The present invention relates generally to improved compositions which can alter a sample and produce plumes of charged molecules from an emitting end useful for analysis by mass spectrometry, and more specifically, it relates to particles entrapped within a polymer capable of producing said plumes, methods of making the compositions, and uses thereof.
Proteomic studies are becoming a very active area of post-genomic research because of the promise of uncovering biological markers to diagnose disease states as well as identifying proteins of therapeutic importance. This great potential for discovery has spurred many to develop new techniques to facilitate the identification of these target proteins in a high-throughput manner. Mass spectrometry has become an important analytical tool for protein studies because of its ability to determine the molecular weight of a protein with sufficient accuracy to enable identification of the protein. Furthermore, mass spectrometry possesses the ability to determine the primary structure of the protein with subsequent collision-induced dissociation (CID) experiments on the intact protein or its digested fragments [(a) Cristoni, S.; Bernardi, L, R.; Mass Spec. Rev. 2003, 22, 369-406. (b) Lill, J. Mass Spec. Rev. 2003, 22, 182-194. (c) Mann, M.; Hendrickson, R. C.; Pandey, A. Annu. Rev. Biochem. 2001, 70, 437-473. (d) Yates, J. R. J. Mass Spec. 1998, 33, 1-19].
In order to collect mass spectral information from protein or peptide samples of the analyte of interest must enter the mass spectrometer in the gas phase. Electrospray ionization provides a technique to facilitate the production of gas phase ions from the atmospheric pressure ionization of highly charged and nonvolatile compounds in a liquid sample. A solution in a capillary or microfluidic device under a strong electric field, in positive ion mode for example, will produce an accumulation of positive charge at the liquid surface located at the end of the device. At this point, the solution leaving the end of the device will undergo a change from spherical to elliptical and finally will form a Taylor cone that emits small droplets. This point occurs when the solution has reached what is called the Rayleigh limit. These smaller droplets then undergo desolvation and division to even smaller droplets until gas phase ions are produced which ultimately enter the mass spectrometer. A sensitive method of detection, which depends on the efficiency of the electrospray process, will maximize the amount of gas phase ions that are formed and reach the detector.
Electrospray techniques such as microelectrospray (microspray) and nanoelectrospray (nanospray) mass spectrometry involve the passage of samples at very low flow rates through capillaries that have been manufactured or pulled to produce a spray tip with a small inner diameter (2-10 micrometres). Flow rates of about 100 nL/min to 1 microlitre/min are generally used for microspray, and flow rates of <100 nL/min are generally used for nanospray.
With the advent of nanospray, it became possible to obtain mass spectral information about molecules such as peptides and proteins from an extremely small sample size, enhancing detection limits to the low femtomole and attomole levels. [(a) Wilm, M.; Mann, M. Anal. Chem. 1996, 68, 1-8. (b) Davis, M. T., Stahl, D. C.; Hefta, S. A.; Lee, T. D. Anal. Chem. 1995, 67, 4549-4556. (c) Valaskovic, G. A.; Kelleher, N, L.; Little, D. P.; Aaserud, D. J.; McLafferty, F. W. Anal. Chem. 1995, 67, 3802-3805]. While mass spectrometry has taken the lead as an analytical tool in proteomic studies because of the sensitivity of the instrument and the ability to gather structural information, the complexity of some samples to be analyzed requires extensive purification before analysis. Borrowing from the drug development process [(a) Hopfgartner, G.; Bourgogne, E. Mass Spec. Rev. 2003, 22, 195-214. (b) Strege, M. A. J. Chromatogr. B 1999, 725, 67-78], research in high-throughput protein analysis has relied on mass spectrometry coupled with automated separation techniques such as nanoliquid chromatography (nanoLC-MS) [Berger, S. J.; Lee, S.; Anderson, G. A.; Pa{hacek over (s)}a-Tolic, L.; Tolic, N.; Shen, Y.; Zhao, R.; Smith, R. D. Anal. Chem. 2002, 74, 4994-5000], and capillary electrophoresis (CE-MS) [Zhang, B.; Foret, F.; Karger, B. Anal, Chem. 2001, 73, 2675-2681].
Liquid chromatography (LC) traditionally utilizes a separation column filled with tightly packed particles with diameters in the low micrometer range. The small particles provide a large surface area, which can be chemically modified and forms a stationary phase. A liquid solvent or eluent, referred to as the mobile phase, is pumped through the column at an optimized flow rate that is based on the particle size and column dimensions. Analytes of a sample injected into the column flow through channels formed by the packed particles. The particles interact with the stationary phase relative to the mobile phase for different lengths of time, and, as a result, the analytes are eluted from the column separately at different times.
Capillary electrophoresis (CE) is a technique that utilizes the electrophoretic nature of molecules and/or the electroosmotic flow of liquids in small capillary tubes to separate analytes within a liquid sample. The capillary tubes are filled with buffer and a voltage is applied across it. It is generally used for separating ions, which move at different speeds when the voltage is applied depending on their size and charge.
Coupling of nanoLC and CE with MS has mostly been performed utilizing a pulled fused silica capillary (tip i.d. 2-10 micrometres), sometimes called a nanocapillary, to provide effective formation of an electrospray ionized (ESI) plume of ions. The main advantage of the pulled capillary is that small droplets are produced at the smaller openings at the end of the capillary. These smaller droplets have a larger surface to volume ratio, which produces a more efficient ionization process. In addition, the relatively small hydrophilic surface at the tip of the capillary reduces wetting of the surface and decreases the voltage needed to produce a stable electrospray. However, pulled silica capillaries have a strong tendency to clog, are difficult to fabricate reproducibly, and are not useful when coupled to separation techniques which require higher than a few microlitre/min flow rates.
Microchip technology (sometimes called lab-on-a-chip technology) has shown promise in the ability to automate many tedious protein purification and preparation steps before analysis. This technology is usually limited to optical detection of the purified proteins, which gives no structural information, and typically comprises a microchip coupled to an optical detector. The components on the microchip are moved from one part of the device to another by electroosmotic flow (EOF) and then pass through the detector. The coupling of such a microchip with a pulled capillary has been attempted in order to create a device that can be used to automate sample purification and analysis of protein or peptide samples by mass spectrometry. However, these first generation devices suffer from disadvantages including the inherent problems of the capillary itself as described above, and the fact that the coupling of the capillary with the chip must be precise in order to create a junction with zero dead volume. Such dead volume could adversely affect the separation efficiency of the device and subsequent sensitivity of the analysis of the sample. In addition, the coupling of a capillary to microchips or similar devices would be an expensive part in any future fabrication process.
