CA2582338A1 - Host-guest enery-absorbing complex - Google Patents

Abstract

The present invention provides an energy-absorbing EAM that is a host-guest complex formed between a molecular host and a guest that is an energy-absorbing molecule (EAM). Exemplary of EAMs of use in the invention are species that absorb energy from a photo-irradiation source and thereby contributes to desorption and ionization of analyte molecules in operative contact therewith. When the complex and analyte are components of a surface, the transfer of energy promotes both the ionization of the analyte and its desorption from the surface. Also provided are methods for using the complexes of the invention and methods of preparing surfaces and devices incorporating a complex or the invention.

Description

HOST-GUEST ENERGY-ABSORBING COMPLEX
BACKGROUND OF THE INVENTION

[0001] Laser desorption/ionization time-of-flight mass spectrometry is a popular method for analyzing biological molecules such as polypeptides and nucleic acids. In this method, the sample to be analyzed is deposited on a solid substrate, referred to as a probe or a target.
Pulses of laser energy are directed against a substrate-bound sample. The laser energy causes the sample molecules to desorb from the probe surface and become ionized. The ion optics of a time-of-flight instrument accelerate the ionized molecules into a free flight tube, where they separate according to mass, and are detected with particle detector.

[0002] Analytes having masses above about 2500 Daltons tend to fragment when struck directly by a laser. This problem needed to be overcome in order to use the technique to analyte large biomolecules. Research was directed to softer ionization methods that would volatilize, but not fragment, large biomolecules. In the 1980's a method was developed referred to as MALDI (matrix assisted laser desorption/ionization) that enabled the detection of intact analytes at mass-to-charge ratios of up to several hundred thousand Daltons.
MALDI was described early on by Karas et al. (Int. J. Mass Spectrom Ion Processes 78 (1987), 53; Anal. Chem 60 (1988) 2299) and Tanaka et al. (Rapid Commun. Mass Spectrom.
2 (1988) 151-153) and Beavis et al. (Rapid Commun Mass Spectrom 4 (1989) 233 and 432-440). In MALDI, the sample is associated on the probe surface with molecules that absorb the laser light and promote desorption/ionization of substantially intact large biomolecules.
Typical laser energy absorbing molecules include sinapinic acid, alpha-cyano-hydroxycinammic acid, 2,5 dihydrobenzoic acid and ferulic acid. In traditional MALDI the sample and the laser energy absorbing molecules are intimately mixed and placed on the probe surface to dry, so that the two co-crystallize. In such a case, the energy absorbing molecules are referred to as "matrix."

[0003] There are a number of problems and limitations with a matrix formed as described above. For example, the energy absorbing molecules tends to be a hydrophobic species, yet many analytes of interest are water-soluble molecules, e.g., peptides, nucleic acids, saccharides. The difference in polarities of the energy absorbing molecule, the analyte and the solvents used for both of these elements, can result in a matrix in which the energy absorbing moleculedistributes into "islands" of irregularly sized crystals.
Moreover, because the matrix is merely "laid down" on the chip, it is challenging to wash away contaminants in the analyte or matrix without perturbing or washing away the matrix. Other problems include formation of analyte-salt ion adducts, less than optimum solubility of analyte in matrix, unknown location and concentration of analyte molecules within the solid matrix, signal (molecular ion) suppression, "poisoning" due to simultaneous presence of multiple components, and selective analyte desorption/ionization.

[0004] In 1993 Hutchens and Yip described an LDI method in which the analyte molecules were not co-crystallized with the energy absorbing molecules. (WO
94/02814.) They referred to the method as SEND. In certain versions of SEND, the energy absorbing molecules, referred to there as "EAMs," were covalently coupled to the probe surface, and the analyte molecules were deposited upon them.

[0005] Although it is a tangible step forward, direct derivatization of the chip substrate with small molecular EAM has some limits in the choice of the constituents of the matrix.
For example, attaching the EAM to the substrate requires the use of EAM and substrate materials having complementary reactive groups, thereby limiting the species that can be used for both the chip and substrate. Moreover, incomplete reaction between EAM and the chip substrate can interfere with the assay for which the chip is intended.
For example, unreacted EAM may adventitiously remain on the chip, or reactive groups on the surface of the chip may remain unfunctionalized with an EAM. Unreacted EAM may itself be ionized during the mass spectrometric analysis, resulting in a high level of background or obscuring data from the analyte. Unfunctionalized groups on the chip may act as affinity moieties, binding the analyte and hindering its desorption from the chip.

[0006] SEND was further refmed by Kitagawa who proposed the incorporation of energy absorbing molecules into polymers that were, themselves, deposited on the probe surface.
See, for example, U.S. Patent Publication 03/0207460, "Monomers And Polymers Having Energy Absorbing Moieties Of Use In Desorption/Ionization Of Analytes." One advantage of the Kitagawa method was the decrease in signal from desorbed energy absorbing molecules, which creates noise in the low mass range in MALDI.

[0007] One problem with EAMs as presently used in the fact that they tend to be hydrophobic molecules and, therefore, cannot be mixed in aqueous solution. A
solution to this problem would be useful in the art.

BRIEF SUMMARY OF THE INVENTION

[0008] A surface combining the advantages of a surface-associtated matrix with the flexibility to alter both the EAM identity and amount according to the parameters of a particular analysis would represent a significant advance in the art. An exemplary surface would incorporate a moiety that interacts non-covalently with an EAM. Such a matrix could be based upon an EAM, including those currently recognized, that does not require derivatization to allow its incorporation into the surface. Accordingly, a surface that provides a receptacle for an EAM would notably simplify the manufacture of EAM-containing surfaces and analytical devices employing these surfaces. Moreover, a matrix that did not require a dry down period and, thus, was of use in high-throughput analyses would greatly speed data acquisition from laser desorption mass spectrometric analyses.

[0009] It has now been discovered that a host-guest complex formed between a molecular host and an EAM transfers energy to an analyte; desorbing the analyte from a surface that includes the complex and ionizing the analyte. The discovery provides access to surfaces and devices incorporating these surfaces that mitigate many of the shortcomings of prior surfaces and devices for use in laser desorption/ionization mass spectrometry.
Moreover, the surfaces and devices of the invention provide analytical formats that were not previously available.

[0010] In contrast to surfaces for laser desorption/ionization mass spectrometry on which a large molar excess of an EAM is randomly distributed in an uncontrolled and unreproducible manner, the present invention provides a surface that includes a matrix that is both readily reproducible and tunable. The matrix is tuned by varying the structure and amount of EAM complexed with the molecular host, providing unparalleled flexibility in the design of a matrix for a selected application.

[0011] In addition to matrix tunability and reproducibility, the complexes, surfaces and devices of the invention provide numerous other advantages. For example, surfaces that include host-guest EAM complexes provide improved washability. EAM is trapped inside the cavity of a host, so the surface can be subjected to intensive washing with buffer solution to remove salts and impurities without losing EAM from the surface.
Furthermore, the physiochemical properties of EAM matrices, such as solubility and stability, can be altered through their formation of inclusion complexes with host. Also, surfaces that include host-guest EAM complexes are well suited for analyses that require the use of water in the presence of the hydrophobic EAM; as the distribution of the EAM is a function of the molecular host, the presence of water on the surface does not adversely affect EAM
distribution. Furthermore, the use of host-guest complexes promotes the controlled distribution of the EAM on the surface, remedying the uneven distribution of EAM that is a common problem with application of an excess of EAM in a solvent that is essentially incompatible with the analyte. The invention also provides access to volatile matrices that used to be disqualified as EAM. For SELDI and MALDI applications, a matrix needs to be stable under vacuum for extended periods of time without being sublimated away. The volatility of the complexed EAM matrices can, however, be reduced to a very low level by complexing it with a host structure.

[0012] High throughput assays are of particular value and relevance in a number of fields, e.g., proteomics, and drug discovery. Tools for use in high throughput analyses are preferably characterized by uniformity and reproducibility in their structure and function. The complexes, surfaces and devices of the present invention are of particular use in high throughput screening. By providing a surface that eliminates the need to mix the sample with the matrix and dry the matrix on the surface, the present invention provides a surface, and devices incorporating that surface, that are quickly and reproducibly prepared.

[0013] The invention also provides surfaces and devices incorporating these surfaces in which the host molecule is not complexed with the EAM. Thus, the uncomplexed host can be contacted with a selected EAM, thereby forming the complex.

[0014] Thus, in a first aspect, the invention provides a host-guest complex that includes a molecular host complexed with an energy absorbing molecule that absorbs energy from a photo-irradiation source and thereby contributes to desorption and ionization of analyte molecules in operative contact therewith. In exemplary einbodiments, the complex is immobilized on a substrate that is a component of a probe for a mass spectrometer, e.g., a substrate that includes a means for engaging a probe interface of a mass spectrometer.

[0015] In exemplary embodiments, the EAM-host complex is essentially water-soluble, or at least more water-soluble than the EAM alone. Thus, the invention provides the first essentially water-soluble matrix for use in laser desorption/ionization mass spectrometry. In practice, an aqueous solution of the water-soluble matrix can be laid down on the analyte in a manner analogous to that presently used with EAM solutions in organic solvent.

[0016] In some embodiments, the host-guest complex is attached to a substrate, forming a surface on the substrate. The complex can be attached to the substrate at a plurality of addressable locations.

[0017] An exemplary surface of the invention is an adsorbent surface, also referred to herein as an "adsorbent film." An adsorbent film of the invention is generally a polymer formed between a polymerizable analogue of the host and a polymerizable monomer that includes a binding functionality.

[0018] In other embodiments, the molecular host includes a binding functionality, through which a population of the host molecules binds an analyte. The functionalized host may also complex an EAM; alternatively, the EAM is complexed by a population of non-functionalized host molecules.

[0019] Exemplary binding functionalities appended to the host or polymerizable monomer include a hydrophobic group, a hydrophilic group, reactive groups such as aldehydes, epoxy, carbonates, thiols and the like; ion exchange moieties, e.g., a carboxyl, a sulfonate, a sulfate, an amino, a substituted amino, a phosphate; a metal chelating group; a thioether, a biotin, a boronate, and other structures such as dyes, nucleic acids, and peptides.

[0020] Exemplary molecular hosts include a macrocyclic species, such a crown ether, calixarene, spherand, cavitand and cyclodextrin. Reference is made to cyclodextrin as an exemplary host of use in the present invention. The cyclodextrin or its EAM
complex may be simply deposited onto a device of the invention or it can be attached to the substrate through a species such as an anchor moiety. A representative anchor moiety is formed by reaction of a reactive functionality on an anchor reagent with a reactive functionality of complementary reactivity on the cyclodextrin. The cyclodextrin is utilized as a monomer, a homopolymer or a copolymer with one or more monomers having a useful property, e.g. the presence of a binding functionality. The cyclodextrin-containing polymer is optionally cross-linked.

