(12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT)
`
`(19) World Intellectual Property Organization ¢ e
`International Bureau
`
`(43) International Publication Date
`10 January 2008 (10.01.2008)
`
`
`
`(10) International Publication Number
`WO 2008/006078 A2
`
`Agents: GARRETT-WACKOWSKI, Eugenia et al.;
`Townsend and Townsend and Crew LLP, Two Em-
`barcadero Center, Lighth Floor, San T'rancisco, CA
`94111-3834 (US).
`
`Designated States (unless otherwise indicated, for every
`kind of national protection available): AE, AG, AL, AM,
`AT, AU, AZ, BA, BB, BG,BII, BR, BW,BY, BZ, CA,CII,
`CN, CO, CR, CU, CZ, DE, DK, DM, DO, DZ, EC, EE, EG,
`ES, FI, GB, GD, GE, GH, GM,GT, HN, HR, HU,IP,IL,
`IN,IS, JP, KE, KG, KM, KN, KP, KR, KZ, LA, LC, LK,
`LR, LS, LT, LU, LY, MA, MD, ME, MG, MK, MN, Mw,
`MX, MY, MZ, NA, NG, NI, NO, NZ, OM, PG, PH, PL,
`PT, RO, RS, RU, SC, SD, SE, SG, SK, SL, SM, SV, SY,
`TJ, TM, TN, TR, TT, TZ, UA, UG, US, UZ, VC, VN, ZA,
`ZM, ZW.
`
`(51) International Patent Classification:
`
`Notclassified
`
`(74)
`
`(21) International Application Number:
`PCT/US2007/072977
`
`(22) International Filing Date:
`
`6 July 2007 (06.07.2007)
`
`(81)
`
`(25) Filing Language:
`
`(26) Publication Language:
`
`(30) Priority Data:
`60/819, 148
`11/773,839
`
`English
`
`English
`
`7 July 2006 (07.07.2006)
`5 July 2007 (05.07.2007)
`
`US
`US
`
`except US):
`all designated States
`(for
`(71) Applicant
`BIO-RAD LABORATORIES,
`INC.
`[US/US]; 1000
`Alfred Nobel Drive, Ilercules, California 94547 (US).
`
`(72) Inventors; and
`(75) Inventors/Applicants (for US only): AGROSKIN, Yury
`[US/US]; 1305 South Stelling Road, Cupertino, Califor-
`nia 95014 (US). GRIMES, Michael, T. [US/US]; 1154
`Garfield Ave, San Jose, California 95125 (US).
`
`(84)
`
`Designated States (unless otherwise indicated, for every
`kind of regional protection available): ARIPO (BW, GH,
`GM, KE, LS, MW, MZ, NA, SD, SL, SZ, TZ, UG, ZM,
`ZW), Eurasian (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM),
`European (AT, BE, BG, CH, CY, CZ, DE, DK, EE, ES, FI,
`
`[Continued on next page]
`
`(54) Title: MASS SPECTROMETRY PROBES HAVING HYDROPHOBIC COATINGS
`
`(57) Abstract: This invention provides
`a mass
`spectrometry probe
`including
`a
`substrate having a
`surface
`and a
`hydrophobic coating that coats the surface.
`The hydrophobic coating includes openings
`that define features for the presentation of
`an analyte.
`‘the hydrophobic coating also
`has a lowersurface tension that the features
`on the substrate surface, and has a water
`contact angle between 115° and 140°.
`
`
`
`
`
`
`WO2008/006078A2IIITIITINNNTINNINGTTIATTUKA
`
`

`

`WO 2008/006078 A2
`
`_[ININIANIIINTMIANIMMITT ANAT TUMI
`
`FR, GB, GR, HU,IE, IS, IT, LT, LU, LV, MC, MT, NL, PL,=For two-letter codes and other abbreviations, refer to the "Guid-
`PT, RO, SE, SI, SK, TR), OAPI (BF, BJ, CF, CG, CI, CM,—ance Notes on Codes and Abbreviations" appearing at the begin-
`GA, GN, GQ, GW, ML, MR, NE, SN, TD, TG).
`ning of each regular issue of the PCT Gazette.
`Published:
`
`— without international search report and to be republished
`upon receipt of that report
`
`

