J. Flow Chem. 2011, 1, 3–12Review

DOI: 10.1556/jfchem.2011.00003 © 2011 Akadémiai Kiadó

Microflow Systems for Chemical Synthesis and Analysis: Approaches to Full Integration of Chemical Process

Kazuma Mawatari1, Yutaka Kazoe1, Arata Aota1, Takehiko Tsukahara2, Kae Sato3 and Takehiko Kitamori1*1 Department of Applied Chemistry, School of Engineering, The University of Tokyo, Bunkyo, Tokyo 115–8656, Japan

2 Research Laboratory of Nuclear Reactors, Tokyo Institute of Technology, Meguro, Tokyo 152–8550, Japan3 Department of Chemical and Biological Sciences, Japan Women’s University, Mejiro, Tokyo 112–8681, Japan

Integrated microchemical systems on microchips, which are based on continuous microflows, are expected to become important tools for analysis and chemical synthesis applications for biological sciences and technologies. For these purposes, general integration concepts have been developed, including microunit operations (MUOs) and continuous-flow chemical processing (CFCP) to create fully functional systems for various chemical processing applications. The general methodology has enabled analysis, synthesis, and fabrication of chemical systems on microchips, and these microsystems have demonstrated superior performance (e.g., rapid, simple, easy operation, and highly efficient pro-cessing) compared to conventional methodologies. Microchemical technology has now entered the phase of practi-cal application. In this review, we discuss the methods for integration of continuous flow-based chemical process on microchips, relevant technologies, and applications.

Keywords: microunit operation (MUO), continuous-flow chemical processing (CFCP), microreactor, chemical synthesis, bioanalysis, environmental analysis

1. Introduction

Recent micro- and nanofabrication technologies have yielded integrated microchemical systems, which are essential to real-ize high-speed, functional, and compact instrumentation. The systems are based on continuous microflows, where the flow is laminar with viscous force, heat and mass transfer is governed mainly by diffusion and rapidly completed by the short diffu-sion distances, and reactions are significantly affected by large interfacial areas with increased surface-to-volume ratios. Such instruments can be used for practical purposes such as chemi-cal analysis and synthesis, and for pure and applied biologi-cal research. In the 1990s, most microchip-based systems were used for gene or protein analysis and employed electropho-retic separation with laser-induced fluorescence (LIF) detection [1,2]. However, other analytical and synthesis methods were required for more general analytical, combinatorial, physical, and biochemical applications that involve complicated chemical processes, organic solvents, neutral species, and nonfluorescent-molecule detection. For these applications, general microinte-gration methods on microchips became quite important.

Kitamori et al. has developed general methods and concepts for microintegration of chemical systems based on system approaches. To describe these concepts, we draw comparisons to the bulk chemical processing plant where bulk-scale oper-ations could include multiphase reaction, mixing, separation, detection, etc. In some cases, an entire chemical process can be miniaturized in a microfluidic system in order to simplify, automate, and improve efficiency. To accomplish this, indi-vidual operations in the bulk chemical processing plant can be scaled down to create discrete miniaturized components that perform similar functions (Figure 1). These unit operations are termed as microunit operations (MUOs), and can be combined in parallel and in serial for continuous-flow chemical process-ing (CFCP). The function of the integrated microchemical chip is therefore analogous to that of a chemical central processing unit. In order to miniaturize such operations in a microdevice, several major hurdles had to be overcome, such as the difficulty of handling multiphase flows in microfluidics, and the difficulty

of performing detection in ultra low volume systems. Because of advances in multiphase flow stabilization and integrated detection approaches, among others, miniaturized chemical plants employing MUOs have been realized. These miniatur-ized chemical plants have demonstrated superior performance with shorter processing times (from days or hours to minutes or seconds), smaller sample and reagent volumes (down to a single drop of blood), easier operation (from skilled operator to general people), and smaller system sizes (from 10-m chemi-cal plants to desktop plants and mobile systems) compared to conventional bulk-scale analysis, diagnosis, and chemical syn-thesis systems. Practical prototype systems have also been real-ized in environmental analysis, clinical diagnosis, cell analysis, gas analysis, drug synthesis, microparticle synthesis, and so on. For example, a portable micro-enzyme-linked immunosorbent assay (micro-ELISA) system was recently constructed where the analysis time was several minutes, rather than several hours as in the conventional technique (see Section 4.2). The sam-ple volume (5 ml) was also considerably smaller than the vol-ume (several milliliters) required for conventional diagnosis. In addition, the operation itself was much easier, while con-ventional ELISA needs much experience. Its correlation with conventional methods was confirmed with real human blood samples. These features are promising for point-of-care (POC) clinical diagnosis.

Now, microtechnology is moving toward practical applica-tion. Although some practical prototype systems have been realized, attaining long-term stability in liquid/liquid and liquid/gas multiphase parallel flow can be sometimes problematic. For robust fluidic control, methods for stabilizing or recover-ing multiphase parallel flow and the fluidic devices are essen-tial. Additional challenges for real-world applications include world-to-chip interfacing, and robust components that allow for flexible and reliable liquid handling for high-throughput chemi-cal processing on a microchip.

In this review, we introduce several basic methodologies and applications. First, we discuss the general concepts for micro-integration (MUOs and CFCP), and general methodologies for their realization. Then, practical applications utilizing the inte-grated microchips are discussed in some detail.* Author for correspondence: kitamori@icl.t.u-tokyo.ac.jp

JFC-D-11-00003.indd 3 8/1/2011 6:30:34 PM

Micro Systems for Chemical Synthesis and Analysis

4

2. MUOs and Continuous-Flow Chemical Processing

2.1. Design and Fabrication Methodology for Integration of Complex Microchemical Systems. Integrated microchemi-cal systems are expected to be applied in various fields. In order to realize general analytical, combinatorial, physical, and bio-chemical applications, general integration methods on micro-chips are quite important. Conventional macroscale chemical plants or analytical systems are constructed by combining unit operations such as mixers, reactors, and separators, and a simi-lar methodology can be applied to microchemical systems. By combining MUOs with different functions in series and in par-allel, various chemical processes can be integrated into micro-chips through use of a multiphase microflow network such as that shown in Figure 2. This methodology is termed CFCP [3]. Miniaturizing conventional unit operations is often not effec-tive and sometimes impossible because many physical proper-ties (e.g., heat and mass transfer efficiency, specific interfacial area, and gravitational force) and dominant factors for fluid-ics and chemistry are significantly different in microspace. Therefore, novel MUOs taking these issues into account are needed. Kitamori et al. have developed MUOs for various pur-poses, such as mixing and reaction [4,5], phase confluence and separation [6–17], solvent extraction [18], gas/liquid extraction [19–22], solid-phase extraction and reaction on surfaces [23–34], heating [35–39], cell culture [40–44], and ultrasensitive detection [45–56] (Figure 3).

2.2. Example of Continuous-Flow Chemical Processing and its Design. Microcobalt wet analysis is an example of CFCP that

has been demonstrated (Figure 4) [3]. Conventional procedures for such analysis consist of a chelating reaction, solvent extraction of the complex, and decomposition and removal of the coexisting metal complex. These procedures are examples of conventional unit operations: mixing and reaction, phase confluence, solvent extraction, and phase separation. These unit operations are anal-ogous to MUOs. CFCP is designed by combining the MUOs in series and in parallel, as shown in Figure 3. The analysis time in this system is only 50 s versus 6 h for conventional devices. Microchemical processing through the use of CFCP has been demonstrated on numerous applications (Table 1).

