Applications In Single-cell Analysis Research Articles

New mass spectrometry technologies contributing towards comprehensive and high throughput omics analyses of single cells.

Mass-spectrometry based omics technologies - namely proteomics, metabolomics and lipidomics - have enabled the molecular level systems biology investigation of organisms in unprecedented detail. There has been increasing interest for gaining a thorough, functional understanding of the biological consequences associated with cellular heterogeneity in a wide variety of research areas such as developmental biology, precision medicine, cancer research and microbiome science. Recent advances in mass spectrometry (MS) instrumentation and sample handling strategies are quickly making comprehensive omics analyses of single cells feasible, but key breakthroughs are still required to push through remaining bottlenecks. In this review, we discuss the challenges faced by single cell MS-based omics analyses and highlight recent technological advances that collectively can contribute to comprehensive and high throughput omics analyses in single cells. We provide a vision of the potential of integrating pioneering technologies such as Structures for Lossless Ion Manipulations (SLIM) for improved sensitivity and resolution, novel peptide identification tactics and standards free metabolomics approaches for future applications in single cell analysis.

Integrating Immunology and Microfluidics for Single Immune Cell Analysis.

The field of immunoengineering aims to develop novel therapies and modern vaccines to manipulate and modulate the immune system and applies innovative technologies toward improved understanding of the immune system in health and disease. Microfluidics has proven to be an excellent technology for analytics in biology and chemistry. From simple microsystem chips to complex microfluidic designs, these platforms have witnessed an immense growth over the last decades with frequent emergence of new designs. Microfluidics provides a highly robust and precise tool which led to its widespread application in single-cell analysis of immune cells. Single-cell analysis allows scientists to account for the heterogeneous behavior of immune cells which often gets overshadowed when conventional bulk study methods are used. Application of single-cell analysis using microfluidics has facilitated the identification of several novel functional immune cell subsets, quantification of signaling molecules, and understanding of cellular communication and signaling pathways. Single-cell analysis research in combination with microfluidics has paved the way for the development of novel therapies, point-of-care diagnostics, and even more complex microfluidic platforms that aid in creating in vitro cellular microenvironments for applications in drug and toxicity screening. In this review, we provide a comprehensive overview on the integration of microsystems and microfluidics with immunology and focus on different designs developed to decode single immune cell behavior and cellular communication. We have categorized the microfluidic designs in three specific categories: microfluidic chips with cell traps, valve-based microfluidics, and droplet microfluidics that have facilitated the ongoing research in the field of immunology at single-cell level.

Pipette Petri Dish Single-Cell Trapping (PP-SCT) in Microfluidic Platforms: A Passive Hydrodynamic Technique

Microfluidics-based biochips play a vital role in single-cell research applications. Handling and positioning of single cells at the microscale level are an essential need for various applications, including genomics, proteomics, secretomics, and lysis-analysis. In this article, the pipette Petri dish single-cell trapping (PP-SCT) technique is demonstrated. PP-SCT is a simple and cost-effective technique with ease of implementation for single cell analysis applications. In this paper a wide operation at different fluid flow rates of the novel PP-SCT technique is demonstrated. The effects of the microfluidic channel shape (straight, branched, and serpent) on the efficiency of single-cell trapping are studied. This article exhibited passive microfluidic-based biochips capable of vertical cell trapping with the hexagonally-positioned array of microwells. Microwells were 35 μm in diameter, a size sufficient to allow the attachment of captured cells for short-term study. Single-cell capture (SCC) capabilities of the microfluidic-biochips were found to be improving from the straight channel, branched channel, and serpent channel, accordingly. Multiple cell capture (MCC) was on the order of decreasing from the straight channel, branch channel, and serpent channel. Among the three designs investigated, the serpent channel biochip offers high SCC percentage with reduced MCC and NC (no capture) percentage. SCC was around 52%, 42%, and 35% for the serpent, branched, and straight channel biochips, respectively, for the tilt angle, θ values were between 10–15°. Human lung cancer cells (A549) were used for characterization. Using the PP-SCT technique, flow rate variations can be precisely achieved with a flow velocity range of 0.25–4 m/s (fluid channel of 2 mm width and 100 µm height). The upper dish (UD) can be used for low flow rate applications and the lower dish (LD) for high flow rate applications. Passive single-cell analysis applications will be facilitated using this method.

