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Recent Developments in Microfluidics for Cell Studies Bin Xiong, Kangning Ren, Yiwei Shu, Yin Chen, Bo Shen, and Hongkai Wu* microscaled samples and reagents and since multiple sample processing steps can be completed on a single microchip, microfluidics has established a revolutionary way for rapid and inexpensive detection in sophisticated chemical and biological analyses, while conventional techniques for these analyses were generally time- and money-consuming because of the complex processing procedures. Microfluidic systems can also be integrated with other techniques for cell analysis and cell manipulation at high temporal and spatial resolution. By fabricating microchips with multiple microchannels and integrating them with ultrasensitive detecting techniques, microfluidics can be used for multiplex analysis with high throughput and high sensitivity. Microfluidic analysis has been applied as a powerful tool for several sophisticated implementations in molecular biology and biomedicine, such as DNA sequencing and biomarker-screening for diseases.[3] As a result, many challenges in biochemical and biological analysis may be overcome using microfluidic technologies. With the advent of single-cell biology, researchers in biology and medicine are interested in the behavior and response of single cells towards variations in the surrounding microenvironment. However, previous techniques in biological research— usually designed for ensemble measurements—cannot reveal the biological heterogeneity of single cells within a population. For example, it is quite difficult to quantitatively determine the expression of immune proteins in single T cells using biological methods for bulk experiments. Microfluidic technology provides a promising solution for addressing this challenge. Versatile microfluidic chips have been developed for cell manipulations involving separating or trapping single cells from a population and quantitatively detecting cellular biomolecules, such as proteins and nucleic acids.[4] Due to the unique properties and versatile features of microfluidic chips, microfluidics holds great promise for numerous applications in chemistry, engineering, biology, biomedicine, and other fields.[2b] One representative example is the application of microfluidics in clinical diagnostics, especially for point-of-care (POC) diagnostics. The advantages of microfluidic analysis over conventional methods include smaller sample sizes, shorter analysis times, higher throughput, and more automation, making microchip-based clinical diagnostics and POC diagnostics readily realizable in both developed and developing countries.[5] In the future, portable microfluidic devices are expected to become a fundamental tool for POC diagnostics with a huge worldwide market.
As a technique for precisely manipulating fluid at the micrometer scale, the field of microfluidics has experienced an explosive growth over the past two decades, particularly owing to the advances in device design and fabrication. With the inherent advantages associated with its scale of operation, and its flexibility in being incorporated with other microscale techniques for manipulation and detection, microfluidics has become a major enabling technology, which has introduced new paradigms in various fields involving biological cells. A microfluidic device is able to realize functions that are not easily imaginable in conventional biological analysis, such as highly parallel, sophisticated high-throughput analysis, single-cell analysis in a well-defined manner, and tissue engineering with the capability of manipulation at the single-cell level. Major advancements in microfluidic device fabrication and the growing trend of implementing microfluidics in cell studies are presented, with a focus on biological research and clinical diagnostics.
1. Introduction Microfluidics has emerged as the science and technology of systems that enables precise control of small amount of fluids; the precise control is made possible using microfluidic chips with microfabricated channels or chamber structures with cross-sections from tens to hundreds of micrometers. The first application of microfluidic technology was a miniature gas chromatographic analysis system demonstrated by Terry et al. in 1975.[1] Their microfluidic system displayed a number of unique merits, including the simultaneous separation and detection of small quantities of sample at high resolution and high sensitivity. The advent of microscale total analysis systems in 1990 resulted in increased interest in microfluidics, and since then, microfluidic technology has experienced explosive developments.[2] Nowadays integrated microfluidic chips are powerful platforms and robust tools for realizing highly sensitive, high-throughput, and low-cost analysis. One distinct feature of microfluidics is the size-effect, which leads to the fluids on microchips having unique physical properties. The transfer distance of mass and heat is relatively small within a microchannel; reactions in the fluids at microscale can be completed in shorter times than those in bulk solution. Since the on-chip processes only require Dr. B. Xiong, Dr. K. Ren, Y. Shu, Y. Chen, B. Shen, Prof. H. Wu Department of Chemistry The Hong Kong University of Science and Technology Clear Water Bay, Kowloon, Hong Kong, China E-mail: [email protected]
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Significant progress has been achieved in microfluidics over the past ten years. The concept and applications of microfluidic chips have been reviewed from different perspectives in several excellent articles.[6] In this Research News, we begin with an overview of the recent advancements in microfluidic device fabrication, and then we focus on the trends and opportunities in using microfluidic platforms for cell studies, which involve cell cultures, tissue engineering, single-cell analysis, and diagnostics.
2. Fabrication of Microfluidic Chips The materials and techniques for microfluidic fabrication have been assessed in depth in several previous reviews;[7] they are not the focus in this article. Nevertheless, because device fabrication is the basis of microfluidics and because it is highly related to the applications, we present an overview of device fabrication before presenting the cell-related applications. The first-generation microfluidic chips were prepared on silicon or glass using standard micromachining or etching, which were mainly used for on-chip capillary electrophoresis (CE). Due to the high thermoconductivity and stable electro-osmotic mobility on its surface, the separation performance of CE is superior on a silicon or glass microchip.[8] However, silicon- or glass-based microdevices are costly. Their microprocessing is labor-intensive and time-consuming, and requires highly specialized skills, costly equipment, and facilities within a cleanroom. With the development of microfluidic device fabrication strategies, various polymers—such as polydimethylsiloxane (PDMS), polyurethane methacrylate (PUMA), thermoset polyester (TPE), poly(methylmethacrylate) (PMMA), polycarbonate (PC), and Teflon polymers—have become common microchip materials for chemical and biological research (Figure 1).[5,9] Using microstructure molds obtained with photolithography or scanning beam lithography, polymer-based microchips can be produced with molding and embossing techniques. Soft lithography, revolutionarily developed by the research group of Whitesides, has been demonstrated to be one of the most robust strategies for polymer-based microdevice fabrication.[10] However, it is also limited because polymer-based soft molds (e.g., PDMS) for soft lithography are easy to distort during molding at higher temperatures. This limitation has been addressed by adjusting the curing formula of PDMS and modifying the treatment procedure in our laboratory. The maximum working temperature of PDMS for transfer molding has been successfully raised to 350 °C, which is applicable for microfluidic fabrication with almost all existing thermoplastics.[9f ] Since microfluidic devices are becoming more common in biological and biomedical studies, hydrogels with excellent biocompatibility—for example, Matrigel, collagen and agarose—have gradually become more common in microfluidic fabrication. Microfluidic channels in hydrogels can be made using molding, printing, and photo-patterning methods according to recent reports.[11] Because hydrogels contain 3D networks of hydrophilic polymer chains, which swell in aqueous medium, the hydrogel devices are generally highly hydrophilic and gas-permeable, and they can be made
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with arbitrary 3D structures, which are widely used for cell cultures in tissue-engineering research.[12] Recently, paper-based microfluidic devices have been produced using lithographic or printing methods based on techniques that had been developed for hydrophobic surface modifications.[13] Because of the easy preparation and low cost, paper-based microchips have great promise as platforms for rapid and convenient off-site diagnosis and bio-assays.[14]
