•Confocal laser-induced fluorescence detector for submicron capillary system.•Laser-induced fluorescence detector with yoctomole detection limit.•2-μm-tubing flow injection system capable of detecting 1.4 zeptomole fluorescein.Laser-induced fluorescence (LIF) detectors for low-micrometer and sub-micrometer capillary on-column detection are not commercially available. In this paper, we describe in details how to construct a confocal LIF detector to address this issue. We characterize the detector by determining its limit of detection (LOD), linear dynamic range (LDR) and background signal drift; a very low LOD (~70 fluorescein molecules or 12 yoctomole fluorescein), a wide LDR (greater than 3 orders of magnitude) and a small background signal drift (~1.2-fold of the root mean square noise) are obtained. For detecting analytes inside a low-micrometer and sub-micrometer capillary, proper alignment is essential. We present a simple protocol to align the capillary with the optical system and use the position-lock capability of a translation stage to fix the capillary in position during the experiment. To demonstrate the feasibility of using this detector for narrow capillary systems, we build a 2-μm-i.d. capillary flow injection analysis (FIA) system using the newly developed LIF prototype as a detector and obtain an FIA LOD of 14 zeptomole fluorescein. We also separate a DNA ladder sample by bare narrow capillary – hydrodynamic chromatography and use the LIF prototype to monitor the resolved DNA fragments. We obtain not only well-resolved peaks but also the quantitative information of all DNA fragments.

•A novel model FFMRB for continuous protein concentration.•Successful prediction of processing capacity, fold and location of protein enrichment via FFMRB.•Up to 125.7 mL/h maximum processing capacity, 95.5% recovery and 12.6 times enrichment factor.In this work, we developed the model and theory of free-flow moving reaction boundary electrophoresis (FFMRB) for continuous protein concentration for the first time. The theoretical results indicated that (i) the moving reaction boundary (MRB) can be quantitatively designed in free-flow electrophoresis (FFE) system; (ii) charge-to-mass ratio (Z/M) analysis could provide guidance for protein concentration optimization; and (iii) the maximum processing capacity could be predicted. To demonstrate the model and theory, three model proteins of hemoglobin (Hb), cytochrome C (Cyt C) and C-phycocyanin (C-PC) were chosen for the experiments. The experimental results verified that (i) stable MRBs with different velocities could be established in FFE apparatus with weak acid/weak base neutralization reaction system; (ii) proteins of Hb, Cyt C and C-PC were well concentrated with FFMRB; and (iii) a maximum processing capacity and recovery ratio of Cyt C enrichment were 126 mL/h and 95.5% respectively, and a maximum enrichment factor was achieved 12.6 times for Hb. All of the experiments demonstrated the protein concentration model and theory. In contrast to other methods, the continuous processing ability enables FFMRB to efficiently enrich diluted protein or peptide in large volume solution.

In this paper, we develop a simple electrokinetic means for fractionating protein samples according to their pI values without using ampholytes. The method uses inexpensive equipment, and its consumables are primarily ammonium acetate buffers. A key component of its apparatus is a dialysis membrane interface that eliminates electrolysis-caused protein oxidation/reduction and constrains proteins in the desired places. We demonstrate its feasibility for fractionating standard proteins and real-world samples. With the elimination of ampholytes, we can analyze the fractionated proteins directly by a matrix assisted laser desorption/ionization time-of-flight mass spectrometer. Important experimental parameters are also discussed in order to obtain good fractionation results.

