Biomedical subjects

Milton L Lee

Publications and source records attributed to Milton L Lee.

At least 19 recordsLinked to original sources

PubMed2006

Sub-2 microm porous and nonporous particles for fast separation in reversed-phase high performance liquid chromatography.

One approach to achieve fast and efficient separations in packed column liquid chromatography (LC) is to reduce eddy diffusion and mass transfer resistance in the mobile phase using short columns packed with small particles. In this study, efficiencies of columns packed with 1.5 and 3.0 microm nonporous and porous particles were compared in reversed-phase LC using nitromethane and a protein as analytes. Nonporous particles provided overall higher efficiency at high linear velocities when nitromethane was used as solute; however, the efficiency difference diminished significantly when the particle size was reduced from 3 to 1.5 microm. Efficiencies for 1.5 microm nonporous particles were considerably higher than those for 1.5 microm porous particles at high linear velocities when a protein, alpha-chymotrypsinogen A (MW 25,000), was used as solute. In addition, the average retention factor for amylbenzene in a column packed with ACQUITY C(18) porous particles was approximately 16 fold higher than for Micra C(18) nonporous particles for aqueous mobile phase compositions containing from 40 to 75% acetonitrile. Pressure drop, sample loading capacity, and separation power were also evaluated and compared for porous and nonporous particles under practical conditions.

Chromatography, High Pressure Liquid↗

PubMed2006

Tandem electric field gradient focusing system for isolation and concentration of target proteins.

Two electric field gradient focusing (EFGF) systems, one based on a hollow dialysis fiber and the other based on a shaped ionically conductive polymer were serially integrated to trap and concentrate selected proteins while simultaneously desalting and removing other unwanted proteins from the sample. A series of experiments were performed to test the EFGF systems individually and after integration. Online concentration of amyloglucosidase indicated a concentration limit of detection of approximately 20 ng mL(-1) (200 pM) from a sample volume of 100 microL. Concentration of human alpha1-acid glycoprotein with simultaneous removal of human serum albumin was also demonstrated. Elimination of small buffer components while concentrating trypsin inhibitor, and selective concentration and separation of myoglobin from a mixture were performed using the integrated EFGF system.

Animals↗

PubMed2006

Fabrication of conductive membrane in a polymeric electric field gradient focusing microdevice.

A novel approach to integrating a buffer ion-permeable membrane in a poly(glycidyl methacrylate-co-methyl methacrylate) micro electric field gradient focusing (muEFGF) device is described. A weir structure on which the membrane was positioned was fabricated between the separation channel and field gradient-generating channel. Before formation of the membrane, the surface of the polymeric microdevice was treated for covalent bonding of the membrane. Following surface modification, a prepolymer solution containing poly(ethylene glycol) acrylate/methacrylate and Tris-HCl buffer was loaded into the microdevice. Low-pressure nitrogen gas was then purged through the separation and field gradient-generating channels to remove the prepolymer solution from these channels. Residual prepolymer solution was retained on the weir structure due to surface tension. Finally, the premembrane was cured in place on the weir using UV radiation. Using a muEFGF device, green fluorescent protein (GFP) was concentrated 4000-fold. Separation of GFP and R-phycoerythrin, and selective elution of GFP from a protein mixture containing GFP, FITC-labeled casein, and FITC-labeled hemoglobin were also demonstrated. It was found that the membrane conductivity and presence of carboxylic acid impurities in the membrane strongly affected the behavior of the muEFGF device.

Electricity↗

PubMed2006

Efficient polymer monolith for strong cation-exchange capillary liquid chromatography of peptides.