3. Microfluidics for Biology As previously described by Whitesides, microfluidics offers so many advantages and has the potential to influence a number of scientific areas.[2b] By integrating various technologies from other fields, such as single-molecule detection and optical trapping for bio-particle manipulation, the rapid development of multifunctional microfluidic devices provides numerous opportunities for biological and biomedical research. Here, we highlight several emerging microfluidic studies applied to biology in recent years. 3.1. Cell Cultures Compared to traditional cell maintenance in culture dishes, culturing cells in microfluidic devices has various advantages, such as providing a controllable micro-environment for guided cell growth and differentiation, facilitating cell manipulation and analysis at the single-cell level, and creating opportunities for studying cellular responses and cell–cell interactions.[15] According to the development of microfluidic cell cultures, cell maintenance on microfluidic devices can be divided into 2D and 3D cell-culture models. The earliest research for microfluidic cell cultures primarily focused on long-term cell cultivation in various types of 2D microchannels and microchambers, in which the mediums can be continuously refreshed throughout the microchannels.[9e] Because the microfluidic channels are suitable for handling samples of microscaled fluids, the local micro-environment around individual cells can be easily controlled; the individual cells in the microdevices can also be readily handled, unlike in traditional culturing methods. By integrating precise manipulation techniques such as optical tweezers, 2D microfluidic cell-culture systems have been developed for patterning and handling cells. Sun and coworkers developed a microfluidic platform that allows simultaneous single-cell manipulation and sorting.[16] With the help of optical tweezers, human embryonic stem cells and yeast cells were individually isolated from mixtures at high accuracy. With cells patterned on microfluidic devices, cell–cell interactions have also been recently investigated. For example, cell pairs that are fixed within microchambers and contacted with each other, can be fused by external electric or chemical stimulation, which facilitates research toward new strategies in cell engineering such as the generation of hybridomas and gene reprogramming of cells.[17] Although significant advancements in microchip-based applications have been achieved in 2D cell-culture systems,
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RESEARCH NEWS Figure 1. A,B) Optical images of six individual microchemostats (A) and the detailed components of a single microchemostat (B) for monitoring the growth of small numbers of cells. C) Fabrication of "whole-Teflon" microchips, in which the channels are made entirely of Teflon, using the hightemperature transfer molding method. D) Sealed Teflon PFA microchips for long-term cell cultures. Reproduced with permission: A,B) Copyright 2005 American Association for the Advancement of Science.[9e] C,D) Copyright 2011 National Academy of Sciences.[9f ]
3D microfluidic cell cultures have begun to attract more and more attention because more in-vivo-like cell-culture conditions can be mimicked by 3D microfluidic systems. Since cells in vivo interact with the extracellular matrix, neighboring cells, and mechanical forces in three dimensions, 3D cell cultures on microchips have been applied to studies investigating the interactions between cells and their surrounding micro-environment. It has been demonstrated that in-vivolike 3D organization and culturing play a critical role in cell behavior and responses towards extracellular stimulation; this is quite different from the behavior and responses of cells in a monolayer culture.[18] For example, Daniela Loessner and coworkers reported that malignant cancer cells cultured in 3D microfluidic systems are more resistant to cytotoxic agents than they are in 2D microfluidic systems.[19] With the development of 3D microfluidic cell-culture systems, more in-vivolike features of cell function can be modeled and revealed in future studies.
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3.2. Tissue Engineering With the rapid development of microfluidic 3D cell-culture techniques, microfluidics has also had significant progress in tissue engineering, e.g., in tissue regeneration and guided tissue development.[20] Compared with conventional techniques, microfluidic tissue engineering, not only provides an in-vivo-like 3D scaffold for the growth and organization of cells, but also facilitates the dynamic control of nutrients supply and signal-factor transportation. One of the most representative examples for microfluidic tissue engineering is the in vitro fabrication of cardiovascular-like tissues. For example, Miller et al. used rigid 3D filament networks as sacrificial templates in order to generate the endothelialization of endothelial cells and form microvascular structures.[21] The blood-perfused vascular microchannels were demonstrated to be capable of sustaining the metabolic function of primary rat hepatocytes in engineered tissue constructs. While many signaling factors at tissue–tissue
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interfaces that are associated with various biological functions (e.g., differentiation) exist as gradients in vivo, gradients mimicking the natural micro-environment can also be realized in microfluidic tissue engineering. Our group has designed a gradient-generating bio-microfluidic device filled with a stemcell-laden agarose hydrogel for steering the simultaneous differentiation of stem cells.[12c] The microfluidic system was divided into five zones, and gradients were generated by injecting a specific culture medium to the assigned zones (Z1, Z2, Z3, Z4, and Z5) as shown in Figure 2A. The adipose-derived mesenchymal stem cells seeded in the microdevice were guided to
simultaneously differentiate into osteoblasts and chondrocytes, which suggests a promising future in clinical tissue-regenerative therapies. 3.3. Single-Cell Analysis 3.3.1. Cellular Contents Because the cells under seemingly identical environmental conditions often display heterogeneous behaviors, increasing
Figure 2. A) Gradient-regulated hydrogel bio-microfluidic device for the guided differentiation of adipose-derived mesenchymal stem cells. B) Integrated microfluidic devices for determining low-copy-number proteins in single cells. Reproduced with permission: A) Copyright 2010 Wiley-VCH.[12c] B) Copyright 2007 American Association for the Advancement of Science.[24]
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3.3.2. Cell Signaling It is believed that the heterogeneous response of single cells towards identical stimulation is related to a discrepancy in the cellular signaling events of each cell; this discrepancy has not been clearly elucidated using conventional biological techniques.[25] Based on the well-developed cell-trapping and cellculture techniques on microchips, microfluidic systems have created numerous opportunities for quantitatively investigating the heterogeneity of biological responses of single cells through recording the bevaviors and detecting the level of cellular signal molecules simultaneously. By determining the dependence of the levels of signal molecules on the high-throughput microfluidic analysis, early research has revealed signaling pathways of individual cells. For example, the group of James R. Heath designed a single-cell proteomic chip to quantify the levels of 11 proteins in signaling pathways under external epidermal growth factor (EGF) stimulation.[26] Their study indicated that microfluidics can be a robust tool for profiling signal transduction networks among single cells. For a better understanding of the relationships between protein–protein interactions and signal processing, it is undoubtedly critical to visualize and quantify the activation of multiple network nodes in individual living cells. Taylor and his colleague developed a high-throughput microfluidic imaging system to study the
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signaling-network responses of living cells under hundreds of combined genetic perturbations and time-varying stimulant sequences.[27] The research group of Mengsu Yang developed a microfluidic device for real-time monitoring of recipient cells in a suspension, which were triggered by mediators released from mechanically stimulated cells in the compressive component of the microchip.[28] These demonstrations of microfluidic imaging platforms for cell-signaling studies have opened a new avenue for further revealing the connections between singlecell responses and the progression of biological functions or pathological mechanisms.
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emphasis has been put on biochemical analysis at the singlecell level.[22] Single-cell analysis of cellular contents on a microchip has become a significant tool for revealing the nature of probabilistic biological functions of individual cells. After the cells are isolated and captured in the chambers of the microchip, individual cells can be easily lysed with the help of chemicals or laser beams; the cellular contents (e.g., nucleic acids, proteins) can be extracted and purified for subsequent biochemical determination. In microchannels, nucleic acids are usually purified using solid-phase extraction methods and then amplified via polymerase chain reaction (PCR).[4] After hybridization with dye-labeled probes, the nucleic acids can be readily sequenced and quantified using fluorescence spectroscopy and microscopy. However, for proteins and other metabolites extracted from individual cells that cannot be chemically amplified, direct identification and qualification in the microchips is usually required. The most commonly used technology for the separation and purification of analytes on a microchip is CE. By using a mixture of ionic and non-ionic surfactants in a PDMS channel, the performance of microchip CE can be optimized for separating and purifying proteins and immunocomplexes.[23] The purified proteins can be selectively detected through specific antibody–antigen interactions or aptamer–proteins interactions, in which the antibodies or aptamers were commonly labeled with dyes or particles (e.g., quantum dots or gold nanoparticles) for optical detection. Recently, Richard Zare’s group has shown a direct counting method for analyzing low-abundance proteins within a confined channel structure (Figure 2B).[24] By integrating cell manipulation, electrophoretic separation, and single-molecule counting techniques into a microfluidic system, they demonstrated the quantitative analysis of the expression amount of the β2 adrenergic proteins at single-cell level.