Multiplex polymerase chain reaction (PCR) has been widely utilized for high-throughput pathogen identification. Often, a dye is used to intercalate the amplified DNA fragments, and identifications of the pathogens are carried out by DNA melting curve analysis or gel electrophoresis. Integrating DNA amplification and identification is a logic path toward maximizing the benefit of multiplex PCR. Although PCR and gel electrophoresis have been integrated, replenishing the gels after each run is tedious and time-consuming. In this technical note, we develop an approach to address this issue. We perform multiplex PCR inside a capillary, transfer the amplified fragments to a bare narrow capillary, and measure their lengths online using bare narrow capillary–hydrodynamic chromatography (BaNC-HDC), a new technique recently developed in our laboratory for free-solution DNA separation. To intercalate the DNA with YOYO-1 (a fluorescent dye) for BaNC-HDC, we flush the capillary column with a YOYO-1 solution; positively charged YOYO-1 is adsorbed (or charged) onto the negatively charged capillary wall. As DNA molecules are driven down the column for separation, they react with the YOYO-1 stored on the capillary wall and are online-intercalated with the dye. With a single YOYO-1 charging, the column can be used for more than 40 runs, although the fluorescence signal intensities of the DNA peaks decrease gradually. Although the dye-DNA intercalation occurs during the separation, it does not affect the retention times, separation efficiencies, or resolutions.

•Using a mixing chamber to produce a smooth gradient for chromatographic elution.•Integrating the above gradient generator with an electroosmotic pump into a miniaturized HPLC.•Coupling the above HPLC with a mass spectrometer for peptide and protein analysis.Toward developing a micro HPLC cartridge, we have recently built a high-pressure electroosmotic pump (EOP). However, we do not recommend people to use this pump to deliver an organic solvent directly, because it often makes the pump rate unstable. We have experimented several approaches to address this issue, but none of them are satisfactory. Here, we develop an innovative approach to address this issue. We first create an abruption (a dead-volume) within a fluid conduit. We then utilize an EOP to withdraw, via a selection valve, a train of eluent solutions having decreasing eluting power into the fluid conduit. When these solutions are further aspirated through the dead-volume, these solutions are partially mixed, smoothening concentration transitions between two adjacent eluent solutions. As these solutions are pushed back, through the dead-volume again, a smooth gradient profile is formed. In this work, we characterize this scheme for gradient formation, and we incorporate this approach with a high-pressure EOP, a nanoliter injection valve, and a capillary column, yielding a micro HPLC system. We then couple this micro HPLC with an electrospray ionization – mass spectrometer for peptide and protein separations and identifications.

•A novel recycling free-flow isoelectric focusing device for protein pre-fractionation.•A reciprocating flow system firstly introduced in a free-flow electrophoresis.•Proper pH gradient obtained in this new recycling free-flow isoelectric focusing device.•Fractionation of Model proteins and serum proteins by the proposed device.The traditional recycling free-flow isoelectric focusing (RFFIEF) suffered from complex structure, tedious operations and poor extensibility as well as high cost. To address these issues, a novel reciprocating free-flow isoelectric focusing device (ReFFIEF) was developed for proteins or peptides pre-fractionation. In the new device, a reciprocating background flow was for the first time introduced into free flow electrophoresis (FFE) system. The gas cushion injector (GCI) used in the previous continuous free-flow electrophoresis (CFFE) was redesigned for the reciprocating background flow. With the GCI, the reciprocating background flow could be achieved between the GCI, separation chamber and transient self-balance collector (tSBC). In a run, process fluid flowed to and from, forming a stable reciprocating fluid flow in the separation chamber. A pH gradient was created within the separation chamber, and at the same time proteins were focused repeatedly when passing through the chamber under perpendicular electric field. The ReFFIEF procedure was optimized for fractionations of three model proteins, and the optimized method was further used for pre-fractionation of model human serum samples. As compared with the traditional RFFIEF devices developed about 25 years ago, the new ReFFIEF system showed several merits, such as simple design and structure, user-friendly operation and easy to extend as well as low cost.

We have microfabricated two functional components toward developing a microchip flow injection analysis (FIA) system, i.e., an open-channel electroosmotic pump and a gas-diffusion chip, consisting of two microfabricated glass wafers and a porous polytetrafluoroethylene membrane. This is the first application of gas-diffusion separation in a microchip FIA system. To demonstrate the feasibility of using these two components for performing gas-diffusion FIA, we have incorporated them together with a regular FIA injection valve and a capillary electrophoresis absorbance detector in a flow injection system for determination of ammonia in environmental water samples. This system has a limit of detection of 0.10 mg L−1 NH3, with a good repeatability (relative standard deviation of less than 5 % for 4.0 mg L−1 NH3). Parameters affecting its performance are also discussed.

