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This book summarizes the various microfluidic-based approaches for single-cell capture, isolation, manipulation, culture and observation, lysis, and analysis. Single-cell analysis reveals the heterogeneities in morphology, functions, composition, and genetic performance of seemingly identical cells, and advances in single-cell analysis can overcome the difficulties arising due to cell heterogeneity in the diagnostics for a targeted model of disease. This book provides a detailed review of the state-of-the-art techniques presenting the pros and cons of each of these methods. It also offers lessons learned and tips from front-line investigators to help researchers overcome bottlenecks in their own studies. Highlighting a number of techniques, such as microfluidic droplet techniques, combined microfluidics-mass-spectrometry systems, and nanochannel sampling, it describes in detail a new microfluidic chip-based live single-cell extractor (LSCE) developed in the editor’s laboratory, which opens up new avenues to use open microfluidics in single-cell extraction, single-cell mass spectrometric analysis, single-cell adhesion analysis and subcellular operations. Serving as both an elementary introduction and advanced guidebook, this book interests and inspires scholars and students who are currently studying or wish to study microfluidics-based cell analysis methods.



Chapter 1. Advances of Single-Cell Analysis on Microfluidics

The advances of microfluidic technologies have promoted researchers to study the inherent heterogeneity of single cells in cell populations. This will be helpful in the acknowledgment of major disease and invention of personalized medicine. Different microfluidic approaches provide varieties of functions in the process of single-cell analysis. In this chapter, we introduce decades of the history in single-cell analysis and give an outline of the mechanisms of various microfluidic-based approaches for cell sorting, single-cell isolation, and single-cell lysis.
Qiushi Huang, Jin-Ming Lin

Chapter 2. Microfluidic Technology for Single-Cell Capture and Isolation

Compared to conventional biological assays that statistically analyze the average response from a large population of cells, single-cell assay can tell the differences between individual cells allowing more precise understanding of single-cell behavior. The challenge of studying single cells is requiring hundreds or thousands of isolated single cells. Numerous microfluidic-based techniques have been developed and are successfully utilized to capture single cells. In this chapter, we summarize technologies integrated onto microfluidic chips for single-cell capture and isolation. According to the principle used, these techniques can be categorized into physical and biochemical approaches. At last, the challenges and future directions about these microfluidic techniques have been remarked.
Jing Wu, Jin-Ming Lin

Chapter 3. Single-Cell Culture and Analysis on Microfluidics

Heterogeneity of cell populations is a major obstacle for understanding complex biological processes. In order to have a more comprehensive quantitative comprehending of cellular processes, it is necessary to quantify the distribution of behavior in a population of individual cells. Analysis of single-cell behaviors requires efficient single-cell capture, controllable single-cell culture performance as well as reliable analysis techniques. The microfluidic system provides advanced technology for single-cell culture and observation. This chapter gives a brief account of single-cell capture by microfluidic methods, long-term single-cell culture on both two-dimensional models and three-dimensional microfluidic systems, as well as single-cell growth and differentiation in a microfluidic environment. Furthermore, the advanced methods used for characterizing on-chip single-cell culture were also discussed.
Weiwei Li, Jin-Ming Lin

Chapter 4. Microfluidic Technology for Single-Cell Manipulation

Single-cell analysis has attracted much attention in the field of biological and biomedical study owing to the heterogeneity among individual cells. This poses significant challenges to conventional bulk assays which would mask rare but important information owing to the assumption of average behavior. To avoid the interference of useless cells and obtain the single cells in the trial of genomics, proteomics, metabonomics, and single-cell behavior study, various cell manipulation techniques have been developed for single-cell research. In this chapter, we introduce the principles of droplet generation and single-cell encapsulation and review the latest achievements of cell manipulation technique by categorizing externally applied manipulation forces: microstructures, electrical, optical, magnetic, acoustic, and mechanical. This chapter will also introduce our latest work and provide important references and ideas for the development of droplet microfluidic-based single-cell manipulation.
Weifei Zhang, Nan Li, Jin-Ming Lin

Chapter 5. Droplet-Based Microfluidics for Single-Cell Encapsulation and Analysis

Droplet microfluidic techniques have been rapidly developed as a powerful tool to perform high-throughput and low-cost analysis of single cells. Microscale droplets can be easily produced by a microfluidic manipulation to encapsulate and manipulate single cells for precise analysis. This offers a new approach to measure genetic and functional heterogeneity of cell division, growth, metabolism, and apoptosis. Functional characteristics of cellular molecules, such as DNA, RNA, and proteins, can be realized at a single-cell level. In this chapter, we will present a general introduction to single-cell analysis involving droplet-based microfluidic techniques. We will highlight the current state of droplet-based microfluidic single-cell analysis for deep insights understanding the biological process at the single-cell level.
Qiushui Chen, Jin-Ming Lin

