After having reviewed some aspects of microfluidic system preparation in the first part (1), in this second part of the review we will cover a number of standard operations (namely: sample preparation, sample injection, sample manipulation, reaction, separation, and detection) as well as some biological applications of micro total analysis systems (namely: cell culture, polymerase chain reaction, DNA separation, DNA sequencing, and clinical diagnostics). As previously, we will include papers issued from different scientific journals as well as useful abstracts from three conference proceedings: MEMS, Transducers, and μTAS. In this second part, we do not include the period covered by the history section (1975-1997) from part 1 but try to cover the relevant examples of the literature published between January 1998 and March 2002. We briefly describe articles that struck us as needing special attention, while more “standard” papers are dutifully reported in groups of interest. An article might be included in more than one section, depending on the ideas developed in it.

Micro total analysis systems. 2. Analytical standard operations and applications.

Micromachining technology was used to prepare chemical analysis systems on glass chips (1 centimeter by 2 centimeters or larger) that utilize electroosmotic pumping to drive fluid flow and electrophoretic separation to distinguish sample components. Capillaries 1 to 10 centimeters long etched in the glass (cross section, 10 micrometers by 30 micrometers) allow for capillary electrophoresis-based separations of amino acids with up to 75,000 theoretical plates in about 15 seconds, and separations of about 600 plates can be effected within 4 seconds. Sample treatment steps within a manifold of intersecting capillaries were demonstrated for a simple sample dilution process. Manipulation of the applied voltages controlled the directions of fluid flow within the manifold. The principles demonstrated in this study can be used to develop a miniaturized system for sample handling and separation with no moving parts.

Micromachining a miniaturized capillary electrophoresis-based chemical analysis system on a chip

According to Genome Sequencing Project statistics (http://www.ncbi.nlm.nih.gov/genomes/static/gpstat.html), as of Feb 16, 2012, complete gene sequences have become available for 2816 viruses, 1117 prokaryotes, and 36 eukaryotes.1–2 The availability of full genome sequences has greatly facilitated biological research in many fields, and has greatly contributed to the growth of proteomics.

Proteins are important because they are the direct bio-functional molecules in the living organisms. The term “proteomics” was coined from merging “protein” and “genomics” in the 1990s.3–4 As a post-genomic discipline, proteomics encompasses efforts to identify and quantify all the proteins of a proteome, including expression, cellular localization, interactions, post-translational modifications (PTMs), and turnover as a function of time, space and cell type, thus making the full investigation of a proteome more challenging than sequencing a genome. There are possibly 100,000 protein forms encoded by the approximate 20,235 genes of the human genome,5 and determining the explicit function of each form will be a challenge.

The progress of proteomics has been driven by the development of new technologies for peptide/protein separation, mass spectrometry analysis, isotope labeling for quantification, and bioinformatics data analysis. Mass spectrometry has emerged as a core tool for large-scale protein analysis. In the past decade, there has been a rapid advance in the resolution, mass accuracy, sensitivity and scan rate of mass spectrometers used to analyze proteins. In addition, hybrid mass analyzers have been introduced recently (e.g. Linear Ion Trap-Orbitrap series6–7) which have significantly improved proteomic analysis.

“Bottom-up” protein analysis refers to the characterization of proteins by analysis of peptides released from the protein through proteolysis. When bottom-up is performed on a mixture of proteins it is called shotgun proteomics,8–10 a name coined by the Yates lab because of its analogy to shotgun genomic sequencing.11 Shotgun proteomics provides an indirect measurement of proteins through peptides derived from proteolytic digestion of intact proteins. In a typical shotgun proteomics experiment, the peptide mixture is fractionated and subjected to LC-MS/MS analysis. Peptide identification is achieved by comparing the tandem mass spectra derived from peptide fragmentation with theoretical tandem mass spectra generated from in silico digestion of a protein database. Protein inference is accomplished by assigning peptide sequences to proteins. Because peptides can be either uniquely assigned to a single protein or shared by more than one protein, the identified proteins may be further scored and grouped based on their peptides. In contrast, another strategy, termed ‘top-down’ proteomics, is used to characterize intact proteins (Figure 1). The top-down approach has some potential advantages for PTM and protein isoform determination and has achieved notable success. Intact proteins have been measured up to 200 kDa,12 and a large scale study has identified more than 1,000 proteins by multi-dimensional separations from complex samples.13 However, the top-down method has significant limitations compared with shotgun proteomics due to difficulties with protein fractionation, protein ionization and fragmentation in the gas phase. By relying on the analysis of peptides, which are more easily fractionated, ionized and fragmented, shotgun proteomics can be more universally adopted for protein analysis. In fact, a hybrid of bottom-up and top-down methodologies and instrumentation has been introduced as middle-down proteomics.14 Essentially, middle-down proteomics analyzes larger peptide fragments than bottom-up proteomics, minimizing peptide redundancy between proteins. Additionally the large peptide fragments yield similar advantages as top-down proteomics, such as gaining further insight into post-translational modifications, without the analytical challenges of analyzing intact proteins. Shotgun proteomics has become a workhorse for the analysis of proteins and their modifications and will be increasingly combined with top-down methods in the future.



Figure 1

Proteomic strategies: bottom-up vs. top-down vs. middle-down. The bottom-up approach analyzes proteolytic peptides. The top-down method measures the intact proteins. The middle-down strategy analyzes larger peptides resulted from limited digestion or ...