Here, we construct an open-channel on-chip electroosmotic pump capable of generating pressures up to ∼170 bar and flow rates up to ∼500 nL/min, adequate for high performance liquid chromatographic (HPLC) separations. A great feature of this pump is that a number of its basic pump units can be connected in series to enhance its pumping power; the output pressure is directly proportional to the number of pump units connected. This additive nature is excellent and useful, and no other pumps can work in this fashion. We demonstrate the feasibility of using this pump to perform nanoflow HPLC separations; tryptic digests of bovine serum albumin (BSA), transferrin factor (TF), and human immunoglobulins (IgG) are utilized as exemplary samples. We also compare the performance of our electroosmotic (EO)-driven HPLC with Agilent 1200 HPLC; comparable efficiencies, resolutions, and peak capacities are obtained. Since the pump is based on electroosmosis, it has no moving parts. The common material and process also allow this pump to be integrated with other microfabricated functional components. Development of this high-pressure on-chip pump will have a profound impact on the advancement of lab-on-a-chip devices.

High-performance liquid chromatography (HPLC) plays an important role in biotechnology, and a majority of chromatographic separations use gradient elution. While gradient generators can be built in different formats, binary pumps or quaternary pumps are most frequently used for gradient generator constructions. We have recently developed a high-pressure electroosmotic pump (EOP); the pump can be manufactured at a cost of a few hundred dollars. However, it is challenging to use this pump to deliver a gradient eluent directly. In this study, we first improve the monolith preparation by applying a pressure to the monomer solution during polymerization. We assemble a binary EOP gradient generator and discuss the relationship between the gradient profile and voltage applied to the EOP. We demonstrate the feasibility of the binary EOP gradient generator for generating a smooth and reproducible nanoflow gradient. After integration of the gradient generator into a miniaturized HPLC system, we use the HPLC system for separating peptide mixtures from trypsin-digested proteins. The performance comparison between the above miniaturized HPLC system and an Agilent 1200 HPLC system exhibits comparable efficiencies, resolutions, and peak capacities.

•We demonstrate the feasibility of a low-cost high-pressure electroosmotic pump for gradient HPLC separation.•We develop a means for isolating a gradient eluent from electroosmotic pump solution, ensuring a stable pump rate.•We develop a means for producing a nanogradient eluent for capillary chromatographic separation.•We develop a total-capillary chromatographic system.•We couple the total-capillary chromatographic system with an MS for protein and peptide analysis.We integrate a high-pressure electroosmotic pump (EOP), a nanoflow gradient generator, and a capillary column into a miniaturized liquid chromatographic system that can be directly coupled with a mass spectrometer for proteomic analysis. We have recently developed a low-cost high-pressure EOP capable of generating pressure of tens of thousands psi, ideal for uses in miniaturized HPLC. The pump worked smoothly when it was used for isocratic elutions. When it was used for gradient elutions, generating reproducible gradient profiles was challenging; because the pump rate fluctuated when the pump was used to pump high-content organic solvents. This presents an issue for separating proteins/peptides since high-content organic solvents are often utilized. In this work, we solve this problem by incorporating our high-pressure EOP with a nano-flow gradient generator so that the EOP needs only to pump an aqueous solution. With this combination, we develop a capillary-based nano-HPLC system capable of performing nano-flow gradient elution; the pump rate is stable, and the gradient profiles are reproducible and can be conveniently tuned. To demonstrate its utility, we couple it with either a UV absorbance detector or a mass spectrometer for peptide separations.