`

`WO 2008/006078
`
`PCT/US2007/072977
`
`MASS SPECTROMETRY PROBES HAVING HYDROPHOBIC
`
`COATINGS
`
`CROSS-REFERENCES TO RELATED APPLICATIONS
`
`[0001] This application is related to U.S. Provisional Application No.
`
`60/819,148, filed July 7, 2006, the disclosure of which is hereby incorporated by reference in
`
`its entirety for all purposes.
`
`10
`
`15
`
`STATEMENTAS TO RIGHTS TO INVENTIONS MADE UNDER
`
`FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
`
`[0002] NOT APPLICABLE
`
`REFERENCETO A "SEQUENCELISTING," A TABLE, OR A COMPUTER
`
`PROGRAM LISTING APPENDIX SUBMITTED ON A COMPACTDISK
`
`[0003] NOT APPLICABLE
`
`BACKGROUND OF THE INVENTION
`
`[0004] Modem laser desorption/ionization mass spectrometry (“LDI-MS’’)
`
`can be practiced in two main variations: matrix assisted laser desorption/ionization
`
`20
`
`(“MALDI’) mass spectrometry and surface-enhanced laser desorption/ionization (““SELDI”).
`
`In MALDI, the analyte, which may contain biological molecules, is mixed with a solution
`
`containing a matrix, and a drop ofthe liquid is placed on the surface of a probe. The matrix
`
`solution then co-crystallizes with the biological molecules. The probeis inserted into the
`
`mass spectrometer. Laser energyis directed to the probe surface where it desorbs and ionizes
`
`25
`
`the biological molecules without significantly fragmenting them. However, MALDI has
`
`limitations as an analytical tool. It does not provide meansfor fractionating the sample, and
`
`the matrix material can interfere with detection, especially for low molecular weight analytes.
`
`See, e.g., U.S. Patent No. 5,118,937 (Hillenkampet al.), and U.S. Patent No. 5,045,694
`
`(Beavis & Chait).
`
`30
`
`[0005] In SELDI,the probe surface is modified so that it is an active
`
`participant in the analyte recovery and/or desorption process. In one variant, the surface is
`
`derivatized with affinity reagents that selectively bind the analyte. In another variant, the
`
`

`

`WO 2008/006078
`
`PCT/US2007/072977
`
`surface is derivatized with energy absorbing moleculesthat are not desorbed when struck
`with the laser. In another variant, the surface is derivatized with molecules that bind the
`
`analyte and that contain a photolytic bond that is broken upon application ofthe laser. In
`each of these methods,the derivatizing agent generally is localized to a specific location on
`the probe surface where the sample is applied. See, e.g., U.S. Patent Nos. 5,719,060 and
`6,225,047, all of which issued to Hutchens and Yip, and PCT International Publication No.
`
`WO 98/59361, also to Hutchens and Yip.
`
`[0006] The two methods can be combinedby, for example, using a SELDI
`affinity surface to capture an analyte and adding matrix-containing liquid to the captured
`analyte to provide the energy absorbing material.
`
`[0007] In the practice of mass spectrometry, localizing the sample onthe
`probesurface provides advantages. Localization provides more concentrated sample at the
`point of laser application.
`In the affinity version of SELDI, localization can be important
`becauseit allowsthe affinity reagent to capture moreofthe analyte, thereby providing greater
`sensitivity of detection. However, if the hydrophilic or hydrophobic characteristics of the
`liquid are similar to that of the probe, liquid samples tend to spread out over the surface of the
`probe, thwarting localization. This especially creates problems when the probe is designed to
`hold multiple samples and the samples cannot be sequestered to specific locations.
`[0008] U.S. Patent No. 6,555,813 (Beecheretal.) discloses a mass
`spectrometry probe comprising a substrate having a surface and a hydrophobic film that coats
`the surface of the substrate. The film includes openings that define features for the
`presentation of an analyte. Thefilm is more hydrophobicthan the surface (lower surface
`tension), thereby localizing the sampleto the defined features.
`
`[0009] Even though muchhas been achieved with mass spectrometry probes
`disclosed in U.S. Patent No. 6,555,813, which have hydrophobic coatings, there is still a need
`for additional means for sequestering a liquid sample to a location on a probe surface.
`
`BRIEF SUMMARYOF THE INVENTION
`
`[0010] This invention provides a mass spectrometry probe capable of
`sequestering liquid samples to specific locations,i.e., openings or features, of the probe
`surface. The probes comprise a substrate having a surface,a first coating ofsilicon dioxide
`or titanium dioxide on the probe surface, a second coating ofa fluoroalkylsilane chemically
`coupled to the first coating, wherein the second coating comprisesa plurality of openings at
`which the polyfloroalkylsilane is not coupled or present, and an adsorbent material
`
`20
`
`25
`
`30
`
`