Multistep synthesis of carbamate is another example of CFCP that has been demonstrated (Figure 5) [57]. Conventional procedures for such synthesis consist of a phase transfer reac-tion between aqueous azide and acid chloride, reaction on sur-faces for formation of aryl isocyanates, and reaction between aryl isocyante and alcohol. These procedures are also examples of conventional unit operations: phase confluence, mixing and reaction, phase separation, decomposition by heating, and reac-tion on surfaces.

The CFCP approach can be applied to develop more com-plicated processing systems. More rapid analysis, bioassays, and immunoassays, as well as more efficient reaction and extraction, can be achieved through CFCP systems compared to con ventional devices. However, development of complicated microchemical processing systems is laborious because of the need to determine appropriate conditions for each reaction and separation process at the miniaturized scale, and also to design and optimize the microchannel structure. In semiconductor integrated circuit design, computer aided design (CAD) is typi-cally used to perform layout, circuit analysis, integrated device analysis, and logical simulation. If CAD is developed specifi-cally for microchemical processing, which includes microfluid simulation, reaction time analysis, extraction time analysis, and microchannel structure design, the development time for these systems would be drastically shortened.

3. Fundamental Technologies: Fluidic Control and Detection Methods

3.1. Multiphase Microflows 3.1.1. Multiphase Microflow Network. For many chemical

processing applications, microchemical systems should include solvent extraction and interfacial reaction components utilizing

Figure 1. Concept for micro-integration of chemical systems: (a) scale down of bulk scale unit operations and (b) integration on micro-chemical chip

Figure 2. Multiphase parallel flow network by CFCP (an example of integration of wet analysis on a single microchip)

JFC-D-11-00003.indd 4 8/1/2011 6:30:34 PM

Kazuma Mawatari et al.

5

Method Analyte LODa Analysis time Reference(s)

Solvent extraction Iron complex 7.7 zmol 60 s 8Cobalt complex 0.13 zmol; 0.72 zmol 90 s; 50 s 3, 9Dye molecules NA 4 s 10Amphetamine 0.5 mg mL−1 5 min 12Uranium (VI) 24 mg mL−1 1 s 13p-chlorophenol NA 2 s 16Dimethylformamide NA 20 s 17

Gas extraction Formaldehyde 8.9 ppb 41 min 19Ammonia 1.4 ppb 16 min 20Hydrogen sulfide 0.1 ppb NA 21

Immunoassay s-IgA <1 mg mL−1 <1 h 23Carcinoembryonic antigen 30 pg mL−1 35 min 24Interferon-g 10 pg mL−1 50 min (4 samples) 25B-type natriuretic peptide 0.1 pg mL−1 10 min 27Staphylococcus enteroxin B 28.5 fg mL−1 20 min 29Tacrolimus 10 pg min−1 15 min 30IgG 244 pg min−1 <30 min 31

Flow injection Fe2+ 6 zmol 150 s 4Ascorbic acid 1 zmol 30 s 5

Enzymatic assay H2O

250 zmol NA 35

aAbbreviations: Ig, immunoglobin; LOD, limit of detection; NA, not available.

Table 1. Examples of microchemical processing

Figure 3. Realization of various chemical processes by MUOs and CFCP

Figure 4. Conventional analytical procedure and conversion to MUOs

both liquid and gas phase reagents, and both aqueous and organic solutions. All of these must be readily manipulated to realize general chemistry in a microchip. In the 1990s, elec-troosmotic flow was used in microchip electrophoresis [1,2]; however, electroosmotic flow control is restricted to mainly aqueous solutions. Therefore, electroosmotic flow is not suitable as a general flow-control method to integrate various chemical processes that require other types of solvents such as organic solvents. Then, simple pressure-driven flow has been developed by many groups as a nonelectrical pumping method [58–60]. Yager et al. have developed diffusion-based extraction of mis-cible solutions and realized separation dependent on diffusion rates [59,60].

Recently, microsegmented flows of immiscible solutions were used for microchemical processes [57,61–63]. Although microsegmented flows are advantageous for mixing and rapid molecular transport, phase separation and more than three-phase contact are difficult. Furthermore, controlling the microsegmented flows in a wide range of flow rates is diffi-cult because the size of the segments changes along with the

JFC-D-11-00003.indd 5 8/1/2011 6:30:37 PM

Micro Systems for Chemical Synthesis and Analysis

6

flow-rate ratio of the two phases. As a result, integration of com-plicated chemical processes by microsegmented flows becomes quite difficult, while it is more suitable for high-throughput applications including simple chemical operations. In order to solve these problems in microsegmented flows, parallel multi-phase microflows were developed [18], where two immiscible solutions flowed side by side (in parallel) along the microchan-nels. Parallel multiphase microflows in the laminar flow regime allow better design and control of microchemical processes because they are stable in a wide range of flow rates and can realize more than three-phase contact, which result in realiza-tion of integration of complicated chemical processes.

3.1.2. Fundamental Physical Properties of Multiphase Microflows. Since multiphase microflows are often needed for applications in chemical processing, control methods to produce stable multiphase microflows in a wide range of flow rates are required, and are an important basic MUO component. However, the methods employed in the conventional size devices cannot be applied to MUOs because the dominant physical properties in the micro space are different from the macro space. In con-ventional macroscale devices, the aqueous and organic phases are separated by gravity. In microspace, however, the fluid is greatly influenced by liquid–solid, liquid–gas, and liquid–liquid interfaces because of the large specific interfacial area. The main physical forces in the microchannels which include vis-cous force and interfacial parameters can be analyzed using the dimensionless Reynolds (Re) and Bond (Bo) numbers, defined as an inertia-to-viscous force ratio and a gravity-to-tension ratio, respectively. Bo is defined as Bo = (Dr)gd2

h/g, where Dr, g, and

dh are the density difference, the interfacial tension between the

two phases, and the equivalent diameter, respectively, and where g is the gravitational acceleration (9.8 m s−2). In microspace, with a d

h of 100 mm, the interfacial tension exceeds gravity by

three orders of magnitude when a water–toluene two-phase flow is formed (Dr = 0.132 × 103 kg m−3, g = 36.3 mN m−1). When water flows in a microchannel with a d

h of 100 mm at a flow rate

of 1 mL min−1, Re becomes 0.05. Therefore, multiphase micro-flows are generally considered to be laminar flow.

Multiphase microflows are dominated by pressure [14,64]. It is important to consider that the pressure driving the fluids decreases in the downstream part of the flow because of the flu-id’s viscosity. When two fluids in contact with one another have different viscosities, the pressure difference (DP

Flow) between

the two phases is a function of the contact length and the flow velocity. Another important parameter is the Laplace pres-sure (DP

Laplace) caused by the interfacial tension between two

phases. The position of the interface is fixed at a position in the

microchannel determined by the balance established between DP

Laplace and DP

Flow.