•A gold nanoparticle-based probe was synthesized for rapid and sensitive detection of bisulfide.•A droplet-based microfluidic fluorescent sensor was constructed by incorporation of the probe.•Continuous monitoring of the bisulfide variation in microdialysis effluents was realized.We demonstrate a novel fluorescent sensor for real-time and continuous monitoring of the variation of bisulfide in microdialysis effluents by using a nanoparticle–glutathione-fluorescein isothiocyanate (AuNP–GSH-FITC) probe coupled with on-line droplet-based microfluidic chip. The AuNP–GSH-FITC fluorescent probe was firstly developed and used for bisulfide detection in bulk solution by quantitative real-time PCR, which achieved a linear working range from 0.1 μM to 5.0 μM and a limit of detection of ~50 nM. The response time was less than 2 min. With the aid of co-immobilized thiol-polyethylene glycol, the probe exhibited excellent stability and reproducibility in high salinity solutions, including artificial cerebrospinal fluids (aCSF). By adding 0.1% glyoxal to the probe solution, the assay allowed quantification of bisulfide in the presence of cysteine at the micro-molarity level. Using the AuNP–GSH-FITC probe, a droplet-based microfluidic fluorescent sensor was further constructed for online monitoring of bisulfide variation in the effluent of microdialysis. By using fluorescence microscope-charge-coupled device camera as the detector, the integrated microdialysis/microfluidic chip device achieved a detection limit of 2.0 μM and a linear response from 5.0 μM to 50 μM for bisulfide in the tested sample. The method was successfully applied for the on-line measurement of bisulfide variation in aCSF and serum samples. It will be a very useful tool for tracking the variation of bisulfide or hydrogen sulfide in extracellular fluids.

We report a novel approach for effectively separating DNA molecules in free solution. The method uses a bare narrow open capillary without any sieving matrices to resolve a wide size-range of DNA fragments at efficiencies of more than a million plates per meter routinely.

In this technical note, we design and fabricate a novel rotary valve and demonstrate its feasibility for performing isoelectric focusing and simultaneous fractionation of proteins, followed by sodium dodecyl–polyacrylamide gel electrophoresis. The valve has two positions. In one position, the valve routes a series of capillary loops together into a single capillary tube where capillary isoelectric focusing (CIEF) is performed. By switching the valve to another position, the CIEF-resolved proteins in all capillary loops are isolated simultaneously, and samples in the loops are removed and collected in vials. After the collected samples are briefly processed, they are separated via sodium dodecyl–polyacrylamide gel electrophoresis (SDS–PAGE, the second-D separation) on either a capillary gel electrophoresis instrument or a slab-gel system. The detailed valve configuration is illustrated, and the experimental conditions and operation protocols are discussed.

Pressure-induced transport of double-stranded DNA (dsDNA) from 10 base pairs (bp) to 1.9 mega base pairs (Mbp) confined in a 750-nm-radius capillary was studied using a hydrodynamic chromatographic technique and four distinct length regions (rod-like, free-coiled, constant mobility, and transition regions) were observed. The transport behavior varied closely with region changes. The rod-like region consisted of DNA shorter than the persistence length (∼150 bp) of dsDNA, and these molecules behaved like polymer rods. Free-coiled region consisted of DNA from ∼150 bp to ∼2 kilo base pairs (kbp), and the effective hydrodynamic radius RHD of these DNA scaled to L0.5 (L is the DNA length in kbp), a characteristic property of freely coiled polymers. Constant mobility region consisted of DNA longer than ∼100 kbp, and these DNA had a constant hydrodynamic mobility and could not be resolved. Transition region existed between free-coiled and constant mobility regions. The transport mechanism of DNA in this region was complicated, and a general empirical equation was established to relate the mobility with DNA length. Understanding of the fundamental principles of DNA transport in narrow capillary channels will be of great interest in the development of “lab-on-chip” technologies and nongel DNA separations.