[0021] The cyclodextrin-EAM complex provides access to novel surfaces and devices and means to incorporate into a device, e.g., a chip, an energy absorbing matrix useful in one or more laser desorption/ionization mass spectrometric analytical technique.
The cavity of the cyclodextrin has properties that are well suited to interaction with the essentially hydrophobic species upon which most art-recognized matrix molecules are based.
The inclusion of the matrix molecule into the cyclodextrin cavity in a host-guest complex eliminates the need to crystallize the hydrophobic matrix on a sample adsorbed onto a chip.
Moreover, the geometric regularity of the cyclodextrin cavity results in matrix components of essentially identical size. In contrast, standard methods of crystallizing the matrix species on the adsorbed sample result in ranges of crystal sizes that can have a deleterious impact on the subsequent mass spectrometric analysis.

[0022] The cyclodextrin-EAM complex is an exemplary water-soluble matrix of the invention.

[0023] Moreover, when the surface is anchored to a substrate, the cyclodextrin-EAM
complex provides a matrix in which multiple EAM are confined to specific, predictable and reproducibly accessed regions of the surface, i.e., the cyclodextrin cavity, providing a reproducible distribution of the EAM on the surface. The reproducibility of EAM
distribution is in distinct contrast to methods in which a water-insoluble matrix is crystallized on the sample; phenomena such as clumping or segregation of the crystals in unrepeatable "island" formations on the surface is avoided.

[0024] In addition, the reversible nature of the complexation of the EAM by cyclodextrin allows the loading/unloading of EAM in the host to be manipulated by a variety of methods, e.g., controlling the polarity of a solvent in contact with the complex. For example, when the host is cyclodextrin and the host is a hydrophobic EAM, contacting the matrix with water will tend to drive the EAM into the cyclodextrin cavity. Conversely, treating the matrix with an organic solvent will tend to extract the complexed EAM out of the cyclodextrin cavity.

[0025] An exemplary cyclodextrin-containing surface of the invention is an "adsorbent film," capable of immobilizing an analyte. In certain embodiments, the film, and thus the device, is capable of selectively binding one or more analyte from a sample.
In an exemplary embodiment, a film includes a cyclodextrin monomer, or a cyclodextrin-containing polymer, and the film is attached to the surface of a substrate. A component of the film may include one or more binding functionality. An array of modalities is available for attaching film components to a substrate surface. In an exemplary embodiment, the attachment is by means of a covalent bond formed between moieties of complementary reactivity on the adsorbent film and on an anchor reagent. Accordingly, the invention provides a cyclodextrin-containing adsorbent film that is grafted to the substrate surface through a chemical reaction that forms a covalent bond. The film can be a hydrogel.

[0026] In addition to the complexes, surfaces and devices of the invention, there are also provided methods for making and using these aspects of the invention.

[0027] Other objects, aspects and embodiments of the invention are set forth in the detailed description that follows.

BRIEF DESCRIPTION OF THE DRAWINGS

[0028] FIG. 1 shows the complexation of a representative EAM compound by a cyclodextrin.

[0029] FIG. 2 is an illustration of constructing cross-linked CD-containing polymer on a chip surface using a cyanate moiety.

[0030] FIG. 3 is an illustration of constructing cross-linked CD-containing polymer on a chip surface using an epoxide moiety.

[0031] FIG. 4 is a mass spectrum of cytochrome C acquired using 1.2 L of cyclodextrin matrix complex solution to assist the desorption and ionization process under typical laser condition.

[0032] FIG. 5 is a mass spectrum of cytochrome C acquired using 4 L of SPA/(3-cyclodextrin matrix complex solution to assist the desorption and ionization process under typical laser condition.

[0033] FIG. 6 is a mass spectrum of ALL-in-1 protein profile in the mass range of 5,000-70,000 Daltons acquired using SPA/0-cyclodextrin polymer matrix complex to assist the desorption and ionization process under typical laser condition.

[0034] FIG. 7 shows an exemplary anchor reagent.

[0035] FIG. 8 shows an exemplary anchor reagent coupled to a glass surface.

[0036] FIG. 9 displays an array of exemplary functionalization chemistries for incorporating binding functionalities into cyclodextrin or a cyclodextrin-containing polymer.

[0037] FIG. 10 is a representative probe format of use in practicing the invention.
DETAILED DESCRIPTION OF THE INVENTION

1. DEFINITIONS

[0038] Unless defined otherwise, all technical and scientific terms used herein generally have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Generally, the nomenclature used herein and the laboratory procedures in cell culture, molecular genetics, organic chemistry, and nucleic acid chemistry and hybridization described below are those well known and commonly employed in the art.
Standard techniques are used for nucleic acid and peptide synthesis. The techniques and procedures are generally performed according to conventional methods in the art and various general references, which are provided throughout this document. The nomenclature used herein and the laboratory procedures in analytical chemistry, and organic synthetic described below are those well known and commonly employed in the art. Standard techniques, or modifications thereof, are used for chemical syntheses and chemical analyses.

[0039] The terms "host" and "molecular host" refer, essentially interchangeably, to a molecule that surrounds or partially surrounds and attractively interacts with a molecular "guest." When the "host" and "guest" interact the resulting species is referred to herein as a "complex."

[0040] Where substituent groups are specified by their conventional chemical formulae, written from left to right, they equally encompass the chemically identical substituents which would result from writing the structure from right to left, e.g., -CH2O- is intended to also recite -OCH2-; -NHS(O)2- is also intended to represent. -S(O)2HN-, etc.

[0041] The term "alkyl," by itself or as part of another substituent, means, unless otherwise stated, a straight or branched chain, or cyclic hydrocarbon radical, or combination thereof, which may be fully saturated, mono- or polyunsaturated and can include di- and multivalent radicals, having the number of carbon atoms designated (i.e. C1-C10 means one to ten carbons). Examples of saturated hydrocarbon radicals include, but are not limited to, groups sucli as methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, isobutyl, sec-butyl, cyclohexyl, (cyclohexyl)methyl, cyclopropylmethyl, homologs and isomers of, for example, n-pentyl, n-hexyl, n-heptyl, n-octyl, and the like. An unsaturated alkyl group is one having one or more double bonds or triple bonds. Examples of unsaturated alkyl groups include, but are not limited to, vinyl, 2-propenyl, crotyl, 2-isopentenyl, 2-(butadienyl), 2,4-pentadienyl, 3-(1,4-pentadienyl), ethynyl, 1- and 3-propynyl, 3-butynyl, and the higher homologs and isomers. The term "alkyl," unless otherwise noted, is also meant to include those derivatives of alkyl defmed in more detail below, such as "heteroalkyl." Alkyl groups, which are limited to hydrocarbon groups are termed "homoalkyl".

[0042] The term "heteroalkyl," by itself or in combination with another term, means, unless otherwise stated, a stable straight or branched chain, or cyclic hydrocarbon radical, or combinations thereof, consisting of the stated number of carbon atoms and at least one heteroatom selected from the group consisting of 0, N, Si and S, and wherein the nitrogen and sulfur atoms may optionally be oxidized and the nitrogen heteroatom may optionally be quaternized. The heteroatom(s) 0, N and S and Si may be placed at any interior position of the heteroalkyl group or at the position at which the alkyl group is attached to the remainder of the molecule. Examples include, but are not limited to, -CH2-CH2-O-CH3, -CH3, -CH2-CHZ-N(CH3)-CH3, -CH2-S-CH2-CH3, -CH2-CH2,-S(O)-CH3, -CH2-CH2-S(O)2-CH3, -CH=CH-O-CH3, -Si(CH3)3, -CH2-CH=N-OCH3, and -CH=CH-N(CH3)-CH3. Up to two heteroatoms may be consecutive, such as, for example, -CH2-NH-OCH3 and -Si(CH3)3. Similarly, the term "heteroalkylene" by itself or as part of another substituent means a divalent radical derived from heteroalkyl, as exemplified, but not limited by, -CH2-CHZ-S-CH2-CH2- and -CH2-S-CH2-CH2-NH-CH2-. For heteroalkylene groups, heteroatoms ca.n also occupy either or both of the chain termini (e.g., alkyleneoxy, alkylenedioxy, alkyleneamino, alkylenediamino, and the like). Still further, for alkylene and heteroalkylene linking groups, no orientation of the linking group is implied by the direction in which the formula of the linking group is written. For example, the formula -C(O)2R'-represents both -C(O)2R'- and -R'C(O)2-.

[0043] Substituents for the alkyl and heteroalkyl radicals (including those groups often referred to as alkylene, alkenyl, heteroalkylene, heteroalkenyl, alkynyl, cycloalkyl, heterocycloalkyl, cycloalkenyl, and heterocycloalkenyl) can be one or more of a variety of groups selected from, but not limited to: -OR', =0, NR', N-OR', -NR'R", -SR', -halogen, -SiR'R"R"', -OC(O)R', -C(O)R', -CO2R', -CONR'R", -OC(O)NR'R", -NR"C(O)R', -NR'-C(O)NR"R"', -NR"C(O)2R', -NR-C(NR'R"R"')-NR"", -NR-C(NR'R")=NR"', -S(O)R', -S(O)2R', -S(O)2NR'R", -NRSO2R', -CN and NOZ in a number ranging from zero to (2m'+l), where m' is the total number of carbon atoms in such radical. R', R", R"' and R"" each preferably independently refer to hydrogen, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, e.g., aryl substituted with 1-3 halogens, substituted or unsubstituted alkyl, alkoxy or thioalkoxy groups, or arylalkyl groups. When a compound of the invention includes more than one R group, for example, each of the R
groups is independently selected as are each R', R", R"' and R"" groups when more than one of these groups is present. When R' and R" are attached to the same nitrogen atom, they can be combined with the nitrogen atom to form a 5-, 6-, or 7-membered ring. For example, -NR'R"
is meant to include, but not be limited to, 1-pyrrolidinyl and 4-morpholinyl.
From the above discussion of substituents, one of skill in the art will understand that the term "alkyl" is meant to include groups including carbon atoms bound to groups other than hydrogen groups, such as haloalkyl (e.g., -CF3 and -CH2CF3) and acyl (e.g., -C(O)CH3, -C(O)CF3, -C(O)CH2OCH3, and the like).

[0044] Each of the above terms is meant to include botli substituted and unsubstituted forms of the indicated radical.

[0045] As used herein, the term "heteroatom" is meant to include oxygen (0), nitrogen (N), sulfur (S) and silicon (Si).

[0046] "Binding functionality" as used herein means a moiety, which has an affinity for a certain substance such as a "substance to be assayed," that is, a moiety capable of interacting with a specific substance to immobilize it on an adsorbent material of the invention. Binding functionalities can be chromatographic or biospecific. Chromatographic binding functionalities bind substances via charge-charge, hydrophilic-hydrophilic, hydrophobic-hydrophobic, van der Waals interactions and combinations thereof. Biospecific binding functionalities generally involve complementary 3-dimensional structures involving one or more of the above interactions. Examples of combinations of biospecific interactions include, but are not limited to, antigens with corresponding antibody molecules, a nucleic acid sequence with its complementary sequence, effector molecules with receptor molecules, enzymes with inhibitors, sugar chain-containing compounds with lectins, an antibody molecule with another antibody molecule specific for the former antibody, receptor molecules with corresponding antibody molecules and the like combinations.
Other examples of the specific binding substances include a chemically biotin-modified antibody molecule or polynucleotide with avidin, an avidin-bound antibody molecule with biotin and the like combinations.

