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

Capillarity and Wetting Phenomena: Drops, Bubbles, Pearls, Waves

Precise and effective control of droplet generation is critical for applications of droplet microfluidics ranging from materials synthesis to lab-on-a-chip systems. Methods for droplet generation can be either passive or active, where the former generates droplets without external actuation, and the latter makes use of additional energy input in promoting interfacial instabilities for droplet generation. A unified physical understanding of both passive and active droplet generation is beneficial for effectively developing new techniques meeting various demands arising from applications. Our review of passive approaches focuses on the characteristics and mechanisms of breakup modes of droplet generation occurring in microfluidic cross-flow, co-flow, flow-focusing, and step emulsification configurations. The review of active approaches covers the state-of-the-art techniques employing either external forces from electrical, magnetic and centrifugal fields or methods of modifying intrinsic properties of flows or fluids such as velocity, viscosity, interfacial tension, channel wettability, and fluid density, with a focus on their implementations and actuation mechanisms. Also included in this review is the contrast among different approaches of either passive or active nature.

Passive and active droplet generation with microfluidics: a review

Fogging occurs when moisture condensation takes the form of accumulated droplets with diameters larger than 190 nm or half of the shortest wavelength (380 nm) of visible light. This problem may be effectively addressed by changing the affinity of a material’s surface for water, which can be accomplished via two approaches: i) the superhydrophilic approach, with a water contact angle (CA) less than 5°, and ii) the superhydrophobic approach, with a water CA greater than 150°, and extremely low CA hysteresis. To date, all techniques reported belong to the former category, as they are intended for applications in optical transparent coatings. A well-known example is the use of photocatalytic TiO2 nanoparticle coatings that become superhydrophilic under UV irradiation. Very recently, a capillary effect was skillfully adopted to achieve superhydrophilic properties by constructing 3D nanoporous structures from layer-by-layer assembled nanoparticles. The key to these two “wet”-style antifogging strategies is for micrometer-sized fog drops to rapidly spread into a uniform thin film, which can prevent light scattering and reflection from nucleated droplets. Optical transparency is not an intrinsic property of antifogging coatings even though recently developed antifogging coatings are almost transparent, and the transparency could be achieved by further tuning the nanoparticle size and film thickness. To our knowledge, the antifogging coatings may also be applied to many fields that do not require optical transparency, including, for example, paints for inhibiting swelling and peeling issues and metal surfaces for preventing corrosion. These types of issues, which are caused by adsorption of moisture, are hard to solve by the superhydrophilic approach because of its inherently “wet” nature. Thus, a “dry”-style antifogging strategy, which consists of a novel superhydrophobic technique that can prevent moisture or microscale fog drops from nucleating on a surface, is desired. Recent bionic researches have revealed that the self-cleaning ability of lotus leaves and the striking ability of a water-strider’s legs to walk on water can be attributed to the ideal superhydrophobicity of their surfaces, induced by special microand nanostructures. To date, the biomimetic fabrication of superhydrophobic microand/or nanostructures has attracted considerable interest, and these types of materials can be used for such applications as self-cleaning coatings and stain-resistant textiles. Although a superhydrophobic technique inspired by lotus leaves is expected to be able to solve such fogging problems because the water droplets can not remain on the surface, there are no reports of such antifogging coatings. Very recently, researchers from General Motors have reported that the surfaces of lotus leaves become wet with moisture because the size of the fog drops are at the microscale—so small that they can be easily trapped in the interspaces among micropapillae. Thus, lotuslike surface microstructures are unsuitable for superhydrophobic antifogging coatings, and a new inspiration from nature is desired for solving this problem. In this communication, we report a novel, biological, superhydrophobic antifogging strategy. It was found that the compound eyes of the mosquito C. pipiens possess ideal superhydrophobic properties that provide an effective protective mechanism for maintaining clear vision in a humid habitat. Our research indicates that this unique property is attributed to the smart design of elaborate microand nanostructures: hexagonally non-close-packed (ncp) nipples at the nanoscale prevent microscale fog drops from condensing on the ommatidia surface, and hexagonally close-packed (hcp) ommatidia at the microscale could efficiently prevent fog drops from being trapped in the voids between the ommatidia. We also fabricated artificial compound eyes by using soft lithography and investigated the effects of microand nanostructures on the surface hydrophobicity. These findings could be used to develop novel superhydrophobic antifogging coatings in the near future. It is known that mosquitoes possess excellent vision, which they exploit to locate various resources such as mates, hosts, and resting sites in a watery and dim habitat. To better understand such remarkable abilities, we first investigated the interaction between moisture and the eye surface. An ultrasonic humidifier was used to regulate the relative humidity of the atmosphere and mimic a mist composed of numerous tiny water droplets with diameters less than 10 lm. As the fog was C O M M U N IC A IO N