An alternative to a capillary fixed to the end of a microchip is a microchip that has the ability to spray a purified sample directly from its end. This has been attempted with glass microchips but has met with limited success due to the large inner diameter of the exit channel of the microchip compared with nanospray capillaries and the hydrophilic nature of glass. Devices have been made with a nanospray nozzle directly fabricated into the microchip but these devices have not been in wide use which is likely due to the difficulty in manufacture and the potential for clogging of the nanospray capillary.
Recently, rigid porous polymer monoliths (PPMs), which are highly crosslinked polymers that have a high porosity, have shown great potential as stationary phases for both LC and CE applications. The PPMs are generally used instead of particles in a column. The pores, which are inherent throughout the PPM, form channels through which sample may flow. Samples are loaded at one end of the column and eluted through the column via the channels with an eluting solvent. Different components of the sample may interact chemically with the PPM for different lengths of time relative to the eluting solvent, which results in the separation of some components. The separated components are eluted from the column at the other end of the column (the eluting end) at different times. The use of PPMs for these systems is attractive because of the ability to modify the physical properties of the stationary phase and the ease at which these monoliths can be prepared. One such property that can be varied is the pore size within the PPM, which has been shown to vary from 0.5-1.5 μM in diameter depending on the properties of the casting solvent [Peters E. C.; Petro, M; Svec, F.; Fréchet, J. M. Anal Chem., 1998, 70, 2288-2295].
The size of the pores defined by PPM at the elating end of such columns have been shown to useful as nanospray emitters. If the sample is eluted at a suitable flow rate, a plume of the sample suitable for analysis by nanospray mass spectrometry is produced. The nanospray emitters prepared using porous polymer monoliths have been shown to function well for generating ESI at a variety of flow rates (Koerner, T.; Turck, K.; Brown, L.; Oleschuk, R. D.; Anal. Chem., 2004, 76, 6456-6460, herein incorporated by reference). However, PPM filled capillaries are not ideal for spraying samples of certain solvent compositions, such as aqueous samples.
The use of a PPM as a stationary phase has disadvantages from a chemical/physical standpoint including (i) the surface area of the PPM available to interact with components of a sample has been shown to be quite low and (ii) it is not amenable to being chemically modified.
The invention provides emitters, compositions, and processes and methods for making emitters and compositions, useful, for example, for emitting sample for mass spectral analysis and/or acting as a stationary phase in chromatographic applications. Compositions according to the invention can comprise particles entrapped by polymeric material such that unoccluded channels are formed and the particles are substantially uncovered and able to interact with sample.
According to an aspect of the present invention, an emitter is provided comprising a plurality of particles collectively forming a plurality of channels, and a polymeric material adhesively disposed between at least a portion of adjacent said particles, wherein the channels are substantially unoccluded by the polymeric material. The polymeric material may form a porous polymer monolith or a substantially non-porous matrix. The polymeric material may be polyolefin, such as polyacrylate, polymethacrylate, polystyrene, or mixtures thereof.
According to another aspect of the present invention, a composition is provided comprising a plurality of particles collectively forming a plurality of channels, and a polymeric material adhesively disposed between at least a portion of adjacent said particles, wherein the channels are substantially unoccluded by the polymeric material. The polymeric material may form a porous polymer monolith or a substantially non-porous matrix. The polymeric material may be polyolefin, such as polyacrylate, polymethacrylate, polystyrene, or mixtures thereof.
In a particularly preferred embodiment of the present invention, a substantial amount of the surface area of the particles is uncovered by the polymer and available to interact with a sample.
According to another embodiment of the present invention, the particles may comprise at least one material selected from the group consisting of inorganic oxides, metal oxides, silica, alumina, titania, zirconia, chemically bonded inorganic oxides, chemically bonded metal oxides, organosiloxane-bonded phases, hydrosilanization/hydrosilation bonded phases, polymer coated inorganic oxides, porous polymers, polyolefin, polystyrene, polymethacrylate, polyacrylate, and styrene-divinylbenzene copolymer. The particles may be metal oxide-coated. The metal oxide-particles may comprise polyolefin, such as polystyrene. These particles may be coated with pepsin enzyme. These particles may be magnetic, such as paramagnetic.
According to another embodiment of the present invention, the particles may be porous or non-porous. The particles may have pores with a diameter in the range of about 100 to about 300 angstroms, greater than 300 angstroms, or less than 100 angstroms.
According to another aspect of the invention, the particles may have a diameter in the range of about 0.1 micrometres to about 1000 micrometres, or a diameter in the range of about 0.3 micrometres to about 600 micrometres, or a diameter in the range of about 0.5 micrometres to about 300 micrometres, or a diameter in the range of about 0.2 micrometres to about 30 micrometres.
According to another embodiment of the present invention, the channels have a diameter in the range of about 0.5 micrometres to about 10 micrometres, or a diameter in the range of about 1.0 micrometres to about 5.0 micrometres.
According to another embodiment of the present invention, the surface of at least one particle is suitable to interact with at least one component of a sample flowing through the channels.
According to another aspect of the invention, the emitter or the composition further comprise a vessel for containing the plurality of particles. The vessel may be a capillary. The inner diameter may about 0.2 to about 1000 micrometres, or about 30 to about 500 micrometres, or about 50 to about 250 micrometres, or about 1 to about 100 micrometres.
According to another aspect of the present invention, a use of an emitter is provided, the emitter comprising a plurality of particles collectively forming a plurality of channels, and a polymeric material adhesively disposed between at least a portion of adjacent said particles, wherein the channels are substantially unoccluded by the polymeric material to provide a sample suitable for analysis by mass spectrometry. The mass spectrometry may be micro-electrospray or nano-electrospray mass spectrometry.
According to another aspect of the present invention, a use of a composition is provided, the composition comprising a plurality of particles collectively forming a plurality of channels, and a polymeric material adhesively disposed between at least a portion of adjacent said particles, wherein the channels are substantially unoccluded by the polymeric material to provide a sample suitable for analysis by a mass spectrometer.
According to another aspect of the present invention, a use of a composition is provided, the composition comprising a plurality of particles collectively forming a plurality of channels, and a polymeric material adhesively disposed between at least a portion of adjacent said particles, wherein the channels are substantially unoccluded by the polymeric material for separating components of a sample.