A stable poly[2-acrylamido-2-methyl-1-propanesulfonic acid-co-poly(ethylene glycol) diacrylate] monolith was synthesized inside a 75-microm-i.d. capillary by photoinitiated copolymerization with water, methanol, and ethyl ether as porogens. The resulting monolith was evaluated for strong cation-exchange capillary liquid chromatography of both synthetic and natural peptides. Although the monolith possessed relatively strong hydrophobicity due to the use of 2-acrylamido-2-methyl-1-propanesulfonic acid as one monomer, the monolith had a high dynamic binding capacity of 157 microequiv of peptide/mL, or 332 mg of cytochrome c/mL. Exceptionally high resolution resulting from extremely narrow peaks was obtained, resulting in a peak capacity of 179 when using a shallow salt elution gradient. Although a second, naturally formed gradient might contribute to the sharp peaks obtained, high efficiency was mainly due to the use of poly(ethylene glycol) diacrylate as a biocompatible cross-linker.

Amino Acid Sequence↗

PubMed2006

New interface plate for microspray ionization mass spectrometry.

A new interface plate was employed in microspray ionization mass spectrometry (microESI-MS) to improve ion transmission from the sprayer into the sampling nozzle of the mass spectrometer at atmospheric pressure. Using a time-of-flight mass spectrometer (TOFMS), a fivefold increase in ion intensity and a sevenfold reduction in method detection limit were observed. The interface plate attenuated the dependence of the ion intensity on the sprayer position. Even when the distance between the sprayer tip and sampling nozzle was 15.0 mm, ion signals were still stronger than when the sprayer tip was positioned 3.0 mm in front of the sampling nozzle with the original interface plate. This enhancement in the performance of microESI-MS was due to the improved shapes of the equipotential lines near the sprayer tip and the long desolvation distance between the sprayer and the sampling nozzle of the MS.

Journal Article↗

PubMed2006

Miniature toroidal radio frequency ion trap mass analyzer.

A miniature ion trap mass analyzer is reported. The described analyzer is a 1/5-scale version of a previously reported toroidal radio frequency (rf) ion trap mass analyzer. The toroidal ion trap operates with maximum rf trapping voltages about 1 kVp-p or less; however despite the reduced dimensions, it retains roughly the same ion trapping capacity as conventional 3D quadrupole ion traps. The curved geometry provides for a compact mass analyzer. Unit-mass resolved mass spectra for n-butylbenzene, xenon, and naphthalene are reported and preliminary sensitivity data are shown for naphthalene. The expected linear mass scale with rf amplitude scan is obtained when scanned using a conventional mass-selective instability scan mode combined with resonance ejection.

Journal Article↗

PubMed2006

Ultrahigh pressure liquid chromatography using elevated temperature.

Fast liquid chromatographic (LC) methods are important for a variety of applications. Reducing the particle diameter (d(p)) is the most effective way to achieve fast separations while preserving high efficiency. Since the pressure drop along a packed column is inversely proportional to the square of the particle size, when columns packed with small particles (<2 microm) are used, ultrahigh pressures (>689 bar) must be applied to overcome the resistance to mobile phase flow. Elevating the column temperature can significantly reduce the mobile phase viscosity, allowing operation at higher flow rate for the same pressure. It also leads to a decrease in retention factor. The advantage of using elevated temperatures in LC is the ability to significantly shorten separation time with minimal loss in column efficiency. Therefore, combining elevated temperature with ultrahigh pressure facilitates fast and efficient separations. In this study, C6-modified 1.0 microm nonporous silica particles were used to demonstrate fast separations using a temperature of 80 degrees C and a pressure of 2413 bar. Selected separations were completed in 30 s with efficiencies as high as 220,000 plates m(-1).

Chromatography, High Pressure Liquid↗

PubMed2006

Pseudolinear gradient ultrahigh-pressure liquid chromatography using an injection valve assembly.