Figure 6a illustrates the pressure balance at the liquid–liquid interface of a two-phase microflow. The liquid–liquid interface curves toward the organic phase with a glass surface because of the hydrophilicity of the glass. DP

Laplace is generated at the

curved liquid–liquid interface. On the basis of the Young–Laplace equation, DP

Laplace is estimated as

R d= =

−g g q2 90sin( °),ΔPLaplace

(1)

where R is the curvature radius of the liquid–liquid interface and q is the contact angle. The contact angle is restricted to the values between the advancing contact angle of the aqueous phase, q

aq, and that of the organic phase, q

org. Therefore, DP

Laplace

is restricted as follows:

2 90 2 90g q g qsin( ) sin( ).aq org

d d− −

P< <Δ Laplace

° °

(2)

When DPFlow

exceeds the maximum value of DPLaplace

, the organic phase flows toward the aqueous phase. When DP

Flow is

lower than the minimum value of DPLaplace

, the aqueous phase flows toward the organic phase. When the flow-rate ratio is changed, the pressure balance is maintained by changing the position of the liquid–liquid interface. This model indicates that the important parameters for micro fluid control are the inter-facial tension, the dynamic contact angle, and the depth of the microchannel. This model can also be applied to gas–liquid microflows. In order to stabilize the interface, DP

Laplace should

be maximized by controlling channel size, shape, and surface hydrophobicity.

3.1.3. Surface Modification for Stabilization of Multiphase Microflows. Disturbance of flow rates can destabilize multi-phase microflows. However, robust and effective control meth-ods have been developed that take advantage of the fact that the microchannel surface structure and surface-free energy can be altered to promote stabilization of two-phase flows. It has been determined that multiphase microflows can be stabilized by altering the structure or adding features to the microchan-nels, specifically by including a guide structure [3] or a pillar structure [65], which permits a wider range of possible contact angles for the fluid interface than a flat surface.

Some groups have proposed selective chemical surface modi-fication to change surface-free energy for stabilization of the multiphase microflows [7,11,14,15,66–70]. Figure 6b illustrates the shape of the liquid–liquid interface in a microchannel with chemically patterned surfaces. The contact angle of water on a hydrophobic surface can be larger than 90°. Therefore, mul-tiphase microflows in microchannels with patterned surfaces

Figure 6. Pressure balance at the liquid–liquid interface of the two-phase parallel microflows for (a) hydrophilic surface and (b) partially hydrophobic and hydrophilic surface

Figure 5. An example of multistep synthesis of carbamate

JFC-D-11-00003.indd 6 8/1/2011 6:30:39 PM

Kazuma Mawatari et al.

7

can be formed under a wider range of conditions than micro-channels with a guide structure or pillar structure. In chemically patterned microchannels, microcounter-current flows can form [11,14]. Because DP

Laplace depends on the depth of the micro-

channels, more effective stabilization of multiphase microflows can be obtained by utilizing shallower microchannels. For example, a microstructure combining shallow and deep micro-channels with both hydrophobic and hydrophilic surfaces can produce a large DP

Laplace. In a shallow 10 mm deep microchannel,

the DPLaplace

of the gas–water interface is 6 kPa. Microchannels having an asymmetric cross section and the patterned surfaces mentioned above are effective not only for the stability of the multiphase microflows, but also for the robustness required for practical applications. Air bubble contamination can also dis-turb the stability of systems. By using microchannels having an asymmetric cross-section with patterned surfaces, one can take advantage of this instability to convert plug flow into two-phase microflow [69,70].

Superhydrophilic and superhydrophobic surfaces are more effective at stabilizing two-phase microflows. Such surfaces can be fabricated by creating roughness with titanium nanoparticles. Titanium nanoparticle modification of a microchannel yields a nanometer-scale surface roughness, and subsequent hydro-phobic treatment produces a superhydrophobic surface [71]. Photocatalytic decomposition of the coated hydrophobic mol-ecules was used to pattern the surface wettability, which was tuned from superhydrophobic to superhydrophilic under con-trolled photoirradiation [71]. This method can also be applied to converting plug flow to two-phase microflows [72].

3.2. Detection Methods3.2.1. Single Molecule Detection Methods. Microsystems

require sophisticated detection technologies due to the micro-channels’ small volumes and short optical path lengths. For example, the time-averaged amount of analyte in 1 mm3 (1 fL) of a 1 nM fluid becomes 10−24 mol, which is less than a single

molecule. Therefore, the detection methods on microchips should be capable of single-molecule sensitivity.

So far, many sensitive detection methods have been devel-oped, and these are mostly dominated by optical and electro-chemical methods which are summarized in Table 2. These methods show excellent performance for some analytes. How-ever, the analytes are usually limited by the detection princi-ple employed (fluorescent molecules for LIF, and SERS active molecules for SERS). The nanopore device is a novel detection platform which can detect single ion passage by current change, although integration with microchemical processing on micro-chips is still challenging.

Kitamori et al. [73] have developed a thermal lens micro-scope (TLM) for the sensitive detection of nonfluorescent mol-ecules in microspace, which is an ultrasensitive absorption spectrophotometer. The basic principle of TLM is illustrated in Figure 7a [73,74]. In TLM, two laser beams are utilized: an excitation laser beam and a probe laser beam. The wavelength of the former is tuned to the light absorption band of the sam-ple, and the wavelength of the latter is adjusted to avoid the absorption band. The excitation laser is tightly focused onto the sample by an objective lens. The diameter of the beam waist is usually ~1 mm. The sample absorbs the excitation beam, and heat is released into the solvent around the molecule, increas-ing the temperature of the solution. The light absorption is lin-early dependent on the light intensity distribution (which is a Gaussian distribution), and the resultant temperature distribu-tion is similar to the intensity distribution. For detection of the thermal lens effect, a probe beam is coaxially introduced into the objective lens and is focused on the sample. When the exci-tation beam is focused on the sample and when the thermal lens effect is induced, the optical path of the probe beam changes. The change in the optical path is detected by measuring the change of the intensity (DI) through a pinhole; DI is usually proportional to the concentration of analytes in dilute solutions.

Method LOD Reference(s)

Optical Laser-induced fluorescence (LIF) Single molecule 51Surface-enhanced Raman scattering (SERS) Single molecule 52, 53Thermal lens microscope (TLM) 0.4 molecule/7 fL 54Differential interference contrast microscopy Single macro molecule (DNA) 55

Electrochemical Scanning electrochemical microscope (SECM) Single molecule 56Current detection through nanopore Single molecule 57

Table 2. Representative detection methods with single molecule sensitivity

Figure 7. Illustration of TLM: (a) basic principle and (b) importance of focal length difference

JFC-D-11-00003.indd 7 8/1/2011 6:30:41 PM

Micro Systems for Chemical Synthesis and Analysis

8

The sign of DI depends on the thermal lens effect on the probe beam, which either converges or diverges, and is determined by the relative focal positions of the probe and excitation beams. When the focus of the probe beam is closer to the objective lens than the excitation beam is, the probe beam diverges.