3.3.3. Cell Secretions Detecting cell secretions at the single-cell level cannot only provide a comprehensive picture of cellular biological functions, but they can also reveal the functional heterogeneity of individual cells. Microfluidics allows dynamic and high-throughput analysis of multiple independent samples, so in recent years, it has attracted increasing attention in the studies of cellular secretions from living cells. The capability of profiling the level of cellular secretions and variations under stimulation over time has been demonstrated by several groups. The research group of Robert Kennedy have presented a dual-chip microfluidic platform that coupled perfusion of cultured adipocytes with an online fluorescence-based enzyme assay to monitor the secretion of glycerol in real time.[29] They also showed the parallel monitoring of insulin release from four islets using an on-chip capillary electrophoresis separation with laser-induced fluorescence detection.[30] These studies indicated that microfluidic analysis of cellular secretions is an effective method for assessing cellular function and dysfunction. Recently quantitative measurements of multiple secreted proteins from single cells on integrated microfluidic devices have been developed to evaluate the functional diversity of cells within a population. Ma et al. reported a microfluidic system for the high-content assessment of the functional heterogeneity of single cells in phenotypically similar T-cells.[31] By quantifying the levels of 12 secreted proteins from individual cells and studying the protein–protein correlations, they found that cellular functions are highly heterogeneous for single immune cells from the same population. By integrating the real-time monitoring of cellular responses with the high-throughput analysis of cell secretions on versatile microfluidic systems, the systematic understanding of the framework of biological events can be expected in the near future.
4. Microfluidics for Diagnostics The prospect of using microfluidic technology for clinical diagnostics is optimistic because of the consumption of miniaturized samples and the high-throughput and automated biochemical analysis.[6a] Clinical diagnostics can be generally divided into four sequential stages: sample collection, sample preparation, analytical processing, and sample detection. Aside from the first stage, the remaining stages can be completed within a microfluidic device that is integrated with other techniques for manipulation and detection. According to the targets
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used in clinical analysis, clinical diagnostics can be divided into immunodiagnostics and genetic diagnostics. Immunodiagnostics is based on the specific interactions between antigens and antibodies, which plays an important role in medicine and life science. Traditional immunoassays have been successfully used for the analysis of antibodies or antigens from biological fluids. However, these tests are generally labor-intensive and time-consuming due to the series of steps for sample processing and analyzing. Microfluidic immunoassays not only allow rapid and convenient detection of biomarkers, but they also create new strategies
for automatically and selectively trapping antigens or cancer cells in the microchips. One application of microfluidic immunoassays is the analysis of biomarkers from whole blood for the diagnosis of cancer or other diseases. Fan et al. presented an integrated barcode microchip for automatically sampling and detecting proteins in microliter quantities of blood.[32] With the integrated barcode microchips, they demonstrated the capability of simultaneous fast sample collection (within 10 min) and sensitive analysis of multiple serum biomarkers from whole blood samples of clinical patients (Figure 3A).
Figure 3. A) Design of an integrated barcode chip for in situ measurements of biomarkers from whole blood samples of clinical patients, where A, B, and C, indicate the type of DNA code and (1)-(5) denote DNA-antibody conjugate, plasma protein, biotin-labeled detection antibody, streptavidin-Cy5, fluorescence probe and complementary DNA-Cy3 reference probe, respectively. RBC, WBC, and DEAL refer to red blood cells, white blood cells, and the DNA-encoded antibody library. B) Digital PCR analysis in microfluidic chips for detecting fetal DNA in maternal plasma. The red- and blue-colored dots represent reaction wells that are positive for two types of zinc-finger proteins genes, respectively. Reproduced with permission: A) Copyright 2008 Nature Publishing Group.[32] B) Copyright 2008 American Association for Clinical Chemistry.[36]
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5. Conclusion and Outlook Microfluidic technologies possess unique advantages for the manipulation and analysis of microscale samples. Many traditional biochemical and biological assays in the macroscale have been transferred onto integrated microfluidic devices, allowing the decreased consumption of samples and reagents, and the enhanced throughput for multiplex analysis. However, microfluidic technologies not only allow the miniaturization of traditional biochemical and biological operations, but they also provide new platforms for generating unconventional strategies for addressing the challenges in biological and biomedical analysis. The advantages of microfluidic technology and the flexibility of integrating techniques from other fields have set the stages for successful applications of microfluidic chips in cell studies in recent years. For the improvement of microfluidic cell studies in the future, more in-vivo-like cell-culture systems on multifunctional and highly integrated microdevices need to be created for the study of cell functions and controlled tissue engineering. Moreover, promoting the automation of
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microfluidic manipulation for more convenient sample pretreatment and high-throughput analysis will advance microfluidics to the next level in cell studies. We anticipate that new insights for a more comprehensive understanding of the nature and heterogeneity of biological functions will be gained from microfluidics-based single-cell analysis that allows quantitative determination of cellular biomolecules and assessment of biological heterogeneity in a high-throughput and automated manner. Nevertheless, there are still great challenges that must be addressed for microfluidic cell studies. For example, the development of microfluidic technologies for real-time analysis of multiple cellular targets in situ is a crucial area because cells are usually subjected to multiple micro-environmental cues that vary in time and space. Also, how to build the linkage between cell heterogeneity and biofunctions is significant and indispensable for a better understanding of life at the singlecell level. We expect that more collaborative work between multidisciplinary researchers will be required in the future in order to addressing this challenge. Because of the rapid, low-cost, and automated microfluidic analysis system, microfluidic clinical diagnostics has attracted more and more interest for commercial POC diagnostics. With the development of the mass production of a microfluidics-based clinical analysis system, it can be anticipated that portable and inexpensive microfluidic devices will become popular and robust tools for POC diagnosis in the future.
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PCR, which is currently a powerful and popular technique for sensitive genetic analysis of diseases, has been realized on microdevices for rapid genetic diagnosis. Due to the increased surface-to-volume ratio inside microchannels or microscale reactors, heat transfer can be greatly accelerated and thus the overall reaction time for microscale PCR has been reduced to several minutes. The group of I-Ming Hsing has demonstrated the capability of microfluidic PCR amplification of DNA for the rapid and multiplexed identification of pathogens.[33] Recently they developed an electrochemical real-time PCR technique on silicon–glass microdevices, which can effectively shorten the analysis time for biomedical diagnosis.[34] Digital PCR on microfluidic devices has also been developed for prenatal diagnosis of fetal genetic diseases, such as chromosomal aneuploidies and single-gene disorders in the laboratory of Dennis Lo.[35] With the high-throughput microchip-based digital PCR analysis shown in Figure 3B, they quantitatively measured circulating fetal DNA in maternal plasma with less quantitative bias, compared to previous methods for genetic diagnosis.[36] Based on microfluidic digital PCR analysis, they presented a non-invasive genetic method for the clinical diagnosis of trisomy 21 and hemophilia.[37] Microfluidic diagnostics has revolutionized the way of traditional clinical diagnostics. With the advent of the commercial home-use glucose meter, microfluidic diagnostics has opened up new areas for POC diagnostics, which are generally described as automated and robust biomedical testing outside of clinical laboratories in a comprehensive format within a short time-frame. The benefits of miniaturization for clinical diagnostic on a microchip—such as the reduced sample and reagent consumption and the rapid, automated, and low-cost analysis—make the integrated microfluidic devices promising for the next generation of POC diagnostics. Quite recently, several paradigms for the commercialization of microfluidics-based POC diagnostics have been demonstrated, which indicates that microfluidic POC diagnostics may one day be common in daily life.[38]
Acknowledgements The authors are grateful for the funding provided by Hong Kong RGC (#605210 and # 604712).