The pressure output of a pump cannot be increased simply by connecting several of them in series. This barrier is eliminated with the micropump developed in this work. The pump is actually an assembly of a number of fundamental pump units connected in series. The maximum pressure output of this pump assembly is directly proportional to the number of serially connected pump units. Theoretically, one can always enhance the pressure output by adding more pump units in the assembly, but in reality the upper pressure is constrained by the microtees or microunions joining the pump components. With commercially available microtees and microunions, pressures of more than 1200 bar have been achieved. We have recently experimented using open capillaries to build this pump, but many capillaries have to be utilized in parallel to produce an adequate flow to drive HPLC separations. In this paper, we synthesize polymer monoliths inside 75 μm i.d. capillaries, use these monoliths to assemble miniaturized pumps, characterize the performance of these pumps, and employ these pumps for HPLC separations of intact proteins. By tuning the experimental parameters for monolith preparations, we obtain both negatively and positively charged submicrometer capillary channels conveniently. Each monolith in a 75 μm i.d. capillary is equivalent to several thousands of open capillaries.

In this article, we introduce a chip-capillary hybrid device to integrate capillary isoelectric focusing (CIEF) with parallel capillary sodium dodecyl sulfate- polyacrylamide gel electrophoresis (SDS-PAGE) or capillary gel electrophoresis (CGE) toward automating two-dimensional (2D) protein separations. The hybrid device consists of three chips that are butted together. The middle chip can be moved between two positions to reroute the fluidic paths, which enables the performance of CIEF and injection of proteins partially resolved by CIEF to CGE capillaries for parallel CGE separations in a continuous and automated fashion. Capillaries are attached to the other two chips to facilitate CIEF and CGE separations and to extend the effective lengths of CGE columns. Specifically, we illustrate the working principle of the hybrid device, develop protocols for producing and preparing the hybrid device, and demonstrate the feasibility of using this hybrid device for automated injection of CIEF-separated sample to parallel CGE for 2D protein separations. Potentials and problems associated with the hybrid device are also discussed.

Capillary gel electrophoresis (CGE) has been used for protein separation for more than two decades. Due to the technology advancement, current CGE methods are becoming more and more robust and reliable for protein analysis, and some of the methods have been routinely used for the analysis of protein-based pharmaceuticals and quality controls. In light of this progress, we survey 147 papers related to CGE separations of proteins and present an overview of this technology. We first introduce briefly the early development of CGE. We then review the methodology, in which we specifically describe the matrices, coatings, and detection strategies used in CGE. CGE using microfabricated channels and incorporation of CGE with two-dimensional protein separations are also discussed in this section. We finally present a few representative applications of CGE for separating proteins in real-world samples.Graphical abstractHighlights► In this review, we survey 100 plus papers related to capillary gel electrophoresis of proteins. ► We present the progress and current status and discuss the pros and cons of this technique. ► We describe the basic components/procedures to implement CGE and exhibit the representative practical applications. ► We expect CGE to be an important analytical technique for protein analysis in the near future.

Numerous micropumps have been developed, but few of them can produce adequate flow rate and pressure for high-performance liquid chromatography (HPLC) applications. We have recently developed an innovative hybrid electroosmotic pump (EOP) to solve this problem. The basic unit of a hybrid pump consists of a +EOP (the pumping element is positively charged) and a −EOP (the pumping element is negatively charged). The outlet of the +EOP is then joined with the inlet of the −EOP, forming a basic pump unit, while the anode of a positive high voltage (HV) power supply is placed at the joint. The inlet and outlet of this pump unit are electrically grounded. With this configuration, we can stack many of such pump units in series to boost the pumping power. In this work, we describe in details how an open-capillary hybrid EOP is constructed and characterize this pump systematically. We also show that a hybrid EOP with ten serially stacked pump units can deliver a maximum pressure of 21.5 MPa (∼3100 psi). We further demonstrate the feasibility of using this hybrid EOP to drive eluents for HPLC separations of proteins and peptides.Highlights► We describe in details how an open-capillary hybrid EOP is constructed. ► We demonstrate that an open-capillary hybrid EOP is capable of delivering >3100 psi pumping pressure. ► We demonstrate the feasibility of using this hybrid EOP to drive HPLC for separations of proteins and peptides.

