Shaorong Liu

Protein separation by capillary gel electrophoresis: A review

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.

Dummy molecularly imprinted mesoporous silica prepared by hybrid imprinting method for solid-phase extraction of bisphenol A

A novel hybrid dummy imprinting strategy was developed to prepare a mesoporous silica for the solid-phase extraction (SPE) of bisphenol A (BPA). A new covalent template-monomer complex (BPAF-Si) was first synthesized with 2,2-bis(4-hydroxyphenyl)hexafluoropropane (BPAF) as the template. The imprinted silica was obtained through the gelation of BPAF-Si with tetraethoxysilane and the subsequent removal of template by thermal cleavage, and then it was characterized by FT-IR spectroscopy, scanning electron microscopy, transmission electron microscopy, and nitrogen adsorption-desorption isotherms. Results showed that the new silica had micron-level particle size and ordered mesoporous structure. The static binding test verified that the imprinted silica had much higher recognition ability for BPA than the non-imprinted silica. The imprinted silica also showed high extraction efficiencies and high enrichment factor for SPE of BPA. Using the imprinted silica, a SPE-HPLC-UV method was developed and successfully applied for detecting BPA in BPA-spiked tap water and lake water samples with a recovery of 99-105%, a RSD of 2.7-5.0% and a limit of detection (S/N=3) of 0.3ng/mL. The new imprinted silica avoided the interference of the residual template molecules and reduced the non-specific binding sites, and therefore it can be utilized as a good sorbent for SPE of BPA in environmental water samples.

Novel surface dummy molecularly imprinted silica as sorbent for solid-phase extraction of bisphenol A from water samples.

A novel surface molecularly imprinted silica composite was prepared by a dummy-template imprinting strategy for the solid-phase extraction (SPE) of bisphenol A (BPA). 2,2-Bis(4-hydroxyphenyl) hexafluoropropane (BPAF) was chosen as the template molecule, and a hybrid technique was used for imprinting procedure. The imprinted silica was characterized by FT-IR spectroscopy, scanning electron microscope, thermo-gravimetric analysis, and nitrogen adsorption-desorption isotherms. The static binding test verified that the imprinted silica had much higher recognition ability for BPA than the non-imprinted silica, and the kinetic adsorption test presented the fast binding kinetics of the surface imprinted silica for BPA. When used as a SPE sorbent, the imprinted silica showed high extraction efficiencies and high enrichment factor for BPA. Based on the imprinted silica, a SPE-HPLC-UV method was developed and successfully applied to the detection of BPA in BPA-spiked lake water, tap water and drinking water samples with a high recovery of 97.3-106.0%, a RSD of 1.2-3.8% (n=3) and a limit of detection (S/N=3) of 0.3 ng/mL. The analysis results of a certified BPA sample also demonstrated the reliability of present method. The new surface dummy molecularly imprinted silica completely avoided the interference of the residual template molecules and greatly improved the binding kinetic of the target molecules. Therefore, it can be used as a good sorbent for SPE of BPA in environmental water samples.

Dummy molecularly imprinted magnetic nanoparticles for dispersive solid-phase extraction and determination of bisphenol A in water samples and orange juice

A novel dummy molecularly imprinted magnetic nanoparticle (MI-MNP) was prepared by a hybrid imprinting technique for dispersive solid-phase extraction (d-SPE) and determination of bisphenol A (BPA). 2,2-Bis(4-hydroxyphenyl)-hexafluoropropane was used as the template molecule and Fe3O4 nanoparticle as the magnetic core. The MI-MNPs were characterized by FT-IR spectroscopy, thermo- gravimetric analysis, X-ray diffraction, transmission electron microscopy, and vibrating sample magnetometer. The adsorption tests showed that the MI-MNPs had a high binding ability for BPA and presented a fast binding kinetics. When used as a d-SPE sorbent, the MI-MNP showed high extraction efficiency, high enrichment factor and good reusability for BPA, and it can be easily recycled by a magnet. Furthermore, the dummy imprinting strategy can completely avoid the interference of the residual template in sorbent to determination of BPA. Using the MI-MNPs as sorbent, a d-SPE-HPLC-UV method was developed and successfully applied to the analysis of BPA spiked in water samples and orange juice and that in a certified sample with recoveries of 95.0-106.2% (RSD=2.5-4.5%), 93.3-100.0% (RSD=1.2-5.0%) and 100.3% (RSD=3.5%), respectively. The limit of detection (S/N=3) for all samples was 0.3ngmL-1. The new MI-MNPs can be utilized as a good d-SPE sorbent for BPA in environmental water samples and beverages.