The dry-style antifogging properties of mosquito compound eyes and artificial analogues prepared by soft lithography: a superhydrophobic state with high adhesive force

Microfluidics consist of microfabricated structures for liquid handling, with cross-sections in the 1–500 μm range, and small volume capacity (fL-nL) Capillary tubes connected with fittings,1 although utilizing small volumes, are not considered microfluidics for the purposes of this paper since they are not microfabricated Likewise, millifluidic systems, made by conventional machining tools, are excluded due to their larger feature sizes (>500 μm)

Though micromachined systems for gas chromatography were introduced in the 1970’s,2 the field of microfluidics did not gain much traction until the 1990’s3 Silicon and glass were the original materials used, but then the focus shifted to include polymer substrates, and in particular, polydimethylsiloxane (PDMS) Since then the field has grown to encompass a wide variety of materials and applications The successful demonstration of electrophoresis and electroosmotic pumping in a microfluidic device provided a nonmechanical method for both fluid control and separation4 Laser induced fluorescence (LIF) enabled sensitive detection of fluorophores or fluorescently labeled molecules The expanded availability of low-cost printing allowed for cheaper and quicker mask fabrication for use in soft lithography5 Commercial microfluidic systems are now available from Abbott, Agilent, Caliper, Dolomite, Micralyne, Microfluidic Chip Shop, Micrux Technologies and Waters, as a few prominent examples For a more thorough description of the history of microfluidics, we refer the reader to a number of comprehensive, specialized reviews,3, 6–11 as well as a more general 2006 review12

The field of microfluidics offers many advantages compared to carrying out processes through bulk solution chemistry, the first of which relates to a lesson taught to every first-year chemistry student Simply stated, diffusion is slow! Thus, the smaller the distance required for interaction, the faster it will be Smaller channel dimensions also lead to smaller sample volumes (fL-nL), which can reduce the amount of sample or reagents required for testing and analysis Reduced dimensions can also lead to portable devices to enable on-site testing (provided the associated hardware is similarly portable) Finally, integration of multiple processes (like labeling, purification, separation and detection) in a microfluidic device can be highly enabling for many applications

Microelectromechanical systems (MEMS) contain integrated electrical and mechanical parts that create a sensor or system Applications of MEMS are ubiquitous, including automobiles, phones, video games and medical and biological sensors13 Micro-total analysis systems, also known as labs-on-a-chip, are the chemical analogue of MEMS, as integrated microfluidic devices that are capable of automating multiple processes relevant to laboratory sciences For example, a typical lab-on-a-chip system might selectively purify a complex mixture (through filtering, antibody capture, etc), then separate target components and detect them