The use of ultrahigh pressures in liquid chromatography (UHPLC) imposes stringent requirements on hardware such as pumps, valves, injectors, connecting tubing, and columns. One of the most difficult components of the UHPLC system to develop has been the sample injector. Static-split injection, which can be performed at pressures up to 6900 bar (100,000 psi), consumes a large sample volume and is very irreproducible. A pressure-balanced injection valve provided better reproducibility, shorter injection time, reduced sample consumption, and greater ease of use; however, it could only withstand pressures up to approximately 1000 bar (15,000 psi). In this study, a new injection valve assembly that can operate at pressures as high as 2070 bar (30,000 psi) was evaluated for UHPLC. This assembly contains six miniature electronically controlled needle valves to provide accurate and precise volumes for introduction into the capillary LC column. It was found that sample volumes as small as several tenths of a nanoliter can be injected, which are comparable to the results obtained from the static-split injector. The reproducibilities of retention time, efficiency, and peak area were investigated, and the results showed that the relative standard deviations of these parameters were small enough for quantitative analyses. Separation experiments using the UHPLC system with this new injection valve assembly showed that this new injector is suitable for both isocratic and gradient operation modes. A newly designed capillary connector was used at a pressure as high as 2070 bar (30,000 psi).

Journal Article↗

PubMed2006

Permanent surface modification of polymeric capillary electrophoresis microchips for protein and peptide analysis.

Because of their surface heterogeneity, proteins readily adsorb on polymeric substrates via various interactions, which adversely affects the performance of polymeric microfluidic devices in electrophoresis-based protein/peptide analysis. Therefore, it is necessary to use surface modification techniques such as dynamic coating or more complicated permanent surface modification, which has broader application and better performance, to render the polymeric microchannels protein-resistant. This manuscript is a review of the surface chemistry of microfluidic devices used for electrophoretic separations of proteins and peptides. The structural complexity of proteins as it relates to adsorption is described, followed by a review of the mechanisms and structural characteristics of protein-resistant surfaces. Permanent surface modification techniques used in grafting protein-resistant materials onto the surfaces of electrophoresis microchannels fabricated from polymer substrates are summarized and successful examples are presented.

Electrophoresis, Capillary↗

PubMed2006

Sacrificial layer microfluidic device fabrication methods.

Over the past 15 years, research in the field of microfluidics has experienced rapid growth due to significant potential advantages such as low cost, short analysis times, and elimination of sources of contamination. Although etched and thermally bonded glass substrates have seen widespread use and offer solid performance, device fabrication still remains cumbersome. Recent advances in sacrificial layer microfabrication methods for microfluidics have overcome many disadvantages of conventional fabrication approaches. Phase-changing sacrificial layers have been implemented in making inexpensive and high-performance polymer microchips for electrophoretic analysis, protein focusing, and sample preconcentration. In addition, novel channel fabrication methods based on standard thin-film processes, which are readily integratable with microfabrication techniques used for electrical components, are being applied increasingly for the creation of microfluidic devices. These new sacrificial layer fabrication approaches will be instrumental in making low-cost and high-quality polymer microchips, and in interfacing electrical and fluidic systems on glass or semiconductor substrates.

Equipment Design↗

PubMed2005

Surface-reactive acrylic copolymer for fabrication of microfluidic devices.

A surface-reactive acrylic polymer, poly(glycidyl methacrylate-co-methyl methacrylate) (PGMAMMA), was synthesized and evaluated for suitability as a substrate for fabrication of microfluidic devices for chemical analysis. This polymer has good thermal and optical properties and is mechanically robust for cutting and hot embossing. A key advantage of this polymeric material is that the surface can be easily modified to control inertness and electroosmotic flow using a variety of chemical procedures. In this work, the procedures for aminolysis, photografting of linear polyacrylamide, and atom-transfer radical polymerization on microchannel surfaces in PGMAMMA substrates were developed, and the performance of resultant microfluidic electrophoresis devices was demonstrated for the separation of amino acids, peptides, and proteins. Separation efficiencies as high as 4.6 x 10(4) plates for a 3.5-cm-long separation channel were obtained. The results indicate that PGMAMMA is an excellent substrate for microfabricated fluidic devices, and a broad range of applications should be possible.