According to another aspect of the present invention, a process for making a composition is provided, the composition comprising a plurality of particles collectively forming a plurality of channels and a polymeric material adhesively disposed between at least a portion of adjacent said particles, wherein the channels are substantially unoccluded by the polymeric material, the process comprising the steps of (a) introducing particles, monomer, and photo-initiator into a containment vessel that at least partly allows the transmittance of light, and (b) exposing the containment vessel to light. The containment vessel may comprise at least one section in which the composition is accessible to ultraviolet light and at least one section in which the composition is protected from the ultraviolet light.
According to another aspect of the present invention, products made by the processes of the present invention are provided.
The invention is illustrated in the figures of the accompanying drawings, which are meant to be exemplary and not limiting, and in which references are intended to refer to like or corresponding parts.
a shows a TIC trace of an electrospray sample of PPG sprayed from a nanospray emitter according to the present invention.
b is an electrospray mass spectral trace corresponding to the TIC trace of
a shows the results of loading a 450 nM leucine enkephalin sample onto a sprayer according to the protocol depicted in
b shows the linear relationship for the amount of leucine enkephalin loaded onto the sprayer and relative ion intensity measured at 556 m/z.
a shows a scanning electron micrograph of silica particles entrapped according to an embodiment of the present invention.
b shows an expanded region of the scanning electron micrograph shown in
a shows a scanning electron micrograph of ODS particles entrapped according to an embodiment of the present invention.
b shows an expanded region of the scanning electron micrograph shown in
a shows a sample extracted ion chromatogram (XIC) showing the analysis of a PPG sample according to an embodiment of the present invention.
b shows an instant electrospray mass spectral trace of the PPG sample generated according to an embodiment of the present invention
The compositions of the present invention comprise particles entrapped in a polymeric material. The plurality of entrapped particles collectively form a plurality of channels. The polymeric material acts as an adhesive and is disposed between at least a portion of adjacent particles which causes the particles to be substantially immobilized relative to each other. The polymer does not substantially block the channels, leaving the channels substantially unoccluded by the polymer. In this composition, a substantial amount of the surface area of the particles is uncovered by the polymer and available to interact with a sample.
The compositions of the present invention are advantageously produced by a photo-initiation process. The particles are loaded into a vessel, as described below, and a solution including monomers, cross-linker and photo-initiator is added. The vessel is at least partly made from a material that allows the transmittance of ultraviolet (U.V.) light. The section of the containment vessel in which the composition of the present invention is desired is left accessible to U.V. light and the other sections are protected from the U.V. light. The methods disclosed herein provide substantially limited surface coverage of the particle to maximize the particle functionality. Such processes, as exemplified below, produce the compositions of the 1 present invention.
The compositions of the present invention are useful as emitters for electrospray mass spectrometry, including nanospray and microspray. Plumes of ions suitable for such analysis can be produced from the surface of the compositions by methods described below.
The compositions of the present invention are also useful as stationary phases for chromatographic procedures such as micro-high performance liquid chromatography (HPLC), gas chromatography (GC), and capillary electrochromatography (CEC). Other uses include solid phase extraction including preconcentration and sample cleanup, solid phase synthesis/catalysis/sample derivatization before analysis, catalyst immobilization (e.g. Pt or Pd spheres or coated particles) for catalyzed digestion of sample, enzyme reactor bed (e.g. trypsin immobilized spheres), and affinity separation (antibody-antigen, protein-affinity column). The particles used in the compositions of the present invention may be selected based on the desired chemical and/or physical characteristics. The stationary phases may be also used as nanospray or microspray emitters, or the composition may be coupled to another emission device for analysis of the components. Alternatively, the eluted compounds are analyzed separately.
The compositions of the present invention may be used with a variety of vessels such as glass capillaries or microchips.
The vessels, such as capillaries, may have inner diameters in the range of about 0.2 to about 1000 micrometres, more preferably in range of about 30 to about 500 micrometres, and even more preferably in the range of about 50 to about 250 micrometres. The vessels may have an inner diameter in the range of about 1 to about 100 micrometres.
Customized vessels are also within the scope of the present invention, wherein particles with different chemical and or physical properties are used in one containment vessel, either in separate sections, or interspersed amongst one another.
The compositions of the present invention may be used for flow-through peptide synthesis, including combinatorial or rational synthesis, or protein enzymatic digestion. This use may include subsequent analysis of products emitted from the channels of composition via electrospray.
The methods disclosed herein allow for patterning of particular particles in specific areas, which generally cannot be done with other methods. The methods associated with entrapping particles are also generally completed in a shorter amount of time compared to other methods of entrapment (hours for polymers to days for sol gel). Sol gel methods can crack during the drying process and create voids in the material. The methods of the present invention are suitable for both small and large capillaries, whereas larger capillaries tend to show unpredictable results in sol gel encapsulation. The methods of the present invention also provide for the use of a wide range of polymerization conditions which enable the entrapment of a variety of different particles.
The methods and apparatus are suitable for scale-up procedures. For example, many vessels may be loaded at once with particles and a polymerization mixture.
Referring now to
In operation, the spray voltage may be in the range of about 0.5 to about 4 kV, more preferably in the range of about 0.6 to about 3 kV, and most preferably in the range about 0.7 to about 2 kV. The voltage on the emitter and the voltage applied to the system are the same and supplied via a liquid junction.
Referring now to
Using the electrospray emitter of the present invention, components of a sample can be detected even when the concentration of the component is in the femtomole or even attomole range.
Referring now to
Sample solution volumes vary, but are in the range of about 50 to about 5000 nL, more preferably in the range of about 100 to about 3000 nL, and most preferably in the range of about 200 to about 1000 nL. Components in the sample solution may be in the concentration of about 1.0×10−18 M to about 1.0×10−2 M, more preferably about 1.0×10−16 M to about 1.0×10−4 M, and most preferably about 1.0×10−15 M to about 1.0×10−4 M. The loading flow rate can range from about 200 to about 5000 nL/min.
The sample flows from sample loading surface 34 to emitting surface 36 by hydrodynamic force provided by such origins as a syringe pump, HPLC pump or nano LC pump. In the case of electroosmotic flow (EOF) experiments the flow is produced from the electroosmotic flow of the solution.
Suitable flow rates of the present invention include rates in the range of about 10 to about 10000 nL/min, more preferably in the range of about 50 to about 1500 nL/min, and most preferably in the range about 200 to about 1000 nL/min.
Pressures applied to entrapped particles 32 of this invention include pressures in the range of about 20 to about 8000 psi, more preferably in the range about 100 to about 4000 psi and most preferably in the range about 300 to about 1500 psi.
