Patent Application
20080023630
“Gas phase ion spectrometer” refers to an apparatus that detects gas phase ions. Gas phase ion spectrometers include an ion source that supplies gas phase ions. As a result, gas phase ion spectrometers include, e.g., mass spectrometers, ion mobility spectrometers, and total ion current measuring devices. “Gas phase ion spectrometry” refers to the use of a gas phase ion spectrometer to detect gas phase ions.
“Mass spectrometer” refers to a gas phase ion spectrometer that measures a parameter that can be translated into mass-to-charge ratios of gas phase ions. Examples of mass spectrometers include: (a) constant-energy gas phase ion laser desorption/ionization TOF mass spectrometers; (b) magnetic sector mass spectrometers; (c) quadrupole filter mass spectrometers; (d) ion trap mass spectrometers; (e) ion cyclotron resonance mass spectrometers; (f) electrostatic sector analyzer mass spectrometers; and (g) hybrids of these. “Mass spectrometry” refers to the process of using a mass spectrometer to detect gas phase ions. Mass spectrometers generally include an ion source, a mass analyzer and a detector.
“Ion source” refers to a sub-assembly of a gas phase ion spectrometer (or mass spectrometer) that provides gas phase ions to the mass analyzer. In one embodiment, the ion source provides ions through a desorption/ionization process. Such embodiments generally comprise a probe interface that positionally engages a probe and aligns the probe with: (a) a source of ionizing energy (e.g., a laser desorption/ionization source) at atmospheric or subatmospheric pressure; and (b) a detector of gas phase ions. Forms of ionizing energy for desorbing/ionizing an analyte from a solid phase include, for example: (1) laser energy; (2) fast atoms (used in fast atom bombardment); (3) high energy particles generated via beta decay of radionucleides (used in plasma desorption); and (4) primary ions generating secondary ions (used in secondary ion mass spectrometry).
The preferred form of ionizing energy for solid phase analytes is a laser (used in laser desorption/ionization), in particular, nitrogen lasers, Nd-Yag lasers and other pulsed laser sources. “Fluence” refers to the energy delivered per unit area of interrogated image. A high fluence source, such as a laser, will deliver about 1 to about 50 mJ per mm2. Typically, a sample is placed on the surface of a probe, the probe is engaged with the probe interface and the probe surface is struck with the ionizing energy from the laser. The energy desorbs analyte molecules from the surface into the gas phase and ionizes them. “Laser desorption mass spectrometer” refers to a mass spectrometer that uses laser energy as a means to desorb, volatilize, and ionize an analyte.
Examples of other forms of ionizing energy for analytes include: (1) electrons that ionize gas phase neutrals; (2) a strong electric field that induces ionization from gas phase, solid phase, or liquid phase neutrals; and (3) a source that applies a combination of ionization particles or electric fields with neutral chemicals to induce chemical ionization of solid phase, gas phase, and liquid phase neutrals.
Mass spectrometers frequently include ion optics to direct the flight of ions. In a parallel extraction TOF instrument, ion optics are included as part of the ion source. More particularly, as shown in
“Mass analyzer” refers to a sub-assembly of a mass spectrometer that comprises means for measuring a parameter that can be translated into mass-to-charge ratios of gas phase ions. In a TOF mass spectrometer the mass analyzer comprises a free flight path. For example, in a linear TOF instrument, the mass analyzer comprises a flight tube. In instruments comprising electric sectors (e.g., U.S. Pat. No. 6,867,414), the mass analyzer includes electric sectors and field free regions.
The ion detector, in a linear TOF instrument is located at the end of the flight tube. The ion detector can include an electron multiplier or a microchannel plate.
As later described in detail, this invention provides conductive mass spectrometer probes that are formed of a polymer doped with a conductive material. “Probe” refers to a device adapted to engage a probe interface of a gas phase ion spectrometer (hereinafter “mass spectrometer”) and to present an analyte sample to ionizing energy for ionization by introduction into a mass spectrometer. Moreover, the probes may be used in mass spectrometry analysis for purposes of research, diagnosis, prediction of purification processes, protein identification, assay, etc. Further, in certain embodiments the probes may be used in a combination of non-mass spectrometry device (e.g., light microscopy, fluorescence, chemiluminescence, etc.) and a mass spectrometry device both of which would be used to analyze a sample on the sample presenting surface. Moreover, with respect to the mass spectrometry device, the probes may be used as, or as part of, a repeller plate in parallel laser ion desorption/ionization TOF mass spectrometers.