Microfluidic devices consist of a core of common components Areas defined by empty space, such as reservoirs (wells), chambers and microchannels, are central to microfluidic systems Positive features, created by areas of solid material, add increased functionality to a chip and can consist of membranes, monoliths, pneumatic controls, beams and pillars Given the ubiquitous nature of negative components, and microchannels in particular, we focus here on a few of their properties Microfluidic channels have small overall volumes, laminar flow and a large surface-to-volume ratio Dimensions of a typical separation channel in microchip electrophoresis (μCE) are: 50 μm width, 15 μm height and 5 cm length for a volume of 375 nL Flow in these devices is normally nonturbulent due to low Reynolds numbers For example, water flowing at 20°C in the above channel at 1 μL/min (222 cm/s) results in a Reynolds number of ~05, where <2000 is laminar flow Since flow is nonturbulent, mixing is normally diffusion-limited Small channel sizes also have a high surface-to-volume ratio, leading to different characteristics from what are commonly found in bulk volumes The material surface can be used to manipulate fluid movement (such as by electroosmotic flow, EOF) and surface interactions For a solution in contact with a charged surface, a double layer of charge is created as oppositely charged ions are attracted to the surface charges This electrical double layer consists of an inner rigid or Stern Layer and an outer diffuse layer An electrostatic potential known as the zeta potential is formed, with the magnitude of the potential decreasing as distance from the surface increases The electrical double layer is the basis for EOF, wherein an applied voltage causes the loosely bound diffuse layer to move towards an electrode, dragging the bulk solution along Charges on the exposed surface also exert a greater influence on the fluid in a channel as its size decreases Larger surface-to-volume ratios are more prone to nonspecific adsorption and surface fouling In particular, non-charged and hydrophobic microdevice surfaces can cause proteins in solution to denature and stick

We focus our review on advances in microfluidic systems since 2008 In doing this, we occasionally must cover foundational work in microfluidics that is considerably less recent We do not focus on chemical synthesis applications of microfluidics although it is an expanding area, nor do we delve into lithography, device fabrication or production costs Our specific emphasis herein is on four areas within microfluidics: properties and applications of commonly used materials, basic functions, integration, and selected applications For each of these four topics we provide a concluding section on opportunities for future development, and at the end of this review, we offer general conclusions and prospective for future work in the field Due to the considerable scope of the field of microfluidics, we limit our discussion to selected examples from each area, but cite in-depth reviews for the reader to turn to for further information about specific topics We also refer the reader to recent comprehensive reviews on advances in lab-on-a-chip systems by Arora et al10 and Kovarik et al14

/pdf/advances-in-microfluidic-materials-functions-integration-and-27mmao3n1o.pdf

Advances in microfluidic materials, functions, integration, and applications.

Flexible microchannels have soft walls which undergo deformation under the influence of fluid flow. The dimensional and flexural similarity of flexible microchannels make them ideal candidates for mimicking biological structures such as blood vessels and air pathway in lungs. The analysis of fluid flow and the dynamics of interaction of cells through flexible arteries provide valuable insights about cardiovascular-related diseases. Flexible microchannels can be instrumental in the in vitro investigation of such diseases. This review discusses the recent developments in pressure-driven flow through flexible microchannels and their applications. Here we present the existing theoretical models that predict the deformation and pressure-flow characteristics of flexible microchannels and the corresponding experimental validations. We compare the models for laminar flow of Newtonian fluids through flexible microchannels with their corresponding experimental validation and enlist their limitations. We discuss in detail the various applications of flexible microchannels and their relevance in cell mechanophenotyping, micropumps, microflow stabilizers, and organ-on-chip devices. The insight into the flow dynamics provided herein will extend using flexible microchannels to develop organs-on-chip and other microfluidic applications.

Pressure-driven flow through PDMS-based flexible microchannels and their applications in microfluidics

Demulsification of droplets stabilized with surfactant is very challenging due to their low surface energy. We report ultralow voltage-based electrocoalescence phenomenon for the demulsification of aqueous droplets with an aqueous stream. In the absence of electric field, due to the disjoining pressure resulting from the tail–tail interaction between the surfactant molecules present on the aqueous droplets and interface, coalescence of aqueous droplets with the aqueous stream is prevented. However, above a critical electric field, the electrical stress overcomes the disjoining pressure, thus leading to the droplet coalescence. The influence of surfactant concentration, droplet diameter, and velocity on the electrocoalescence phenomena is studied. The macroscopic contact between the aqueous droplet with the aqueous stream enables droplet coalescence at much lower voltage (10 to 90 V), which is at least two orders of magnitude smaller than voltages used in prior works (1.0 to 3.0 kV). The electrocoalescence...

Droplet Demulsification Using Ultralow Voltage-Based Electrocoalescence.