Received: October 28, 2013 Revised: December 9, 2013 Published online:
[1] S. C. Terry, J. H. Jerman, J. B. Angell, IEEE Trans. Electron Devices 1979, 26, 1880. [2] a) A. Manz, N. Graber, H. M. Widmer, Sens. Actuators B 1990, 1, 244; b) G. M. Whitesides, Nature 2006, 442, 368. [3] a) R. G. Blazej, P. Kumaresan, R. A. Mathies, Proc. Natl. Acad. Sci. USA 2006, 103, 7240; b) P. S. Dittrich, A. Manz, Nat. Rev. Drug Discov. 2006, 5, 210. [4] R. N. Zare, S. Kim, Annu. Rev. Biomed. Eng. 2010, 12, 187. [5] J. El-Ali, P. K. Sorger, K. F. Jensen, Nature 2006, 442, 403. [6] a) L. Gervais, N. de Rooij, E. Delamarche, Adv. Mater. 2011, 23, H151; b) M. Abdelgawad, A. R. Wheeler, Adv. Mater. 2009, 21, 920. [7] a) K. Ren, J. Zhou, H. Wu, Acc. Chem. Res. 2013, 46, 2396; b) D. J. Beebe, G. A. Mensing, G. M. Walker, Annu. Rev. Biomed. Eng. 2002, 4, 261; c) B. D. Gates, Q. Xu, M. Stewart, D. Ryan, C. G. Willson, G. M. Whitesides, Chem. Rev. 2005, 105, 1171. [8] A. T. Woolley, R. A. Mathies, Anal. Chem. 1995, 67, 3676. [9] a) X. Zhou, L. Lau, W. W. L. Lam, S. W. N. Au, B. Zheng, Anal. Chem. 2007, 79, 4924; b) J. S. Kuo, Y. Zhao, L. Ng, G. S. Yen, R. M. Lorenz, D. S. W. Lim, D. T. Chiu, Lab Chip 2009, 9, 1951; c) X. Gong, X. Yi, K. Xiao, S. Li, R. Kodzius, J. Qin, W. Wen, Lab Chip 2010, 10, 2622; d) J. C. McDonald, G. M. Whitesides, Acc. Chem. Res. 2002, 35, 491;
© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
wileyonlinelibrary.com
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www.MaterialsViews.com
[10] [11] [12]
[13] [14] [15] [16] [17] [18] [19] [20] [21]
[22]
8
e) F. K. Balagaddé, L. You, C. L. Hansen, F. H. Arnold, S. R. Quake, Science 2005, 309, 137; f) K. Ren, W. Dai, J. Zhou, J. Su, H. Wu, Proc. Natl. Acad. Sci. USA 2011, 108, 8162; g) J. P. Rolland, R. M. Van Dam, D. A. Schorzman, S. R. Quake, J. M. DeSimone, J. Am. Chem. Soc. 2004, 126, 2322. G. M. Whitesides, E. Ostuni, S. Takayama, X. Jiang, D. E. Ingber, Annu. Rev. Biomed. Eng. 2001, 3, 335. G. Y. Huang, L. H. Zhou, Q. C. Zhang, Y. M. Chen, W. Sun, F. Xu, T. J. Lu, Biofabrication 2011, 3, 012001. a) N. Annabi, S. Selimovic, J. P. Acevedo Cox, J. Ribas, M. Afshar Bakooshli, D. Heintze, A. S. Weiss, D. Cropek, A. Khademhosseini, Lab Chip 2013, 13, 3569; b) N. W. Choi, M. Cabodi, B. Held, J. P. Gleghorn, L. J. Bonassar, A. D. Stroock, Nat. Mater. 2007, 6, 908; c) X. Shi, S. Chen, Y. Zhao, C. Lai, H. Wu, Adv. Healthcare Mater. 2013, 2, 846. X. Li, D. R. Ballerini, W. Shen, Biomicrofluidics 2012, 6, 011301. A. W. Martinez, S. T. Phillips, M. J. Butte, G. M. Whitesides, Angew. Chem. Int. Ed. 2007, 46, 1318. E. W. K. Young, D. J. Beebe, Chem. Soc. Rev. 2010, 39, 1036. X. Wang, S. Chen, M. Kong, Z. Wang, K. D. Costa, R. A. Li, D. Sun, Lab Chip 2011, 11, 3656. A. M. Skelley, O. Kirak, H. Suh, R. Jaenisch, J. Voldman, Nat. Methods 2009, 6, 147. M.-H. Wu, S.-B. Huang, G.-B. Lee, Lab Chip 2010, 10, 939. D. Loessner, K. S. Stok, M. P. Lutolf, D. W. Hutmacher, J. A. Clements, S. C. RizziBiomaterials 2010, 31, 8494. L. E. Freed, G. C. Engelmayr, J. T. Borenstein, F. T. Moutos, F. Guilak, Adv. Mater. 2009, 21, 3410. J. S. Miller, K. R. Stevens, M. T. Yang, B. M. Baker, D.-H. T. Nguyen, D. M. Cohen, E. Toro, A. A. Chen, P. A. Galie, X. Yu, R. Chaturvedi, S. N. Bhatia, C. S. Chen, Nat. Mater. 2012, 11, 768. a) L. Cai, N. Friedman, X. S. Xie, Nature 2006, 440, 358; b) J. Yu, J. Xiao, X. Ren, K. Lao, X. S. Xie, Science 2006, 311, 1600.
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[23] a) B. Huang, S. Kim, H. Wu, R. N. Zare, Anal. Chem. 2007, 79, 9145; b) S. Kim, B. Huang, R. N. Zare, Lab Chip 2007, 7, 1663. [24] B. Huang, H. Wu, D. Bhaya, A. Grossman, S. Granier, B. K. Kobilka, R. N. Zare, Science 2007, 315, 81. [25] D. G. Spiller, C. D. Wood, D. A. Rand, M. R. H. White, Nature 2010, 465, 736. [26] Q. Shi, L. Qin, W. Wei, F. Geng, R. Fan, Y. Shik Shin, D. Guo, L. Hood, P. S. Mischel, J. R. Heath, Proc. Natl. Acad. Sci. USA 2012, 109, 419. [27] R. J. Taylor, D. Falconnet, A. Niemistö, S. A. Ramsey, S. Prinz, I. Shmulevich, T. Galitski, C. L. Hansen, Proc. Natl. Acad. Sci. USA 2009, 106, 3758. [28] T. Xu, W. Yue, C.-W. Li, X. Yao, G. Cai, M. Yang, Lab Chip 2010, 10, 2271. [29] A. M. Clark, K. M. Sousa, C. Jennings, O. A. MacDougald, R. T. Kennedy, Anal. Chem. 2009, 81, 2350. [30] J. F. Dishinger, R. T. Kennedy, Anal. Chem. 2007, 79, 947. [31] C. Ma, R. Fan, H. Ahmad, Q. Shi, B. Comin-Anduix, T. Chodon, R. C. Koya, C.-C. Liu, G. A. Kwong, C. G. Radu, A. Ribas, J. R. Heath, Nat. Med. 2011, 17, 738. [32] R. Fan, O. Vermesh, A. Srivastava, B. K. H. Yen, L. Qin, H. Ahmad, G. A. Kwong, C.-C. Liu, J. Gould, L. Hood, J. R. Heath, Nat. Biotechnol. 2008, 26, 1373. [33] S.-W. Yeung, T. M.-H. Lee, H. Cai, I.-M. Hsing, Nucleic Acids Res. 2006, 34, e118. [34] S. S. W. Yeung, T. M. H. Lee, I. M. Hsing, Anal. Chem. 2008, 80, 363. [35] Y. D. Lo, R. W. Chiu, Nat. Rev. Genet. 2006, 8, 71. [36] F. M. Lun, R. W. Chiu, K. A. Chan, T. Y. Leung, T. K. Lau, Y. D. Lo, Clin. Chem. 2008, 54, 1664. [37] a) Y. K. Tong, S. Jin, R. W. K. Chiu, C. Ding, K. C. A. Chan, T. Y. Leung, L. Yu, T. K. Lau, Y. M. D. Lo, Clin. Chem. 2010, 56, 90; b) N. B. Y. Tsui, R. A. Kadir, K. C. A. Chan, C. Chi, G. Mellars, E. G. Tuddenham, T. Y. Leung, T. K. Lau, R. W. K. Chiu, Y. M. D. Lo, Blood 2011, 117, 3684. [38] C. D. Chin, V. Linder, S. K. Sia, Lab Chip 2012, 12, 2118.