[0047] "Molecular binding partners" and "specific binding partners" refer to pairs of molecules, typically pairs of biomolecules that exhibit specific binding.
Molecular binding partners include, without limitation, receptor and ligand, antibody and antigen, biotin and avidin, and biotin and streptavidin.

[0048] "Adsorbent film" as used herein means an area where a substance to be assayed is immobilized and a specific binding reaction occurs. The reaction optionally has a distribution along the flow direction of a test sample.

[0049] As used herein, the terms "polymer" and "polymers" include "copolymer"
and "copolymers," and are used interchangeably with the terms "oligomer" and "oligomers."

[0050] "Attached," as used herein encompasses interaction including, but not limited to, covalent bonding, ionic bonding, chemisorption, physisorption and combinations thereof.

[0051] "Independently selected" is used herein to indicate that the groups so described can be identical or different.

[0052] "Analyte" refers to any component of a sample that is desired to be detected. The term can refer to a single component or a plurality of components in the sample. Analytes include, for example, biomolecules. Biomolecules can be sourced from any biological material.

[0053] "Biomolecule" or "bioorganic molecule" refers to an organic molecule typically made by living organisms. This includes, for example, molecules comprising nucleotides, amino acids, sugars, fatty acids, steroids, nucleic acids, polypeptides, peptides, peptide fragments, carbohydrates, lipids, and combinations of these (e.g., glycoproteins, ribonucleoproteins, lipoproteins, or the like).

[0054] "Biological material" refers to any material derived from an organism, organ, tissue, cell or virus. This includes biological fluids such as saliva, blood, urine, lymphatic fluid, prostatic or seminal fluid, milk, etc., as well as extracts of any of these, e.g., cell extracts or lysates (from, e.g., primary tissue or cells, cultured tissue or cells, normal tissue or cells, diseased tissue or cells, benign tissue or cells, cancerous tissue or cells, salivary glandular tissue or cells, intestinal tissue or cells, neural tissue or cells, renal tissue or cells, lymphatic tissue or cells, bladder tissue or cells, prostatic tissue or cells, urogenital tissues or cells, tumoral tissue or cells, tumoral neovasculature tissue or cells, or the like), cell culture media, fractionated samples (e.g., serum or plasma), or the like. For example, cell lysate samples are optionally derived.

[0055] "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. 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.

[0056] "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.
Mass spectrometers generally include an ion source and a mass analyzer. Examples of mass spectrometers are time-of-flight, magnetic sector, quadrupole filter, 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 phase ions.

[0057] "Laser desorption mass spectrometer" refers to a mass spectrometer that uses laser energy as a means to desorb, volatilize, and ionize an analyte.

[0058] "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 time-of-flight mass spectrometer the mass analyzer comprises an ion optic assembly, a flight tube and an ion detector.

[0059] "Ion source" refers to a sub-assembly of a gas phase ion spectrometer that provides gas phase ions. 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 in 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.

[0060] 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 mJ / mm2 to about 50 mJ
/ 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 exposed to the ionizing energy. The energy desorbs analyte molecules from the surface into the gas phase and ionizes them.

[0061] Other forms of ionizing energy for analytes include, for example: (1) 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 solid phase, gas phase, and liquid phase neutrals.

[0062] "Surface-enhanced laser desorption/ionization" or "SELDI" refers to a method of desorption/ionization gas phase ion spectrometry (e.g., mass spectrometry) in which the analyte is captured on the surface of a SELDI probe that engages the probe interface of the gas phase ion spectrometer. In "SELDI MS," the gas phase ion spectrometer is a mass spectrometer. SELDI technology is described in, e.g., U.S. patent 5,719,060 (Hutchens and Yip) and U.S. patent 6,225,047 (Hutchens and Yip).

[0063] "Surface-Enhanced Affinity Capture" ("SEAC") or "affinity gas phase ion spectrometry" (e.g., "affinity mass spectrometry") is a version of the SELDI
method that uses a probe comprising an absorbent surface (a "SEAC probe"). "Adsorbent surface"
refers to a sample presenting surface of a probe to which an adsorbent (also called a "capture reagent"
or an "affinity reagent") is attached. An adsorbent is any material capable of binding an analyte (e.g., a target polypeptide or nucleic acid). "Chromatographic adsorbent" refers to a material typically used in chromatography. "Biospecific adsorbent" refers 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 of these (e.g., a glycoprotein, a lipoprotein, a glycolipid, a nucleic acid (e.g., DNA)-protein conjugate). Further examples of adsorbents for use in SELDI can be found in U.S. Patent 6,225,047 (Hutchens and Yip, "Use of retentate chromatography to generate difference maps," May 1, 2001).

[0064] In some embodiments, a SEAC probe is provided as a pre-activated surface that can be modified to provide an adsorbent of choice. For example, certain probes are provided with a reactive moiety that is capable of binding a biological molecule through a covalent bond. Epoxide and carbodiimidizole are useful reactive moieties to covalently bind biospecific adsorbents such as antibodies or cellular receptors.

[0065] In a preferred embodiment affinity mass spectrometry involves applying a liquid sample comprising an analyte to the adsorbent surface of a SELDI probe.
Analytes, such as polypeptides, having affinity for the adsorbent bind to the probe surface.
Typically, the surface is then washed to remove unbound molecules, and leaving retained molecules. The extent of analyte retention is a function of the stringency of the wash used.
An energy absorbing material (e.g., matrix) is then applied to the adsorbent surface.
Retained molecules are then detected by laser desorption/ionization mass spectrometry.

[0066] SELDI is useful for protein profiling, in which proteins in a sample are detected using one or several different SELDI surfaces. In turn, protein profiling is useful for difference mapping, in which the protein profiles of different samples are compared to detect differences in protein expression between the samples.

[0067] "Surface-Enhanced Neat Desorption" or "SEND" is a version of SELDI that involves the use of probes ("SEND probe") comprising a layer of energy absorbing molecules attached to the probe surface. Attachment can be, for example, by covalent or non-covalent chemical bonds. Unlike traditional MALDI, the analyte in SEND is not required to be trapped within a crystalline matrix of energy absorbing molecules for desorption/ionization.

[0068] The phrase, "operative contact," refers to a state in which the analyte and the EAM are sufficiently proximate each other that the excited EAM transfers energy to the analyte.

[0069] SEAC/SEND is a version of SELDI in which both a capture reagent and an energy absorbing molecule are attached to the sample presenting surface. SEAC/SEND
probes therefore allow the capture of analytes through affinity capture and desorption without the need to apply external matrix. The C18 SEND chip is a version of SEAC/SEND, comprising a C 18 moiety which functions as a capture reagent, and a CHCA moiety that functions as an energy absorbing moiety.

[0070] "Surface-Enhanced Photolabile Attachment and Release" or "SEPAR" is a version of SELDI that involves the use of probes having moieties attached to the surface that can covalently bind an analyte, and then release the analyte through breaking a photolabile bond in the moiety after exposure to light, e.g., laser light. SEPAR is further described in United States patent 5,719,060.

[0071] "Eluant" or "wash solution" refers to an agent, typically a solution, which is used to affect or modify adsorption of an 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.

[0072] "Monitoring" refers to recording changes in a continuously varying parameter.

[0073] Data generation in mass spectrometry begins with the detection of ions by an ion detector. A typical laser desorption mass spectrometer can employ a nitrogen laser at 337.1 nm. A useful pulse width is about 4 nanoseconds. Generally, power output of about 1-25 J is used. Ions that strike the detector generate an electric potential that is digitized by a high speed time-array recording device that digitally captures the analog signal.
Ciphergen's ProteinChip system employs an analog-to-digital converter (ADC) to accomplish this. The ADC integrates detector output at regularly spaced time intervals into time-dependent bins.
The time intervals typically are one to four nanoseconds long. Furthermore, the time-of-flight spectrum ultimately analyzed typically does not represent the signal from a single pulse of ionizing energy against a sample, but rather the sum of signals from a number of pulses.
This reduces noise and increases dynamic range. This time-of-flight data is then subject to data processing. In Ciphergen's ProteinCliip software, data processing typically includes TOF-to-M/Z transformation, baseline subtraction, high frequency noise filtering.

[0074] TOF-to-M/Z transfornlation involves the application of an algorithm that transforms times-of-flight into mass-to-charge ratio (M/Z). In this step, the signals are converted from the time domain to the mass domain. That is, each time-of-flight is converted into mass-to-charge ratio, or M/Z. Calibration can be done internally or externally. In internal calibration, the sample analyzed contains one or more analytes of known M/Z.
Signal peaks at times-of-flight representing these massed analytes are assigned the known M/Z. Based on these assigned M/Z ratios, parameters are calculated for a mathematical function that converts times-of-flight to M/Z. In external calibration, a function that converts times-of-flight to M/Z, such as one created by prior internal calibration, is applied to a time-of-flight spectrum without the use of internal calibrants.

[0075] Baseline subtraction improves data quantification by eliminating artificial, reproducible instrument offsets that perturb the spectrum. It involves calculating a spectrum baseline using an algorithm that incorporates parameters such as peak width, and then subtracting the baseline from the mass spectrum.

[0076] High frequency noise signals are eliminated by the application of a smoothing function. A typical smoothing function applies a moving average function to each time-dependent bin. In an improved version, the moving average filter is a variable width digital filter in which the bandwidth of the filter varies as a function of, e.g., peak bandwidth, generally becoming broader with increased time-of-flight. See, e.g., WO
00/70648, November 23, 2000 (Gavin et al., "Variable Width Digital Filter for Time-of-flight Mass Spectrometry").

[0077] A computer can transform the resulting spectrum into various formats for displaying. In one format, referred to as "spectrum view or retentate map," a standard spectral view can be displayed, wherein the view depicts the quantity of analyte reaching the detector at each particular molecular weight. In another format, referred to as "peak map,"
only the peak height and mass information are retained from the spectrum view, yielding a cleaner image and enabling analytes with nearly identical molecular weights to be more easily seen. In yet another format, referred to as "gel view," each mass from the peak view can be converted into a grayscale image based on the height of each peak, resulting in an appearance similar to bands on electrophoretic gels. In yet another format, referred to as "3-D overlays," several spectra can be overlaid to study subtle changes in relative peak heights.
In yet another format, referred to as "difference map view," two or more spectra can be compared, conveniently highlighting unique analytes and analytes that are up-or down-regulated between samples.