As hereafter explained, the invention not only relates to a probe that is mechanically structured for engagement with a corresponding apparatus in a mass spectrometer, but also to the type of materials from which the probe may be manufactured, the type of materials that may be applied to the probe to facilitate maintaining a sample thereon, the method by which the probe is manufactured, and a method of mass spectrometry using such a probe.
Typically, a probe will comprise a solid substrate that includes a sample presenting surface on which an analyte is presented to the source of ionizing energy. Particular target properties for the probe according to the present invention include: (a) optional optical transparency of at least portions of the probe; (b) electrical conductivity; (c) low outgassing; (d) presence of abstractable hydrogen atoms on the surface of the probe's polymeric substrate for reacting with photoreactive chemistries such as benzophenone; (e) good sample retention characteristics for direct application of a sample onto the probe or onto a binding group layered on the probe; (f) hydrophobicity; (g) moldability; (h) mechanical stability; and (i) chemical stability in the presence of, and compatibility with, solvents used for sample application.
As a result of the conductive doping material in the polymer, the probe maintains the consistency of the electromagnetic field applied to the sample in a mass spectrometer. The probe also displays low outgassing and high chemical stability, thereby enabling it to be used repetitively. A hydrophobic surface of the probe can be configured to receive a hydrophilic hydrogel. Microstructures, which may be in the form of moats formed in the sample presenting surface, and/or the surface's hydrophobicity can be used to maintain a sample on the sample presenting surface or on a hydrogel on the sample presenting surface. The microstructures, which may be on the order of about 200 μm in width and about 100 μm in depth, may be in the form of channels and/or indentation in the sample presenting surface.
In an embodiment of the present invention, the probe may be designed to serve as a chip onto which samples comprising the analyte (e.g., biomolecules such as proteins, peptides, nucleic acids, lipids, complex carbohydrates, etc.) are placed. Moreover, in some embodiments, the probe may become part of the repeller plate in a gas phase ion mass spectrometer. In other embodiments, the probe may interface with a carrier plate that, in turn, interfaces with the repeller plate of a gas phase ion mass spectrometer. To facilitate its being positioned in a sample chamber of a mass spectrometer, the probe includes an engagement mechanism that is configured to engage a complementary structure of a probe interface of either the repeller plate or the carrier plate. The term “positioned” is generally understood to mean that the probe can be moved into a position within the sample chamber in which it resides in appropriate alignment with the energy source for the duration of a particular desorption/ionization cycle.
A first embodiment of a mass spectrometer probe according to the present invention will hereafter be described with reference to
As shown in
The perimeters of the sample presenting surface 108 and the underside 110 are connected by two sidewalls 112 and two endwalls 114. Whereas the endwalls 114 are generally planar in shape, the sidewalls 112 are substantially v-shaped, thereby defining channels 102. The channels 102 are sized and configured to receive rails 2102 formed on either a repeller plate 2010 or a carrier plate 200. The sidewalls 112 also include notch projections 109 that are sized to be received in channels 2109 formed on either a repeller plate 2010 or a carrier plate 200. As a result of the engagement between the v-shaped channels 102 and the notch projections 109 of the probe 100 and the rails 2102 and the channels 2109, respectively, of the repeller plate 2010 (or the carrier plate 200), the probe 100 can be immobilized in the repeller plate 2010 (or the carrier plate 200), as hereafter discussed with respect to
In a manner similar to the probe 100, the repeller plate 2010 includes a topside 2108, an underside 2110, two sidewalls 2112, and two endwalls 2114. As shown, a probe receiving section 2020 is formed in the topside 2108 of the repeller plate 2010. The probe receiving section 2020 includes two rails 2102 and two channels 2109, which jointly serve as a probe interface. The rails 2102 and channels 2109 are sized such that the probe 100 may slide into the repeller plate 2010 in a direction parallel to a longitudinal axis LA of the probe 100 (shown in
As shown in
A second embodiment of a mass spectrometer probe according to the present invention will hereafter be described with reference to
As shown in
In a manner similar to the probe 300, the repeller plate 4010 includes a topside 4108 and an underside 4110. As shown, a probe receiving section 4020 is formed in the topside 4108 of the repeller plate 4010. The probe receiving section 4020 includes a curved, oval-shaped wall 4102 that defines two overhangs 4109, which jointly serve as a probe interface. The curved wall 4102 and the overhangs 4109 are sized such that the probe 300 may slide into the repeller plate 4010 in a direction parallel to a longitudinal axis LA′ of the probe 300 (shown in