In the past decade shotgun proteomics has been widely used by biologists for many different research experiments, advancing biological discoveries. Some applications include, but are not limited to, proteome profiling, protein quantification, protein modification, and protein-protein interaction. There have been several reviews nicely summarizing mass spectrometry history,15 protein quantification with mass spectrometry,16 its biological applications,5,17–26 and many recent advances in methodology.27–32 In this review, we try to provide a full and updated survey of shotgun proteomics, including the fundamental techniques and applications that laid the foundation along with those developed and greatly improved in the past several years.

Protein Analysis by Shotgun/Bottom-up Proteomics

Polymer microfluidic devices

A large part of the excitement behind microfluidics is in its potential for producing practical devices, but surprisingly few lab-on-a-chip based technologies have been successfully introduced into the market. Here, we review current work in commercializing microfluidic technologies, with a focus on point-of-care diagnostics applications. We will also identify challenges to commercialization, including lessons drawn from our experience in Claros Diagnostics. Moving forward, we discuss the need to strike a balance between achieving real-world impact with integrated devices versus design of novel single microfluidic components.

http://mnbs-2014.sciencesconf.org/conference/mnbs-2014/pages/Chin_Lab_Chip_2012_commercialisation_of_PoC.pdf

Commercialization of microfluidic point-of-care diagnostic devices

Electrochemistry at liquid/liquid interfaces: methodology and potential applications

Our recent developments concerning the fabrication of polymer microchips and their applications for biochemical analyses are reviewed. We first describe two methods of fabrication of polymer microfluidic chips, namely UV-laser photoablation and plasma etching that are well suited for the prototyping and mass fabrication of microchannel networks with integrated microelectrodes. These microanalytical systems can be coupled with various detection means including mass spectrometry, and their applications in capillary electrophoresis are presented here. We also present how UV laser photoablation can be used for the patterning of biomolecules on polymer surfaces for generating two-dimensional arrays of microspots to carry out affinity assays. Finally, the use of the microchips for the development of fast affinity and immunological assays with electrochemical detection is presented, demonstrating the potential of these polymer microchips for medical diagnostics and drug discovery.

Polymer microfluidic chips for electrochemical and biochemical analyses.

The present review deals with a number of prefractionation protocols in preparation for two-dimensional map analysis, both in the fields of chromatography and in the field of electrophoresis In the first case, Fountoulaki’s groups has reported just about any chromatographic procedure useful as a prefractionation step, including affinity, ion-exchange, and reversed-phase resins As a result of the various enrichment steps, several hundred new species, previously undetected in unfractionated samples, could be revealed for the first time Electrophoretic prefractionation protocols include all those electrokinetic methodologies which are performed in free solution, essentially all relying on isoelectric focusing steps The devices here reviewed include multichamber apparatus, such as the multicompartment electrolyzer with Immobiline membranes, Off-Gel electrophoresis in a multicup device and the Rotofor, an instrument also based on a multichamber system but exploiting the conventional technique of carrier-ampholyte-focusing Other instruments of interest are the Octopus, a continuous-flow device for isoelectric focusing in a upward flowing liquid curtain, and the Gradiflow, where different pI cuts are obtained by a multistep passage through two compartments buffered at different pH values It is felt that this panoply of methods could offer a strong step forward in “mining below the tip of the iceberg” for detecting the “unseen proteome”

Prefractionation techniques in proteome analysis.

A new protein fractionation technique based on off-gel isoelectric focusing (IEF) is presented, where the proteins are separated according to their isoelectric point (pI) in a multiwell device with the advantage to be directly recovered in solution for further analysis. The protein fractions obtained with this technique have then been characterized with polymer nanoelectrospray for mass spectrometry (MS) analyses or with Bioanalyzer for mass identification. This methodology shows the possibility of developing alternatives to the classical two-dimensional (2-D) gel electrophoresis. One species numerical simulation of the electric field distribution during off-gel separation is also presented in order to demonstrate the principle of the purification. Experiments with pI protein markers have been carried out in order to highlight the kinetics and the efficiency of the technique. Moreover, the resolution of the fractionation was shown to be 0.1 pH unit for the separation of beta-lactoglobulin A and B. In addition, the isoelectric fractionation of an Escherichia coli extract was performed in standard solubilization buffer to demonstrate the performances of the technique, notably for proteomics applications.

Protein fractionation in a multicompartment device using Off-Gel isoelectric focusing.

This paper presents a fabrication of polymer microchips with homogeneous material technique due to surface treatment by plasma before sealing. UV laser photoablation was used for fast prototyping of microstructures, and oxygen plasma was used as a surface treatment for both the microfabricated substrate and the polymer cover. It was found that with an oxidative plasma treatment, successful bonding could be achieved without adhesive material between polymer sheets substantially below the glass transition temperature of the polymer. Homogeneous polyethylene terephthalate (PET) microstructures were characterized by scanning electron microscopy (SEM) and analyzed by X-ray photoelectron spectroscopy (XPS) surface analyses after different surface treatments. The electroosmotic flow characteristics including the velocity and the stability over 20 days have been tested and compared to composite channels, in which the cover presents a polyethylene (PE) adhesive layer. Capillary zone electrophoresis in both homogeneous and composite microanalytical devices were then performed and compared in order to evaluate the separation efficiency. In preliminary experiments, a plate height of 0.6 m has been obtained with homogenous microchannels. The surface analysis pointed out that the surface chemistry is of prime importance for the performance of microfluidic separation.

Frédéric Reymond

Papers

Electrochemistry at liquid/liquid interfaces: methodology and potential applications

Polymer microfluidic chips for electrochemical and biochemical analyses.

Prefractionation techniques in proteome analysis.

Protein fractionation in a multicompartment device using Off-Gel isoelectric focusing.

Polymer microchips bonded by O2-plasma activation