[0078] Analysis generally involves the identification of peaks in the spectrum that represent signal from an analyte. Peak selection ca.n, of course, be done by eye. However, software is available as part of Ciphergen's ProteinChip software that can automate the detection of peaks. In general, this software functions by identifying signals having a signal-to-noise ratio above a selected threshold and labeling the mass of the peak at the centroid of the peak signal. In one useful application many spectra are compared to identify identical peaks present in some selected percentage of the mass spectra. One version of this software clusters all peaks appearing in the various spectra within a defined mass range, and assigns a mass (M/Z) to all the peaks that are near the mid-point of the mass (M/Z) cluster.
2. COMPLEXES OF CYCLODEXTRIN AND ENERGY ABSORBING
MOLECULES
2.1. Introduction [0079] In a first aspect, the invention provides a host-guest complex that includes a molecular host complexed with an energy-absorbing molecule that absorbs photo-irradiation from an energy source to promote desorption/ionization of an analyte from a surface. While not wishing to be limited by theory, it is believed that energy absorbing molecules absorb photo-irradiation, such as laser energy, and generate thermal energy. The thermal energy is believed to be transferred to an analyte in operative contact with the energy-absorbing molecule. The transferred energy is believed to promote desorption from the surface and ionization of the analyte. In this way, large biomolecules can be desorbed essentially intact from the surface of a probe. In exemplary embodiments, the complex is immobilized on a substrate that is a component of a probe for a mass spectrometer, e.g., a substrate that includes a means for engaging a probe interface of a mass spectrometer.

2.2. Host Molecules [0080] Exemplary hosts include chelating agents, crown ethers, cryptands, calixarenes, spherands and a polymeric, cyclic structure, e.g., cyclodextrin. The use of host-guest chemistry to prepare a matrix allows an unprecedented degree of flexibility to be engineered into a matrix, surface and device of the invention. The use of host molecules to bind guest compounds ("inclusion") is well known to those of skill in the art. See, for example, Pitt et al. "The Design of Chelating Agents for the Treatment of Iron Overload," In, INORGANIC

CHEMISTRY IN BIOLOGY AND MEDICINE; Martell, A.E., Ed.; American Chemical Society, Washington, D.C., 1980, pp. 279-312; Lindoy, L.F., THE CHEMISTRY OF
MACROCYCLIC
LIGAND COMPLEXES; Cambridge University Press, Cambridge, 1989; Dugas, H., BIOORGANIC CHEMISTRY; Springer-Verlag, New York, 1989, and references contained therein.

[0081] Additionally, a number of routes allowing the attachment of chelating agents, crown ethers and cyclodextrins to other molecules are available to those of skill in the art.
See, for example, Meares et al., "Properties of In vivo Chelate-Tagged Proteins and Polypeptides." In, MODIFICATION OF PROTEINS: FOOD, NUTRITIONAL, AND
PHARMACOLOGICAL ASPECTS;" Feeney, R.E., Whitaker, J.R., Eds., American Chemical Society, Washington, D.C., 1982, pp.370-387; Kasina et al. Bioconjugate Chem.
9:108-117 (1998); Song et al., Bioconjugate Chem. 8:249-255 (1997).

[0082] In an exemplary embodiment, the host is a cyclodextrin or modified cyclodextrin (FIG. 1). Cyclodextrins are a group of cyclic oligosaccharides produced by numerous microorganisms. Cyclodextrins have a ring structure that has a basket-like shape. This shape allows cyclodextrins to include many kinds of molecules into their internal cavity. See, for example, Szejtli, J., CYCLODEXTRINS AND THEIR INCLUSION COMPLEXES; Akademiai Klado, Budapest, 1982; and Bender et al., CYCLODEXTRIN CHEMISTRY, Springer-Verlag, Berlin, 1978. Cyclodextrins are able to form inclusion complexes with an array of organic molecules including, for example, drugs, pesticides, herbicides and agents of war. See, Tenjarla et al., J.
Pharm. Sci. 87:425-429 (1998); Zughul et al., Pharm. Dev. Technol. 3:43-53 (1998); and Albers et al., Crit. Rev. Ther. Drug Carrier Syst. 12:311-337 (1995).
Cyclodextrins are also able to discriminate between enantiomers of compounds in their inclusion complexes. See, Koppenhoefer et al. J Chromatogr. A 793:153-164 (1998). The cyclodextrin binding functionality can be attached to a spacer arm or directly to the substrate.
See, Yamamoto et al., J. Phys. Chem. B 101:6855-6860 (1997). Methods to attach cyclodextrins to other molecules are well known to those of skill in the chromatographic and pharmaceutical arts.
See, Sreenivasan, K. J. Appl. Polym. Sci. 60:2245-2249 (1996). Because of their well-studied, diverse and robust complexation chemistry, cyclodextrins provide an EAM having properties that are unique and generally superior to a matrix prepared by applying a solution of the, generally hydrophobic, EAM to the sample.

[0083] In general, EAMs used in laser desorption/ionization mass spectrometry are essentially insoluble in water. However, the physiochemical properties of EAMs, such as solubility and stability, can be altered through their formation of inclusion complexes witli cyclodextrins. For example, when the EAM is complexed by a water-soluble host, e.g., cyclodextrin, it is possible to prepare an aqueous solution that includes an EAM. Thus, the present invention also provides a water-soluble EAM that includes an EAM-host complex that is more soluble in water than the uncomplexed EAM. Exemplary EAM
according to this embodiment of the invention are set forth herein.

[0084] In embodiments in which a water-soluble EAM, as described above, it utilized, the EAM can be applied to an analyte in a manner that is generally similar to methods used with uncomplexed EAM. This embodiment of the invention includes a notable distinction from prior methods; the EAM is applied as an aqueous solution, rather than as a solute in an essentially organic solvent or organic/water co-solvent. Because the solvents for the analyte and the EAM are both aqueous they are essentially freely miscible. Thus, difficulties associated with the immiscibility of the EAM solvent with the analyte solvent, e.g., EAM
aggregation, irregular crystal size, and the like, are minimized or avoided entirely.

[0085] In addition to the complex formed between the host and the EAM, the invention also provides a mass spectrometric probe, e.g., a chip that is configured to allow it to be positioned within the sample chamber of a mass spectrometer. The chips include a host-guest (EAM) complex on their surface. Alternatively, the chips include the host on the surface, providing a device on which a EAM can be formed by the operator prior to use.

[0086] The complexes, surfaces and devices of the invention can be used in the acquisition of data regarding an analyte in a laser assisted desorption/ionization mass spectrometric modality. For example chips that incorporate a EAM of the invention can be used to analyze a single peptide (FIG. 4 and FIG. 5), and mixtures of peptides (FIG. 6). Due to its readily controlled and reproduced structure, the EAM of the invention provides reproducible results in mass spectrometric analyses.

[0087] In the discussion that follows, the materials of the device incorporating the host-guest (EAM) concept of the invention are illustrated by reference to the use of cyclodextrin as the host. The focus on cyclodextrin is for clarity of illustration and is not limiting, nor are the exemplified mono-molecular and polymeric motifs in which the host is incorporated.
Similarly, the present invention is furtller explained and illustrated in the following sections, by reference to a representative embodiment using detection by mass spectrometry. The focus on mass spectrometric detection is for purposes of clarity and simplicity of illustration only, and should not be construed as limiting the scope of the present invention or circumscribing the types of methods in which the present invention finds application. Those of skill in the art will recognize that the methods set forth herein are broadly applicable to a number of chip formats and assays using these chips for the detection of a wide range of target moieties.

2.3. Energy Absorbing Molecules [0088] Energy absorbing molecules include those molecules capable of absorbing photo-irraditation and promoting desorption/ionization of analyte molecules in operative contact therewith. For example, in laser desorption/ionization processes that employ ultraviolet lasers, the EAM can absorb ultraviolet light. In LDI processes using infrared lasers, the EAM
can absorb infrared light. In an exemplary embodiment, the EAM has the formula:

Ar Rl-C(O)Rla in which Ar is a member selected from aryl (e.g., substituted or unsubstituted phenyl) and heteroaryl (e.g., substituted or unsubstituted indolyl and substituted or unsubstituted pyridyl).
The symbol Rl represents a bond, substituted or unsubstituted alkyl or substituted or unsubstituted heteroalkyl. Ria is H, OH or substituted or unsubstituted alkyl.

[0089] Exemplary aryl and heteroary moieties of use as Ar groups include:

Jõlv .n n , .nn, .~vti V~LAJ

::: / OR4 / N
I ;and OR2 R2O \ N \

wherein the symbols R2, R3, and R4 independently represent H or substituted or unsubstituted alkyl, e.g., C1-C6 unsubstituted alkyl.

[0090] In another embodiment, R' has the formula:
CR5=CR6-~
wherein R5 and R6 independently represent H, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, or CN. Exemplary R5 and R6 groups include:

~ -c-H ~ ; and ~HH I
CN

[0091] Exemplary EAMs include a-cyano-4-hydroxycinnamic acid; sinapic acid, 2-(4-hydroxyphenylazo) benzoic acid; 2-Mercapto-benzothiazole; succinic acid; 2,6-hihydroxy acetophenone; ferulic acid; caffeic acid; 4-nitroaniline; 2,4,6-trihydroxy acetophenone; 3-hydroxy picolinic acid; anthranilic acid; nicotinic acid; salicylamide; trans-3-indoleacrylic acid; dithranol; 2,5-Dihydroxy benzoic acid; succinic acid; 2,5-dihydroxy benzoic acid;
isovanillin; 2,5-dihydroxybenzoic acid; 3-aminoquinoline; 2,4,6-; dithranol ;
T-2-(3-(4-t-butyl-phenyl)- 2-methyl- 2-propenylidene) malononitrile; and 1-isoquinolinone, and any other molecule typically used for matrix in MALDI.

2.4. Polymeric Cyclodextrin [0092] In an exemplary embodiment, the complex of this invention is comprised in a polymer in which the cyclodextrin is a component of the polymer. The polymer is selected from cyclodextrin homopolymers and cyclodextrin copolymers; either of which is optionally cross-linked by one or more cross-linking moieties. Selected cyclodextrin polymers are commercially available (e.g., CarboMer, Inc. Cat. # 4-00236). The cyclodextrin is covalently bonded to the polymer, either in the polymer backbone or as a substituent along the polymer chain. The art is replete with developed methods for preparing cyclodextrin polymers. Those of skill in the art will readily appreciate that essentially any cyclodextrin polymer capable of forming a complex with an EAM is of use in the device and methods of the invention.

[0093] Methods for synthesizing cyclodextrin polymers, in which cyclodextrin itself constitutes the backbone, are known. Such methods are often based on the use of different bifunctional agents such as epichlorhydrin, dialdehydes, dibasic acids, diesters, dibasic acid dichlorides, diepoxides, diisocyanates or dihalogenated derivatives, polyisocyanates, ethylene glycol bis(epoxypropyl)ether, dibasic carboxylic acid dihalides in an organic solvent, or phytic acid. See, for example, Comprehensive Supramolecular Chemistry, Volume 3, J.L.
Atwood et al., eds. Pergamon Press (1996); Cserhati et al., Anal. Chim. Acta 279: 107-113 (1993); U.S. Patent No. 5,608,015; and U.S. Patent No. 5,276,088.