As shown in
A third embodiment of a mass spectrometer probe according to the present invention will hereafter be described with reference to
As shown in
The circular sidewall 512 is sized such that the probe 500 can slide into a probe receiving section 620, 6020 of a carrier plate 600 or a repeller plate 6010, while maintaining an engagement between the probe 500 and the carrier plate 600 or the repeller plate 6010, as hereafter described with respect to
In a manner similar to the probe 500, the repeller plate 6010 includes a topside 6108 and an underside 6110. As shown, a probe receiving section 6020 is formed in the topside 6108 of the repeller plate 6010. The probe receiving section 6020 includes a circular sidewall 6102 that is configured to circumscribe the circular sidewall 512 of the probe 500 in a concentric fashion. The circular wall 6102 is sized such that the probe 500 may slide into the repeller plate 6010 in a direction parallel to a longitudinal axis LA″ of the probe 500 (shown in
As shown in
In the foregoing embodiments, as a result of the top, sample presenting surface of the probe being substantially coplanar with the topside of the repeller plate (and the topside of the carrier plate if one is provided), the consistency of the electromagnetic field around the combination of the conductive probe and the repeller plate is substantially maintained.
In addition, it should be readily recognized that some technicians may opt to use the repeller plate 7010 shown in
To enable the mass spectrometer probe to be mass-produced at low cost, plastic was considered, especially as most plastics are hydrophobic. However, it was also recognized that plastics typically experience outgassing in mass spectrometers and are typically nonconductive, both of which can negatively affect mass spectrometry in constant energy ion laser desorption/ionization TOF mass spectrometers. As a result, the present invention aimed to create a probe, which is conductive and which optionally includes transparent portions, out of a polymer that exhibits low outgassing, that affords a high amount of abstractable hydrogen atoms, that exhibits both chemical and mechanical stability, and that can be easily molded into a probe of the types previously described in
The create such a probe, an appropriate amount (1-50% in weight, e.g., 30% in weight) of conductive solute material is added to a nonconductive solvent polymer (e.g., polymethylpentene, polybutyltherephtalate, etc.) that in the absence of such a solute may otherwise be transparent. Although the conductive material may be either non-metallic (e.g., graphite, metal oxides such as antimony-tin oxide, indium-tin oxide, etc.) or metallic (e.g., antimony, aluminum, etc.), non-metallic materials may be preferable. The mixture is then molded (e.g., injection molding, over-molding, etc.) into a mass spectrometer probe, possibly with a microstructure imprint of an array and/or a moat sized for sample containment. The molded probe is then cooled and removed from the molding apparatus. By doping the polymer with the conductive material, the resultant probe is conductive. The conductive nature of the doped-polymer probe is not to be confused with conductive polymers (e.g., polypyrrole). Moreover, in certain embodiments, the probe may be substantially free (e.g., comprising less than about 1% of a metal) or completely free of metal.
Table 1 compares various physical properties of six nonconductive plastic polymers that were identified as potential polymer solvents for use in creating a mass spectrometer probe.
With respect to Table 1, a polymer is generally determined to have “low” outgassing if its pump down time to 5.5 mV in a Ciphergen PCS 4000 mass spectrometer is less than about 150 seconds and “not low” outgassing if its pump down time is greater than 150 seconds. Similarly, a polymer is generally understood to have “low” hydrophobicity when the contact angle of deionized water is less than 60°, “average” hydrophobicity when the contact angle of deionized water is between 60° and 85°, and “high” hydrophobicity when the contact angle of deionized water is greater than 85° (typically up to about 120°).
As can be seen in Table 1, both polymethylpentene and polybutyltherephtalate exhibit: (a) easy moldability; (b) low outgassing; (c) at least an average hydrophobicity; (d) a semicrystalline morphology; (e) ability to bond with benzophenone (i.e., a chemistry used to cross-link polymers and couple them to the surface of a mass spectrometer probe); and (f) at least a moderate amount of abstractable hydrogen atoms. As a result of these positive attributes, polymethylpentene and polybutyltherephtalate were further tested for chemical and mechanical stability when exposed to various solvents. A polymer is considered to have “chemical stability” when it does not deteriorate when exposed to typical solvents such as organic solvents (e.g., methanol, ethanol, hexane, chloroform, etc.). Similarly, a polymer is considered to have “mechanical stability” when it does not scar, break-apart, or become damaged during insertion into, and removal from, a mass spectrometer. Both polymethylpentene and polybutyltherephtalate were determined to have mechanical stability; the results of their chemical stability are shown in Table 2.