Amines↗

PubMed2005

Design and evaluation of a coupled monolithic pre-concentrator-capillary zone electrophoresis system for the extraction of immunoglobulin G from human serum.

The analysis of proteins in biological fluids by capillary electrophoresis (CE) is of interest in clinical chemistry. However, due to low analyte concentrations and poor concentration limits of detection (CLOD), protein analysis by this technique is frequently challenging. Coupling preconcentration techniques with CE greatly improves the CLOD. An on-line preconcentration-CE method that can selectively pre-concentrate any protein for which an antibody is available would be very useful for the analysis of low abundance proteins and would establish CE as a major tool in biomarker discovery. To accomplish this, the development of an on-line protein G monolithic pre-concentrator-CE device is proposed. To generate active groups for protein immobilization, glycidyl methacrylate (GMA) was used to prepare polymer monoliths. A 1.5-2 cm monolith was cast inside a 75 microm I.D. fused silica capillary that had previously been coated with alternating layers of negatively (dextran) and positively (polybrene) charged polymers. Protein G was covalently bound to GMA. Monoliths from different formulations were prepared and evaluated for binding capacity to optimize the monolith formulation for protein preconcentration. The physical properties of the column considered best for preconcentration were determined by mercury intrusion porosimetry. The total pore area was 4.8m(2)/g, the average pore diameter was 3.3 microm and the porosity was 82%. The monolith had a low flow resistance and was macroscopically homogeneous. The effectiveness of the monolith to rapidly pre-concentrate proteins at flow rates as high as 10 microL/min was demonstrated using a 1.8 microM IgG solution. This system proved effective for on-line sample extraction, clean-up, preconcentration, and CE of IgG in human serum. IgG from diluted (500 and 65,000 times) human serum samples was successfully analyzed using this system. The approach can be applied to the on-line preconcentration and analysis of any protein for which an antibody is available.

Adsorption↗

PubMed2005

Superimposition of a magnetic field around an ion guide for electron ionization time-of-flight mass spectrometry.

A new electron ionization source was developed for orthogonal acceleration time-of-flight mass spectrometry (TOFMS) based on the superimposition of a magnetic field around a radio frequency-only (rf-only) ion guide. The cylindrically symmetric magnetic field compresses the electron beam from the electron source into a long narrow volume along the ion guide axis. The magnetic field also helps to maintain a narrow energy distribution of electrons that penetrate the full length of the ion guide despite the influence of the radial rf field. Ionization occurs inside the ion guide with improved efficiency resulting from efficient use of electrons, prolonged interaction time, and nontraditionally large ionization volume. At the same time, the rf field effectively focuses ions radially and confines them to the axis of the ion guide by collisional focusing, leading to high ion transmission efficiency. Furthermore, the source can also be operated in a trap-and-pulse mode to improve the ion sampling duty cycle of orthogonal acceleration TOFMS. To validate the design concept of this new ion source, a simple prototype using a single set of cylindrical rods was constructed and retrofitted to an orthogonal acceleration TOFMS. A significant increase in ion signal intensity was observed by operating the source in a pulsed ion extraction mode. Low detection limits (for example, 12 fg for toluene) were determined at 12.5 spectra s(-1) in the full spectrum mode.

Journal Article↗

PubMed2005

Electron ionization in superimposed magnetic and radio frequency quadrupolar electric fields.

An improved design of a novel electron ionization source for orthogonal acceleration time-of-flight mass spectrometry is described, based on the superimposition of an axial magnetic field with cylindrical symmetry around a radio frequency-only quadrupole. A tubular permanent magnet was designed to generate the required strong magnetic field and field profile. An axial electric field along the ion guide for efficient ion extraction was introduced using segmented quadrupole rods. Details of the source design and the effects of various operating parameters are described. The source produces high-quality mass spectra with regard to fragmentation, relative abundances, and isotopic ratios. Preliminary results have shown excellent sensitivity, with limits of detection in the subfemtogram range (octafluoronaphthalene, full spectrum acquisition) in gas chromatography/mass spectrometry operation.