[0094] In one embodiment, the present invention utilizes a cyclodextrin copolymer that is formed using epichlorhydrin. See, e.g., Nestle (NETH 6505361) and by Solms and Egli (Helv. Chim. Acta 48: 1225 (1965); U.S. Patent No. 3,420,788); GB 1,244,990;
Wiedenhof et al., Die Starke 21(5): 119-123 (1969); Hoffman, J. Macromol. Sci-Chem., A7(5): 1147-1157 (1973); and Japanese patents JP-A-58171404 and JP 61283601.

[0095] Other cyclodextrin polymers of use are prepared using a process using a dialdehyde, a dibasic acid, a diester, a dibasic acid dichloride, a diepoxide, a diisocyanate or a dihalogenated derivative is described in U.S. Pat. No. 3,472,835.

[0096] Also of use are cyclodextrin polymers prepared by a process using polyisocyanates in organic aprotic solvents (U.S. Patent No. 4,917,956;
Asanuma et al., Chem. Commun., 1971 (1997) and in WO-A-98 22197).

[0097] Additional useful cyclodextrin polymers are those that include ethylene glycol moieties witliin their structural framework. An exemplary process for preparing such polymers uses ethylene glycol bis(epoxypropyl) ether (Fenyvesi et al. (Ann.
Univ. Sci.
Budapest, Rolando Eotvos Nominatae, Sect. Chim. 15: 13-22 (1979)). A process using other diepoxy compounds has also been described by Sugiura et al.(Bull. Chem. Soc.
Jpn., 62:
1643-1651 (1989)).

[0098] A second type of polymer, where the cyclodextrin is a pendent group from a polymer chain is of use in the present invention. Exemplary polymers having this motif are produced by grafting cyclodextrin(s) or cyclodextrin derivative(s) to a pre-existing polymer chain. In an exemplary embodiment, a reactive dextran intermediates, e.g., a halotriazine and halopyrimidine derivative of cyclodextrins are used to attach the cyclodextrin moieties to the backbone. See, e.g., DE 19520989.

[0099] Cyclodextrins can also be functionalized with aldehyde groups, and then grafted onto an amine-containing polymer, e.g., chitosan by a reductive amination reaction are of use.
See, for example, Tomoya et al., J. Polym. Sci., PartA: Polyin. Chem., 36 (11): 1965-1968 (1998).

[0100] A number of versatile cyclodextrin intermediates are also of use for the preparation of polymers and the substrate attachment of both cyclodextrin polymers and monomers. For example, cyclodextrin that is functionalized with a polymerizable functional group, e.g., acryloyl or methacryloyl groups, can be used to prepare polymers or to attach a cyclodextrin-containing species to a surface through interaction of the polymerizable group with a group of complementary reactivity on the surface or an anchor moiety attached to the surface. See, e.g., DE-A-4 009 825; Wimmer et al., Minutes Int. Symp.
Cyclodextrins, 6Ih 106-109, (1992), Ed: Hedges A. L. Sante Paris; EP-A-0780401; and Harada et al., Macromolecules 9(5): 701-704, (1976).

[0101] In an exemplary embodiment, a methacryloyloylated cyclodextrin is homo-polymerized or copolymerized. In certain embodiments, co-polymerization proceeds with a crosslinking agent, such as poly(ethylene glycol)dimethacrylate, to form a cross-linked polymer. The polymerization can be initiated by the addition of a peroxide, such as lauroyl peroxide. The polymer is purified by methods known in the art, e.g., extraction of unreacted monomers, precipitation, crystallization, fractional crystallization, size exclusion chromatography, dialysis and the like. The polymer is also characterized by art-recognized metliods, e.g., NMR, IR, size exclusion chromatography, elemental analysis and the like.

[0102] In another embodiment, the cyclodextrin is functionalized with a binding moiety, as described further below. In this embodiment, at least one of the hydroxyl moieties of the cyclodextrin is derivatized, providing a group of the formula:

~wherein R7 is a moiety that comprises a binding functionality.
2.4.1.1. Cross-Linking [0103] In certain embodiments, a percentage of the polymer is cross-linked.
Any cross-linking agent, useful to crosslink the components of the polymer can be used to prepare the host-polymer of the invention. In an exemplary embodiment, the cross-linking agent is a polyol or a polyisocyanate having. Alternatively, the crosslinking agent is a polymerizable monomer. Members of this class are those wherein at least one and preferably most of such linkages are conjugated with a double bonded carbon, including carbon double bonded to carbon and to such heteroatoms as nitrogen, oxygen and sulfur. Also included are such materials wherein the ethylenically unsaturated groups, especially the vinylidene groups, are conjugated with ester or amide structures and the like.

[0104] In certain embodiments, this group may be used to bind the complex or polymers to the surface of a substrate, e.g., through an anchor moiety via reaction of complementary reactive functional groups on the cyclodextrin-containing species and an anchor reagent. For example, a reactive functional group on the anchor reagent is used to tether the cyclodextrin-containing species to the substrate. An exemplary anchor moiety of use in immobilizing the polymers of the invention on a substrate is shown in FIG. 7, and its attachment to a glass surface is shown in FIG. 8. For example, by providing free vinyl groups or benzophenone groups in the polymer, it can be coupled to a surface on which free vinyl groups are provided, for example methacryl groups.

[0105] An exemplary cross-linked polymer of the invention is a hydrogel.
2.5. Functionalized Complexes and Polymers [0106] In certain embodiments the host-guest complex or the host-guest complex polymer further comprise a functional group for binding a target. In certain embodiments the functional groups are reactive functional groups that covalently bind the target. In other embodiments, the functional groups are binding functional groups that bind a target non-covalently. Exemplary functional groups and functionalization chemistries of use in the present invention are set forth in FIG. 9.

2.5.1.1. Reactive Functional Groups [0107] Exemplary reactive functional groups include:

(a) carboxyl groups and various derivatives thereof including, but not limited to, N-hydroxysuccinimide esters, N-hydroxybenztriazole esters, acid halides, acyl imidazoles, thioesters, p-nitrophenyl esters, alkyl, alkenyl, alkynyl and aromatic esters;

(b) hydroxyl groups, which can be converted to esters, ethers, aldehydes, etc.;

(c) haloalkyl groups wherein the halide can be later displaced with a nucleophilic group such as, for example, an amine, a carboxylate anion, thiol anion, carbanion, or an alkoxide ion, thereby resulting in the covalent attachment of a new group at the site of the halogen atom;

(d) dienophile groups, which are capable of participating in Diels-Alder reactions such as, for example, maleimido groups;

(e) aldehyde or ketone groups such that subsequent derivatization is possible via formation of carbonyl derivatives such as, for example, imines, hydrazones, semicarbazones or oximes, or via such mechanisms as Grignard addition or alkyllithium addition;

(f) sulfonyl halide groups for subsequent reaction with amines, for example, to form sulfonamides;

(g) thiol groups, which can be converted to disulfides or reacted with acyl halides;
(h) amine or sulfhydryl groups, which can be, for example, acylated or alkylated;
(i) alkenes, which can undergo, for example, cycloadditions, acylation, Michael addition, etc; and (j) epoxides, which can react with nucleophiles, for example, amines and hydroxyl compounds.

[0108] The reactive functional groups can be chosen such that they do not participate in, or interfere with reactions in which they are not intended to participate.
Alternatively, the reactive functional group can be protected from participating in the reaction by the presence of a protecting group. Those of skill in the art will understand how to protect a particular functional group from interfering with a chosen set of reaction conditions.
For examples of useful protecting groups, See, Greene et al., PROTECTIVE GROUPS IN ORGANIC
SYNTHESIS, John Wiley & Sons, New York, 1991.

[0109] One skilled in the art will readily appreciate that many of these linkages may be produced in a variety of ways and using a variety of conditions. For the preparation of esters, see, e.g., March supra at 1157; for thioesters, see, March, supra at 362-363, 491, 720-722, 829, 941, and 1172; for carbonates, see, March, supra at 346-347; for carbamates, see, March, supra at 1156-57; for amides, see, March supra at 1152; for ureas and thioureas, see, March supra at 1174; for acetals and ketals, see, Greene et al. supra 178-210 and March supra at 1146; for acyloxyalkyl derivatives, see, PRODRUGS: TOPICAL AND OCULAR
DRUG
DELIVERY, K. B. Sloan, ed., Marcel Dekker, Inc., New York, 1992; for enol esters, see, March supra at 1160; for N-sulfonylimidates, see, Bundgaard et al., J. Med.
Chem., 31:2066 (1988); for anhydrides, see, March supra at 355-56, 636-37, 990-91, and 1154;
for N-acylamides, see, March supra at 379; for N-Mannich bases, see, March supra at 800-02, and 828; for hydroxymethyl ketone esters, see, Petracek et al. Annals NYAcad.
Sci., 507:353-54 (1987); for disulfides, see, March supra at 1160; and for phosphonate esters and phosphonamidates, see, e.g., copending application Ser. No. 07/943,805, which is expressly incorporated herein by reference.

[0110] Those of skill in the art understand that the reactive functional groups discussed herein represent only a subset of functional groups that are useful in assembling the chips of the invention. Moreover, those of skill understand that the reactive functional groups are also of use as binding functionality components of the adsorbent film and the linker arms.

2.5.1.2. Binding Functionalities [0111] An exemplary complex or polymer of the invention can include a binding functionality. Binding functionalities (which also can be attached through reactive functionalities) are useful for capturing analytes from a sample for further analysis. Binding functionalities may be grouped into two classes - chromatographic binding groups and biospecific binding groups.

[0112] Chromatographic binding functionalities include those typically used in chromatography, and they tend to bind substances via charge-charge, hydrophilic-hydrophilic, hydrophobic-hydrophobic, van der Waals interactions and combinations thereof.
They include, for exainple, ion exchange functionalities (anion and cation), hydrophobic functionalities, hydrophilic functionalities, metal chelate functionalities and dye functionalities.

[0113] In an exemplary embodiment, the binding functionality is selected from the group consisting of a positively charged moiety, a negatively charged moiety, an anion exchange moiety, a cation exchange moiety, a metal ion complexing moiety, a metal complex, a polar moiety and a hydrophobic moiety.

[0114] Ion exchange moieties of use as binding functionalities in the polymers of the invention are, e.g., diethylaminoethyl, triethylamine, sulfonate, tetraalkylammonium salts and carboxylate.

[0115] In an exemplary embodiment, the binding functionality is a polyaminocarboxylate chelating agent such as ethylenedianminetetraacetic acid (EDTA) or diethylenetriaminepentaacetic acid (DTPA), which is attached to an amine on the substrate, or spacer arm, by utilizing the commercially available dianhydride (Aldrich Chemical Co., Milwaukee, WI). When complexed with a metal ion, the metal chelate binds to tagged species, such as polyhistidyl-tagged proteins, which can be used to recognize and bind target species. Alternatively, the metal ion itself, or a species complexing the metal ion can be the target.