DNA molecules are usually separated by gel electrophoresis. While gels help resolve DNA, the polymers bring problems to the analysis (e.g., difficulty in automating and poor reproducibility). One way to eliminate these problems completely is to separate DNA in free solutions. However, DNA fragments cannot normally be resolved in a free solution by electrophoresis because the electrophoretic mobilities of these molecules have similar charge-to-mass ratios, especially when the fragments are large.Efforts have been invested in free-solution DNA separations and a number of approaches (e.g., high-performance liquid chromatography, end-labeled free-solution electrophoresis, entropic traps, and DNA prism) have been successfully developed. Very recently, a new technique, called bare narrow capillary-open tubular chromatography (BaNC-OTC), was developed and can resolve a wide range of sizes of DNA in a few minutes.In this article, we review the significant progress on free-solution DNA separations, with emphasis on the most recent, advanced developments.Highlights► We review progress in DNA separations in free solution. ► In particular, we introduce a new technique for DNA separations in free solution. ► We discuss potential applications of developments of DNA separations in free solution.

A micropump provides flow and pressure for a lab-on-chip device, just as a battery supplies current and voltage for an electronic system. Numerous micropumps have been developed, but none is as versatile as a battery. One cannot easily insert a micropump into a nonterminal position of a fluidic line without affecting the rest of the fluidic system, and one cannot simply connect several micropumps in series to enhance the pressure output, etc. In this work we develop a flow battery (or pressure power supply) to address this issue. A flow battery consists of a +EOP (in which the liquid flows in the same direction as the field gradient) and a −EOP (in which the liquid flows opposite to the electric field gradient), and the outlet of the +EOP is directly connected to the inlet of the −EOP. An external high voltage is applied to this outlet−inlet joint via a short gel-filled capillary that allows ions but not bulk liquid flow, while the +EOP’s inlet and the −EOP’s outlet (the flow battery’s inlet and outlet) are grounded. This flow battery can be deployed anywhere in a fluidic network without electrically affecting the rest of the system. Several flow batteries can be connected in series to enhance the pressure output to drive HPLC separations. In a fluidic system powered by flow batteries, a hydraulic equivalent of Ohm’s law can be applied to analyze system pressures and flow rates.

Sodium dodecyl sulfate (SDS)−polyacrylamide gel electrophoresis (PAGE) is a fundamental analytical technique for proteomic research, and SDS−capillary gel electrophoresis (CGE) is its miniaturized version. Compared to conventional slab-gel electrophoresis, SDS−CGE has many advantages such as increased separation efficiency, reduced separation time, and automated operation. SDS−CGE is not widely accepted in proteomic research primarily due to the difficulties in identifying the well-resolved proteins. MALDI-TOF-MS is an outstanding platform for protein identifications. Coupling the two would solve the problem but is extremely challenging because the MS detector has no access to the SDS−CGE-resolved proteins and the SDS interferes with MS detection. In this work we introduce an approach to address these issues. We discover that poly(tetrafluoroethylene) (PTFE) membranes are excellent materials for collecting SDS−CGE-separated proteins. We demonstrate that we can wash off the SDS bound to the collected proteins and identify these proteins on-membrane with MALDI-TOF-MS. We also show that we can immunoblot and Coomassie-stain the proteins collected on these membranes.

Gel electrophoresis is commonly used to separate DNA, but narrow capillaries or microchannels desired for high throughput efficient separations are difficult to fill with gels. We report here that a narrow capillary can be used to hydrodynamically separate a wide size range of DNA fragments in a single run without the need for gels, wall coatings, or an electric field. We also demonstrate that attractive separation is possible in a few minutes and that the separated DNA can be collected into individual fractions that remain viable for amplification via the polymerase chain reaction.