Metal/Matrix Enhanced Time-of-flight Secondary Ion Mass Spectrometry for Single Cell Lipids Analysis

Abstract The chemical components analysis of single cell is important for the understanding of physiological processes such as cell growth, signal transduction and apoptosis. Time-of-flight secondary ion mass spectrometry (ToF-SIMS) is a sensitive surface analysis technique with high spatial resolution, which has been used for single cell and micro-area analysis. However, relatively low ionization yield of biomolecules limited its wide applications in single cell analysis. Herein, we used metal substrate and matrix material to enhance the ionization yield of lipids. The signal intensity of phosphatidylcholine (PC 40:0) casted on the matrix/gold-coated silicon substrate was 65 times higher than that on the silicon wafer. The signal enhancement of phosphatidylcholine (PC 34:1) on single cell surface cultured on matrix/gold-coated silicon substrate was observed as well. Owing to the influence of irregular topography and complex chemical environment of cell, the increase of lipids signal was smaller. Delayed extraction mode of ToF-SIMS overcame the effects of cell topography, leading to further enhancement of the signal intensity of lipids. Meanwhile, simultaneous high spatial resolution of chemical imaging and high mass resolution of the mass spectra of single cells were obtained. Our strategies provided new insights into the study of cell metabolism and cell-environment interactions.

Development of a facile droplet-based single-cell isolation platform for cultivation and genomic analysis in microorganisms

Wider application of single-cell analysis has been limited by the lack of an easy-to-use and low-cost strategy for single-cell isolation that can be directly coupled to single-cell sequencing and single-cell cultivation, especially for small-size microbes. Herein, a facile droplet microfluidic platform was developed to dispense individual microbial cells into conventional standard containers for downstream analysis. Functional parts for cell encapsulation, droplet inspection and sorting, as well as a chip-to-tube capillary interface were integrated on one single chip with simple architecture, and control of the droplet sorting was achieved by a low-cost solenoid microvalve. Using microalgal and yeast cells as models, single-cell isolation success rate of over 90% and single-cell cultivation success rate of 80% were demonstrated. We further showed that the individual cells isolated can be used in high-quality DNA and RNA analyses at both gene-specific and whole-genome levels (i.e. real-time quantitative PCR and genome sequencing). The simplicity and reliability of the method should improve accessibility of single-cell analysis and facilitate its wider application in microbiology researches.

Get to Understand More from Single-Cells: Current Studies of Microfluidic-Based Techniques for Single-Cell Analysis.

This review describes the microfluidic techniques developed for the analysis of a single cell. The characteristics of microfluidic (e.g., little sample amount required, high-throughput performance) make this tool suitable to answer and to solve biological questions of interest about a single cell. This review aims to introduce microfluidic related techniques for the isolation, trapping and manipulation of a single cell. The major approaches for detection in single-cell analysis are introduced; the applications of single-cell analysis are then summarized. The review concludes with discussions of the future directions and opportunities of microfluidic systems applied in analysis of a single cell.

Recent patent applications in single cell analysis.

Recent patent applications in single cell analysis.

Improved Laser Manipulation for On-chip Fabricated Microstructures Based on Solution Replacement and Its Application in Single Cell Analysis

In this paper, we present the fabrication and assembly of microstructures inside a microfluidic device based on a photocrosslinkable resin and optical tweezers. We also report a method of solution replacement inside the microfluidic channel in order to improve the manipulation performance and apply the assembled microstructures for single cell cultivation. By the illumination of patterned ultraviolet (UV) through a microscope, microstructures of arbitrary shape were fabricated by the photocrosslinkable resin inside a microfluidic channel. Based on the microfluidic channel with both glass and polydimethylsiloxane (PDMS) surfaces, immovable and movable microstructures were fabricated and manipulated. The microstructures were fabricated at the desired places and manipulated by the optical tweezers. A rotational microstructure including a microgear and a rotation axis was assembled and rotated in demonstrating this technique. The improved laser manipulation of microstructures was achieved based on the on-chip solution replacement method. The manipulation speed of the microstructures increased when the viscosity of the solvent decreased. The movement efficiency of the fabricated microstructures inside the lower viscosity solvent was evaluated and compared with those microstructures inside the former high viscosity solvent. A novel cell cage was fabricated and the cultivation of a single yeast cell ( w303) was demonstrated in the cell cage, inside the microfluidic device.

Non-biased and efficient global amplification of a single-cell cDNA library

Analysis of single-cell gene expression promises a more precise understanding of molecular mechanisms of a living system. Most techniques only allow studies of the expressions for limited numbers of gene species. When amplification of cDNA was carried out for analysing more genes, amplification biases were frequently reported. A non-biased and efficient global-amplification method, which uses a single-cell cDNA library immobilized on beads, was developed for analysing entire gene expressions for single cells. Every step in this analysis from reverse transcription to cDNA amplification was optimized. By removing degrading excess primers, the bias due to the digestion of cDNA was prevented. Since the residual reagents, which affect the efficiency of each subsequent reaction, could be removed by washing beads, the conditions for uniform and maximized amplification of cDNAs were achieved. The differences in the amplification rates for randomly selected eight genes were within 1.5-folds, which could be negligible for most of the applications of single-cell analysis. The global amplification gives a large amount of amplified cDNA (>100 μg) from a single cell (2-pg mRNA), and that amount is enough for downstream analysis. The proposed global-amplification method was used to analyse transcript ratios of multiple cDNA targets (from several copies to several thousand copies) quantitatively.