Chapter 6. Microfluidics for Single-Cell Genomics

Genomics is the systematic study of entire deoxyribonucleic acid (DNA) sequencing of an organism or virus. A single-cell DNA sequencing explores the heterogeneity among a cellular population of a biological sample and also predicts the growth and function of a living entity. However, efficient extraction of chromosomes from a single living cell requires sophisticated moods for sample preparation. Microfluidic devices offer several improvements including, effective heat transfer (enhanced the multiplication of DNA) and small volume (enabled the accurate quantification of DNA molecules) within the lysate of a single cell. However, at present, only one step such as single-cell isolation, cell lysis, or chromosome isolation from an individual cell and its amplification can be performed on-chip. Besides, microfluidics relies on external techniques for analysis of DNA. Therefore, the integration of multi-microfluidic systems is required for automated genome investigation. This chapter describes the advancement, limitations, and future prospects of microfluidic/nanofluidic for single-cell analysis.
Mashooq Khan, Jin-Ming Lin

Chapter 7. Microfluidics-Mass Spectrometry Combination Systems for Single-Cell Analysis

Due to the existence of heterogeneities in individual cells, analysis of intercellular contents at the single-cell level has become an important direction in modern bioanalytical chemistry. The advances in miniaturized analytical systems and emerging microfluidic tools bring a new opportunity for single-cell analysis. Microfluidic systems have abilities to the single cell and reagents manipulation with minimal dilution, automatic and parallel sample preparation, and compatible with different detection techniques, which made them powerful tools for single-cell analysis. Mass spectrometry (MS) is one of the most popular analytical methods for the detection of unknown chemicals because of its unique advantages, such as label-free detection, high sensitivity, high chemical specificity, and board detection range. Recently, the coupling of microfluidics to MS for single-cell analysis has attracted substantial interests and developments. Nowadays, different types of ionization methods including electrospray ionization (ESI), laser desorption ionization (LDI), secondary ionization (SI), and inductively coupled plasma (ICP) have been coupled to a mass spectrometer. Owing to these ionization methods, a board range of chemicals can be detected by MS, such as proteins, metabolites, lipids, peptides, glycomics, elements, and so on. Recent progress in the fields of technologies and applications in the microfluidics-MS combination systems for single-cell analysis is described. Several analytical procedures integrated on the microfluidics such as single-cell manipulation and sample pretreatment before introduction into the mass spectrometer are reviewed. The future research opportunities by focusing on key performances of throughput, multiparametric target detection, and highly automated analysis are also discussed.
Dan Gao, Chao Song, Jin-Ming Lin

Chapter 8. Micro/Nano fluidics Enabled Single-Cell Biochemical Analysis

In the last 20 years, micro-fluidic technique has emerged as an important enabling tool for single-cell chemical analysis, owing to the miniaturization of the fluidic environment. These methodologies made various applications in single-cell analysis fields, and their superior performances such as rapid, simple, and high-efficient processing have been proved. Recently, the space is further downscaling to the 10–1000 nm scale (nano-space). The nano-space is located between conventional nanotechnology (10–1000 nm) and microtechnology (>1 mm), and the research tools are not well established. For these purposes, a new research field is now being created which are quite different from those in micro-space. In this chapter, we focus on the basic researches in nano-fluidic space and survey the fundamental technologies for nano-fluidic space. Then, recent developments of nano-fluidic technologies for single-cell analysis are reported. Finally, the potential of nano-fluidics-based single-cell analysis is discussed.
Ling Lin

Chapter 9. Microfluidic Chip-Based Live Single-Cell Probes

Single-cell analysis provides critical information to understand key disease processes and disease diagnosis. However, nearly all of the current methods carry out single-cell analysis in suspension, which not only destroy extracellular context but also may perturb the intracellular metabolites. It is essential to develop new methods to meet the requirements of understanding individual cell behaviors and their relations in adherent tissue culture. Advances in single-cell methodologies have highlighted the single-cell biology and are opening new vistas for scientists to explore. How to realize precise operation of single cells and subcellular molecule infusion is a critical question that researchers face. And, as the technologies to study single cells expand, sophisticated analytical tools are required to make sense of various behaviors and components of single cells as well as their relations in adherent tissue culture. Microfluidic chip has been proved an outstanding approach for single-cell analysis. Recently, the developments of single-cell probes open up new avenues to operate open microfluidics to perform single-cell extraction, single-cell mass spectrometric analysis, single-cell adhesion analysis, and subcellular operations.
Sifeng Mao, Jin-Ming Lin


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