`

`WO 2008/006078
`
`PCT/US2007/072977
`
`physisorbed or chemisorbedto thefirst coating at the plurality of openings. The second
`coating is advantageous more hydrophobic than the openingsorfeatures of the probe (lower
`surface tension) and, thus, the samples used in mass spectrometry, which are typically
`dissolved in aqueoussolutions, are selectively sequestered to the openings or features of the
`probe.
`
`[0011] The fluoroalkylsilane coatings of the present invention provideseveral
`advantages compared with mechanical borders. First, they avoid electrical field perturbations
`that hamper mass resolving power and mass accuracy. Second, they avoidareas ofpossible
`sample pooling and preferential crystallization in regions other than the probed area. Third,
`they avoid the need for maintaining strict mechanical tolerances such as in the case of
`elevated sample ridges or depressed sample wells, which can result in poor molecular weight
`determination accuracy and precision. Fourth, they avoid, unlike elevated margins, an optical
`stop which limits the probed area. Moreover, since an adsorbent material is physisorbed or
`chemisorbed to the first coating at the plurality of openings in a stable and robust way, the
`probesof the present invention can be advantageously used to capture analytes by providing
`adsorbentmaterials that are derivatized in any numberofways to allow non-covalentaffinity
`interaction (adsorption) between the adsorbent material and the analyte ofinterest.
`[0012] As such, in one aspect, the present invention provides a probe
`comprising: (a) a solid substrate; (b) a first coating of silicon dioxide ortitanium dioxide on
`a surface of the substrate; (c) a second coating of a polyfluoroalkylsilane chemically coupled
`to the first coating, wherein the second coating comprises a plurality of openings at which the
`polyfluoroalkylsilane is not coupled; and (d) an adsorbent material physisorbed and/or
`chemisorbed to thefirst coating at the plurality of openings.
`[0013] In one embodiment, the probe takes the shapeofa flat strip or plate.
`another embodiment, the probe further comprises means for engaging a probeinterfaceofa
`laser desorption mass spectrometer. In certain embodiments, the solid substrate comprises a
`conductive material. Examples of suitable conductive materials include, but are not limited
`to, aluminum, iron or gold. In other embodiments, the solid substrate comprises a conductive
`polymeror a polymer doped with a material that renders it conductive.
`In other
`
`In
`
`10
`
`20
`
`25
`
`30
`
`embodiments, the solid substrate comprises a non-conductive material. Examplesofsuitable
`non-conductive materials include, but are not limited to, glass, plastic and ceramic oxide.
`
`[0014] The second coating preferably comprisesa fluoroalkylsilane.
`certain embodiments, the fluoroalkylsilane comprises (heptadecafluoro-1,1,2,2-tetra-
`
`In
`
`