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Recent Developments in Microfluidics for Cell Studies Bin Xiong, Kangning Ren, Yiwei Shu, Yin Chen, Bo Shen, and Hongkai Wu* microscaled samples and reagents and since multiple sample processing steps can be completed on a single microchip, microfluidics has established a revolutionary way for rapid and inexpensive detection in sophisticated chemical and biological analyses, while conventional techniques for these analyses were generally time- and money-consuming because of the complex processing procedures. Microfluidic systems can also be integrated with other techniques for cell analysis and cell manipulation at high temporal and spatial resolution. By fabricating microchips with multiple microchannels and integrating them with ultrasensitive detecting techniques, microfluidics can be used for multiplex analysis with high throughput and high sensitivity. Microfluidic analysis has been applied as a powerful tool for several sophisticated implementations in molecular biology and biomedicine, such as DNA sequencing and biomarker-screening for diseases.[3] As a result, many challenges in biochemical and biological analysis may be overcome using microfluidic technologies. With the advent of single-cell biology, researchers in biology and medicine are interested in the behavior and response of single cells towards variations in the surrounding microenvironment. However, previous techniques in biological research— usually designed for ensemble measurements—cannot reveal the biological heterogeneity of single cells within a population. For example, it is quite difficult to quantitatively determine the expression of immune proteins in single T cells using biological methods for bulk experiments. Microfluidic technology provides a promising solution for addressing this challenge. Versatile microfluidic chips have been developed for cell manipulations involving separating or trapping single cells from a population and quantitatively detecting cellular biomolecules, such as proteins and nucleic acids.[4] Due to the unique properties and versatile features of microfluidic chips, microfluidics holds great promise for numerous applications in chemistry, engineering, biology, biomedicine, and other fields.[2b] One representative example is the application of microfluidics in clinical diagnostics, especially for point-of-care (POC) diagnostics. The advantages of microfluidic analysis over conventional methods include smaller sample sizes, shorter analysis times, higher throughput, and more automation, making microchip-based clinical diagnostics and POC diagnostics readily realizable in both developed and developing countries.[5] In the future, portable microfluidic devices are expected to become a fundamental tool for POC diagnostics with a huge worldwide market.
As a technique for precisely manipulating fluid at the micrometer scale, the field of microfluidics has experienced an explosive growth over the past two decades, particularly owing to the advances in device design and fabrication. With the inherent advantages associated with its scale of operation, and its flexibility in being incorporated with other microscale techniques for manipulation and detection, microfluidics has become a major enabling technology, which has introduced new paradigms in various fields involving biological cells. A microfluidic device is able to realize functions that are not easily imaginable in conventional biological analysis, such as highly parallel, sophisticated high-throughput analysis, single-cell analysis in a well-defined manner, and tissue engineering with the capability of manipulation at the single-cell level. Major advancements in microfluidic device fabrication and the growing trend of implementing microfluidics in cell studies are presented, with a focus on biological research and clinical diagnostics.
1. Introduction Microfluidics has emerged as the science and technology of systems that enables precise control of small amount of fluids; the precise control is made possible using microfluidic chips with microfabricated channels or chamber structures with cross-sections from tens to hundreds of micrometers. The first application of microfluidic technology was a miniature gas chromatographic analysis system demonstrated by Terry et al. in 1975.[1] Their microfluidic system displayed a number of unique merits, including the simultaneous separation and detection of small quantities of sample at high resolution and high sensitivity. The advent of microscale total analysis systems in 1990 resulted in increased interest in microfluidics, and since then, microfluidic technology has experienced explosive developments.[2] Nowadays integrated microfluidic chips are powerful platforms and robust tools for realizing highly sensitive, high-throughput, and low-cost analysis. One distinct feature of microfluidics is the size-effect, which leads to the fluids on microchips having unique physical properties. The transfer distance of mass and heat is relatively small within a microchannel; reactions in the fluids at microscale can be completed in shorter times than those in bulk solution. Since the on-chip processes only require Dr. B. Xiong, Dr. K. Ren, Y. Shu, Y. Chen, B. Shen, Prof. H. Wu Department of Chemistry The Hong Kong University of Science and Technology Clear Water Bay, Kowloon, Hong Kong, China E-mail: [email protected]
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Significant progress has been achieved in microfluidics over the past ten years. The concept and applications of microfluidic chips have been reviewed from different perspectives in several excellent articles.[6] In this Research News, we begin with an overview of the recent advancements in microfluidic device fabrication, and then we focus on the trends and opportunities in using microfluidic platforms for cell studies, which involve cell cultures, tissue engineering, single-cell analysis, and diagnostics.
2. Fabrication of Microfluidic Chips The materials and techniques for microfluidic fabrication have been assessed in depth in several previous reviews;[7] they are not the focus in this article. Nevertheless, because device fabrication is the basis of microfluidics and because it is highly related to the applications, we present an overview of device fabrication before presenting the cell-related applications. The first-generation microfluidic chips were prepared on silicon or glass using standard micromachining or etching, which were mainly used for on-chip capillary electrophoresis (CE). Due to the high thermoconductivity and stable electro-osmotic mobility on its surface, the separation performance of CE is superior on a silicon or glass microchip.[8] However, silicon- or glass-based microdevices are costly. Their microprocessing is labor-intensive and time-consuming, and requires highly specialized skills, costly equipment, and facilities within a cleanroom. With the development of microfluidic device fabrication strategies, various polymers—such as polydimethylsiloxane (PDMS), polyurethane methacrylate (PUMA), thermoset polyester (TPE), poly(methylmethacrylate) (PMMA), polycarbonate (PC), and Teflon polymers—have become common microchip materials for chemical and biological research (Figure 1).[5,9] Using microstructure molds obtained with photolithography or scanning beam lithography, polymer-based microchips can be produced with molding and embossing techniques. Soft lithography, revolutionarily developed by the research group of Whitesides, has been demonstrated to be one of the most robust strategies for polymer-based microdevice fabrication.[10] However, it is also limited because polymer-based soft molds (e.g., PDMS) for soft lithography are easy to distort during molding at higher temperatures. This limitation has been addressed by adjusting the curing formula of PDMS and modifying the treatment procedure in our laboratory. The maximum working temperature of PDMS for transfer molding has been successfully raised to 350 °C, which is applicable for microfluidic fabrication with almost all existing thermoplastics.[9f ] Since microfluidic devices are becoming more common in biological and biomedical studies, hydrogels with excellent biocompatibility—for example, Matrigel, collagen and agarose—have gradually become more common in microfluidic fabrication. Microfluidic channels in hydrogels can be made using molding, printing, and photo-patterning methods according to recent reports.[11] Because hydrogels contain 3D networks of hydrophilic polymer chains, which swell in aqueous medium, the hydrogel devices are generally highly hydrophilic and gas-permeable, and they can be made
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with arbitrary 3D structures, which are widely used for cell cultures in tissue-engineering research.[12] Recently, paper-based microfluidic devices have been produced using lithographic or printing methods based on techniques that had been developed for hydrophobic surface modifications.[13] Because of the easy preparation and low cost, paper-based microchips have great promise as platforms for rapid and convenient off-site diagnosis and bio-assays.[14]