The amount of pressure used to pump the solution through the entrapped particles is proportional to the length of the path through the composition, i.e., the amount of entrapped particles through which the sample passes. Generally speaking, the longer the path, the higher the pressure.
Suitable inner and outer diameters of emitting end 60 include outer diameters in the range about 100 to about 5000 μm and inner diameters in the range about 5 to about 2500 μm, more preferably, the outer diameters are in the range about 100 to about 3000 μm and inner diameters are in the range about 20 to about 100, and most preferably the outer diameters are in the range about 150 to about 360 μm and inner diameters are in the range about 30 to about 75 μm. The surface area of emitting surface 36 will cover the entire area within the inner diameter of the capillary.
As used herein, the term “particles” refers to spheres, such as microspheres or spheres of any size, beads, cubes, and other three-dimensional structures of generally regular or irregular shape, and the like, and are generally commercially available, although modifications may be made before use. The particles may comprise a substrate of materials such as metal oxides, such as iron oxide, inorganic oxides, silica, alumina, titania and zirconia, chemically bonded inorganic oxides, such as organosiloxane-bonded phases hydrosilanization/hydrosilation bonded phases, polymer coated inorganic oxides or metal oxides, porous polymers, such as styrene-divinylbenzene copolymer, polyolefins, such as polyacrylates, polymethacrylates, and polystyrene. Particles may include, for example, octadecyl silane (ODS) particles, agarose beads, fluorinated beads, and silica based particles. The particles may be porous, mesoporous, or non-porous, or a combination. Porous or mesoporous particles may have pores of less than about 100 angstroms in diameter, in the range of about 100 to about 300 angstroms in diameter, or greater than about 300 angstroms in diameter, or a combination.
The particles may optionally bear substituents that confer desirable chemical properties, e.g. affinity, to the particles so that the particles are suitable for chromatography. Substituents may include, e.g., ketone groups, aldehyde groups, carboxyl groups, such as carboxylic acid, ester, amide, and acid halide groups, chloromethyl groups, cyanuric groups, polyglutaraldehyde groups, epoxide groups, thiol groups, amine groups, silanol groups, hydroxyl groups, sulphonic acid groups, phosphonic acid groups, and/or unsubstituted or substituted aliphatic or aromatic hydrocarbons. For example, for reversed-phase chromatography, alkyl, fluoroalkyl and phenyl bonded materials may be added; for ion-exchange chromatography, sulfonic acid, carboxylic acid, quaternary amine bonded or other materials may be added; for size-exclusion chromatography, glycerol bonded materials, poly(saccharide) and poly (dextran) gels may be added; for affinity chromatography, enzyme, antibody, lectin, and metal ion immobilized materials may be added. For example, particles may comprise nickel to attract molecules with histidine groups, or lectin to attract proteins with glycosylation sites.
The particles may be modified chemically and/or physically in order to be suitable for chromatography. The particles may be used without modification if they already have chemical and/or physical properties desirable for chromatography.
Different properties may be demonstrated by the same particles in different conditions, such as different solvent conditions.
The particles can comprise a magnetic material, such as a paramagnetic material, so that a magnet can be used to position the particles in a vessel before the photo-initiation step. Particles suitable for this application or other applications include metal oxide-coated polyolefin particles, such as iron oxide-polystyrene or magnetite-polystyrene particles.
The particles, including the magnetic particles, may be coated with pepsin, for example, such as the particles PMPE-4 (paramagnetic pepsin coated particles, 4 micrometres, Kisker Biotech, Steinfurt, Germany). The particles may also be coated with such groups as, e.g., avidin, streptavidin, albumin antibodies, such as goat anti-mouse IgG, papain, protein A, protein G, PEG-COOH, or PEG-NH2 groups (all such magnetic particles available from, for example, Kisker Biotech, Steinfurt, Germany).
In one embodiment of the present invention, the entrapped particles can be used to digest proteins. In this embodiment, the materials must be stable to reagents used to digest proteins, such as enzymes, and suitable buffers such as trypsin.
Particle diameters may be in the range of about 0.1 to about 1000 micrometres, more preferably in the range of about 0.3 to about 600 micrometres, and most preferably in the range of about 0.5 to about 300 micrometres. Larger particles may be considered for specialized applications.
It is also contemplated that particles useful for peptide synthesis and/or combinatorial synthesis are applicable to other embodiments of the invention. In this case, particles for peptide synthesis and/or combinatorial synthesis can be entrapped within a vessel, such as a column or capillary, so that flow-through synthesis can be performed. A variety of active species attached to the particles and/or part of the solution, such as nucleophilic amino acids or amino acids with activated esters. Alternatively or in addition, solutions could be passed through a catalytic bed for continuous synthesis applications. It will be understood that such a process can also be adapted for syntheses such as small molecule synthesis or polynucleotide synthesis.
Entrapped particles 32 can function by chemically and/or physically interacting with components of an injected sample. Such interaction can result in a change in the relative composition and/or characteristics of the components of the injected sample from injection surface 34 to emitting surface 36.
The surface chemistry of the particles can be performed “off-line” and then integrated into the device or capillary. Possible interactions with components of the sample include hydrophobic, when the particles are functionalized with carbon 18 (C18), for example, and hydrophilic and/or electrostatic, when the particles are functionalized with sulfonic acids, for example. Other interactions include size exclusion interactions, where the particles comprise cavities or pores of varying sizes which interact with components of varying size within the sample and separates the components based on size.
Entrapped particles 32 need not chemically or physically interact with the sample at all, and may only function as providing suitable channels and/or pores for emitting the sample as a microspray or nanospray (described further below).
Electrospray emitter 30 comprises vessel 70, which may be a capillary suitable for the entrapment of particles in accordance with the present invention. Other suitable containment vessels include portions of a microchip as described below, Glass, such as fused silica, capillaries are preferred. Vessels which are commercially available may be used as received or may be modified by such techniques as pulling with a laser or manually with a microtorch to change its size or shape. For sufficient conductivity, the vessels may be sputter-coated with conductive material, e.g. gold, or a thin metal wire may be inserted into the capillary during operation of nanospray mass spectrometry system 20. The vessel should be made of a material which allows the passage of U.V. light in order to allow induction of the polymerization process (described below). The vessels may be made of material including glass, such as fused silicon, and plastics, such as polymethylmethacrylate (PMMA), polycarbonate and the like.