`

`WO 2008/006078
`
`PCT/US2007/072977
`
`hydrodecyl)trichlorosilane (FDTS)or(tridecafluoro-1,1,2,2-tetrahydrooctyl)trichlorosilane
`(FOTS).
`In a preferred embodiment, the fluoroalkylsilane comprises FDTS.
`[0015] The adsorbent material can be any material capable of binding an
`analyte. The adsorbent material is attached to the first coating by physisorption or
`chemisorption. In certain embodiments, the adsorbent material is coupled to the surface
`through polymerization with a vinyl group ofan acrylate or a methacrylate group of an
`alkylsilane coupled to the first coating. Examplesof suitable adsorbent materials include, but
`are notlimited to, a hydrophilic material, a hydrophobic material, an anion exchange
`material, a cation exchange material, a metal chelating material, a dye, an epoxide, a
`carboimidizole, an affinity ligand or a biospecific material. In certain embodiments, the
`adsorbent material comprises a polymeric hydrogel. Suitable hydrogels comprise acrylate,
`methacrylate, polyurethane or polysaccharide polymers.
`
`[0016] In certain embodiments, the plurality of openings take the form of
`spots arranged in a line or a grid having the same dimensionsas a line or grid of spots in a
`96-well or 384-wellplate.
`
`15
`
`[0017] In another aspect, the present invention provides a method of making a
`probe, the method comprising:
`(a) coating a surface of a solid substrate with a first coating of
`silicon dioxide ortitanium dioxide (by molecular vapor deposition); (b) coating thefirst
`coating with a second coating of a polyfluoroalkylsilane; (c) covering the second coating with
`20
`a mask comprising a plurality of openings that exposeaplurality of areas on the second
`coating; (d) exposing the probe to UV ozone to remove the polyfluoroalkylsilane from the
`exposed areas; and (e) attaching by physisorption or chemisorption an adsorbent material at
`each of the exposed areas.
`
`25
`
`30
`
`[0018] In yet another aspect, the present invention provides a probe ofthis
`invention that is removably insertable into a gas phase ion detector(e.g., a mass
`spectrometer).
`
`[0019] In another aspect, the present invention provides a system comprising:
`a gas phase ion detector comprising aninlet port; and a probeofthis invention inserted into
`the inlet port.
`
`[0020] In a further aspect, the present invention provides a method of
`detecting an analyte, the method comprising: a) placing the analyte on a feature of a surface
`of a probeofthis invention; b) inserting the probe into an inlet port of a gas phase ion
`detector comprising: i) an ionization source that desorbsthe analyte from the probe surface
`into a gas phase and ionizes the analyte; andii) an ion detector in communication with the
`
`

`

`WO 2008/006078
`
`PCT/US2007/072977
`
`probe surfacethat detects desorbed ions; c) desorbing and ionizing the analyte with the
`ionization source; and d) detecting the ionized analyte with the ion detector.
`
`BRIEF DESCRIPTION OF THE DRAWINGS
`
`[0021] FIG. 1 shows a sample mass spectrometry probe with of this invention,
`wherein 101 is the substrate, 102 is the lip ofthe substrate 101, 103 is the spot or feature, 104
`is the roughenedsurface, 105 is the glass layer or coating, 106 is the fluoroalkylsilane (e.g.,
`FTDS)layer, 107 is the silane anchor; and 108 is the adsorbent.
`
`[0022] FIG.2 illustrates a flow-diagram of a method used to generate the
`probesof the present invention in accordance with the embodimentillustrated in FIG. 1.
`
`10
`
`DETAILED DESCRIPTION OF THE INVENTION
`
`L
`
`DEFINITIONS
`
`15
`
`20
`
`25
`
`30
`
`[0023] Unless defined otherwise, all technical and scientific terms used herein
`
`have the meaning commonly understood by a person skilled in the art to which this invention
`belongs. The following references provide oneofskill with a general definition of many of
`the terms used in this invention: Singleton et al., Dictionary ofMicrobiology and Molecular
`Biology (24 ed. 1994); The Cambridge Dictionary ofScience and Technology (Walker ed.,
`1988); The Glossary ofGenetics, 5" Ed., R. Riegeret al. (eds.), Springer Verlag (1991); and
`Hale & Marham, The Harper Collins Dictionary ofBiology (1991). As used herein, the
`following terms have the meaningsascribed to them unless specified otherwise.
`[0024] “Attached,”as used herein, encompassesinteractions including
`chemisorption and physisorption.
`
`[0025] “Biomolecule” or “bioorganic molecule”refers to an organic molecule
`typically made by living organisms. This includes, for example, molecules comprising
`nucleotides, aminoacids, sugars, fatty acids, steroids, nucleic acids, polypeptides, peptides,
`peptide fragments, carbohydrates, lipids, and combinationsofthese (e.g., glycoproteins,
`ribonucleoproteins, lipoproteins, or the like). Biomolecules can be sourced from any
`biological material.
`
`[0026] “Coupled,” as used herein, encompasses chemisorption interactions,
`wherein one entity is chemically bound to another entity.
`[0027] “Probe,” as used herein, refers to a device that comprises a substrate
`having a surface adapted for the presentation of an analyte for detection and that includes
`
`