3. Microfluidics for Biology As previously described by Whitesides, microfluidics offers so many advantages and has the potential to influence a number of scientific areas.[2b] By integrating various technologies from other fields, such as single-molecule detection and optical trapping for bio-particle manipulation, the rapid development of multifunctional microfluidic devices provides numerous opportunities for biological and biomedical research. Here, we highlight several emerging microfluidic studies applied to biology in recent years. 3.1. Cell Cultures Compared to traditional cell maintenance in culture dishes, culturing cells in microfluidic devices has various advantages, such as providing a controllable micro-environment for guided cell growth and differentiation, facilitating cell manipulation and analysis at the single-cell level, and creating opportunities for studying cellular responses and cell–cell interactions.[15] According to the development of microfluidic cell cultures, cell maintenance on microfluidic devices can be divided into 2D and 3D cell-culture models. The earliest research for microfluidic cell cultures primarily focused on long-term cell cultivation in various types of 2D microchannels and microchambers, in which the mediums can be continuously refreshed throughout the microchannels.[9e] Because the microfluidic channels are suitable for handling samples of microscaled fluids, the local micro-environment around individual cells can be easily controlled; the individual cells in the microdevices can also be readily handled, unlike in traditional culturing methods. By integrating precise manipulation techniques such as optical tweezers, 2D microfluidic cell-culture systems have been developed for patterning and handling cells. Sun and coworkers developed a microfluidic platform that allows simultaneous single-cell manipulation and sorting.[16] With the help of optical tweezers, human embryonic stem cells and yeast cells were individually isolated from mixtures at high accuracy. With cells patterned on microfluidic devices, cell–cell interactions have also been recently investigated. For example, cell pairs that are fixed within microchambers and contacted with each other, can be fused by external electric or chemical stimulation, which facilitates research toward new strategies in cell engineering such as the generation of hybridomas and gene reprogramming of cells.[17] Although significant advancements in microchip-based applications have been achieved in 2D cell-culture systems,
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RESEARCH NEWS Figure 1. A,B) Optical images of six individual microchemostats (A) and the detailed components of a single microchemostat (B) for monitoring the growth of small numbers of cells. C) Fabrication of "whole-Teflon" microchips, in which the channels are made entirely of Teflon, using the hightemperature transfer molding method. D) Sealed Teflon PFA microchips for long-term cell cultures. Reproduced with permission: A,B) Copyright 2005 American Association for the Advancement of Science.[9e] C,D) Copyright 2011 National Academy of Sciences.[9f ]
3D microfluidic cell cultures have begun to attract more and more attention because more in-vivo-like cell-culture conditions can be mimicked by 3D microfluidic systems. Since cells in vivo interact with the extracellular matrix, neighboring cells, and mechanical forces in three dimensions, 3D cell cultures on microchips have been applied to studies investigating the interactions between cells and their surrounding micro-environment. It has been demonstrated that in-vivolike 3D organization and culturing play a critical role in cell behavior and responses towards extracellular stimulation; this is quite different from the behavior and responses of cells in a monolayer culture.[18] For example, Daniela Loessner and coworkers reported that malignant cancer cells cultured in 3D microfluidic systems are more resistant to cytotoxic agents than they are in 2D microfluidic systems.[19] With the development of 3D microfluidic cell-culture systems, more in-vivolike features of cell function can be modeled and revealed in future studies.
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3.2. Tissue Engineering With the rapid development of microfluidic 3D cell-culture techniques, microfluidics has also had significant progress in tissue engineering, e.g., in tissue regeneration and guided tissue development.[20] Compared with conventional techniques, microfluidic tissue engineering, not only provides an in-vivo-like 3D scaffold for the growth and organization of cells, but also facilitates the dynamic control of nutrients supply and signal-factor transportation. One of the most representative examples for microfluidic tissue engineering is the in vitro fabrication of cardiovascular-like tissues. For example, Miller et al. used rigid 3D filament networks as sacrificial templates in order to generate the endothelialization of endothelial cells and form microvascular structures.[21] The blood-perfused vascular microchannels were demonstrated to be capable of sustaining the metabolic function of primary rat hepatocytes in engineered tissue constructs. While many signaling factors at tissue–tissue
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interfaces that are associated with various biological functions (e.g., differentiation) exist as gradients in vivo, gradients mimicking the natural micro-environment can also be realized in microfluidic tissue engineering. Our group has designed a gradient-generating bio-microfluidic device filled with a stemcell-laden agarose hydrogel for steering the simultaneous differentiation of stem cells.[12c] The microfluidic system was divided into five zones, and gradients were generated by injecting a specific culture medium to the assigned zones (Z1, Z2, Z3, Z4, and Z5) as shown in Figure 2A. The adipose-derived mesenchymal stem cells seeded in the microdevice were guided to
simultaneously differentiate into osteoblasts and chondrocytes, which suggests a promising future in clinical tissue-regenerative therapies. 3.3. Single-Cell Analysis 3.3.1. Cellular Contents Because the cells under seemingly identical environmental conditions often display heterogeneous behaviors, increasing
Figure 2. A) Gradient-regulated hydrogel bio-microfluidic device for the guided differentiation of adipose-derived mesenchymal stem cells. B) Integrated microfluidic devices for determining low-copy-number proteins in single cells. Reproduced with permission: A) Copyright 2010 Wiley-VCH.[12c] B) Copyright 2007 American Association for the Advancement of Science.[24]
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3.3.2. Cell Signaling It is believed that the heterogeneous response of single cells towards identical stimulation is related to a discrepancy in the cellular signaling events of each cell; this discrepancy has not been clearly elucidated using conventional biological techniques.[25] Based on the well-developed cell-trapping and cellculture techniques on microchips, microfluidic systems have created numerous opportunities for quantitatively investigating the heterogeneity of biological responses of single cells through recording the bevaviors and detecting the level of cellular signal molecules simultaneously. By determining the dependence of the levels of signal molecules on the high-throughput microfluidic analysis, early research has revealed signaling pathways of individual cells. For example, the group of James R. Heath designed a single-cell proteomic chip to quantify the levels of 11 proteins in signaling pathways under external epidermal growth factor (EGF) stimulation.[26] Their study indicated that microfluidics can be a robust tool for profiling signal transduction networks among single cells. For a better understanding of the relationships between protein–protein interactions and signal processing, it is undoubtedly critical to visualize and quantify the activation of multiple network nodes in individual living cells. Taylor and his colleague developed a high-throughput microfluidic imaging system to study the
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signaling-network responses of living cells under hundreds of combined genetic perturbations and time-varying stimulant sequences.[27] The research group of Mengsu Yang developed a microfluidic device for real-time monitoring of recipient cells in a suspension, which were triggered by mediators released from mechanically stimulated cells in the compressive component of the microchip.[28] These demonstrations of microfluidic imaging platforms for cell-signaling studies have opened a new avenue for further revealing the connections between singlecell responses and the progression of biological functions or pathological mechanisms.
RESEARCH NEWS
emphasis has been put on biochemical analysis at the singlecell level.[22] Single-cell analysis of cellular contents on a microchip has become a significant tool for revealing the nature of probabilistic biological functions of individual cells. After the cells are isolated and captured in the chambers of the microchip, individual cells can be easily lysed with the help of chemicals or laser beams; the cellular contents (e.g., nucleic acids, proteins) can be extracted and purified for subsequent biochemical determination. In microchannels, nucleic acids are usually purified using solid-phase extraction methods and then amplified via polymerase chain reaction (PCR).[4] After hybridization with dye-labeled probes, the nucleic acids can be readily sequenced and quantified using fluorescence spectroscopy and microscopy. However, for proteins and other metabolites extracted from individual cells that cannot be chemically amplified, direct identification and qualification in the microchips is usually required. The most commonly used technology for the separation and purification of analytes on a microchip is CE. By using a mixture of ionic and non-ionic surfactants in a PDMS channel, the performance of microchip CE can be optimized for separating and purifying proteins and immunocomplexes.[23] The purified proteins can be selectively detected through specific antibody–antigen interactions or aptamer–proteins interactions, in which the antibodies or aptamers were commonly labeled with dyes or particles (e.g., quantum dots or gold nanoparticles) for optical detection. Recently, Richard Zare’s group has shown a direct counting method for analyzing low-abundance proteins within a confined channel structure (Figure 2B).[24] By integrating cell manipulation, electrophoretic separation, and single-molecule counting techniques into a microfluidic system, they demonstrated the quantitative analysis of the expression amount of the β2 adrenergic proteins at single-cell level.