The focal length difference between the excitation and probe beams, as shown in Figure 7b [50], is the most important factor in achieving sensitive TLM. When the focal lengths of the two beams are the same, the probe beam is focused at the center of the thermal lens. In this case, the optical path of the probe beam does not change, and the signal intensity is almost zero. An adequate focal length difference (Dz) is also necessary to attain optimum sensitivity. Some research groups attempted to realize quasi-TLM by using a noncoaxial beam configuration due to the difficulty of performing sensitive thermal lens measurement under an objective lens [75]. Kitamori et al. [73] found that the low-grade microscope used in the first TLM had a 2.2-mm chro-matic aberration for the two coaxial beams, and sensitive TLM was possible.

The first large TLMs required laborious optical alignment. They have since been integrated and commercialized as desktop- [76] and palm-sized TLMs [46], as shown in Figure 8. In the palm-sized TLM, laser diodes are used for the excitation and probe beams, which are introduced into a microchannel via a light mixer and a single-mode optical fiber. The palm-sized TLM can also be integrated into a system incorporating a microchip. For example, there has been a report of an allergen-analysis system employing a palm-sized TLM [28], wherein all the components including lock-in amplifier and power supply have been integrated in a mobile format.

Thermal lens detection under a microscope allows ultrasen-sitive detection of nonfluorescence molecules. An example of dye solution detection in a microchannel at the single-molecule level has been reported [45]. In this experiment, the microchan-nel was 150 mm wide and 100 mm deep. The authors used ben-zene as the solvent and determined that the molar absorption coefficient of the porphyrin complex was 3.2 × 104 M−1 cm−1. The expectation value of number of molecules in detection volume was calculated by multiplying the concentration by the detection volume (7 fL). The time constant of the lock-in amplifier was 1 s, which was longer than the molecular resi-dence time (~1 ms) in the detection volume. The signal inten-sity showed good linear dependency on the expectation value. The lower limit of detection (LOD) was estimated to be 0.32 molecules, given a signal-to-noise ratio of 2 and a signal-to-background ratio of 0.1 for an averaging time of several sec-onds. This ultrahigh-sensitivity technique has wide applications for nonfluorescent molecules and promises to become a power-ful tool for various microchip-based analytical or chemical syn-thesis applications.

Functional TLMs have also been developed. The excitation wavelengths range from deep ultraviolet to near infrared by special optical design for ultraviolet laser beams [49]. In addi-tion to concentration determination, many other functions have been realized. For example, TLM can sensitively and selectively detect chirality of the molecules through phase modulation of the excitation beam [48]. The sensitivity thus attained is 1 mil-lion times higher than that of a conventional detector (e.g., a circular-dichroism spectrometer). One can also use TLM for in situ flow-velocity determination by tuning the wavelength of the excitation beam to the infrared (1480 nm) region and by detect-ing the light absorption of water molecules downstream via the thermal lens effect [77]. TLM can also be utilized for imaging through scanning the laser beams. Direct imaging of a single neuron cell was demonstrated by measuring cytochrome c to monitor apoptosis. In situ imaging of a single cell with a spatial resolution of nearly 1 mm was also successfully realized [50]. Many other applications, such as in situ immunoassays on bio-logical cells and membranes, have also been reported [78]. Development of new methods for single-molecule detection is under way. TLM is a powerful analytical tool for chemistry and biochemistry in both micro- and nanospace, and may become an important component in functional and compact instrumen-tation for analysis, diagnosis, and chemical synthesis.

3.2.2. Interfaces of Conventional Detection Methods. Cou-pling with conventional analytical instruments is another way to create analytical functions for microchips. For example, in micro-chip chemical synthesis applications, postsynthesis quantitative or qualitative analysis techniques are required to identify unknown products. Electrospray ionization-mass spectrometry (ESI-MS) is widely used for quantitative analysis, and many researchers have reported interfaces between MS and microchips [79]. Although ESI-MS is known to be a highly sensitive analytical technique, the structural information which it provides is not as useful as that provided by nuclear magnetic resonance (NMR).

Massin et al. [80] have developed a microcoil-based micro-fluidic NMR probe consisting of electroplated planar micro-coils integrated on a microfabricated glass substrate with etched microchannels. However, this method requires expensive, spe-cially modified probes, and the frequency resolution is not very high. Another recently reported microfluidic device, a micro-channel cell for synthesis monitoring (MICCS) [81], works simultaneously as a microreaction device and as an interface to a conventional NMR instrument. In this study, the authors con-nected the microchip to PEEK™ parts, which were in turn con-nected with syringe pumps. The channels included both reaction and detection components. The channel size was 300 mm wide and 100 mm deep. The channel length was 60 mm for reaction and 240 mm for detection, and the channel volumes were approxi-mately 4.4 mL for reaction and 7.2 mL for detection, respectively. The microchip was inserted into standard 5-mm j solution NMR probes, and d-solvent was filled into the probes for NMR lock. The flow-rate ratio was altered to change the reaction time. The authors demonstrated real-time monitoring of the Grignard reaction, and they observed peaks from the intermediate of this reaction. Thus, the MICCS method will likely become an indis-pensable tool for chemistry and biochemistry in microspace, especially for optimization of reaction conditions, acquisition of structural information on unstable intermediates, and determina-tion of reaction pathways and the kinetics of reactions.

4. Development of Applications: Microchip-Based Chemical Synthesis and Analysis

4.1. Chemical Synthesis4.1.1. Chemical Reactions on Microchip. Microchemical

systems have been developed for chemical syntheses in drug

Figure 8. Picture of TLM systems: (a) desktop-TLM and (b) micro-TLM system

JFC-D-11-00003.indd 8 8/1/2011 6:30:42 PM

Kazuma Mawatari et al.

9

discovery and other inorganic/organic chemical engineering. Since flows in microscale are in a regime of laminar flows, where the viscous force is dominant (Re << 1), heat and mass transfer is mainly governed by diffusion. Small diffusion length decreases a time until the transfer over the channel size is com-pleted: diffusion time of 50 ms (coefficient: 10−9 m2 s−1) for a channel of 10 mm. In addition, increased surface-to-volume ratio extremely increases the contact area of reactant flows. Therefore, rapid, stable and high efficiency/yield chemical reactions can be provided in microchemical systems.

Kitamori et al. have developed phase transfer synthesis based on immiscible liquid–liquid flow formed in a micro-channel chip [82]. The large specific interfacial areas and short molecular diffusion distances simultaneously realize effective phase transfer synthetic reaction and avoiding an undesirable side reaction. The reaction based on continuous parallel multi-phase flow is expected to synthesize large amounts of chemicals with high conversion. A phase transfer diazo coupling reaction was demonstrated as illustrated in Figure 9. Conversion of the diazonium salt of close to 100% was achieved with a residence time of starting materials of 2.3 s, while the conversion of 80% in maximum with the residence time of around 1 h for mac-roscale. The concept was extended to alkylation reactions [83] and amide formation reactions [84,85].

The large specific interfacial areas of microchannel can provide high efficiency multiphase catalytic reactions of gas–liquid, gas–liquid–liquid, or gas–liquid–solid. A system for tri-phase reactions using a microchannel reactor was developed and applied to hydrogenenation and deprotection reactions (Figure 10) [86]. A solid catalyst is immobilized on the wall, and liquid and gas are flown through the microchannel. The gas passes through the center of the channel, and the liquid flows along the inner surface of the channel, by optimal control of gas pressure. By using a palladium (Pd) catalyst, hydrogenation reactions could be achieved within a reaction time of 2 min.