`

`WO 2008/006078
`
`PCT/US2007/072977
`
`means for engaging a probe interface of a mass spectrometer, which positions the probe so
`that the probe surface can beinterrogated by an energy source. Thus, the probe is removably
`insertable into a gas phase ion detector (e.g., a mass spectrometer).
`[0028] “Substrate” refers to a solid material that is capable of supporting an
`
`analyte.
`
`substrate.
`
`[0029] “Surface” refers to the exterior or upper boundaryofa bodyor a
`
`[0030] “Coating”refers to a thin film orlayer ofsilicon dioxide ortitanium
`dioxide or of a fluoroalkylsilane coating on the surface of a substrate.
`
`[0031] “Surface tension,” as used herein, refers to the reversible work
`required to create a unit surface area at constant temperature and pressure and composition.
`Surface tension is measured by: g = (dG/dA)T,P,n where g = the surface tension; G = Gibbs
`free energyof the system; A = surface area; T = temperature; P = pressure; and N =
`composition.
`
`[0032] “Contact angle,” as used herein, refers to the angle between the plane
`of the solid surface and the tangential line to the liquid boundaryoriginating at the point of
`three phase contact (solid/liquid/vapor).
`
`[0033] “Strip” refers to a long narrow piece of a material that is substantially
`
`flat or planar.
`
`[0034] “Plate” refers to a thin piece of material that is substantially flat or
`planar, and that can be in any suitable shape (e.g., rectangular, square, oblong,circular, etc.).
`[0035] “Substantially flat” refers to a substrate having the major surfaces
`essentially parallel and distinctly greater than the minor surfaces(e.g., a strip or a plate).
`[0036] “Gas phase ion spectrometer” refers to an apparatus that detects gas
`phase ions. Gas phase ion spectrometers include an ion sourcethat supplies gas phaseions.
`Gas phase ion spectrometers include, for example, 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.
`[0037] “Mass spectrometer” refers to a gas phase ion spectrometer that
`measures a parameter that can be translated into mass-to-chargeratios of gas phase ions.
`Massspectrometers generally include an ion source and a mass analyzer. Examples of mass
`spectrometers are time-of-flight, magnetic sector, quadrupolefilter, ion trap, ion cyclotron
`resonance, electrostatic sector analyzer and hybrids of these. “Mass spectrometry”refers to
`the use of a mass spectrometer to detect gas phaseions.
`
`15
`
`20
`
`25
`
`30
`
`

`

`WO 2008/006078
`
`PCT/US2007/072977
`
`[0038] “Laser desorption mass spectrometer” refers to a mass spectrometer
`that uses laser energy as a means to desorb,volatilize, and ionize an analyte. Laser
`desorption/ionization in a single TOF instrumenttypically is performed in linear extraction
`mode. Tandem mass spectrometers can employ orthogonal extraction modes.
`
`[0039] “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 phaseions. In a time-of-flight mass spectrometer the mass analyzer comprises an ion
`optic assembly that accclerates ionsinto theflight tube,a flight tube and an ion detector.
`
`[0040] “Ion source”refers to a sub-assembly of a gas phase ion spectrometer
`that provides gas phase ions.
`In one embodiment, the ion source providesions through a
`desorption/ionization process. Such embodiments generally comprise a probeinterface that
`positionally engages a probein an interrogatable relationship to a source of ionizing energy
`(e.g., a laser desorption/ionization source) and in concurrent communication at atmospheric
`or subatmospheric pressure with a detector of a gas phase ion spectrometer.
`
`[0041] Formsof 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
`plasmadesorption); 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-Yaglasers 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 mJ / mm” to about 50 mJ/
`mm’. Typically, a sample is placed on the surface of a probe, the probe is engaged with the
`probeinterface and the probe surface is exposed to the ionizing energy. The energy desorbs
`analyte molecules from the surface into the gas phase and ionizes them.
`
`(1)
`[0042] Other forms of ionizing energy for analytes include, for example:
`electrons that ionize gas phase neutrals; (2) strong electric field to induce 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
`
`15
`
`20
`
`25
`
`30
`
`solid phase, gas phase, and liquid phase neutrals.
`
`[0043] This invention is directed to probes useful for a mass spectrometric
`technique knownas “Surface Enhanced Laser Desorption and Ionization”or “SELDI,”as
`described, for example, in U.S. Patent Nos. 5,719,060 and 6,225,047, both to Hutchensetal.
`This refers to a method of desorption/ionization gas phase ion spectrometry (e.g., mass
`
`