3.3.3. Cell Secretions Detecting cell secretions at the single-cell level cannot only provide a comprehensive picture of cellular biological functions, but they can also reveal the functional heterogeneity of individual cells. Microfluidics allows dynamic and high-throughput analysis of multiple independent samples, so in recent years, it has attracted increasing attention in the studies of cellular secretions from living cells. The capability of profiling the level of cellular secretions and variations under stimulation over time has been demonstrated by several groups. The research group of Robert Kennedy have presented a dual-chip microfluidic platform that coupled perfusion of cultured adipocytes with an online fluorescence-based enzyme assay to monitor the secretion of glycerol in real time.[29] They also showed the parallel monitoring of insulin release from four islets using an on-chip capillary electrophoresis separation with laser-induced fluorescence detection.[30] These studies indicated that microfluidic analysis of cellular secretions is an effective method for assessing cellular function and dysfunction. Recently quantitative measurements of multiple secreted proteins from single cells on integrated microfluidic devices have been developed to evaluate the functional diversity of cells within a population. Ma et al. reported a microfluidic system for the high-content assessment of the functional heterogeneity of single cells in phenotypically similar T-cells.[31] By quantifying the levels of 12 secreted proteins from individual cells and studying the protein–protein correlations, they found that cellular functions are highly heterogeneous for single immune cells from the same population. By integrating the real-time monitoring of cellular responses with the high-throughput analysis of cell secretions on versatile microfluidic systems, the systematic understanding of the framework of biological events can be expected in the near future.
4. Microfluidics for Diagnostics The prospect of using microfluidic technology for clinical diagnostics is optimistic because of the consumption of miniaturized samples and the high-throughput and automated biochemical analysis.[6a] Clinical diagnostics can be generally divided into four sequential stages: sample collection, sample preparation, analytical processing, and sample detection. Aside from the first stage, the remaining stages can be completed within a microfluidic device that is integrated with other techniques for manipulation and detection. According to the targets
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used in clinical analysis, clinical diagnostics can be divided into immunodiagnostics and genetic diagnostics. Immunodiagnostics is based on the specific interactions between antigens and antibodies, which plays an important role in medicine and life science. Traditional immunoassays have been successfully used for the analysis of antibodies or antigens from biological fluids. However, these tests are generally labor-intensive and time-consuming due to the series of steps for sample processing and analyzing. Microfluidic immunoassays not only allow rapid and convenient detection of biomarkers, but they also create new strategies
for automatically and selectively trapping antigens or cancer cells in the microchips. One application of microfluidic immunoassays is the analysis of biomarkers from whole blood for the diagnosis of cancer or other diseases. Fan et al. presented an integrated barcode microchip for automatically sampling and detecting proteins in microliter quantities of blood.[32] With the integrated barcode microchips, they demonstrated the capability of simultaneous fast sample collection (within 10 min) and sensitive analysis of multiple serum biomarkers from whole blood samples of clinical patients (Figure 3A).
Figure 3. A) Design of an integrated barcode chip for in situ measurements of biomarkers from whole blood samples of clinical patients, where A, B, and C, indicate the type of DNA code and (1)-(5) denote DNA-antibody conjugate, plasma protein, biotin-labeled detection antibody, streptavidin-Cy5, fluorescence probe and complementary DNA-Cy3 reference probe, respectively. RBC, WBC, and DEAL refer to red blood cells, white blood cells, and the DNA-encoded antibody library. B) Digital PCR analysis in microfluidic chips for detecting fetal DNA in maternal plasma. The red- and blue-colored dots represent reaction wells that are positive for two types of zinc-finger proteins genes, respectively. Reproduced with permission: A) Copyright 2008 Nature Publishing Group.[32] B) Copyright 2008 American Association for Clinical Chemistry.[36]
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5. Conclusion and Outlook Microfluidic technologies possess unique advantages for the manipulation and analysis of microscale samples. Many traditional biochemical and biological assays in the macroscale have been transferred onto integrated microfluidic devices, allowing the decreased consumption of samples and reagents, and the enhanced throughput for multiplex analysis. However, microfluidic technologies not only allow the miniaturization of traditional biochemical and biological operations, but they also provide new platforms for generating unconventional strategies for addressing the challenges in biological and biomedical analysis. The advantages of microfluidic technology and the flexibility of integrating techniques from other fields have set the stages for successful applications of microfluidic chips in cell studies in recent years. For the improvement of microfluidic cell studies in the future, more in-vivo-like cell-culture systems on multifunctional and highly integrated microdevices need to be created for the study of cell functions and controlled tissue engineering. Moreover, promoting the automation of
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microfluidic manipulation for more convenient sample pretreatment and high-throughput analysis will advance microfluidics to the next level in cell studies. We anticipate that new insights for a more comprehensive understanding of the nature and heterogeneity of biological functions will be gained from microfluidics-based single-cell analysis that allows quantitative determination of cellular biomolecules and assessment of biological heterogeneity in a high-throughput and automated manner. Nevertheless, there are still great challenges that must be addressed for microfluidic cell studies. For example, the development of microfluidic technologies for real-time analysis of multiple cellular targets in situ is a crucial area because cells are usually subjected to multiple micro-environmental cues that vary in time and space. Also, how to build the linkage between cell heterogeneity and biofunctions is significant and indispensable for a better understanding of life at the singlecell level. We expect that more collaborative work between multidisciplinary researchers will be required in the future in order to addressing this challenge. Because of the rapid, low-cost, and automated microfluidic analysis system, microfluidic clinical diagnostics has attracted more and more interest for commercial POC diagnostics. With the development of the mass production of a microfluidics-based clinical analysis system, it can be anticipated that portable and inexpensive microfluidic devices will become popular and robust tools for POC diagnosis in the future.
RESEARCH NEWS
PCR, which is currently a powerful and popular technique for sensitive genetic analysis of diseases, has been realized on microdevices for rapid genetic diagnosis. Due to the increased surface-to-volume ratio inside microchannels or microscale reactors, heat transfer can be greatly accelerated and thus the overall reaction time for microscale PCR has been reduced to several minutes. The group of I-Ming Hsing has demonstrated the capability of microfluidic PCR amplification of DNA for the rapid and multiplexed identification of pathogens.[33] Recently they developed an electrochemical real-time PCR technique on silicon–glass microdevices, which can effectively shorten the analysis time for biomedical diagnosis.[34] Digital PCR on microfluidic devices has also been developed for prenatal diagnosis of fetal genetic diseases, such as chromosomal aneuploidies and single-gene disorders in the laboratory of Dennis Lo.[35] With the high-throughput microchip-based digital PCR analysis shown in Figure 3B, they quantitatively measured circulating fetal DNA in maternal plasma with less quantitative bias, compared to previous methods for genetic diagnosis.[36] Based on microfluidic digital PCR analysis, they presented a non-invasive genetic method for the clinical diagnosis of trisomy 21 and hemophilia.[37] Microfluidic diagnostics has revolutionized the way of traditional clinical diagnostics. With the advent of the commercial home-use glucose meter, microfluidic diagnostics has opened up new areas for POC diagnostics, which are generally described as automated and robust biomedical testing outside of clinical laboratories in a comprehensive format within a short time-frame. The benefits of miniaturization for clinical diagnostic on a microchip—such as the reduced sample and reagent consumption and the rapid, automated, and low-cost analysis—make the integrated microfluidic devices promising for the next generation of POC diagnostics. Quite recently, several paradigms for the commercialization of microfluidics-based POC diagnostics have been demonstrated, which indicates that microfluidic POC diagnostics may one day be common in daily life.[38]
Acknowledgements The authors are grateful for the funding provided by Hong Kong RGC (#605210 and # 604712).