Methods and Applications of Raman Microspectroscopy to Single-Cell Analysis

Raman spectroscopy is a powerful biochemical analysis technique that allows for the dynamic characterization and imaging of living biological cells in the absence of fluorescent stains. In this review, we summarize some of the most recent developments in the noninvasive biochemical characterization of single cells by spontaneous Raman scattering. Different instrumentation strategies utilizing confocal detection optics, multispot, and line illumination have been developed to improve the speed and sensitivity of the analysis of single cells by Raman spectroscopy. To analyze and visualize the large data sets obtained during such experiments, sophisticated multivariate statistical analysis tools are necessary to reduce the data and extract components of interest. We highlight the most recent applications of single cell analysis by Raman spectroscopy and their biomedical implications that have enabled the noninvasive characterization of specific metabolic states of eukaryotic cells, the identification and characterization of stem cells, and the rapid identification of bacterial cells. We conclude the article with a brief look into the future of this rapidly evolving research area.

Single-Cell Analysis in Biotechnology, Systems Biology, and Biocatalysis

Single-cell analysis (SCA) has been increasingly recognized as the key technology for the elucidation of cellular functions, which are not accessible from bulk measurements on the population level. Thus far, SCA has been achieved by miniaturization of established engineering concepts to match the dimensions of a single cell. However, SCA requires procedures beyond the classical approach of upstream processing, fermentation, and downstream processing because the biological system itself defines the technical demands. This review characterizes currently available microfluidics and microreactors for invasive (i.e., chemical) and noninvasive (i.e., biological) SCA. We describe the recent SCA omics approaches as tools for systems biology and discuss the role of SCA in genomics, transcriptomics, proteomics, metabolomics, and fluxomics. Furthermore, we discuss applications of SCA for biocatalysis and metabolic engineering as well as its potential for bioprocess optimization. Finally, we define present and future challenges for SCA and propose strategies to overcome current limitations.

Multifunctional Picoliter Droplet Manipulation Platform and Its Application in Single Cell Analysis

We developed an automated and multifunctional microfluidic platform based on DropLab to perform flexible generation and complex manipulations of picoliter-scale droplets. Multiple manipulations including precise droplet generation, sequential reagent merging, and multistep solid-phase extraction for picoliter-scale droplets could be achieved in the present platform. The system precision in generating picoliter-scale droplets was significantly improved by minimizing the thermo-induced fluctuation of flow rate. A novel droplet fusion technique based on the difference of droplet interfacial tensions was developed without the need of special microchannel networks or external devices. It enabled sequential addition of reagents to droplets on demand for multistep reactions. We also developed an effective picoliter-scale droplet splitting technique with magnetic actuation. The difficulty in phase separation of magnetic beads from picoliter-scale droplets due to the high interfacial tension was overcome using ferromagnetic particles to carry the magnetic beads to pass through the phase interface. With this technique, multistep solid-phase extraction was achieved among picoliter-scale droplets. The present platform had the ability to perform complex multistep manipulations to picoliter-scale droplets, which is particularly required for single cell analysis. Its utility and potentials in single cell analysis were preliminarily demonstrated in achieving high-efficiency single-cell encapsulation, enzyme activity assay at the single cell level, and especially, single cell DNA purification based on solid-phase extraction.

Single amino acid resolution of proteolytic fragments generated in individual cells

The complex nature of enzyme regulation mandates that enzyme activity profiles be measured in the context of the intact cell. Single-cell capillary electrophoresis (CE) coupled with laser-induced fluorescence is a powerful approach for quantitation and separation of analytes present in small samples and single live cells; however, it does not allow for the definitive identification of the reaction products. On the other hand, mass spectrometry (MS) is able to identify analytes but still lacks the requisite sensitivity for most single-cell analysis applications. Thus, it follows that by determining the relative amounts of reaction products generated in single cells using CE and by producing larger quantities of these products using bulk cell populations to identify them using MS, it is possible to determine enzyme activity profiles in single cells. In this study, the applicability of this approach was demonstrated by examining the intracellular fate of a protease substrate derived from the beta-amyloid precursor protein (beta-APP). In single live TF-1 cells, three distinct fragments were generated from the beta-APP peptide, which differed by a single uncharged amino acid. The CE measurements indicated that the proteolytic fragment profiles (i.e., the relative amounts of each fragment) were consistent from cell to cell but that they were different from those obtained in cell lysates. Furthermore, measurements obtained at the single cell level made it possible to observe a modest but statistically significant negative correlation between the total amount of beta-APP peptide loaded in cells and the fraction of peptide that remained intact. This study demonstrates how single-cell CE, MS, and peptide substrates can be combined to identify and measure enzyme activities in single live cells.