The stable thermal condition in microchip realizes chemical reactions with specific fluid regimes. Supercooled microflows, where the fluid remains in a liquid state at a temperature below the melting point, were achieved for water at −15°C and −28°C with hydrophilic glass surface and hydrophobic surface modi-fied by octadecylsilane, respectively [87]. The supercooled micro-flow was applied to an asymmetric aqueous/organic two-phase reaction with a chiral phase-transfer catalyst. A higher enantio-meric excess with decreasing temperature was confirmed.

4.1.2. Developments of Microchemical Plant. Applications of microchip have a great advantage in syntheses of many spe-cies in small volumes for industries such as personalized medi-cine. On the other hand, a chemical plant of mass production

can be developed by piling up microchips to magnify the prod-uct. While development of macroscale chemical plants require a process to scale up reactions in laboratory considering fluid dynamics, microchemical system can skip this rearchitecting process and simplify the development. The product of amide formation reactions as mentioned above was successfully mag-nified by this ‘pile-up’ approach [85].

Particle/droplet synthesis has been one of the typical appli-cations of microchemical system because flows and interfaces in microchannels are repeatable and precisely controllable. A mass production system of nearly monodisperse diameter gel particles, which are available to various applications (ion exchange resin, LCD spacer, liquid chromatography, for exam-ple), was developed by piling up Y-junction microchannel reac-tors [88]. The gel particles were formed by flows of organic phase (Buthyl acetate) and water phase (3%-polyvinyl alcohol aqueous solution) at the Y-junction. The formation of homo-geneous organic droplets of the diameter dispersion degree less than 10% without any class selection was achieved by the simple microchannel. The gel particle production system with piled-up 3,000 microchannels had a production rate of 1.8 L h−1 for 200 mm gel particles.

4.2. Chemical Analysis4.2.1. Integration of Immunoassays on Microchips. Immu-

noassays are important analytical methods for clinical diagno-sis and can be used for quantitative or qualitative postreaction analysis in CFCP systems. The heterogeneous immunoassay is a particularly popular approach, wherein captured antibod-ies or antigens fixed on a solid surface react with serum tar-get antigens or antibodies. Labeled secondary antibodies then capture the targets, and the target amounts are quantified by detecting labels. Heterogeneous immunoassays feature easy and accurate bound-free separation, which is important for improving limits of detection. Shortening analysis time is nec-essary for many applications including diagnostics and chemi-cal reaction monitoring. In conventional methods, a microtiter plate well (typically 0.65 mm in diameter) is used, and the spe-cific interfacial area for 50-mL volumes is 13 cm−1. However, when a microchannel (200 mm deep × 200 mm wide) is uti-lized for immobilizing the antibodies (antigens), the interfacial area, which decreases reaction time and improves efficiency, increases to 200 cm−1.

Kitamori et al. [23] reported the successful integration of an ELISA on a microchip. The authors developed a new MUO for the heterogeneous reaction through the use of bead-packed microchannels (Figure 11). Antibodies (antigens) were immo-bilized on the bead surface via physisorption or chemisorp-tion. After immobilization, bovine serum albumin or MPC

Figure 10. Solid–liquid–gas triphase flow for hydrogenation systemFigure 9. Phase transfer diazocoupling reaction on a microchip

JFC-D-11-00003.indd 9 8/1/2011 6:30:44 PM

Micro Systems for Chemical Synthesis and Analysis

10

(2-methacryloyloxyethyl phosphorylcholine) polymer coat-ing was applied to minimize nonspecific adsorption. A micro-bead suspension with captured antibodies was introduced and stopped at a microchannel dam structure (with a 10-mm gap), and a 5−10-mm long area of packed beads area was formed. The target sample solution was introduced and captured by the antibodies (antigens). Enzyme-conjugated secondary (HRP or ALP) antibodies were then introduced to capture the antigens. Finally, substrates were applied, and the dye molecules pro-duced by the enzymatic reaction were detected by TLM, down-stream from the microbeads.

The developed method features the utilization of microbeads for immunoreactions, and by simply introducing new micro-beads, new measurements can easily be made. Recently, a por-table micro-ELISA system for clinical diagnosis was developed by designing a simple microfluidic system with integrated flu-idics, optics, and electronics [28]. The fully integrated system with multiple MUOs is illustrated in Figure 12. The samples and reagents are stocked in tubes on a moving tray. A tube, under the control of four syringe pumps, aspirates and injects the samples and reagents (microliter volume) into the micro-chip. The diversion of flow, to either waste or microchip, is con-trolled by microvalves. The tube is connected to the microchip through a hydrophobic connector with pressure resistance of >1 MPa. An immunoassay is then conducted on the microchip

and detected by an on-chip TLM [46]. In this case, stopped-flow conditions were utilized for the enzymatic reaction, and signals were obtained as a peak for each channel.

The application to real human blood samples is quite impor-tant, while a lot of conventional methods on microchip are use-ful for standard solution and sometimes face difficulties for real samples. The ELISA system was tested with real samples of 85 allergy patients using the same microchip, and a good cor-relation (r 2 = 0.939) with a conventional method (College of American Pathology method) was demonstrated. Importantly, sample volumes (5 mL) were approximately 10 times smaller and analysis time (12 min) was more than 30 times faster than the conventional method. With this stable and simple microflu-idic system, all the procedures were fully automated, from bead introduction to bead removal for new measurement, resulting in system ease-of-operation. In addition to the sandwich assay format, excellent results were obtained for competitive immuno-assays, such as methamphetamine analysis in hair. The exam-ples are summarized in Table 1.

There were many other reports on integration of immuno-assays by CFCP. Yager et al. developed a rapid diffusion immu-noassay [89]. Diffusion of a labeled probe molecule during continuous flow of two fluid streams (antigens and antibodies) was used for the immunoassay. Excellent performance in a small sample volume (<1 mL), rapid analysis time (<1 min) and high sensitivity (sub nM for small molecules) were demonstrated. A similar approach utilizing two fluid streams was also dem-onstrated by Hosokawa et al.; however, detection was instead accomplished through an amplification reaction on the channel surface [90]. A microfabricated flow cytometer has been devel-oped for the analysis of micron-sized polymer beads onto which fluorescently labeled proteins have been immobilized [91]. In a continuous flow, an immunoreaction was induced on the bead surface, and the reaction was monitored by measuring the fluo-rescence signals downstream. A high throughput micromosaic immunoassay using CFCP was also demonstrated. Antibodies were patterned on planar surfaces, and the microfluidic net-work was used for the immunoassay [92]. Sixty-four simulta-neous assays were successfully demonstrated. A new approach for integration of various MUOs using a capillary-assembled microchip is one of the promising methods for general and mul-tiplexed immunoassays [93].

4.2.2. DNA/RNA Analysis Using Amplification Reaction of Nucleic Acids. Genetic analysis employing microchemical

Figure 12. A practical micro ELISA system

Figure 11. Conventional procedure of ELISA and conversion to MUOs

JFC-D-11-00003.indd 10 8/1/2011 6:30:45 PM

Kazuma Mawatari et al.