The compositions may be formed in any vessel, including, for example, a void in a device, such as a void in a microdevice. The void may be of any suitable shape, including, for example, a cubic void. Such compositions can be used for reactions in situ. For example, a composition of the present invention comprising trypsin enzyme coated particles may be made in a void or reservoir on the surface of a microdevice. In this case, the void or reservoir may be used as a digestion bed. The composition may be made by placing the particles in the void and then mixing with the polymerization mixture. Once the particles have settled by way of gravity or centrifugal force, to the bottom of the void, the composition may be formed by photo-intitiation. It is desirable in such a reaction to minimize the amount of oxygen available to react with the polymerization mixture. One method of minimizing the oxygen exposure is to degas the solvents before using them. The stationary composition could then be exposed to a solution of a suitable amount of protein for a suitable amount of time until a substantial amount of digestion products are left in the solution. The solution with the digestion products may then be removed via means known in the art, such as decantation or suction.
The particles are entrapped within the vessel by a polymer. Polymers suitable to use in accordance with this invention include any polymer or co-polymer mixture that can form a matrix. The matrix may be a porous polymer monolith including polyolefins, such as polyacrylates, polymethacrylates, polystyrenes, and the like. Alternatively, the matrix may be a substantially non-porous material, such as a material that is made by the polymerization of dimethacrylate without an additional polymer.
The polymer can advantageously be formed by exposing monomers to U.V. light in the presence of an appropriate solvent and photo-initiator. In this way, only selected portions of the vessel, such as a capillary may be submitted to the polymerization process, and therefore, only the selected portions of the vessel would contain the entrapped particles. The unreacted polymerization mixture can be washed away from the non-selected portions of the vessel. This process is referred to as “photo-patterning”.
Referring now to
Suitable channel diameters with the present compositions include diameters in the range of about 0.2 to about 30 micrometres, more preferably in the range of about 0.5 to about 10 micrometres and most preferably in the range of about 1.0 to about 5.0 micrometres.
The channel diameters at emitting end 36 may be controlled by particle size. When the particles are tightly packed, the spaces between the particles form the channels which act as the electrospray emitters. The larger the spheres the larger the spaces between the spheres.
It will be understood by one skilled in the relevant arts that not all polymerization compositions or conditions will be suitable for use with all particles. For example, polystyrene-based particles, such as polystyrene spheres, may swell in the presence of certain polymerization compositions. However, it would not cause a skilled person to undertake undue experimentation to learn that using monomers and solvent conditions that are more hydrophilic can decrease the swelling of the polystyrene particles.
Embodiments of the present invention will now be described by way of examples. It will be understood that the scope of the invention is not limited by the specific embodiments exemplified herein.
1.1 Materials and Equipment
Fused-silica capillaries (about 75 μm i.d., about 363 μm o.d.) with a ultraviolet (U.V.)-transparent coating were obtained from Polymicro Technologies, L.L.C. (Phoenix, Ariz., US). Polymerization was performed using a Mineralight UV lamp, UVG-11 254 nm (Upland, Calif., US). A Harvard Apparatus 11 plus syringe pump (Holliston, Mass. US) was used to drive liquid through capillary or microchip. A Nikon Eclipse ME600 microscope (Tokyo, Japan) was used to monitor the particles packing and polymerization in the capillaries and microchip channels. Scanning electron microscopy (SEM) analyses were performed on a Jeol JSM-840 Scanning Microscope (Tokyo, Japan). All experiments were conducted at ambient temperatures.
Butyl acrylate monomer was obtained from Aldrich and filtered through freshly activated alumina to remove inhibitor. 3-(trimethoxysilyl)propyl methacrylate, 2-acrylamido-2-methyl-1-propanesulfonic acid (AMPS), 1,3-butanediol diacrylate (BDDA), and benzoin methyl ether (BME) were obtained from Aldrich and used as received. Buffer salt Tris was purchased from Fisher Scientific, while Tricine was obtained from Sigma. Buffers were prepared using ˜18.2 MΩ·cm deionized water filtered through a Milli-Q Gradient water purification system (Millipore S.A. Molsheim, France). Ethanol was purchased from Commercial Alcohols Inc. (Brampton, ON, Canada). Glacial acetic acid and HPLC grade acetonitrile and methanol were obtained from Fisher Scientific. 3 micrometre (μm) octadecyl silane (ODS) particles Microsorb 100-3 C18) were received as a gift from Varian Canada Inc. (Mississauga, ON, Canada).
All experiments were performed on an API 3000 triple quadrupole mass spectrometer (MDS-Sciex, Concord, Canada) fitted with a nanoelectrospray source (Proxeon, Odense, Denmark) consisting of a x-y-z stage and two Charge Coupled Device (CCD) camera kits to aid in the positioning of the capillary. A micro-Tee union (Scientific Products, Toronto, ON, Canada) was used to couple the solution transfer line, the electrospray capillary and the electrode necessary to supply the electrospray voltage. A syringe was filled with the solution to be analyzed and fitted to the transfer line of the micro-Tee union. The entire assembly was fixed to the x-y-z stage and the capillary was directed to the entrance of the mass spectrometer with the aid of CCD cameras. In most experiments the capillary was maintained approximately 5 mm from the orifice of the mass spectrometer (MS). The electrospray (ES) voltage was supplied through a liquid junction by connecting the MS power supply to a platinum electrode inserted within the micro-Tee.
1.2 Nanospray Emitter Fabrication
1.2.1 Particle Retaining Frit Fabrication
The nanospray emitters were prepared by first fabricating an outlet frit. The capillary was treated with 3-(trimethoxysilyl)propyl methacrylate for 8 hours to provide an anchor to the capillary wall. Following this, the polymerization mixture was introduced into the capillary or microchip channel with a syringe pump. The entire capillary or microchip was then masked leaving only 1.5 mm of the UV-transparent capillary or microchip exposed. The polymerization reaction was initiated by illuminating the exposed regions with 254 nm U.V. light for 1.5 minutes.