Incorporating high-pressure electroosmotic pump and a nano-flow gradient generator into a miniaturized liquid chromatographic system for 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.

Stacking open-capillary electroosmotic pumps in series to boost the pumping pressure to drive high-performance liquid chromatographic separations.

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.

A microfabricated electroosmotic pump coupled to a gas-diffusion microchip for flow injection analysis of ammonia

AbstractWe 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 0.10xa0mgxa0L−1 NH3, with a good repeatability (relative standard deviation of less than 5xa0% for 4.0xa0mgxa0L−1 NH3). Parameters affecting its performance are also discussed.n Graphical AbstractA gas-diffusion microchip was fabricated for the first time and incorporated in a flow injection analysis (FIA) system with an open-channel electroosmotic pump, which was used successfully for the determination of ammonia in environmental water samples.

Integrated Bare Narrow Capillary–Hydrodynamic Chromatographic System for Free‐Solution DNA Separation at the Single‐Molecule Level

We report an integrated bare narrow-capillary–hydrodynamic chromatographic system (BaNC-HDC) for rapid, high-resolution, and repeatable separations of a wide size range of DNA at the single-molecule level in free solution. DNA separation is a common task in molecular biological research. Traditionally, DNA molecules are separated using slab-gel electrophoresis,[1] including pulsed field gel electrophoresis (PFGE).[2] To improve resolution, reduce running time, and increase throughput, capillary gel electrophoresis (CGE) and later capillary array electrophoresis (CAE) have been developed, and high resolutions have been achieved.[3] However, both CGE and CAE require viscous polymer sieving matrices, which can be difficult to work with, especially when narrow capillaries are employed. Researchers have experimented with separating DNA in free solutions, but DNA cannot be easily resolved in these media because all DNA molecules have similar mass-to-charge ratios (m/z), and, as a result, similar electrophoretic mobilities. In 1992, Noolandi[4] proposed an approach to solve this problem by attaching a monodisperse entity to each DNA fragment, generating varying m/z values for the DNA to be separated. The idea was experimentally validated in the late 1990s[5] and termed end-labeled free-solution electrophoresis (ELFSE). This method has proven to be effective for separating DNA fragments shorter than a few hundreds of base pairs.[6] Other gel-free approaches for DNA separation include radial migration,[7] liquid chromatography,[8] entropic trapping,[9] and DNA prism.[10] These approaches overcome the problems brought by viscous gels and offer promising alternatives for resolving DNA, but their resolutions are not competitive compared to that of gel electrophoresis. Recently, we have developed a new technique, called BaNC-HDC,[11] for free-solution DNA separations. When DNA molecules are transported inside a narrow capillary under pressure-driven conditions, the DNA molecules move as particles.[12] Larger DNA fragments have greater effective diameters and cannot go as close to the capillary wall (the slow-moving region) as smaller fragments can and, therefore, they move faster. On the basis of this principle, samples of DNA fragments with a wide size range have been separated with resolutions comparable to gel electrophoresis.[11] The minimal waste generation and low operation costs make BaNC-HDC an attractive alternative to gel-based techniques, particularly to PFGE for separating large DNA fragments. Herein, we integrate a high-pressure electroosmotic pump (EOP) and a microfabricated chip-injector with BaNC-HDC; the integrated system enables us to inject samples at low-picoliter (pL) volumes reliably, elute analytes at hundreds of pLmin−1 flow-rates or lower reproducibly, and resolve a wide size range of DNA fragments rapidly in free solution at the single-molecule level.

Confocal laser-induced fluorescence detector for narrow capillary system with yoctomole limit of detection

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.

Resolving DNA at efficiencies of more than a million plates per meter using bare narrow open capillaries without sieving matrices

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.