As a result of the similar chemical and mechanical stability of both polymethylpentene and polybutyltherephtalate when exposed to the solvents in Table 2, probes were fashioned from polymethylpentene doped with carbon black (i.e., graphite) and polybutyltherephtalate doped with carbon black. Each of these carbon black doped probes was then compared against similar non-doped probes for the properties shown in Table 3.
The hydrophobicity of non-carbon black doped polymethylpentene is such that the surface of a probe formed of polymethylpentene is capable of containing 1-2 μl of an energy absorbing molecule (“EAM”), even without a moat. Further, regardless of the polymer and regardless of carbon black doping, it is clear that a moat enhances the ability of the probe to contain a sample.
In light of the conductivity of both polymethylpentene and polybutyltherephtalate when doped with carbon black, mass spectrometry of a peptide sample was performed using the probes formed of: (a) polybutyltherephtalate both with and without carbon black doping; and (b) polymethylpentene both with and without carbon black doping. The result of the mass spectrometry of both of the polybutyltherephtalate probes are shown in
In
Similar results were obtained using the polymethylpentene probes (with and without carbon black doping). Accordingly, a duplicative discussion of the mass spectrometry results will be omitted.
To illuminate the accuracy of probes formed of a nonconductive polymer doped with conductive material, two pH comparisons were performed between a carbon black doped polybutyltherephtalate probe and a standard Q10 ProteinChip® array probe. Specifically,
In certain embodiments of this invention the surface of a laser desorption/ionization probe is enhanced to selectively bind analytes from a sample. Such probes are referred to as SELDI (Surface-Enhanced Laser Desorption/Ionization) probes. Probe surfaces are enhanced by attaching analyte binding moieties to them. Analyte binding moieties include both chromatographic and biospecific adsorbent materials. Chromatographic materials include, for example, hydrophobic moieties, hydrophilic moieties, anion exchange materials, cation exchange materials, immobilized metal chelates and dyes. Biospecific adsorbent materials include, for example, antibodies and binding portions thereof, receptors and nucleic acids (DNA and RNA). Such analyte binding moieties will selectively bind analytes from a sample to which they are attracted. Unbound materials can be washed away from the surface. This allows for on-chip fractionation of a sample. The analyte binding moieties can be attached to the surface of the probe by chemisorption or physisorption. In preferred embodiments, the analyte binding moieties are provided in the form of a hydrogel. Hydrogels are preferred because their volume allows them to bind increased amounts of an analyte from a sample. In one embodiment, the hydrogel is formed using a photoreactive chemistry, such as benzophenone, to cross-link linear polymers with each other and, optionally, to the surface of the probe.
Benzophenone is attractive for this use because it can couple to abstractable hydrogen atoms on the surface of the probe and because it is compatible with most plastics when creating an activated surface. Examples of both blended polymers and copolymers comprising analyte binding moieties and formed through the use of benzophenone chemistry are described in U.S. Patent Application Publication Nos. 2004-0124149 and 2005-0059086. Because of their versatility, blended polymers are particularly attractive. Briefly, in the case of blended polymers a first polymer, such as dextran, is derivatized with benzophenone groups. A second polymer, also, for example, dextran, is derivatized with analyte binding moiety group (e.g., hydrophilic groups, hydrophobic groups, metal chelates (e.g., IMAC), anion exchange groups, cation exchange groups, dyes or chemically reactive groups). The two polymers are mixed, placed, or coated (optionally in the form of a predetermined pattern) on the surface of the probe and exposed to light. This causes the photoreactive benzophenone groups to react with abstractable hydrogens in both polymers and the polymeric material in the probe. This results in a cross-linked hydrophilic polymer attached to the surface of the probe. A layered polymer also may be applied to the probe. An example of such a blended copolymer hydrogel, which is shown in
Other polymers that may be used to create an activated surface include ultraviolet (“UV”) sensitive benzophenone-Q polymer (quarternary ammonium), benzophenone-DEAE dextran, benzophenone-CM polymer (carboxymethylate), benzophenone-immobilized metal interaction chromatography (“IMAC”) polymer, benzophenone-H50 polymer, polysaccharides (e.g., dextran), and synthetic polymers (e.g., acrylic soluble copolymers). Moreover, certain of these polymers may be used based on certain desired properties (e.g., benzophenone-dextran or DEAE-dextran could be used to obtain an anion exchange surface).