1.2.2 Particle Entrapment
Following frit formation, ODS particles entrapped in porous polymer matrix devices were prepared using the following procedure: ODS particles were introduced into either a capillary or microchip channel by a slurry packing method. This was followed by the introduction of the polymerization mixture into the capillary or microchip channel with a syringe pump. After several column volumes of the polymerization mixture had passed through the capillary or microchip channel, the packed beads were immobilized by exposing a specified region to about 254 nm U.V. light for about 2 minutes. The polymerization was followed by a washing step with a mixture of 80:20 v/v acetonitrile/5 mM tris buffer, pH 8 which was flushed through the capillary column with a syringe pump or nano-HPLC pump. The retaining frit was then removed by cutting the capillary in the bead-entrapped region. To observe the cross-section of the entrapped beads, a short length of the capillary column was cut off, coated with gold and observed by SEM. Results are shown in
1.3 Preliminary Electrospray Performance
a shows a total ion current (TIC) trace and
The sprayer was tested with a number of different flow rates by examining the TIC traces and associated mass spectrum. Electrospray ionization (ESI) could be conducted over a very wide flow rate range. At flow rates ranging from about 100 nL-about 200 nL/min a single stable Taylor cone was observed which generated a stable TIC trace. Below about 200 nL/min a “mist” presumably due to multiple Taylor cones yielding a stable TIC signal. Below 50 nL/min the trace became significantly noisier however sufficient ions were still produced to enable mass spectral acquisition. A “clean” spectrum of leucine enkephalin was produced even at 10 nL/min.
The generation of an electrospray at these minimal flow rates shows the benefit of using the compositions of the present invention in microfluidic chips coupled to a mass spectrometer. Typically, microfluidic devices that utilize electroosmotic pumping deliver less than about 50 nL/minute flow rates.
The surface chemistry of the particles can be exploited to perform sample preparation procedures to aid in MS analysis. To demonstrate the sample preparation capabilities of the composition of an embodiment of the present invention, solid phase extraction experiments were conducted. A schematic diagram depicting the SPE protocol is shown in
a shows the results of loading a 450 nM leucine enkephalin sample onto the sprayer at a flow rate of 800 nL/min according to the protocol depicted in
Although SPE with ODS functionalized particles was performed, a variety of commercially available particles possessing a variety of surface chemistries could be utilized.
A series of solid-phase extraction (SPE) experiments were conducted with of trace amount of BODIPY® and BODIPY®FL. The composition of this embodiment of the present invention fabricated in a capillary showed better performance than another two particle immobilization technologies (the packed column with a single frit, the packed column with an inlet and outlet frit) in terms of reproducibility and robustness.
3.1 Apparatus and Reagents
All the CEC experiments in capillaries were performed on a Beckman Coulter P/ACE MDQ capillary electrophoresis system (Fullerton, Calif., US) equipped with a laser-induced fluorescence (LIF) detector (about 488 nm excitation, about 520 nm emission). Fused-silica capillaries (about 75 μm i.d., about 363 μm o.d.) with a UV-transparent coating were obtained from Polymicro Technologies, L.I.C. (Phoenix, Ariz., US). Polymerization was performed using a Mineralight UV lamp, UVG-11 254 nm (Upland, Calif., US). A Harvard Apparatus 11 plus syringe pump (Holliston, Mass., US) was used to drive liquid through the capillary. A Nikon Eclipse ME600 microscope (Tokyo, Japan) was utilized to inspect the particles packing and polymerization in the capillaries. Scanning electron microscopy (SEM) analyses were performed on a Jeol JSM-840 Scanning Microscope (Tokyo, Japan). All experiments were conducted at ambient temperature.
Butyl acrylate monomer was obtained from Aldrich and filtered through freshly activated alumina to remove inhibitor (monomethyl ether hydroquinone). 3-(trimethoxysilyl)propyl methacrylate, 3-methacryloxypropyltrimethoxysilane, 2-acrylamido-2-methyl-1-propanesulfonic acid (AMPS), 1,3-butanediol diacrylate (BDDA), and benzoin methyl ether (BME) were all obtained from Aldrich and used as received. The buffer salt, Tris, was purchased from Fisher Scientific, while Tricine was obtained from Sigma. Buffers were prepared using −18.2 MS2·cm deionized water filtered through a Milli-Q Gradient water purification system (Millipore S. A. Molsheim, France).
Ethanol was purchased from Commercial Alcohols Inc. (Brampton, ON, Canada). Glacial acetic acid and HPLC grade acetonitrile and methanol were obtained from Fisher Scientific. 31.tm ODS particles (Microsorb 100-3 C18) were received as gift from Varian Canada Inc. (Mississauga, ON, Canada). 4,4-difluoro-1, 3, 5, 7, 8-penta methyl-4-bora-3a,4a-diaza-(S)-indacene, (BODIPY 493/503) and 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionic acid (BODIPY®FL) were purchased from Molecular Probes, Inc. (Eugene, Oreg., US).
3.2 Packed Column Fabrication
Packed column with one frit: To prepare an outlet fit, a short length of porous polymer monolith was prepared in a way similar to the method previously described by Ngola et al. [S. M. Ngola, Y. Fintschenko, W. Y. Choi, and T. J. Shepodd, Anal. Chem, 73 (2001) 849]. The capillary walls were first pretreated by grafting with vinyl groups to ensure that formed polymer will be covalently attached to the wall: the capillary was filled with a solution of 3-methacryloxypropyltrimethoxysilane (about 20%, all quantities are volume percent unless otherwise stated), glacial acetic acid (about 30%), and deionized water (about 50%) and left to react for 12 h, then washed and stored in a solution consisting ethanol (about 20%), acetonitrile (about 60%), and 5 mM phosphate buffer, pH 6.8 (about 20%). The polymerization mixture consisting of about 23% butyl acrylate monomer, about 10% BDDA as the cross-linker, about 0.2% AMPS to support electroosmotic flow, about 0.1% 3-methacryloxypropyltrimethoxysilane as additional adhesion promoter, about 0.2% (g/ml) BME as initiator, about 13.25% ethanol, about 40% acetonitrile, and about 13.25% 5 mM phosphate buffer, pH 6.8 as porogenic solvent, was introduced into the capillary with a syringe pump. The capillary was then covered by aluminum foil, leaving about 1.5 mm of the UV-transparent capillary exposed to the 254 nm UV light for about 1.5 min. To prepare a packed column with only one frit in a capillary, a slurry of 31.tm ODS particles in acetonitrile was then introduced into the capillary with pressure (immersed in a ultrasonic bath) to create a 2 cm long column.
Packed column with two retaining frits: After fabricating the outlet frit and 2 cm long column, the polymerization mixture was again introduced into the capillary with a syringe pump. After several column volumes of the polymerization mixture had passed through, the capillary was covered by aluminum foil, leaving a about 1.5 mm region of the capillary just at the open end of the packed particles exposed to the 254 mm UV light for about 1.5 min. Then a mixture of 80:20 v/v acetonitrile/5 mM tris buffer, pH 8 was flushed through the column with syringe pump to remove residual monomeric materials and porogenic solvent.