This work presents design, fabrication and test of a microfluidic device which employs Fahraeus-Lindqvist and Zweifach-Fung effects for cell concentration and blood cell-plasma separation. The device design comprises a straight main channel with a series of branched channels placed symmetrically on both sides of the main channel. The design implements constrictions before each junction (branching point) in order to direct cells that would have migrated closer to the wall (naturally or after liquid extraction at a junction) towards the centre of the main channel. Theoretical and numerical analysis are performed for design of the microchannel network to ensure that a minimum flow rate ratio (of 2.5:1, main channel-to-side channels) is maintained at each junction and predict flow rate at the plasma outlet. The dimensions and location of the constrictions were determined using numerical simulations. The effect of presence of constrictions before the junctions was demonstrated by comparing the performances of the device with and without constrictions. To demonstrate the performance of the device, initial experiments were performed with polystyrene microbeads (10 and 15 μm size) and droplets. Finally, the device was used for concentration of HL60 cells and separation of plasma and cells in diluted blood samples. The cell concentration and blood-plasma purification efficiency was quantified using Haemocytometer and Fluorescence-Activated Cell Sorter (FACS). A seven-fold cell concentration was obtained with HL60 cells and a purification efficiency of 70 % and plasma recovery of 80 % was observed for diluted (1:20) blood sample. FACS was used to identify cell lysis and the cell viability was checked using Trypan Blue test which showed that more than 99 % cells are alive indicating the suitability of the device for practical use. The proposed device has potential to be used as a sample preparation module in lab on chip based diagnostic platforms.

Development of a microfluidic device for cell concentration and blood cell-plasma separation

This paper reports a novel hydrodynamic technique for sorting of droplets and cells based on size and deformability. The device comprises two modules: a focusing and spacing control module and a sorting module. The focusing and spacing control module enables focusing of objects present in a sample onto one of the side walls of a channel with controlled spacing between them using a sheath fluid. A 3D analytical model is developed to predict the sheath-to-sample flow rate ratio required to facilitate single-file focusing and maintain the required spacing between a pair of adjacent objects. Experiments are performed to demonstrate focusing and spacing control of droplets (size 5–40 μm) and cells (HL60, size 10–25 μm). The model predictions compare well with experimental data in terms of focusing and spacing control within 9%. In the sorting module, the main channel splits into two branch channels (straight and side branches) with the flow into these two channels separated by a “dividing streamline”. A sensing channel and a bypass channel control the shifting of the dividing streamline depending on the object size and deformability. While resistance offered by individual droplets of different sizes has been studied in our previous work (P. Sajeesh, M. Doble and A. K. Sen, Biomicrofluidics, 2014, 8, 1–23), here we present resistance of individual cells (HL60) as a function of size. A theoretical model is developed and used for the design of the sorter. Experiments are performed for size-based sorting of droplets (sizes 25 and 40 μm, 10 and 15 μm) and HL60 cells (sizes 11 μm and 19 μm) and deformability-based sorting of droplets (size 10 ± 1.0 μm) and polystyrene microbeads (size 10 ± 0.2 μm). The performance of the device for size- and deformability-based sorting is characterized in terms of sorting efficiency. The proposed device could be potentially used as a diagnostic tool for sorting of larger tumour cells from smaller leukocytes.

A microfluidic device with focusing and spacing control for resistance-based sorting of droplets and cells

We report controlled motion of aqueous droplets on soot-wax coated superhydrophobic surface of a PDMS-iron particle composite substrate. The surface profile around a droplet is altered due to magnetic force acting on the iron particles embedded in the substrate, which leads to droplet motion. The contact angle and roll off angle depend on the soot deposit time and particle distribution. Endurance test is performed to confirm stability of the surface. The deformation behaviour of the substrate is studied with particle size, surface to magnet gap and the cone angle of the magnet. The proposed surface could find applications in the manipulation of sample drops in biological and chemical assays.

Ashis Kumar Sen

Papers

Pressure-driven flow through PDMS-based flexible microchannels and their applications in microfluidics

Droplet Demulsification Using Ultralow Voltage-Based Electrocoalescence.

Development of a microfluidic device for cell concentration and blood cell-plasma separation

A microfluidic device with focusing and spacing control for resistance-based sorting of droplets and cells

Magnetic field assisted droplet manipulation on a soot-wax coated superhydrophobic surface of a PDMS-iron particle composite substrate