[0116] Metal ion complexing moieties include, but are not limited to N-hydroxyethylethylenediaminoe-triacetic acid (NTA), N,N-bis(carboxymethyl)-L-lysine, iminodiacetic acid, aminohydroxamic acid, salicylaldehyde, 8-hydroxy-quinoline, N,N,N'-tris(carboxytrimethyl)ethanolamine, and N-(2-pyridylmethyl) aminoacetate. The metal ion complexing agents can complex any useful metal ion, e.g., copper, iron, nickel, cobalt, gallium and zinc.

[0117] In another exemplary embodiment, the binding functionality is a biomolecule, e.g., a natural or synthetic peptide, antibody (including binding fragments thereof, such as Fab' or F(ab')2), nucleic acid, saccharide, lectin, member of a receptor/ligand binding pair, antigen, cell or a combination thereof. Thus, in an exemplary embodiment, the binding functionality is an antibody raised against a target or against a species that is structurally analogous to a target. In another exemplary embodiment, the binding functionality is avidin, or a derivative thereof, which binds to a biotinylated analogue of the target.
In still another exemplary embodiment, the binding functionality is a nucleic acid, which binds to single- or double-stranded nucleic acid target having a sequence complementary to that of the binding functionality. In another embodiment, the binding functionality is selected from nucleic acid species, such as aptamers and aptazymes that recognize specific targets.

[0118] In another exemplary embodiment, the binding functionality is a drug moiety or a pharmacophore derived from a drug moiety. The drug moieties can be agents already accepted for clinical use or they can be drugs whose use is experimental, or whose activity or mechanism of action is under investigation. The drug moieties can have a proven action in a given disease state or can be only hypothesized to show desirable action in a given disease state. In a preferred embodiment, the drug moieties are compounds, which are being screened for their ability to interact with a target of choice. As such, drug moieties, which are useful in practicing the instant invention include drugs from a broad range of drug classes having a variety of pharmacological activities.

3. DEVICES
3.1. Introduction [0119] This invention contemplates devices in which the surface of a substrate is coated with the monomeric or polymeric complexes of this invention. The complexes can be bound to the surface by any means, including covalent or non-covalent chemical bonding, or simply physical attachment by applying the complex to the substrate surface where it sticks.
Depending on the nature of the substrate, the devices of this invention can come in the form of chips, resins (e.g., beads), microtiter plates or membranes.

3.2. Substrate [0120] In selected devices of the invention (e.g., chips, chromatographic supports, microtiter plates, membranes), the complex is immobilized on a substrate, either directly or through linker arms that are interposed between the substrate and the adsorbent film. The nature and intended use of the device influences the configuration of the substrate. For example, a chip of the invention is typically based upon a planar substrate format. In contrast, a chromatographic support of the invention generally makes use of a spherical or approximately spherical substrate, while a membrane of the invention is formed using a porous substrate. A microtiter plate is generally a plastic article of manufacture comprising wells in which reactions can be performed.
3.2.1.1. Chip [0121] Exemplary chips of the invention are formed using a planar substrate.
The complex is applied directly to the substrate or is bound to an anchor moiety that is bound to the substrate surface, or to a feature on the substrate surface, such as a region that is raised (e.g., island) or depressed (e.g., a well, trough, etc.).

[0122] The complex, in either polymer or monomer form, is generally immobilized on the chip substrate. The interaction between the polymer and the substrate can be a covalent, electrostatic, ionic, hydrogen bonding, hydrophobic-hydrophobic, hydrophilic-hydrophilic interaction or physisorption or physical adhesion.

[0123] Substrates that are useful in practicing the present invention can be made of any stable material, or combination of materials. Moreover, useful substrates can be configured to have any convenient geometry or combination of structural features. The substrates can be either rigid or flexible and can be either optically transparent or optically opaque. The substrates can also be electrical insulators, conductors or semiconductors.
When the sample to be applied to the chip is water based, the substrate preferable is water insoluble.

[0124] In a preferred embodiment, the substrate material is essentially non-reactive with the analyte, thus preventing non-specific binding between the substrate and the analyte or other components of an assay mixture. Methods of coating substrates with materials to prevent non-specific binding are generally known in the art. Exemplary coating agents include, but are not limited to cellulose, bovine serum albumin, and poly(ethylene glycol).
The proper coating agent for a particular application will be apparent to one of skill in the art.

[0125] In an exemplary embodiment, the substrate includes an aluminum support that is coated with a layer of silicon dioxide. In yet a further preferred embodiment, the silicon dioxide layer is from about 1000-3000 A in thickness. In other embodiments, the substrate comprises a polymeric material, such as cellulose or a plastic.

3.2.1.2. Water-Soluble EAM

[0126] In exemplary embodiments, the EAM-host complex is not anchored to a surface and it is essentially water-soluble, or at least more water-soluble than the EAM alone. Thus, the invention provides the first essentially water-soluble EAM for use in laser desorption/ionization mass spectrometry. In practice, an aqueous solution of the water-soluble EAM can be used in a manner analogous to that presently used with EAM
solutions in organic solvent. For example, the complex, either in monomeric or polymeric forms, can be mixed with the analyte before application to the probe surface.
Alternatively, either the complex or the analyte can be applied to the probe surface and the other can be applied subsequently.

3.2.1.3. Anchor reagents (moieties) [0127] When the host-guest EAM complex is covalently bound to the surface, it can be tethered to the surface by means of an anchor moiety that is formed by reacting a group on an anchor reagent with a group of complementary reactivity on a substrate surface; and reacting a group on the free or surface-tethered anchor reagent with a group of complementary reactivity on the host; these reactions are performed in any useful sequence.
An exemplary anchor moiety is set forth in FIG. 7. FIG. 8 provides a putative structure of the anchor moiety bound to a glass surface. Both monomeric and polymeric complexes can be tethered to the surface.

[0128] In the case of a representative substrate, which is coated with a glass-like material, presenting a surface with reactive Si-OH bonds, the anchor moiety will generally include a first functional group of reactivity complementary to the bonds at the surface of the glass, and a second reactive functionality that forms a covalent bond with a reactive functionality of complementary reactivity on the host. As discussed above, in an exemplary embodiment the complex includes a host that is a cylcodextrin moiety.

[0129] Regarding the first functional group, a number of siloxane functionalizing reagents can form bonds to the substrate. Exemplary reagents include, but are not limited to:
1. hydroxyalkyl siloxanes (Silylate surface, functionalize with diborane, and H202 to oxidize the alcohol) a. allyl trichlorosilane -~ -~ 3-hydroxypropyl, b. 7-oct-l-enyl trichlorchlorosilane -->->8-hydroxyoctyl;
2. diol (dihydroxyalkyl) siloxanes (silylate surface and hydrolyze to diol) a. (glycidyl trimethoxysilane -+ -> (2,3-dihydroxypropyloxy)propyl;

3. aminoalkyl siloxanes (amines requiring no intermediate functionalizing step) a. 3-aminopropyl trimethoxysilane -> aminopropyl;

4. dimeric secondary aminoalkyl siloxanes -a. bis (3-trimethoxysilylpropyl) amine -> bis(silyloxylpropyl)amine; and 4. unsaturated species (e.g., acryloyl, methacryloyl, styryl, etc.).

[0130] Exemplary anchor reagents according to motif 4, above, include styrylethyltrimethoxysilane, styrylethylmethyldimethoxysilane, styrylethyldimethylmethoxysilane, styrylethyltrichlorosilane, styrylethylmethyldimethoxysilane, styrylethyldimethylmethoxysilane, (3-acryloxypropyl)trimethoxysilane, (3-acryloxypropyl)methyldimethoxysilane, (3-acryloxypropyl)dimethylmethoxysilane, (3-acryloxypropyl)trichlorosilane, (3-acryloxypropyl)methyldichlorosilane, (3-acryloxypropyl)dimethylchlorosilane, (3-methacryloxypropyl)trimethoxysilane, (3-methacryloxypropyl)methyldimethoxysilane, (3-methacryloxypropyl)dimethylmethoxysilane, (3-methacryloxypropyl)trichlorosilane, (3-methacryloxypropyl)methyldichlorosilane, (3-methacryloxypropyl)dimethylchlorosilane and combinations thereof.

[0131] In an exemplary embodiment, an anchor reagent having a polymerizable moiety set forth above is coupled to a host that is derivatized with a polymerizable moiety of complementary reactivity.

3.2.1.4. Patterning [0132] The surface of a substrate of use in practicing the present invention can be smooth, rough and/or patterned. The surface can be engineered by the use of mechanical and/or chemical techniques. For example, the surface can be roughened or patterned by rubbing, etching, grooving, stretching, and the oblique deposition of metal films. The substrate can be patterned using techniques such as photolithography (Kleinfield et al., J.
Neurosci. 8: 4098-120 (1998)), photoetching, chemical etching and microcontact printing (Kumar et al., Langmuir 10: 1498-511 (1994)). Other techniques for forming patterns on a substrate will be readily apparent to those of skill in the art.

[0133] The size and complexity of the pattern on the substrate is controlled by the resolution of the technique utilized and the purpose for which the pattern is intended. For example, using microcontact printing, features as small as 200 nm have been layered onto a substrate. See, Xia, Y.; Whitesides, G., J. Am. Chem. Soc. 117: 3274-75 (1995). Similarly, using photolithography, patterns with features as small as 1 m have been produced. See, Hickman et al., J. Vac. Sci. Technol. 12: 607-16 (1994). Patterns that are useful in the present invention include those which comprise features such as wells, enclosures, partitions, recesses, inlets, outlets, channels, troughs, diffraction gratings and the like.

[0134] In an exemplary embodiment, the patterning is used to produce a substrate having a plurality of adjacent addressable features, wherein each of the features is separately identifiable by a detection means. In another exemplary embodiment, an addressable feature does not fluidically communicate with other adjacent features. Thus, an analyte, or other substance, placed in a particular feature remains essentially confined to that feature. In another preferred embodiment, the patterning allows the creation of channels through the device whereby fluids can enter and/or exit the device.

[0135] Using recognized techniques, substrates with patterns having regions of different chemical characteristics can be produced. Thus, for example, an array of adjacent, isolated features is created by varying the hydrophobicity/hydrophilicity, charge or other chemical characteristic of a pattern constituent. For example, hydrophilic compounds can be confined to individual hydrophilic features by patterning "walls" between the adjacent features using hydrophobic materials. Similarly, positively or negatively charged compounds can be confined to features having "walls" made of compounds with charges similar to those of the confined compounds. Similar substrate configurations are also accessible through microprinting a layer with the desired characteristics directly onto the substrate. See, Mrkish, M.; Whitesides, G.M., Ann. Rev. Biophys. Biomol. Struct. 25:55-78 (1996).

[0136] The specificity and multiplexing capacity of the chips of the invention is improved by incorporating spatial encoding (e.g., spotted microarrays) into the chip substrate. Spatial encoding can be introduced into each of the chips of the invention. In an exemplary embodiment, binding functionalities for different analytes can be arrayed across the chip surface, allowing specific data codes (e.g., target-binding functionality specificity) to be reused in each location. In this case, the array location is an additional encoding parameter, allowing the detection of a virtually unlimited number of different analytes.