3.3 Entrapped Column Fabrication
Capillaries with ODS particles entrapped in a porous polymer matrix were prepared using the following procedure after constructing the outlet frit, ODS particles were introduced into the capillary by slurry packing method to yield a about 2 cm long column. The polymerization mixture was introduced into the capillary again with a syringe pump. After several column volumes of the polymerization mixture had passed through the capillary, the packed beads were immobilized by exposing the 2 cm packed region to the 254 nm UV light for about 2 minutes. Then a mixture of about 80:20 v/v acetonitrile/5 mM tris buffer, pH 8 was flushed through the capillary column with a syringe pump to remove unreacted monomeric materials and porogenic solvent. To observe the cross-section of the column bed, a short length of the capillary column was cut with ceramic cutter, and allowed to dry in a desiccator to remove all water and solvents. Cross sectional images were then captured with a scanning electron microscope after the sample was sputter coated with gold.
3.4 Solid-Phase Extraction
Two analytes were chosen to demonstrate solid-phase extraction with columns described above. The 0.10 mM stock solutions of BODIPY 493/503 and BODIPY®FL were prepared in HPLC grade methanol, then were diluted in 10 mM tricine buffer, pH8 to desired concentrations.
SPE was carried out in three steps: first, diluted samples were loaded onto the chromatographic bed using pressure. Secondly, aqueous buffer was flushed through the capillary to wash sample remaining within the capillary onto the column. The analyte retained on the bed was then eluted with 80% acetonitrile in aqueous buffer. The fluorescence of BODIPY or BODIPY®FL was detected with a LIF detection system (488 nm excitation, 520 nm emission) of Beckman P/ACE MDQ CE placed just the downstream of the chromatographic bed. Between each extraction, the device was equilibrated by rinsing with aqueous buffer before a new loading step commenced.
3.5 Breakthrough Curves
In order to determine the total capacity of the SPE bed, breakthrough curves were obtained with a 10 nM solution of BODIPY and BODIPY®FL dye by injecting them individually onto an aqueous buffer equilibrated capillary column. Fluorescent signal of BODIPY or BODIPY®FL was recorded just the downstream of the bed using the LIF detector (488 nm excitation, 520 nm emission).
3.6 Results
The UV photopolymerization of solution of butyl acrylate, BDDA and AMPS required only few minutes at room temperature to complete which reduces the column fabrication time compared to thermal polymerization. An additional advantage was the ability to readily pattern the material through appropriate masking.
The sample preparation process resulted in the particles being scattered on the capillary surface. In contrast, ODS particles entrapped with organic polymer remained “packed” following capillary cutting (as shown in
To test the mechanical strength of the entrapped beads, a 0.5 cm long bed with no outlet frit was fabricated and found to withstand a pressure more that 4,400 psi which is the maximum pressure generated by the HPLC pump. The high strength is attributed to the covalent attachment of the beads to one another and the surface of the capillary. As a result the packed capillary should be robust enough for most high pressure chromatographic and electrochromatographic applications.
To demonstrate the SPE capability of the ODS columns prepared with different methods, a dilute solution of BODIPY was concentrated on them. BODIPY is a highly hydrophobic dye showing a strong affinity to ODS particles in an aqueous environment and affords an intensive fluorescence emission at 520 nm, so it was chosen as the starting analyte to investigate the SPE characteristics of the different types of columns.
The ODS beads retained with one frit showed irreproducible SPE properties because the open end of the packed bed allowed the movement of chromatographic material. Although the packed bed with two retaining frits was reproducible in the first few days of use, the reproducibility gradually deteriorated in the further runs. After 4 days of use, the relative standard deviation (RSD) of integrated peak area of eluted analyte increased from 4.8% to 6.2%, while the R2 of a linear regression of peak area versus sample loading time decreased from 0.9816 to 0.8479. This is due to the accumulated migration of particles resulting in void formation within the column. In contrast, the entrapped column showed much better reproducibility in SPE experiments. The RSD of integrated peak area of eluted analyte still remained at 4.2% after 5 days of use, while the R2 of linear regression of peak area versus sample loading time remained at 0.9934. This is again believed to be due to organic polymer immobilizing the packed particles in place, preventing movement, resulting in a robust continuous extraction bed.
BODIPY®FL is more hydrophilic than BODIPY because of the caboxylic acid group in its chemical structure. In the same breakthrough experiment conditions as BODIPY, 10 nM BODIPY®FL showed a rapid and steep breakthrough (
In the SPE experiment of BODIPY®FL at pH8.0, BODIPY®FL was observed to partially wash out during the aqueous buffer wash step (
A SPE experiment of leucine enkephalin has been done with a composition of the present invention in microchip using Microfluidic Tool Kit (Micralyne, Edmonton, Canada). The kit consisted of a high-voltage power supply coupled with a laser-induced fluorescence (LIF) detection system (about 635 nm diode laser with 670 nm band pass filer). Since leucine enkephalin has no fluorescence emission at about 675 nm, it was labeled by Cy5 fluorescent dye in 0.1M sodium carbonate-sodium bicarbonate buffer, pH9.3 to make it detectable with a 675 nm LIF detector, and was then diluted to 180 mmol/L in 5 mM, pH8 phosphate buffer. SPE was carried out in three steps: (1) diluted samples were loaded onto the chromatographic bed with an electroosmotic flow (EOF) generated by a about 2.5 kV power applied across the microchip channel, (2) aqueous buffer was flushed through the channel to wash sample remaining within the channel onto the bed, and (3) the analyte retained on the bed was eluted with 80% acetonitrile in aqueous buffer. The fluorescence of Cy5 labeled leucine enkephalin was detected with the LIF detection system placed just the downstream of the chromatographic bed. Between each extraction, the device was equilibrated by rinsing with aqueous buffer before a new loading step commenced.
Particles are entrapped at the end of a plastic or glass microdevice according to methods of the present invention.
Silica particles of about 3 micrometers in diameter were trapped as generally described for ODS beads in Examples 3.1 to 3.3. To trap the silica particles in this example, some adjustments to the monomer conditions were made in order to make the mixture more hydrophilic. This was accomplished by increasing the sulfonic acid component from 1 to 40 percent of the monomer mixture.
6.1 Column Preparation
Fused-silica capillaries (363 μm o.d., 75 μm i.d.) with a U.V.-transparent coating (Polymicro Technologies Inc.) were pretreated by methods known in the art. A frit was then prepared in the capillary in a manner similar to the previous entrapment procedure.