Interfacing Microfluidics and Laser Desorption/Ionization Mass Spectrometry by Continuous Deposition for Application in Single Cell Analysis

We present a simple method for continuous deposition of effluent originating from a microfluidic device on a flat metal surface for subsequent analysis by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). The sample is delivered using a microscale fused silica capillary and passed onto the surface of a stainless steel plate coated with a layer of a standard matrix. The key parameters optimized in order to obtain high quality and reproducible sample traces are: i) sampleflow rate, ii) speed of the XY-stage movement, and iii) distance of the capillary tip from the plate. Tapering the capillary end as well as surface functionalization to induce hydrophobicity were shown to further enhance the deposition process. The described continuous deposition method is compared with a previously published mass spectrometric method utilizing a piezoelectric microdispenser for microspotting onto the MALDI plates which enabled detection of primary metabolites at the singlecell level. Research is underway to adapt the continuous deposition as an interface for single cell metabolite detection and enhancement of quantitative abilities of the MALDI methodology. We envisage that the presented continuous deposition method may also be suitable for sensitive detection of analytes using other surface analysis tools.

The potential of autofluorescence for the detection of single living cells for label‐free cell sorting in microfluidic systems

A novel method for studying unlabeled living mammalian cells based on their autofluorescence (AF) signal in a prototype microfluidic device is presented. When combined, cellular AF detection and microfluidic devices have the potential to facilitate high-throughput analysis of different cell populations. To demonstrate this, unlabeled cultured cells in microfluidic devices were excited with a 488 nm excitation light and the AF emission (> 505 nm) was detected using a confocal fluorescence microscope (CFM). For example, a simple microfluidic three-port glass microstructure was used together with conventional electroosmotic flow (EOF) to switch the direction of the fluid flow. As a means to test the potential of AF-based cell sorting in this microfluidic device, granulocytes were successfully differentiated from human red blood cells (RBCs) based on differences in AF. This study demonstrated the use of a simple microfabricated device to perform high-throughput live cell detection and differentiation without the need for cell-specific fluorescent labeling dyes and thereby reducing the sample preparation time. Hence, the combined use of microfluidic devices and cell AF may have many applications in single-cell analysis.

Nanobiosensors: probing the sanctuary of individual living cells.

Recently, nanotechnology has been revolutionizing important areas in molecular biology, especially diagnostics and therapy at the molecular and cellular level. The combination of nanotechnology, biology, and photonics opens the possibility of detecting and manipulating atoms and molecules using nanodevices, which have the potential for a wide variety of medical uses at the cellular level. The nanoprobes were fabricated with optical fibers pulled down to tips with distal ends having sizes of approximately 30-50 nm. The nanoscale size of this new class of sensors, allows for measurements in the smallest of environments. One such environment that has evoked a great deal of interest is that of individual cells. Using these nanobiosensors, it has become possible to probe individual chemical species in specific locations throughout a cell. This article provides an overview of the principle, development, and applications of optical nanosensor systems for in vivo bioanalysis at the single-cell level. The fiberoptics nanoprobes were covalently bound with antibodies that are selective to target analyte molecules. Excitation light is launched into the fiber and the resulting evanescent field at the tip of the fiber is used to excite target molecules bound to the antibody molecules. The fluorescence emission from the analyte molecules is then collected via a microscope. The usefulness and potential of this nanotechnology-based biosensor systems in biological research and applications in single-cell analysis are discussed.

High resolution nuclear and X-ray microprobes and their applications in single cell analysis

Investigations of uptake of different chemical compounds by single cells have become a very popular topic and have been performed with different microanalytical techniques using electron, laser, ion and X-ray beams. With the advent of nuclear microprobes with submicron resolution it has been possible to investigate distribution of different elements inside individual cell with sensitivity of parts-per-million (ppm). Recently, with X-ray beams focused to 150 nm and high intensity obtained from third generation synchrotron facilities it became possible to perform analysis on a single cell with sensitivity of parts-per-billion (ppb). Synchrotron radiation offers also a unique possibility to investigate an oxidation state for different elements. In this paper, authors will present and discuss data from experiments on single cells performed with high resolution nuclear and X-ray microprobes.