`

`WO 2008/006078
`
`PCT/US2007/072977
`
`spectrometry) in which an analyte (here, one or moreofthe analytes) is captured on the
`surface of a SELDI massspectrometry probe.
`
`[0044] SELDIalso has been called “affinity capture mass spectrometry” or
`“Surface-Enhanced Affinity Capture” (‘SEAC”). This version involves the use of probes
`that have a material on the probe surface that captures analytes through a non-covalent
`affinity interaction (adsorption) between the material and the analyte. The material is
`
`variously called an “adsorbent,” a “capture reagent,” an “affinity reagent” a “binding
`functionality or a “binding moiety.” Such probes can be referred to as “affinity capture
`probes”and as having an “adsorbent surface.” The capture reagent can be any material
`capable of binding an analyte. The capture reagentis attached to the probe surface by
`physisorption or chemisorption.
`In certain embodimentsthe probes havethe capture reagent
`alreadyattachedto the surface.
`In other embodiments, the probes are pre-activated and
`include a reactive moiety that is capable of bindingthe capture reagent,e.g., through a
`reaction forming a covalent or coordinate covalent bond. Epoxide and acyl-imidizole are
`useful reactive moieties to covalently bind polypeptide capture reagents such as antibodies or
`
`cellular receptors. Nitrilotriacetic acid and iminodiacetic acid are useful reactive moieties
`
`that function as chelating agents to bind metalionsthat interact non-covalently with histidine
`containing peptides. Adsorbents are generally classified as chromatographic adsorbents and
`biospecific adsorbents.
`
`[0045] “Binding functionalities,” as used herein, refer to a functional group(s)
`that binds an analyte of interest. Binding functionalities include, but are not limited to, a
`carboxyl group, a sulfonate group, a phosphate group, an ammonium group, a hydrophilic
`group, a hydrophobic group, a reactive group, a metal chelating group,a thioether group, a
`biotin group, a boronate group, a dye group, a cholesterol group,derivatives thereof, or any
`combinations thereof. Binding functionalities can further include other functionalities that
`
`can bind analytes based on individual structural properties, such as the interaction of
`
`antibodies with antigens, enzymes with substrate analogs, nucleic acids with binding proteins,
`and hormoneswith receptors.
`[0046] “Chromatographic adsorbent”refers to an adsorbent material typically
`used in chromatography. Chromatographic adsorbentsinclude, for example, ion exchange
`materials, metal chelators (e.g., nitrilotriacetic acid or iminodiacetic acid), immobilized metal
`chelates, hydrophobic interaction adsorbents, hydrophilic interaction adsorbents, dyes, simple
`biomolecules(e.g., nucleotides, amino acids, simple sugars and fatty acids) and mixed mode
`adsorbents(e.g., hydrophobic attraction/electrostatic repulsion adsorbents).
`
`15
`
`20
`
`25
`
`30
`
`