We report a highly effective on-chip preconcentration method by combining field-amplified sample injection (FASI) and bovine serum albumin (BSA) sweeping for ultrasensitive detection of green fluorescent protein (GFP) on a simple cross-channel microchip device. With the formation of a stagnant sample/running buffer boundary by balancing the hydrodynamic flow and the electro-osmotic flow (EOF), GFP molecules can be continuously injected into the sample loading channel and stacked. We have also demonstrated that BSA is a very effective pseudo-stationary phase for sweeping concentration of proteins in comparison to the commonly used micelles. The combination of FASI and BSA sweeping yields a concentration factor of 3570 and a limit of detection of 8.4 pM for GFP. Using this method, we have separated GFP and GFP-insulin-like growth factor-I (GFP-IGF-I) fusion protein. The entire assay (GFP concentration, matrix elimination, and electrophoretic separation) can be completed within <5 min. Furthermore, we have successfully applied this method for the detection of GFP expression of E. coli cells and the GFP content in single E. coli cells.

We have recently examined the potential of bare nanocapillaries for free solution DNA separations and demonstrated efficiencies exceeding 106 theoretical plates/m. In the present work, we demonstrate the use of bare and hydroxypropylcellulose (HPC) coated open tubular nanocapillaries for protein separations. Using 1.5 μm inner diameter (i.d.) capillary columns, hydrodynamically injecting femto- to picoliter volumes of fluorescent or fluorescent dye labeled protein samples, utilizing a pneumatically pressurized chamber containing 1.0 mM sodium tetraborate solution eluent (typically 200 psi) as the pump, and performing on-column detection using a simple laser-induced fluorescence detector, we demonstrate efficiencies of close to a million theoretical plates/m while generating single digit microliter volumes of waste for a complete chromatographic run. We achieve baseline resolution for a protein mixture consisting of transferrin, α-lactalbumin, insulin, and α-2-macroglobulin.

Electroosmotic pumping is receiving increasing attention in recent years owing to the rapid development in micro total analytical systems. Compared with other micropumps, electroosmotic pumps (EOPs) offer a number of advantages such as creation of constant pulse-free flows and elimination of moving parts. The flow rates and pumping pressures of EOPs matches well with micro analysis systems. The common materials and fabrication technologies make it readily integrateable with lab-on-a-chip devices. This paper reviews the recent progress on EOP fabrications and applications in order to promote the awareness of EOPs to researchers interested in using micro- and nano-fluidic devices. The pros and cons of EOPs are also discussed, which helps these researchers in designing and constructing their micro platforms.

In this work, we demonstrate DNA separation and genotyping analysis in gel-free solutions using a nanocapillary under pressure-driven conditions without application of an external electric field. The nanocapillary is a ∼50-cm-long and 500-nm-radius bare fused-silica capillary. After a DNA sample is injected, the analytes are eluted out in a chromatographic separation format. The elution order of DNA molecules follows strictly with their sizes, with the longer DNA being eluted out faster than the shorter ones. High resolutions are obtained for both short (a few bases) and long (tens of thousands of base pairs) DNA fragments. Effects of key experimental parameters, such as eluent composition and elution pressure, on separation efficiency and resolution are investigated. We also apply this technique for DNA separations of real-world genotyping samples to demonstrate its feasibility in biological applications. PCR products (without any purification) amplified from Arabidopsis plant genomic DNA crude preparations are directly injected into the nanocapillary, and PCR-amplified DNA fragments are well resolved, allowing for unambiguous identification of samples from heterozygous and homozygous individuals. Since the capillaries used to conduct the separations are uncoated, column lifetime is virtually unlimited. The only material that is consumed in these assays is the eluent, and hence, the operation cost is low.

We report a novel approach for effectively separating DNA molecules in free solution. The method uses a bare narrow open capillary without any sieving matrices to resolve a wide size-range of DNA fragments at efficiencies of more than a million plates per meter routinely.