge of device function; however, the synthetic biologist designs a synthetic organism using a quantitative approach for the genes and biological pathways. While electronics perfectly controls signal transmission by restricting the signal line, the biological counterparts are disturbed by multiple bypass pathways due to the stochastic behavior of each component [74]. To solve these problems, new techniques that can offer stable and robust tools for precise control of microenvironments and reactions on the cellular level are highly required. Thus, microfluidic techniques are a vital and key technology in synthetic biology [9]. The advantages of microfluidics are obvious. Compared with typical pipette-based lab-scale equipments that deal with milliliter to microliter volumes of fluid, the microfluidic technique deals with just nanoliter to picoliter volumes and hence requires lesser reagents. In addition, flow in a microchannel is laminar rather than turbulent due to the size of the microchannel (typically, a few micrometers) and thus favors a highly predictable and controllable flow. Thanks to the recent developments in microfabrication technology, physical and geographical interactions between cells and environments can be studied in ways that were previously not possible with conventional technologies. With these advantages, microfluidics technology revolutionizes the way we study cellular environments. The technology has been successfully applied to many biological problems, especially high-speed PCR [75,76], gene sequencing [77,78], high-throughput screening [79–81], and quantitative analysis of multiple or single cells [82–84]. The technologies used for monitoring synthetic bacterial cell-to-cell signaling [85,86], screening of biomass-to-biofuel conversion enzyme [87,88], and in situ monitoring of synthetic organisms [30,69,89] have shown great promise with the combination of these two fields.

A paradigm shift from macroscale methods to microscale analysis of biological parts has been a boon for synthetic biology. Microfluidics is one of the best ways to accomplish automation and multiplexing of biological parts. The importance of microfluidics in synthetic biology will be more appreciated if the process of handling the microchips is simplified in order that it could even be managed by a non-expert.