`

`WO 2008/006078
`
`PCT/US2007/072977
`
`[0047] “Biospecific adsorbent” refers to an adsorbent comprising a
`biomolecule, e.g., a nucleic acid molecule (e.g., an aptamer), a polypeptide, a polysaccharide,
`a lipid, a steroid or a conjugate ofthese (e.g., a glycoprotein, a lipoprotein, a glycolipid, a
`nucleic acid (e.g., DNA)-protein conjugate).
`In certain instances, the biospecific adsorbent
`
`can be a macromolecular structure such as a multiprotein complex, a biological membrane or
`
`a virus. Examples of biospecific adsorbents are antibodies, receptor proteins and nucleic
`
`acids. Biospecific adsorbents typically have higherspecificity for a target analyte than
`
`chromatographic adsorbents. Further examples of adsorbents for use in SELDI can be found
`
`in U.S. Patent No. 6,225,047. A “bioselective adsorbent”refers to an adsorbent that binds to
`an analyte with an affinity ofat least 10° M.
`
`10
`
`[0048] Various chemistries for adsorbents are further described in: U.S.
`
`Patent No. 6,579,719 (Hutchensef al., “Retentate Chromatography,” June 17, 2003); U.S.
`
`Patent 6,897,072 (Rich etal., “Probes for a Gas Phase Ion Spectrometer,” May 24, 2005);
`
`U.S. Patent Publication No. U.S. 2003 0032043 A1 (Pohl et al, “Latex Based Adsorbent
`
`15
`
`Chip,” July 16, 2002); PCT International Publication No. WO 03/040700 (Um et al.,
`
`“Hydrophobic Surface Chip,” May 15, 2003); U.S. Patent Publication No. US 2003/0218130
`
`Al (Boschetti e¢ ai, “Biochips With Surfaces Coated With Polysaccharide-Based
`
`Hydrogels,” April 14, 2003); and U.S. Patent No. 7,045,366, (Huanget al., “Photocrosslinked
`
`20
`
`25
`
`30
`
`Hydrogel Surface Coatings,” May 16, 2006).
`
`[9049] In general, a probe with an adsorbent surface is contacted with the
`
`sample for a period oftime sufficient to allow the analyte or analytes that may be presentin
`the sample to bind to the adsorbent. After an incubation period, the substrate is washed to
`
`remove unbound material. Any suitable washing solutions can be used; preferably, aqueous
`solutions are employed. The extent to which molecules remain bound can be manipulated by
`adjusting the stringency of the wash. The elution characteristics of a wash solution can
`
`depend, for example, on pH,ionic strength, hydrophobicity, degree of chaotropism, detergent
`strength, and temperature. Unless the probe has both SEAC and SENDproperties (as
`
`described herein), an energy absorbing molecule then is applied to the substrate with the
`
`bound analytes.
`
`[0050] In yet another method, one can capture the analytes with a solid-phase
`bound immuno-adsorbentthat has antibodies that bind the analytes. After washing the
`
`adsorbent to remove unbound material, the analytes are eluted from the solid phase, applied
`
`to a SELDIbiochip that binds the analytes and analyzed by SELDI.
`
`

`

`WO 2008/006078
`
`PCT/US2007/072977
`
`[0051] The analytes bound to the substrates are detected in a gas phase ion
`
`spectrometer such as a time-of-flight mass spectrometer. The analytes are ionized by an
`ionization source suchas a laser, the generated ions are collected by an ion optic assembly,
`and then a mass analyzer disperses and analyzes the passing ions. The detector then
`
`translates information of the detected ions into mass-to-charge ratios. Detection of a analyte
`typically will involve detection of signal intensity. Thus, both the quantity and massof the
`
`analyte can be determined.
`
`[0052] Another method oflaser desorption mass spectrometry is called
`
`Surface-Enhanced Neat Desorption (“SEND”). SEND involvesthe use of probes comprising
`
`energy absorbing molecules that are chemically bound to the probe surface (“SEND probe”).
`The phrase “energy absorbing molecules” (EAM) denotes molecules that are capable of
`
`absorbing energy from a laser desorption/ionization source and, thereafter, contribute to
`
`desorption and ionization of analyte molecules in contact therewith. The EAM category
`includes molecules used in MALDI, frequently referred to as “matrix,” and is exemplified by
`cinnamic acid derivatives, sinapinic acid (SPA), cyano-hydroxy-cinnamic acid (CHCA) and
`
`10
`
`15
`
`dihydroxybenzoic acid, ferulic acid, and hydroxyaceto-phenone derivatives. In certain
`
`embodiments, the energy absorbing molecule is incorporated into a linear or cross-linked
`
`polymer,e.g., a polymethacrylate. For example, the composition can be a co-polymerof «-
`
`cyano-4-methacryloyloxycinnamic acid and acrylate. In another embodiment,the
`
`20
`
`composition is a co-polymer of a-cyano-4-methacryloyloxycinnamic acid, acrylate and 3-(tri-
`
`ethoxy)silyl propyl methacrylate. In another embodiment, the composition is a co-polymer of
`
`a-cyano-4-methacryloyloxycinnamic acid and octadecylmethacrylate (“C18 SEND”). SEND
`
`is further described in U.S. Patent No. 6,124,137 and PCT International Publication No. WO
`
`03/64594 (Kitagawa, “Monomers And Polymers Having Energy Absorbing Moieties of Use
`
`25
`
`In Desorption/Ionization Of Analytes,” August 7, 2003).
`
`[0053] SEAC/SENDis a version of SELDI in which both a capture reagent
`
`and an energy absorbing molecule are attached to the sample presenting surface.
`
`SEAC/SENDprobestherefore allow the capture of analytes through affinity capture and
`ionization/desorption without the need to apply external matrix. The C18 SENDbiochipis a
`version of SEAC/SEND, comprising a C18 moiety which functionsas a capture reagent, and
`
`30
`
`a CHCA moiety which functions as an energy absorbing moiety.
`
`[0054] Another version of LDI is called Surface-Enhanced Photolabile
`
`Attachment and Release (“SEPAR”). SEPARinvolvesthe use ofprobes having moieties
`
`10
`
`