11

chips has elicited enormous interest because the amount of original matter available for genetic analysis is often limited. Originally, this field started as a Micro Total Analysis System (mTAS) based on the technology of electrophoretic separation with laser-induced fluorescence (LIF) detection. Analysis of pL volume samples with short analysis time (seconds or minutes) was successfully demonstrated. However, the genetic analy-sis usually requires many other chemical operations such as the amplification of nucleic acids for sensitive detection. For example, polymerase chain reaction (PCR) can be used for amplification. In addition to this function, total integration of multiple functions is required for DNA/RNA analysis, includ-ing purification (e.g., via electrophoresis, solid or liquid phase extraction), amplification, separation, and detection. For the complicated chemical processes, the methodologies of CFCP will be advantageous.

In the first step, the PCR process was integrated on micro-chemical chips by CFCP. Integration of PCR on microchemical chips results in advantages such as high thermal cycling speed and low reagent consumption, which benefit from the intrinsic high surface-to-volume ratio. Manz et al. developed continu-ous flow PCR chips [58]. Three well-defined zones were kept at 95°C, 77°C, and 60°C, and the sample was hydrostatically pumped through a single channel etched into the glass chip. The channel passing through the three temperature zones induced the thermal cycling process. The process time was shortened to several minutes. Kitamori et al. developed a photothermal temperature control method by IR laser heating [36]. Extremely fast heating (67°C s−1) and cooling (53°C s−1) rates were dem-onstrated for liquid volumes as small as 5 nL. Other approaches can be illustrated in a recent review focusing on continuous-flow microfluidic PCR devices [94].

Recently, total integration for DNA/RNA analysis was also developed. Landers et al. developed a fully integrated microflu-idic genetic analysis system by integrating purification, ampli-fication, separation, and detection functions [95]. Excellent performance was demonstrated in a small sample volume (<1 mL) and short analysis time (<30 min). Ease of operation will also create important advantages for microchemical chips. Mathies et al. developed a microfluidic device for mRNA anal-ysis from a single cell utilizing reverse transcription polymerase chain reaction [96]. Integration of multiple functions, such as cell selection, localization, enzymatic reaction, and quantita-tive detection, were demonstrated; therefore, this method is expected to be a very sensitive and versatile analytical tool for single cell research. There are many other reports on the inte-gration of DNA/RNA analysis on microchemical chips which can be seen in a recent review [97].

4.2.3. Analysis Methods Using Solvent Extraction. Solvent extraction is widely utilized in analytical fields such as environ-mental analysis. Integration of chemical processes utilizing sol-vent extraction has been demonstrated for various applications [8–13,15–17]. Here, we focus on two such examples.

Urine analysis for illegal drugs is increasingly performed in forensic laboratories. Gas chromatography mass spectrometry (GC-MS) is extensively used because of its versatility and reli-ability. By way of sample preparation for GC analysis, conven-tional liquid–liquid extraction has a widespread use, but it is not only laborious but also environmentally unfriendly due to the consumption of considerable amounts of organic solvents. Therefore, microintegration of the sample preparation proce-dure is required. Kitamori et al. demonstrated the integration of the sample preparation procedure using CFCP [12]. Ease of operation and very small sample and reagent volumes were demonstrated.

Solvent extraction was also applied to nuclide separation and analysis of high-level radioactive waste [13]. Short analysis

time (<1 s) was demonstrated, and reduction of sample and reagent volume to sub-microliter levels can be expected, which provides a means for safe treatment of radioactive waste.

4.2.4. Other Analysis Methods. Gas analysis is also impor-tant in environmental analysis and the semiconductor industry. Microchemical chips possess some advantages for gas analysis. For example, continuous monitoring becomes easier because rapid analysis is possible with small reagent consumption. The miniaturized analytical device will also allow on-site moni-toring. As an example, the functions of gas extraction (gas–liquid parallel microflow) were integrated with reactions and detection by CFCP [19,20]. There are other examples of micro-integration of gas analysis system including gas chromatogra-phy, which are summarized in a recent review [98].

5. Summary and Outlook

In this review, the basic concept and the supporting meth-odologies for development of integrated microchemical sys-tems based on continuous flows have been described. MUOs of mixing, reaction, separation, extraction, heating, cell culture and detection are connected in series or parallel by CFCP to integrate chemical process on a microchip. Mutiphase paral-lel microflows are used to produce MUOs and CFCP systems, which have been applied to various chemical processes. These systems possess many advantages, including high efficiency/yield reactions, shorter reaction/analysis time, reduction of sample and reagents, integration of multiple functions and min-iaturization of the systems. The ease of operation inherent in continuous flow is also a great advantage for widespread use of the devices. Taking advantage of this wide applicability, many examples of analytical and synthetic applications were intro-duced in organic/inorganic syntheses, environmental analysis, clinical diagnosis and other fields. For chemical synthesis, mass production systems were developed by piling up microchan-nel chips. In various applications for analysis and synthesis, the microchannel chip works as a central chemical processing unit. Although more technical advancement of methodologies is required for both fluidic devices and design tools for micro-chemical processes, the microtechnology will become a useful tool for the analytical and synthetic fields.

6. References

1. Reyes, D. R.; Lossifidis, D.; Auroux, P. A.; Manz, A. Anal. Chem. 2002, 74, 2623–2636.

2. Auroux, P. A.; Lossifidis, D.; Reyes, D. R.; Manz, A. Anal. Chem. 2002, 74, 2637–2652.

3. Tokeshi, M.; Minagawa, T.; Uchiyama, K.; Hibara, A.; Sato, K.; Hisamoto, H.; Kitamori, T. Anal. Chem. 2002, 74, 1565–1571.

4. Sato, K.; Tokeshi, M.; Kitamori, T.; Sawada, T. Anal. Sci. 1999, 15, 641–645.

5. Sorouraddin, H. M.; Hibara, A.; Proskrunin, M. A.; Kitamori, T. Anal. Sci. 2000, 16, 1033–1037.

6. Hisamoto, H.; Horiuchi, T.; Uchiyama, K.; Tokeshi, M.; Hibara, A.; Kitamori, T. Anal. Chem. 2001, 73, 5551–5556.

7. Hibara, A.; Nonaka, M.; Hisamoto, H.; Uchiyama, K.; Kikutani, Y.; Tokeshi, M.; Kitamori, T. Anal. Chem. 2002, 74, 1724–1728.

8. Tokeshi, M.; Minagawa, T.; Kitamori, T. Anal. Chem. 2000, 72, 1711–1714. 9. Tokeshi, M.; Minagawa, T.; Kitamori, T. J. Chromatogr. A 2000, 894,

19–23.10. Surmeian, M.; Slyadnev, M. N.; Hisamoto, H.; Hibara, A.; Uchiyama,

K.; Kitamori, T. Anal. Chem. 2002, 74, 2014–2020.11. Aota, A.; Nonaka, M.; Hibara, A.; Kitamori, T. Angew. Chem. Int. Ed.