Received: October 28, 2013 Revised: December 9, 2013 Published online:
[1] S. C. Terry, J. H. Jerman, J. B. Angell, IEEE Trans. Electron Devices 1979, 26, 1880. [2] a) A. Manz, N. Graber, H. M. Widmer, Sens. Actuators B 1990, 1, 244; b) G. M. Whitesides, Nature 2006, 442, 368. [3] a) R. G. Blazej, P. Kumaresan, R. A. Mathies, Proc. Natl. Acad. Sci. USA 2006, 103, 7240; b) P. S. Dittrich, A. Manz, Nat. Rev. Drug Discov. 2006, 5, 210. [4] R. N. Zare, S. Kim, Annu. Rev. Biomed. Eng. 2010, 12, 187. [5] J. El-Ali, P. K. Sorger, K. F. Jensen, Nature 2006, 442, 403. [6] a) L. Gervais, N. de Rooij, E. Delamarche, Adv. Mater. 2011, 23, H151; b) M. Abdelgawad, A. R. Wheeler, Adv. Mater. 2009, 21, 920. [7] a) K. Ren, J. Zhou, H. Wu, Acc. Chem. Res. 2013, 46, 2396; b) D. J. Beebe, G. A. Mensing, G. M. Walker, Annu. Rev. Biomed. Eng. 2002, 4, 261; c) B. D. Gates, Q. Xu, M. Stewart, D. Ryan, C. G. Willson, G. M. Whitesides, Chem. Rev. 2005, 105, 1171. [8] A. T. Woolley, R. A. Mathies, Anal. Chem. 1995, 67, 3676. [9] a) X. Zhou, L. Lau, W. W. L. Lam, S. W. N. Au, B. Zheng, Anal. Chem. 2007, 79, 4924; b) J. S. Kuo, Y. Zhao, L. Ng, G. S. Yen, R. M. Lorenz, D. S. W. Lim, D. T. Chiu, Lab Chip 2009, 9, 1951; c) X. Gong, X. Yi, K. Xiao, S. Li, R. Kodzius, J. Qin, W. Wen, Lab Chip 2010, 10, 2622; d) J. C. McDonald, G. M. Whitesides, Acc. Chem. Res. 2002, 35, 491;
© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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[10] [11] [12]
[13] [14] [15] [16] [17] [18] [19] [20] [21]
[22]
8
e) F. K. Balagaddé, L. You, C. L. Hansen, F. H. Arnold, S. R. Quake, Science 2005, 309, 137; f) K. Ren, W. Dai, J. Zhou, J. Su, H. Wu, Proc. Natl. Acad. Sci. USA 2011, 108, 8162; g) J. P. Rolland, R. M. Van Dam, D. A. Schorzman, S. R. Quake, J. M. DeSimone, J. Am. Chem. Soc. 2004, 126, 2322. G. M. Whitesides, E. Ostuni, S. Takayama, X. Jiang, D. E. Ingber, Annu. Rev. Biomed. Eng. 2001, 3, 335. G. Y. Huang, L. H. Zhou, Q. C. Zhang, Y. M. Chen, W. Sun, F. Xu, T. J. Lu, Biofabrication 2011, 3, 012001. a) N. Annabi, S. Selimovic, J. P. Acevedo Cox, J. Ribas, M. Afshar Bakooshli, D. Heintze, A. S. Weiss, D. Cropek, A. Khademhosseini, Lab Chip 2013, 13, 3569; b) N. W. Choi, M. Cabodi, B. Held, J. P. Gleghorn, L. J. Bonassar, A. D. Stroock, Nat. Mater. 2007, 6, 908; c) X. Shi, S. Chen, Y. Zhao, C. Lai, H. Wu, Adv. Healthcare Mater. 2013, 2, 846. X. Li, D. R. Ballerini, W. Shen, Biomicrofluidics 2012, 6, 011301. A. W. Martinez, S. T. Phillips, M. J. Butte, G. M. Whitesides, Angew. Chem. Int. Ed. 2007, 46, 1318. E. W. K. Young, D. J. Beebe, Chem. Soc. Rev. 2010, 39, 1036. X. Wang, S. Chen, M. Kong, Z. Wang, K. D. Costa, R. A. Li, D. Sun, Lab Chip 2011, 11, 3656. A. M. Skelley, O. Kirak, H. Suh, R. Jaenisch, J. Voldman, Nat. Methods 2009, 6, 147. M.-H. Wu, S.-B. Huang, G.-B. Lee, Lab Chip 2010, 10, 939. D. Loessner, K. S. Stok, M. P. Lutolf, D. W. Hutmacher, J. A. Clements, S. C. RizziBiomaterials 2010, 31, 8494. L. E. Freed, G. C. Engelmayr, J. T. Borenstein, F. T. Moutos, F. Guilak, Adv. Mater. 2009, 21, 3410. J. S. Miller, K. R. Stevens, M. T. Yang, B. M. Baker, D.-H. T. Nguyen, D. M. Cohen, E. Toro, A. A. Chen, P. A. Galie, X. Yu, R. Chaturvedi, S. N. Bhatia, C. S. Chen, Nat. Mater. 2012, 11, 768. a) L. Cai, N. Friedman, X. S. Xie, Nature 2006, 440, 358; b) J. Yu, J. Xiao, X. Ren, K. Lao, X. S. Xie, Science 2006, 311, 1600.
wileyonlinelibrary.com
[23] a) B. Huang, S. Kim, H. Wu, R. N. Zare, Anal. Chem. 2007, 79, 9145; b) S. Kim, B. Huang, R. N. Zare, Lab Chip 2007, 7, 1663. [24] B. Huang, H. Wu, D. Bhaya, A. Grossman, S. Granier, B. K. Kobilka, R. N. Zare, Science 2007, 315, 81. [25] D. G. Spiller, C. D. Wood, D. A. Rand, M. R. H. White, Nature 2010, 465, 736. [26] Q. Shi, L. Qin, W. Wei, F. Geng, R. Fan, Y. Shik Shin, D. Guo, L. Hood, P. S. Mischel, J. R. Heath, Proc. Natl. Acad. Sci. USA 2012, 109, 419. [27] R. J. Taylor, D. Falconnet, A. Niemistö, S. A. Ramsey, S. Prinz, I. Shmulevich, T. Galitski, C. L. Hansen, Proc. Natl. Acad. Sci. USA 2009, 106, 3758. [28] T. Xu, W. Yue, C.-W. Li, X. Yao, G. Cai, M. Yang, Lab Chip 2010, 10, 2271. [29] A. M. Clark, K. M. Sousa, C. Jennings, O. A. MacDougald, R. T. Kennedy, Anal. Chem. 2009, 81, 2350. [30] J. F. Dishinger, R. T. Kennedy, Anal. Chem. 2007, 79, 947. [31] C. Ma, R. Fan, H. Ahmad, Q. Shi, B. Comin-Anduix, T. Chodon, R. C. Koya, C.-C. Liu, G. A. Kwong, C. G. Radu, A. Ribas, J. R. Heath, Nat. Med. 2011, 17, 738. [32] R. Fan, O. Vermesh, A. Srivastava, B. K. H. Yen, L. Qin, H. Ahmad, G. A. Kwong, C.-C. Liu, J. Gould, L. Hood, J. R. Heath, Nat. Biotechnol. 2008, 26, 1373. [33] S.-W. Yeung, T. M.-H. Lee, H. Cai, I.-M. Hsing, Nucleic Acids Res. 2006, 34, e118. [34] S. S. W. Yeung, T. M. H. Lee, I. M. Hsing, Anal. Chem. 2008, 80, 363. [35] Y. D. Lo, R. W. Chiu, Nat. Rev. Genet. 2006, 8, 71. [36] F. M. Lun, R. W. Chiu, K. A. Chan, T. Y. Leung, T. K. Lau, Y. D. Lo, Clin. Chem. 2008, 54, 1664. [37] a) Y. K. Tong, S. Jin, R. W. K. Chiu, C. Ding, K. C. A. Chan, T. Y. Leung, L. Yu, T. K. Lau, Y. M. D. Lo, Clin. Chem. 2010, 56, 90; b) N. B. Y. Tsui, R. A. Kadir, K. C. A. Chan, C. Chi, G. Mellars, E. G. Tuddenham, T. Y. Leung, T. K. Lau, R. W. K. Chiu, Y. M. D. Lo, Blood 2011, 117, 3684. [38] C. D. Chin, V. Linder, S. K. Sia, Lab Chip 2012, 12, 2118.
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