`

`WO 2008/006078
`
`PCT/US2007/072977
`
`attached to the surface that can covalently bind an analyte, and then release the analyte
`through breaking a photolabile bond in the moiety after exposureto light, e.g., to laser light
`(see, U.S. Patent No. 5,719,060). SEPAR and other forms of SELDIare readily adapted to
`detecting a analyte or analyte profile, pursuant to the present invention.
`
`[0055] “Analyte” refers to any componentof a sample that to be detected
`and/or separated from a contaminant. The term can refer to a single componentora plurality
`of components in the sample. Analytes include, for example, biomolecules.
`
`[0056] “Eluant” or “wash solution”refers to an agent, typically a solution,
`whichis used to affect or modify adsorption ofan analyte to an adsorbent surface and/or
`
`remove unbound materials from the surface. The elution characteristics of an eluant can
`
`depend, for example, on pH,ionic strength, hydrophobicity, degree of chaotropism, detergent
`strength and temperature.
`
`[0057] As used herein, “contaminant,”refers to species removed from a
`sample or assay mixture. The contaminant can be an extraneousspecies not ofinterest in the
`assay, or it can be materialof interest that is present in excess of the amount needed to
`perform the assay. When the excess “contaminating” analyte negatively affects the dynamic
`range of detection in the assay, its removal provides a method of enhancing properties of the
`assay including, but not limited to, its sensitivity.
`
`[0058] The terms, “assay mixture” and “sample,”are used interchangeable to
`refer to a mixture that includes the analyte and other components. The other components are,
`for example, diluents, buffers, detergents, and contaminating species, debris and the like that
`are found mixed with the target. Illustrative examples include urine, sera, blood plasma,total
`blood, saliva, tear fluid, cerebrospinal fluid, secretory fluids from nipples and the like. Also
`includedaresolid, gel or sol substances such as mucus, bodytissues, cells and the like
`suspendedordissolved in liquid materials such as buffers, extractants, solvents and the like.
`[0059] “Adsorb”refers to the detectable binding between binding
`functionalities and an analyte either before or after washing with an eluant(selectivity
`threshold modifier).
`
`[0060] “Resolve,” “resolution,” or “resolution of analyte” refers to the
`detection ofat least one analyte in a sample. Resolution includes the detection ofa plurality
`of analytes in a sample by separation and subsequentdifferential detection. Resolution does
`not require the complete separation of an analyte from all other analytes in a mixture. Rather,
`any separation that allows the distinction between at least two analytes suffices.
`
`15
`
`20
`
`25
`
`30
`
`11
`
`

`

`WO 2008/006078
`
`PCT/US2007/072977
`
`[0061] “Detect” refers to identifying the presence, absence or amountof the
`
`object to be detected.
`
`[0062] “Energy absorbing molecule” or “EAM”refers to a molecule that
`
`absorbs energy from an energy source in a mass spectrometer thereby enabling desorption of
`analyte from a probe surface. Energy absorbing molecules used in MALDIare frequently
`referred to as “matrix.” Cinnamic acid derivatives, sinapi