In order to trap silica beads with minimal surface coverage, a more hydrophilic monomer solution was needed. This was accomplished by increasing the amount of sulfonic acid from 1 to 40 percent. This solution consisted of 2 mL casting solvent (1:1:3 ethanol: buffer pH=7: acetonitrile), 297 μL BDDA, 3 μL z-6030, 0.25 g AMPS, 516 μL butyl acrylate and 5 mg of benzoyl methyl ether. This solution was flushed through the capillaries and the beads were entrapped using 254 nanometer (nm) light for 90 seconds.
a is a scanning electron micrograph showing the silica particles entrapped by the method of Example 6. It can be seen from this figure that most of each particle is not covered with the polymer.
The protocol described herein in Example 3 was used for this experiment, except in this experiment monomer was not included. In order to determine if ODS beads could be entrapped using only cross-linker (BDDA), all other components except for the casting solvent and initiator were removed from the system. This method used BDDA (300 μL) in 3.0 mL of casting solvent (1:1:3 ethanol: buffer pH=7: acetonitrile). This solution was flushed through the capillaries containing ODS beads and the polymerization was initiated using 254 nanometer light for 90 seconds,
a shows a scanning electron micrograph showing the beads with unoccluded openings that in the great majority of cases lead to channels. Gold coating was used to make the beads visible by SEM.
The experiment was performed to demonstrate the separation and solid phase extraction of two hormones. Hormones are a group of compounds that show important biological effects in living organisms. However, in many real applications, such as biological or environmental analysis, the low sample concentration usually impedes the accurate detection and quantitation of these compounds.
In this experiment, stock solutions of 4 millimolar (mM) beta-estradiol and 5 millimolar progesterone were prepared in HPLC grade acetonitrile (ACN), and were diluted in 3 millimolar tricine buffer, pH 7 to desired concentrations, Solid phase extraction was carried out in three steps on a 6 centimetre long column containing entrapped ODS particles (Microsorb 100-3 C18), First, diluted samples were loaded onto the chromatographic bed electrokinetically. Second, aqueous buffer was flushed through the capillary onto the column by applying voltage. Third, the analyte retained on the bed was then eluted by 70% ACN in aqueous buffer with EOF. The ultraviolet absorbance of beta-estradiol and progesterone was detected with a PDA (Photo Diode Array) detection system placed downstream of the chromatographic bed. Between each extraction, the device was equilibrated by rinsing with aqueous buffer before a new loading step commenced.
Following bed equilibration with aqueous buffer, diluted samples were loaded onto the bed electrokinetically by applying a voltage of 5 kilovolts for 5 minutes. After a 6 minute rinse step with aqueous buffer using voltage, 70% of ACN in aqueous buffer was then used to elute the preconcentrated beta-estradiol and progesterone with EOF.
Different sample loading times were performed, and the result is shown in
This experiment showed the electrochromatography of sixteen polyaromatic hydrocarbons, namely naphthalene, acenaphthylene, fluorene, acenaphthene, phenanthrene, anthracene, fluoranthene, pyrene, benzo(a)anthracene, chrysene, benzo(b)fluoranthene, benzo(k)fluoranthene, benzo(a)pyrene, dibenzo(a,h)anthracene, indeno(1,2,3-cd)pyrene and benzo(ghi)perylene.
This experiment demonstrated that multiple particle-entrapped vessels can be prepared and used simultaneously. A 1×10−6 molar sample of PPG was injected into each of twelve capillaries and the results were analyzed by ESI-MS. The sample was injected into the capillaries using a constant infusion method. The experiment details are set forth below.
10.1: Materials and Protocols
Capillary: UV transparent, 360 μm OD, 75 μm ID
Number of capillary: 12 (use packing manifold)
Total length of capillary; 5.5 cm (2 capillaries are 4.5 cm and 4.7 cm long respectively)
Nanosphere: 3 μm ODS bead (100 Å)
Immobilized bead length: 1.1 cm
UV exposure time: 1.5 min
Distance between UV lamp to capillary: 3.5 cm
Power of UV lamp: 254 nm, 0.16 AMPS
Concentration of polypropylene glycol (PPG) solution: 1×10−6 M (solvent: ACN)
Parameters of ESI-MS
70% ACN flow rate: 500 nL min
IS voltage: 3 kV
Scan range: 400-1100 amu
Ion extraction range: 539.5-541 amu
10.2: Packing Manifold
10.3 Results
The results are shown in Table 1. The average intensities of the PPG ions and the standard deviations demonstrate that the use of the polymer entrapped particles can be scaled up in manufacture and use without a substantial loss of reproducibility.
This experiment demonstrated the reproducibility of the results of a 1×10−6 M sample of PPG emitted from two different nano-sprayer capillaries. The experimental details are set forth below.
11.1 Materials and Protocols
Nano-sprayer test
Capillary: UV transparent, 360 μm OD, 75 μm ID
Number of capillary: 2
Total length of capillary: 5.5 cm
Nanosphere: 3 μm ODS bead (100 Å)
Immobilized bead length: 1.1 mm
UV exposure time: 1.5 min
Distance between UV lamp to capillary: 3.5 cm
Power of UV lamp: 254 mm, 0.16 AMPS
Concentration of PPG solution: 1×10 M (solvent: ACN)
Parameters of ESI-MS
70% ACN flow rate: 500 nL/min
IS voltage: 3 kV
Scan range: 400-1100 amu
Ion extraction range: 539.5-541 amu
Back pressure: around 30 psi
11.2 Results
a shows an extracted ion chromatogram (XIC) in the range of 539.5-541 showing the analysis of the PPG (1×10 M) emitted from one emitter.
The data in Table 3 demonstrate a selection of suitable parameters in accordance with the present invention. The conditions used to obtain these data are described in Example 1.
NA - standard deviation exceeded approx. 50%
The protocol called for this experiment was the same as that described in Example 7.
In this study two solvent systems were used to determine how solvent hydrophobicity would affect the polymerization. One method used BDDA (300 μL) in 3.0 mL of casting solvent (1:1:3 ethanol: buffer pH=7: acetonitrile) while the other method used BDDA (300 μL) in 3.0 mL of octanol. These solutions were flushed through the capillaries containing ODS beads and the polymerization was initiated using 254 nanometer light for 90 seconds.
Comparison of Present Invention with Art-Recognized Methods and Compositions
All third party documents referred to herein are hereby incorporated by reference
While specific exemplary embodiments have been discussed herein, other variations, combinations and embodiments will now occur to those of skill in the art and are encompassed by the invention.
Number | Date | Country | Kind |
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2,499,657 | Mar 2005 | CA | national |