Journal Article↗

PubMed2005

Preparation and evaluation of poly(polyethylene glycol methyl ether acrylate-co-polyethylene glycol diacrylate) monolith for protein analysis.

A poly(polyethylene glycol methyl ether acrylate-co-polyethylene glycol diacrylate) monolith was prepared by UV-initiated polymerization. Methanol and ethyl ether were selected as porogens from a variety of organic solvents to achieve the desirable characteristics of the monolith. The preparation of the monolith could be achieved within 10 min. The monolith was macroscopically homogeneous, had low flow resistance, and did not swell or shrink significantly in tetrahydrofuran. Inverse size-exclusion data indicate that the monolith had a total porosity of 75.4% and an internal porosity of 9.1%. The monolith could be used for size-exclusion separation of peptides, although it could not separate proteins with molecular masses between 10 and 100 K due to its unique pore size distribution. It was found to resist adsorption of proteins in capillary liquid chromatography when using 100 mM phosphate buffer (pH 7.0) containing 0.5 M NaCl. Complete recovery of both acidic and basic proteins was achieved. The monolith can be used for applications in which inert materials are required for protein analysis.

Acrylates↗

PubMed2005

Compact detector for proteins based on two-photon excitation of native fluorescence.

A compact, inexpensive detector for proteins has been constructed based on two-photon excitation of fluorescence from phenylalanine, tyrosine, and tryptophan. The fluorescence was excited by a solid-state microchip laser operating at 532 nm. Detection limits for phenylalanine, tyrosine, and tryptophan were 62 microM, 2.0 microM, and 470 nM, respectively, in a volume of 3 fL. The detection limit for a test protein, bovine serum albumin, was 130 nM.

Animals↗

PubMed2005

Field gradient electrophoresis.

The class of equilibrium gradient methods utilizes the opposition of two forces, at least one of which changes in magnitude with position, to separate and concentrate analytes. The drawback of many methods of this type is that the production of two opposing forces requires in comparison to standard methods, such as capillary electrophoresis, a relatively complex apparatus. In addition, for techniques such as electric field gradient focusing, hydrodynamic flow leads to Taylor dispersion, which limits the attainable concentration factor. We propose a new method, gradient field electrophoresis, which achieves analyte separation and focusing with only one spatially varying force, an electric field gradient. A model for the method is developed and used to analyze peak capacity. Experimental results for a protein (R-phycoerythrin) are given and compared to the model.

Electrophoresis↗

PubMed2004

Surface-modified poly(methyl methacrylate) capillary electrophoresis microchips for protein and peptide analysis.

Polymeric materials have emerged as appealing alternatives to conventional inorganic substrates for the fabrication of microscale analytical systems; however, native polymeric surfaces typically require covalent modification to ensure optimum biocompatibility. 2-Bromoisobutyryl bromide was immobilized on poly(methyl methacrylate) (PMMA) substrates activated using an oxygen plasma. Atom-transfer radical polymerization was then performed to graft poly(ethylene glycol) (PEG) on the PMMA surface. PMMA microcapillary electrophoresis (muCE) devices made with the covalently modified surfaces exhibited substantially reduced electroosmotic flow and nonspecific adsorption of proteins on microchannel surfaces. Experiments using fluorescein isothiocyanate-conjugated bovine serum albumin indicated that both column efficiency and migration time reproducibility were 1 order of magnitude better with derivatized compared to untreated PMMA muCE chips. Fast, reproducible, and efficient separations of proteins and peptides were demonstrated using the PEG-grafted PMMA muCE chips. All analyses were completed in less than 60 s, and separation efficiencies as high as 5.2 x10(4) plates for a 3.5-cm-long separation channel were obtained. These results demonstrate the general applicability of surface-grafted PMMA microdevices for a broad range of protein analyses.

Animals↗