2007, 46, 878–880.12. Miyaguchi, H.; Tokeshi, M.; Kikutani, Y.; Hibara, A.; Inoue, H.;

Kitamori, T. J. Chromatogr. A 2006, 1129, 105–110.13. Hotokezaka, H.; Tokeshi, M.; Harada, M.; Kitamori, T.; Ikeda, Y. Prog.

Nucl. Energy 2005, 47, 439–447.14. Aota, A.; Hibara, A.; Kitamori, T. Anal. Chem. 2007, 79, 3919–3924.15. Hibara, A.; Nonaka, M.; Tokeshi, M.; Kitamori, T. J. Am. Chem. Soc.

2003, 125, 14954–14955.16. Maruyama, T.; Uchida, J.; Ohkawa, T.; Futami, T.; Katayama, K.;

Nishizawa, K.; Sotowa, K.; Kubora, F.; Kamiya, N.; Goto, M. Lab Chip 2003, 3, 308–312.

JFC-D-11-00003.indd 11 8/1/2011 6:30:48 PM

Micro Systems for Chemical Synthesis and Analysis

12

17. Kralj, J. G.; Sahoo, H. R.; Jensen, K. F. Lab Chip 2007, 7, 256–263.18. Hibara, A.; Tokeshi, M.; Uchiyama, K.; Hisamoto, H.; Kitamori, T. Anal.

Sci. 2001, 17, 89–93.19. Hachiya, H.; Matsumoto, T.; Kanda, K.; Tokeshi, M.; Yoshida, Y.;

Kitamori, T. In Proc. Micro Total Anal. Syst. 2004, 99–101.20. Aota, A.; Mawatari, K.; Kihira, Y.; Sasaki, M.; Kitamori, T. In Proc.

Micro Total Anal. Syst. 2009, 609–611.21. Ohira, S. I.; Toda, K. Lab Chip 2005, 5, 1374–1379.22. Timmer, B.; Olthuis, W.; van den Berg, A. Lab Chip 2004, 4, 252–255.23. Sato, K.; Tokeshi, M.; Odake, T.; Kimura, H.; Ooi, T.; Kitamori, T. Anal.

Chem. 2000, 72, 1144–1147.24. Sato, K.; Tokeshi, M.; Kimura, H.; Kitamori, T. Anal. Chem. 2001, 73,

1213–1218.25. Sato, K.; Yamanaka, M.; Takahashi, H.; Tokeshi, M.; Kimura, H.;

Kitamori, T. Electrophoresis 2002, 23, 734–739.26. Sato, K.; Yamanaka, M.; Hagino, T.; Tokeshi, M.; Kimura, H.; Kitamori,

T. Lab Chip 2004, 4, 570–575.27. Kakuta, M.; Takahashi, H.; Kazuno, S.; Murayama, K.; Ueno, T.; Tokeshi,

M. Meas. Sci. Technol. 2006, 17, 3189–3194.28. Ohashi, T.; Mawatari, K.; Sato, K.; Tokeshi, M.; Kitamori, T. Lab Chip

2009, 9, 991–995.29. Haes, A. J.; Terray, A.; Collins, G. E. Anal. Chem. 2006, 78, 8412–8420.30. Murakami, Y.; Endo, T.; Yamamura, N.; Nagatani, N.; Takamura, Y.;

Tamiya, E. Anal. Biochem. 2004, 334, 111–116.31. Sung, K.; Park, J. K. Lab Chip 2005, 5, 657–664.32. Legendre, L. A.; Bienvenue, J. M.; Roper, M. G.; Ferrance, J. P.; Landers,

J. P. Anal. Chem. 2006, 78, 1444–1451.33. Tan, A.; Benetton, S.; Henion, J. D. Anal. Chem. 2003, 75, 5504–5511.34. Oleschuk, R. D.; Shultz-Lockyear, L. L.; Ning, Y.; Harrison, D. J. Anal.

Chem. 1999, 72, 585–590.35. Tanaka, Y.; Slyadnev, M. N.; Hibara, A.; Tokeshi, M.; Kitamori, T.

J. Chromatogr. A 2000, 894, 45–51.36. Slyadnev, M. N.; Tanaka, Y.; Tokeshi, M.; Kitamori, T. Anal. Chem.

2001, 73, 4037–4044.37. Goto, M.; Sato, K.; Murakami, A.; Tokeshi, M.; Kitamori, T. Anal. Chem.

2005, 77, 2125–2131.38. Burns, M. A.; Johnson, B. N.; Brahmasandra, S. N.; Handique, K.;

Webster, J. R.; Krishnan, M.; Sammarco, T. S.; Man, P. M.; Jones, D.; Heldsinger, D.; Mastrangelo, C. H.; Burke, D. T. Science 1998, 282, 484–487.

39. Yoon, D. S.; Lee, Y. S.; Lee, Y.; Cho, H. J.; Sung, S. W.; Oh, K. W.; Cha, J.; Lim, G. J. Micromech. Microeng. 2002, 12, 813–823.

40. Tanaka, Y.; Sato, K.; Yamato, M.; Okano, T.; Kitamori, T. Anal. Sci. 2004, 20, 411–423.

41. Tanaka, Y.; Sato, K.; Yamato, M.; Okano, T.; Kitamori, T. J. Chromatogr. A 2006, 1111, 233–237.

42. Goto, M.; Tsukahara, T.; Sato, K.; Kitamori, T. Anal. Bioanal. Chem. 2008, 390, 817–823.

43. Breslauer, D. N.; Lee, P. J.; Lee, L. P. Mol. Biosyst. 2006, 2, 97–112.44. El-Ali, J.; Sorger, P. K.; Jensen, K. F. Nature 2006, 442, 403–411.45. Tokeshi, M.; Uchida, M.; Hibara, A.; Sawada, T.; Kitamori, T. Anal.

Chem. 2001, 73, 2112–2116.46. Tokeshi, M.; Yamaguchi, J.; Hattori, A.; Kitamori, T. Anal. Chem. 2005,

77, 626–630.47. Mawatari, K.; Tokeshi, M.; Kitamori, T. Anal. Sci. 2006, 22, 781–784.48. Yamauchi, M.; Mawatari, K.; Hibara, A.; Tokeshi, M.; Kitamori, T. Anal.

Chem. 2006, 78, 2646–2650.49. Hiki, S.; Mawatari, K.; Hibara, A.; Tokeshi, M.; Kitamori, T. Anal. Chem.

2006, 78, 2859–2863.50. Tamaki, E.; Sato, K.; Tokeshi, M.; Sato, K.; Aihara, M.; Kitamori, T.

Anal. Chem. 2002, 74, 1560–1564.51. Ambrose, W. P.; Goodwin, P. M.; Jett, J. H.; Orden, A. V.; Werner, J. H.;

Keller, R. A. Chem. Rev. 1999, 99, 2929–2956.52. Park, T.; Lee, S.; Seong, G. H.; Choo, J.; Lee, E. K.; Kim, Y. S.; Ji, W. H.;

Hwang, S. Y.; Gweon, D.-G.; Lee, S. Lab Chip 2005, 5, 437–442.53. Kneipp, J.; Kneipp, H.; Kneipp, K. Chem. Soc. Rev. 2008, 37, 1052–1060.54. Kang, S. H.; Lee, S.; Yeung, E. S. Anal. Chem. 2004, 76, 4459–4464.55. Fan, F. R. F.; Bard, A. J. Science 1997, 277, 1791–1793.56. Zhao, Q.; Sigalov, G.; Dimitrov, V.; Dorvel, B.; Mirsaidov, U.; Sligar, S.;

Aksimentiev, A.; Timp, G. Nano Lett. 2007, 7, 1680–1685.57. Sahoof, H. R.; Kralj, J. G.; Jensen, K. F. Angew. Chem. Int. Ed. 2007,

46, 5704–5708.