[0137] In the embodiments of the invention in which spatial encoding is utilized, they preferably utilize a spatially encoded array comprising m binding functionalities distributed over m regions of the substrate. Each of the m binding functionalities can be a different functionality or the same functionality, or different functionalities can be arranged in patterns on the surface. For example, in the case of matrix array of addressable locations, all the locations in a single row or colunm can have the same binding functionality.
The m binding functionalities are preferably patterned on the substrate in a manner that allows the identity of each of the m locations to be ascertained. In another embodiment, the m binding functionalities are ordered in a p by q matrix of (p x q) discrete locations, wherein each of the (p x q) locations has bound thereto at least one of the m binding functionalities. The microarray can be patterned from essentially any type of binding functionality.

3.2.1.5. Mass spectrometry probe [0138] In preferred embodiments the chip of this invention is designed in the form of a probe for a gas phase ion spectrometer, such as a mass spectrometry probe. To facilitate its being positioned in a sample chamber of a mass spectrometer, the substrate of the chip is generally configured to comprise.means that engage a complementary structure within the interface. The term "positioned" is generally understood to mean that the chip 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. There are many commercially available laser desorption/ionization mass spectrometers. Vendors include Ciphergen Biosystems, Inc., Waters, Micromass, MDS, Shimadzu, Applied Biosystems and Bruker Biosciences.

[0139] An exemplary structure according to this description is a chip that includes means for slidably engaging a groove in an interface, such as that used in the Ciphergen probes (FIG. 10). In this figure, the means to position the probe in the sample chamber is integral to substrate 101, which includes a lip 102 that engages a complementary receiving structure in the probe.

[0140] In another example, the probe is round and is typically attached to a holder /
actuator using a magnetic coupler. The target is then pushed into a repeller and makes intimate contact to insure positional and electrical certainty.

[0141] Other probes are rectangular and they either marry directly to a carrier using a magnetic coupling or physically attach to a secondary carrier using pins or latches. The secondary carrier then magnetically couples to a sample actuator. This approach is generally used by systems which have autoloader capability and the actuator is generally a classical x,y 2-d stage.

[0142] In yet another exemplary embodiment, the probe is a barrel. The barrel was used to support gel pieces o r blots. By rotating and moving in the vertical plane, a 2-d stage is created.

[0143] Still a further exemplary embodiment the probe is a disk. The disk is rotated and moved in either a vertical or horizontal position to create an r-theta stage.
Such disks are typically engaged using either magnetic or compression couplers.

3.2.1.6. Resin [0144] In accordance with one aspect of the invention, the solid substrate is an organic material. Preferably, the organic material is cellulose, agarose, dextran, polyacrylates, polystyrene, polyacrylamide, polymethacrylamide, copolymers of styrene and divinylbenzene, or mixtures thereof. Alternatively, the solid support is an inorganic material, preferably one selected from hydrogel-containing silica, zirconia, alumina, titania, ceramics, and mixtures thereof.

4. METHODS OF MAKING A DEVICE

[0145] In another exemplary embodiment, the invention provides methods of making complexes and devices of the invention. An advantage of the method set forth herein for forming a matrix is the ease of preparation of industrially relevant amounts of a compound that includes a host moiety as a component of its structure, referred to herein as a "host-containing species," or an EAM complex of this species. Access to batches of the components necessary to form the complexes, surfaces and chips of the invention allows the formation of a multitude of chips having essentially identical properties.
Moreover, as discussed above, the methods of the invention provide a matrix with EAM
loadings that are freely tunable. Furthermore, the matrix is not hampered by the irregularities of matrix crystal distribution resulting from present methods of layering a solution of the matrix onto the sample. Thus, the chips are amenable to reproducible, high throughput analysis.

[0146] Complexes of the invention are prepared by bringing a host and guest into contact under conditions that promote the formation of the complex. General conditions for forming such complexes are art-recognized; developing appropriate conditions for a selected application is well within the abilities of those of skill in the art. An exemplary host-guest complex is a cyclodextrin, or a cyclodextrin homopolymer or co-polymer complexed with an EAM such as those described above. In addition to the vast knowledge in the art regarding forming cyclodextrin complexes, the present application includes examples of forming the complexes.

[0147] In another embodiment, the invention provides a method for making a plurality of adsorbent devices that include a host or a host-guest EAM complex. Each member of the plurality of devices includes: (a) a solid support having a surface; and (b) an adsorbent film that includes a host or a host-guest complex on the surface. In a preferred method, each solid support is contacted with an aliquot of a single preparation, i.e., batch, of a compound that includes a host moiety as a component of its structure, referred to herein as a "host-containing species." The aliquot is sampled from a single batch of the host-containing species or an EAM complex of the host-containing species.

[0148] The use of a single batch of a component used to form a species of the invention minimizes chip-to-chip and lot-to-lot variations. A preferred size for a single batch of the host-containing species is from about 0.5 liters and 5 liters. The single batch is preferably of sufficient volume to prepare a total area of addressable locations of least about 500,000 mma preferably from about 500,000 mm2 to about 50,000,000 mm2, more preferably from about 100,000 to about 5,000,000 addressable locations.

[0149] The surface, once laid down on the substrate, is amenable to functionalization by a variety of chemical reactions known to those skilled in the art. For example, in order to produce an anion exchange host-containing species, a reactive host-containing species is reacted with a suitable amine (e.g. dimethylethanol amine or trimethyl amine), producing a quaternary ion exchange site. The discussion above regarding reactive functional groups and binding functionalities is generally relevant here as well.

5. METHODS OF USING THE COMPLEXES AND DEVICES

[0150] The host-guest EAM complexes of this invention are useful for detecting analytes, in particular in comiection with mass spectrometry, but also with other methods of surface detection. In one embodiment, the analyte, in, e.g., an aqueous solution, is mixed witli a complex of the invention in either monomeric or polymeric form. The mixture is applied to the surface of a probe, engaged with an interface of a mass spectrometer, and then detected by laser desorption/ionization mass spectrometry. In another embodiment, a chip coated with a monomeric or polymeric complex of the invention is provided. A
sample comprising an analyte is applied to the chip surface. Then the analyte is detected by mass spectrometry. In other embodiments, the chip also comprises binding moieties that can bind the analyte non-covalently. After capture of the analyte, non-binding molecules are washed away. Then the analyte can be detected by mass spectrometry. In other embodiments, the analyte can be detected by other surface detection methods as well. Generally, these are suited to examine biospecific interactions between, for example, receptor and ligand.
.[0151] Upon capture on a chip, analytes can be detected by a variety of detection methods selected from, for example, a gas phase ion spectrometry method, an optical method, an electrochemical method, atomic force microscopy and a radio frequency method. Gas phase ion spectrometry methods are described herein. Of particular interest is the use of mass spectrometry and, in particular, SELDI. Optical methods include, for example, detection of fluorescence, luminescence, chemiluminescence, absorbance, reflectance, transmittance, birefringence or refractive index (e.g., surface plasmon resonance, ellipsometry, a resonant mirror method, a grating coupler waveguide method (e.g., wavelength-interrogated optical sensor) or interferometry). Optical methods include microscopy (both confocal and non-confocal), imaging methods and non-imaging methods. Immunoassays in various formats (e.g., ELISA) are popular methods for detection of analytes captured on a solid phase.
Electrochemical methods include voltametry and amperometry methods. Radio frequency methods include multipolar resonance spectroscopy.

Example 1: Complex Formation of a-Cyano-4-Hydroxylcinnamic Acid/(i-Cyclodextrin [0152] 1.26 g of 0-cyclodextrin (Aldrich) was dissolved in 30 g of DI H20/DMF
mixture (w/w, 7.5:1) at 60 C. To this clear solution was added 0.21 g of a-cyano -4-hydroxylcinnamic acid (CHCA, Aldrich). The suspension was shaken for 12 h at 60 C. An optically clear solution then was obtained. The solvent mixture was frozen and removed by lyophilyzer, and a yellowish solid product was obtained.

[0153] Control experiment: to a solution of 30 g of DI H20/DMF mixture (w/w, 7.5:1) was added 0.21 g of CHCA. The suspension was shaken for 12 h at 60 C. The CHCA in the suspension remained undissolved.

[0154] The above experiments confirmed that the CHCA guest was included into a complex with cyclodextrin. Disappearance of the guest in the aqueous solution in the presence of 0-cyclodextrin host is confirmatory of the formation of the complex. In contrast, CHCA remained insoluble in the absence of [3-cyclodextrin in the control experiment.
Example 2: Complex Formation of Sinapinic Acid/(3-Cyclodextrin [0155] 1.3 g of (3-cyclodextrin (Aldrich) was dissolved in 30 g of DI H2O/DMF
mixture (w/w, 7.5:1) at 60 C. To this clear solution was added 0.257 g of sinapinic acid (SPA, Aldrich). The suspension was shaken for 12 h at 60 C. An optically clear solution was then obtained. The solvent mixture was frozen and removed by lyophilyzer, and a solid product was obtained.

[0156] Control experiment: to a solution of 30 g of DI H2O/DMF mixture (w/w, 7.5:1) was added 0.257 g of SPA. The suspension was shaken for 12 h at 60 C. The SPA
in the suspension remained undissolved.

[0157] The above experiments confirmed that the SPA guest was included into a complex with cyclodextrin. The disappearance of the guest into the aqueous solution in the presence of P-cyclodextrin host is confirmatory of complex formation. In contrast, in the absence of (3-cyclodextrin, SPA remained insoluble in the control experiment.

Example 3: Complex Formation of a-Cyano-4-Hydroxylcinnamic Acid/(3-Cyclodextrin Polymer [0158] 1.5 g of 0-cyclodextrin polymer (CarboMer, Inc. Cat. # 4-00236) was dissolved in 25 g of DI H20/DMF mixture (w/w, 85:15) at 60 C. To this clear solution was added 0.21 g of CHCA. The suspension was shaken for 12 h at 60 C. An optically clear solution then was obtained. The solvent mixture was frozen and removed by lyophilyzer, and a yellowish solid product was obtained.

[0159] Control experiment: to a solution of 25 g of DI H2O/DMF mixture (w/w, 85:15) was added 0.21 g of CHCA. The suspension was shaken for 12 h at 60 C. The CHCA in the suspension remained undissolved.

[0160] The above experiments confinned that the CHCA guest was included into a complex with cyclodextrin. The disappearance of the guest into the aqueous solution in the presence of 0-cyclodextrin polymer host is confirmatory of complex formation.
In contrast, CHCA remained insoluble in the control experiment with the absence of 0-cyclodextrin polymer.

Example 4: Complex Formation of Sinapinic Acid/(3-Cyclodextrin Polymer [0161] 1.5 g of 0-cyclodextrin polymer (CarboMer, Inc. Cat. # 4-00236) was dissolved in g of DI H20/DMF mixture (w/w, 85:15) at 60 C. To this clear solution was added 0.257 g of SPA. The suspension was shaken for 12 h at 60 C. An optically clear solution was then 20 obtained. The solvent mixture was frozen and removed by lyophilyzer, and the solid product was obtained.