As shown in
Various advantages are afforded by the conductive probes of the present invention. For example, the mass spectrometer probes may be at least partially transparent, thereby enabling a technician to visualize a sample on the probe. More specifically, the probes may have one or more transparent locations on the sample presenting surface onto which the analyte is positioned for analysis. To create the transparent locations, after the probe is molded (in Step S1), small holes may be drilled through the probe. Subsequently, the holes may be filled with a transparent polymer (which may be the same nonconductive polymer that was used to mold the probe) that is not doped with conductive material. The probe may then be re-molded. Although, the overall conductivity of the probe may be slightly degraded, the degradation should be to such a limited degree that it will have a negligible affect on the resultant mass spectrometry. As a result, the probe will have transparent channels therethrough, thereby enabling a technician to view (e.g., by light microscopy, fluorescence, chemiluminence, etc.) samples positioned on locations on the sample presenting surface that are aligned with the channels.
In other embodiments, the holes may be formed during probe formation. For example, before the probe is molded in Step S1, micropipettes (or other similar structure) could be positioned in the mold in locations that are to be transparent. Accordingly, after the polymer is added, the polymer will fill all portions of the mold except for those portions that are already occupied by the micropipettes. As a result, after the polymer is molded, the micropipettes could be removed, thereby yielding holes through the probe.
It should be readily recognized that the same drilling and polymer filling steps may used to add transparent locations to conventional metal (e.g., aluminum) probes. As a result of such transparent locations in conventional probes, the conventional probes could be used in other analysis protocols such as light microscopy, fluorescence, chemiluminence, etc.
By way of another example, the conductivity of the probes maintains the consistency of the electromagnetic field generated in the mass spectrometer, thereby ensuring the accuracy of the mass spectrometry.
By way of another example, the probes exhibit low outgassing, thereby preventing impurities from negatively affecting the mass spectrometry.
By way of still another example, the probes may be mass-produced at low cost. Moreover, the probes may be readily molded into any desired shape, i.e., the probes may be molded into shapes other than rectangular, tubular, and disk-shaped.
By way of yet another example, samples may be positioned directly on the probe, which, in turn, can be automatically received by the probe interface of the mass spectrometer, thereby significantly reducing production costs and enhancing reproducibility.
Although the aforementioned describes embodiments of the invention, the invention is not so restricted. It will be apparent to those skilled in the art that various modifications and variations can be made to the disclosed embodiments of the present invention without departing from the scope or spirit of the invention.
For instance, the polymer probe may be formed of nonconductive polymer that is coated with conductive suspension (e.g., graphite suspension).
By way of another example, the probe may be formed using an over-molding technique by which zones of conductivity and zones of transparency may be established. More specifically, the probe may be formed of two or more polymers, e.g., a first polymer layer could have conductive mass spectrometer properties and a second layer could be better configured to bind samples (e.g., protein) on a surface thereof by way of a specific function. The surface of the second layer could be functionalized, e.g., for porosity, transparency, ion exchange, hydrophobicity interaction, mix-mode interaction, IMAC, affinity for a particular target sample (e.g., a protein or family of proteins), reactivity for the rapid immobilization of a particular target sample, specific affinity biologicals (e.g., antibodies, lectins, receptors, and nucleic acids), small affinity ligands (e.g., dyes, peptides, oligonucleotides, and sugars), etc. In this embodiment, the first layer would be molded (e.g., injection molding, over-molding, etc.) and then the second layer would be molded onto the first layer (e.g., injection molding, over-molding, etc.). The subsequent steps for creating the probe may be substantially the same as steps S2-S5.
Accordingly, these other conductive mass spectrometer probes, mass spectrometers using such probes, methods of probe fabrication, and methods of sample analysis using such mass spectrometer probes are fully within the scope of the claimed invention. Therefore, it should be understood that the devices and methods described herein are illustrative only and are not limiting upon the scope of the invention, which is indicated by the following claims.
| Number | Date | Country | |
|---|---|---|---|
| 60738575 | Nov 2005 | US |