58. Kopp, M. U.; de Mello, A. J.; Manz, A. Science 1998, 280, 1046–1048.59. Brody, J. P.; Yager, P. Sens. Actuators A 1997, 58, 13–18.60. Weigl, B. H.; Yager, P. Science 1999, 283, 346–347.61. Burns, J. R.; Ramshaw, C. Lab Chip 2001, 1, 10–15.62. Song, H.; Tice, J. D.; Ismagilov, R. F. Angew. Chem. Int. Ed. 2003, 42,

767–772.63. Kralj, J. G.; Schmidt, M. A.; Jensen, K. F. Lab Chip 2005, 5, 531–535.64. Aota, A.; Mawatari, K.; Takahashi, S.; Matsumoto, T.; Kanda, K.;

Anraku, R.; Hibara, A.; Tokeshi, M.; Kitamori, T. Microchim. Acta 2009, 164, 249–255.

65. Maruyama, T.; Kaji, T.; Ohkawa, T.; Sotowa, K.; Matsushita, H.; Kubota, F.; Kamiya, N.; Kusakabe, K.; Goto, M. Analyst 2004, 129, 1008–1013.

66. Zhao, B.; Moore, J. S.; Beebe, D. J. Science 2001, 291, 1023–1026.67. Zhao, B.; Moore, J. S.; Beebe, D. J. Anal. Chem. 2002, 74, 4259–4268.68. Van Der Linden, H. J.; Jellema, L. C.; Holwerda, M.; Verpoorte, E. Anal.

Bioanal. Chem. 2006, 385, 1376–1383.69. Hibara, A.; Kasai, K.; Miyaguchi, H.; Kitamori, T. In Proc. Micro Total

Anal. Syst. 2008, 1326–1328.70. Hibara, A.; Iwayama, S.; Matsuoka, S.; Ueno, M.; Kikutani, Y.; Tokeshi,

M.; Kitamori, T. Anal. Chem. 2005, 77, 943–947.71. Takei, G.; Nonogi, M.; Hibara, A.; Kitamori, T.; Kim, H. B. Lab Chip

2007, 7, 596–602.72. Takei, G.; Aota, A.; Hibara, A.; Kitamori, T.; Kim, H. B. In Proc. Micro

Total Anal. Syst. 2007, 1213–1215.73. Uchiyama, K.; Hibara, A.; Kimura, H.; Sawada, T.; Kitamori, T. Jpn. J.

Appl. Phys. 2000, 39, 5316–5322.74. Bialkowski, S. E. Photothermal Spectroscopy Methods for Chemical

Analysis; Wiley: New York, 1996.75. Nolan, T. G.; Dovichi, N. J. IEEE Circ. Dev. Mag. 1986, 1, 54–56.76. Hiki, S.; Tokeshi, M.; Hibara, A.; Kitamori, T. Bunseki Kagaku 2003,

52, 569–574.77. Kikutani, Y.; Mawatari, K.; Katayama, K.; Fukuzawa, T.; Tokeshi, M.;

Kitamori, T. Sens. Actuators B 2008, 133, 91–96.78. Kitamori, T.; Tokeshi, M.; Sato, K.; Hibara, A. Anal. Chem. 2004, 76,

52A–60A.79. Andrew, J. Lab Chip 2001, 1, 7N–12N.80. Massin, C.; Vincent, F.; Homsy, A.; Ehrmann, K.; Boero, G.; Besse,

P.-A.; Daridon, A.; Verpoorte, E.; de Rooij, N. F.; Popovic, R. S. J. Magn. Reson. 2003, 164, 242–255.

81. Takahashi, Y.; Nakakoshi, M.; Sakurai, S.; Akiyama, Y.; Suematsu, H.; Utsumi, H.; Kitamori, T. Anal. Sci. 2007, 23, 395–400.

82. Hisamoto, H.; Saito, T.; Tokeshi, M.; Hibara, A.; Kitamori, T. Chem. Commun. 2001, 2662–2663.

83. Ueno, M.; Hisamoto, H.; Kitamori, T.; Kobayashi, S. Chem. Commun. 2003, 936.

84. Kikutani, Y.; Horiuchi, T.; Uchiyama, K.; Hisamoto, H.; Tokeshi, M.; Kitamori, T. Lab Chip 2002, 2, 188–192.

85. Kikutani, Y.; Hibara, A.; Uchiyama, K.; Hisamaoto, H.; Tokeshi, M.; Kitamori, T. Lab Chip 2002, 2, 193–196.

86. Kobayashi, J.; Mori, Y.; Okamoto, K.; Akiyama, R.; Ueno, M.; Kitamori, T.; Kobayashi, S. Science 2004, 305, 1305–1308.

87. Matsuoka, S.; Hibata, A.; Ueno, M.; Kitamori, T. Lab Chip 2006, 6, 1236–1238.

88. Kawai, A.; Futami, T.; Kiriya, H.; Katayama, K.; Nishizawa, K. Proc. Micro Total Anal. Syst. 2002, 1, 368–370.

89. Hatch, A.; Kamholz, A. E.; Hawkins, K. R.; Munson, M. S.; Schilling, E. A.; Weigl, B. H.; Yager, P. Nat. Biotechnol. 2001, 19, 461–465.

90. Hosokawa, K.; Omata, M.; Maeda, M. Anal. Chem. 2007, 79, 6000–6004.91. Holmes, D.; She, J. K.; Roach, P. L.; Morgan, H. Lab Chip 2007, 7,

1048–1056.92. Bernard, A.; Michel, B.; Delamarche, E. Anal. Chem. 2001, 73, 8–12.93. Henares, T. G.; Funano, S.; Terabe, S.; Mizutani, F.; Sekizawa, R.;

Hisamoto, H. Anal. Chim. Acta 2007, 589, 173–179.94. Zhang, Y.; Ozdemir, P. Anal. Chim. Acta 2009, 638, 115–125.95. Easley, C. J.; Karlinsey, J. M.; Bienvenue, J. M.; Legendre, L. A.; Roper,

M. G.; Feldman, S. H.; Hughes, M. A.; Hewlett, E. L.; Merkel, T. J.; Ferrance, J. P.; Landers, J. P. PNAS 2006, 103, 19272–19277.

96. Toriello, N. M.; Douglas, E. S.; Thaitrong, N.; Hsiao, S. C.; Francis, M. B.; Bertozzi, C. R.; Mathies, R. PNAS 2008, 105, 20173–20178.

97. Liu, P.; Mathies, R. A. Trends Biotechnol. 2009, 27, 572–581.98. Ohira, S.; Toda, K. Anal. Chim. Acta 2008, 619, 143–156.

JFC-D-11-00003.indd 12 8/1/2011 6:30:48 PM