[0162] Control experiment: to a solution of 25 g of DI H20/DMF mixture (w/w, 85:15) was added 0.257 g of SPA. The suspension was shaken for 12 h at 60 C. The SPA
suspension remained undissolved.

25 [0163] The above experiments confirmed that the SPA guest was included into a complex with cyclodextrin. The disappearance of the guest into the aqueous solution in the presence of R-cyclodextrin polymer host is confirmatory of complex formation. In contrast, in the absence of the absence of 0-cyclodextrin polymer, SPA remained insoluble in the control experiment.

Example 5: SELDI of Cytochrome C Using SPA/(3-cyclodextrin [0164] SPA/0-cyclodextrin complexes were used as an EAM in a SELDI analysis to assist the desorption and ionization of cytochrome C under typical laser condition. For instructions of using SELDI and ProteinChip, see, for example, WO 00/66265 (Rich et al., "Probes for a Gas Phase Ion Spectrometer," November 9, 2000). The EAM complex of the invention was applied to an analyte in the same way as a non-complexed matrix.

[0165] The following is an example of a protocol for using an exemplary EAM
complex.
(1) DI H20 (200 L) was added to 2 mg of SPA/(3-cyclodextrin complex to make a clear EAM solution;
(2) To a NP 20 ProteinChip, each spot was added with various amount of the EAM
solution;
(3) After the EAM solution was dried, -10 pmol of cytochrome C dissolved in 0.1 wt%
of trifluoroacetic acid (TFA) was added onto the spot;
(4) After the spot is dried, 2 L of ethanol was added onto each spot to extract SPA out of the cavity of (3-cyclodextrin;
(5) Dried the array and read in PBS IIC instrument.
[0166] FIG. 4 and FIG. 5 show the mass spectrum of cytochrome C using a SPA/[3-cyclodextrin EAM complex to assist the desorption and ionization under typical analytical parameters. The results indicate that the SPA/0-cyclodextrin EAM complex acts as a EAM
to promote the desorption and ionization of cytochrome C. The sample was treated with ethanol to extract SPA out of the cavity before the desorption and ionization experiment.
Example 6: SELDI of Protein Mixture Using SPA/[3-Cyclodextrin [0167] In another SELDI analysis, a SPA/0-cyclodextrin polymer complex prepared according to Example 4 was used as an EAM to promote the desorption and ionization of ALL-in-1 protein standard under typical laser condition. For a description of SELDI and ProteinChip, see, for example, WO 00/66265 (Rich et al., "Probes for a Gas Phase Ion Spectrometer," November 9, 2000). ALL-in-1 protein standard is a product available from Ciphergen Biosystems, Inc. The components of the All-in-1 protein are hirudin BHVK (MW
7,034 Dalton), cytochrome C (MW 12,230 Dalton), myoglobin (MW 16,951 Dalton), carbonic anhydrase (MW 29,023 Dalton), enolase (MW 46,671 Dalton), albumin (MW
66,433 Dalton), IgG (MW 147,300 Dalton).

[0168] The following is an example of a protocol using a host-guest EAM
complex.

(1) To 200 L of 0.1 wt% of TFA aqueous solution was added to 2 mg of SPA/0-cyclodextrin polymer complex to make a clear EAM solution;
(2) One vial of ALL-in-1 protein was dissolved in 25 L of 20 mM ammonium acetate;
(3) To a NP 20 ProteinChip, each spot was added with 1 L of ALL-in-1 protein solution followed with 1 L of DI H20;
(4) After the solution was dried, 2.5 L of EAM solution was added onto each spot twice.
(5) After the spot is dried, 2 L of ethanol was added onto each spot to extract SPA out of the cavity of (i-cyclodextrin polymer;
(6) Dried the array and read in PBS II C instrument.
[0169] FIG. 6 shows the mass spectrum of ALL-in-1 protein profile in the mass range 5,000-70,000 Daltons using an SPA/(3-cyclodextrin polymer EAM complex to promote the desorption and ionization process under typical laser conditions. The spectrum included the expected protein peaks. The results indicate that SPA/[i-cyclodextrin polymer acts as a EAM
to promote the desorption and ionization of the components of ALL-in-1 protein. The sample was treated with ethanol to extract SPA out of the cavity before the desorption and ionization experiment.

[0170] It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes.

Claims (44)

1. A complex comprising:
molecular host complexed with an energy absorbing molecule that absorbs energy from a photo-irradiation source and thereby contributes to desorption and ionization of analyte molecules in operative contact therewith.

2. The complex according to claim 1 wherein said host is a macrocycle.

3. The complex according to claim 2 wherein said macrocycle is a member selected from crowns, cryptands and cyclodextrin.

4. The complex according to claim 1, wherein said energy absorbing molecule comprises the structure:
Ar~R1~C(O)R1a wherein Ar is a member selected from aryl and heteroaryl;
R1 is a member selected from a bond, substituted or unsubstituted alkyl and substituted or unsubstituted heteroalkyl; and R1a is a member selected from H, OH and substituted or unsubstituted alkyl.

5. The complex according to claim 4, wherein Ar is a member selected from substituted or unsubstituted phenyl, substituted or unsubstituted indolyl and substituted or unsubstituted pyridyl.

6. The complex according to claim 5, wherein Ar is a member selected from:
wherein R2, R3, and R4 are members independently selected from H and substituted or unsubstituted alkyl.

7. The complex according to claim 6, wherein R2, R3, and R4 are members independently selected from H and C1-C6 unsubstituted alkyl.

8. The complex according to claim 4, wherein R1 has the formula:

wherein R5 and R6 are members independently selected from H, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, and CN.

9. The complex according to claim 8, wherein R1 has a formula that is a member selected from:

10. The complex according to claim 6, wherein said energy absorbing molecule is a member selected from ferulic acid, caffeic acid, cinnamic acid, .alpha.-cyano-4-hydroxycinnamic acid, sinapic acid, picolinic acid, nicotinic acid, 2,5-dihydroxybenzoic acid, 2-aminobenzoic acid, acetamide, salicylamide, isovanillin and trans-3-indoleacrylic acid.

11. The complex according to claim 1 wherein said complex is a component of a polymer.

12. The complex according to claim 11, wherein said polymer comprises a member selected from a binding functionality and a reactive functionality.

13. The complex according to claim 12, wherein said binding functionality and said reactive functionality are members independently selected from an electrostatic functionality, a hydrophobic functionality, a hydrogen bonding functionality, a coordinate covalent bonding functionality, a covalent bonding functionality, an epoxide functionality, a carbodiimidizole functionality, a biospecific bonding functionality and combinations thereof.

14. The complex according to claim 11, wherein said polymer is a blend comprising said complex and a monomer or polymer comprising a binding functionality.

15. The complex according to claim 11, wherein said polymer is a cross-linked polymer.

16. The complex according to claim 15, wherein said cross-linked polymer comprises a binding functionality.

17. The complex according to claim 11, wherein said polymer comprises a cyclodextrin moiety.

18. The complex according to claim 1, wherein said complex is immobilized on a solid support.

19. The complex according to claim 1, wherein said molecular host comprises a cyclodextrin moiety and at least one hydroxyl group of said cyclodextrin moiety is functionalized as:

wherein R7 is a moiety that comprises a binding functionality.

20. The complex according to claim 19, wherein an analyte is immobilized on said binding functionality.

21. A kit comprising:
(a) a complex according to claim 1; and (b) a substrate adapted to be positioned within a sample chamber of a mass spectrometer.

22. A device comprising:
(a) a substrate having a surface comprising a complex, said complex comprising a molecular host complexed with an energy absorbing molecule that absorbs energy from a photo-irradiation source and thereby contributes to desorption and ionization of analyte molecules in operative contact therewith.

23. The device according to claim 22, wherein said energy absorbing molecule comprises a moiety comprising an aryl nucleus having a substituent thereon, said substituent comprising a carbonyl or carboxyl group conjugated to said .pi.-system of said aryl nucleus.

24. The device according to claim 23, wherein said molecular host is attached to said substrate.

25. The device according to claim 24, wherein said molecular host is attached to said surface by physical adhesion.

26. The device according to claim 24, wherein said molecular host is attached to said surface by covalent attachment.

27. The device according to claim 26, wherein said covalent attachment results from a polymerization reaction between a polymerizable moiety on said substrate surface and a polymerizable moiety of said molecular host.

28. The device according to claim 22, further comprising an analyte adsorbed onto said molecular host.

29. The device according to claim 28, wlierein said analyte is adsorbed onto said molecular host through an interaction between said analyte and a binding functionality on said molecular host.

30. The device according to claim 22, wherein said complex is a component of a polymer.

31. The device according to claim 30, wherein said polymer comprises a member selected from a binding functionality and a reactive functionality.

32. The device according to claim 31, wherein said binding functionality and said reactive functionality are members independently selected from an electrostatic functionality, a hydrophobic functionality, a hydrogen bonding functionality, a coordinate covalent bonding functionality, a covalent bonding functionality, an epoxide functionality, a carbodiimidizole functionality, a biospecific bonding functionality and combinations thereof.

33. The device according to claim 30, wherein said polymer is a blend comprising said complex and a monomer or polymer comprising a binding functionality.

34. The device according to claim 30, wherein said polymer is a cross-linked polymer.

35. The device according to claim 34, wherein said cross-linked polymer comprises a binding functionality.

36. The device according to claim 30, wherein said polymer comprises a cyclodextrin moiety.

37. The device according to claim 22, wherein said substrate comprises an electrically conductive material.

38. The device according to claim 22, wherein said substrate comprises means for engaging a probe interface of a mass spectrometer.

39. The device according to claim 30, wherein said polymer is distributed on said substrate in a plurality of addressable locations.

40. A method of detecting an analyte comprising:
(a) desorbing said analyte from a surface on which said analyte is adsorbed, and ionizing said analyte;
said surface comprising a host-guest complex, said complex comprising a molecular host complexed with an energy absorbing molecule that absorbs energy from a photo-irradiation source and thereby contributes to desorption and ionization of analyte molecules in operative contact therewith;
and (c) detecting the desorbed, ionized analyte.

41. The method of claim 40, comprising detecting said analyte by laser desorption/ionization mass spectrometry.

42. A water-soluble energy-absorbing EAM for laser desorption/ionization mass spectrometry comprising an energy-absorbing molecule complexed to a water-soluble molecular host.

43 43. ~The EAM according to claim 42 wherein said water-soluble molecular host is a cyclodextrin.

44. ~The EAM according to claim 43 wherein said energy absorbing molecule is a member selected from ferulic acid, caffeic acid, cinnamic acid, .alpha.-cyano-4-hydroxycinnamic acid, sinapic acid, picolinic acid, nicotinic acid, 2,5-dihydroxybenzoic acid, 2-aminobenzoic acid, acetamide, salicylamide, isovanillin and trans-3-indoleacrylic acid.